research & patent summary for blis oral-cavity probiotics ... · 6) di pierro f, adami t,...
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Research Summary
Research & Patent Summary for BLIS Oral-cavity Probiotics: BLIS K12™ & BLIS M18™
Published: Wednesday, August 22, 2017
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stratumnutrition.com [email protected] (800) 970-4479
*These statements have not been evaluated by the Food and Drug Administration. This product is not intended to diagnose, treat, cure, or prevent any disease.
Research & Patent Portfolio for BLIS Probiotics: BLIS K12™ & BLIS M18™ BLIS K12 and BLIS M18 are highly advanced probiotics for the oral cavity and upper respiratory tract. BLIS K12 colonizes in the mouth and helps to maintain ear, nose and throat (ENT) immune health, while BLIS M18 supports tooth and gum health. BLIS probiotics were originally discovered as scientists studied the oral microbiome of a child with exceptional throat health. Both BLIS K12 and BLIS M18, or Streptococcus salivarius (S. salivarius), were isolated from the mouths of healthy humans and can help to establish or re-establish a healthy balance of beneficial bacteria in the oral cavity and upper respiratory tract.
Published Research in Peer-reviewed Journals:
ENT Health1) Di Pierro F, Colombo M, Giuliani MG, Danza ML, Basile I, Bollani, T, Conti AM, Zanvit A, and Rottoli AS. (2016) Effect of administration of Streptococcus salivarius K12 on the occurrence of streptococcal pharyngo-tonsillitis, scarlet fever, and acute otitis media in 3 years old children. European Review for Medical and Pharmacological Sciences, 20:4601-4606.
2) Di Pierro F, Colombo M, Zanvit A, and Rottoli AS. (2016) Positive clinical outcomes derived from using Streptococcus salivarius K12 to prevent streptococcal pharyngitis in children: a pilot investigation. Drug Healthcare and Patient Safety, 8:77-81.
3) Gregori G, Righi O, Risso P, Boiardi G, Demuru G, Ferzetti A, Galli A, Ghisoni M, Lenzini S, Marenghi C, Mura C, Sacchetti R and Suzzani L. (2016) Reduction of group A beta-hemolytic streptococcus pharyngo-tonsillar infections asso-ciated with use of the oral probiotic Streptococcus salivarius K12: a retrospective observational study. Therapeutics and Clinical Risk Management, 12:87-92.
4) Di Pierro F, Di Pasquale D and Di Cicco M. (2015) Oral use of Streptococcus salivarius K12 in children with secretory otitis media: preliminary results of a pilot, uncontrolled study. International Journal of General Medicine, 8:303-308.
5) Di Pierro F, Colombo M, Zanvit A, Risso P and Rottoli A. (2014) Use of Streptococcus salivarius K12 in the prevention of streptococcal and viral pharyngotonsillitis in children. Drug, Healthcare and Patient Safety, 6:15-20.
6) Di Pierro F, Adami T, Rapacioli G, Giardini N and Streitberger C. (2013) Clinical evaluation of the oral probiotic Strep-tococcus salivarius K12 in the prevention of recurrent pharyngitis and/or tonsillitis caused by Streptococcus pyogenes in adults. Expert Opinion Biological Therapy, 13(3):339-343.
7) Di Pierro F, Donato G, Fomia F, Adami T, Careddu D, Cassandro C and Albera R. (2012) Preliminary pediatric clinical evaluation of the oral probiotic Streptococcus salivarius K12 in preventing recurrent pharyngitis and/or tonsillitis caused by Streptococcus pyogenes and recurrent acute otitis media. International Journal of General Medicine, 5:991-997.
stratumnutrition.com [email protected] (800) 970-4479
*These statements have not been evaluated by the Food and Drug Administration. This product is not intended to diagnose, treat, cure, or prevent any disease.
Fresh Breath8) Jamali Z, Aminabadi N A, Samiei M, Deljavan A S, Shokravi M and Shirazi S. (2016) Impact of Chlorhexidine Pretreat-ment Followed by Probiotic Streptococcus salivarius Strain K12 on Halitosis in Children: A Randomised Controlled Clinical Trial. Oral Health & Preventive Dentistry, 14(4):305-313.
9) Masdea L, Kulik E, Hauser-Gerspach I, Ramseier A, Filippi A and Waltimo T. (2012) Antimicrobial activity of Strepto-coccus salivarius K12 on bacteria involved in oral malodour. Archives of Oral Biology, 57(8):1041-1047.
Patent Portfolio:
US 8,057,790 “Treatment of malodour” (2011). This invention relates to methods for inhibiting growth of anaerobic bac-teria, particularly halitosis causing bacteria. The methods use BLIS-producing Streptococcus salivarius strains, extracts thereof, and compositions comprising same in the prevention or treatment of halitosis.
US 7,595,041 “Treatment of Malodour (2009). This invention relates to methods for inhibiting growth of anaerobic bac-teria, particularly halitosis causing bacteria. The methods use BLIS-producing Streptococcus salivarius strains, extracts thereof, and compositions comprising same in the prevention or treatment of halitosis.
EP 1 169 340 “Lantibiotic” (2007). US 6,773,912 “Lantibiotic” (2004). This invention provides an antibacterial protein, Salivaricin B. Salivaricin B is bacteriocidal with respect to, inter alia, S. pyogenes and therefore has numerous therapeutic applications. These applications include, but are not limited to, forming part of therapeutic formulations for use in treating or preventing streptococcal infections of the throat.
Published Research in Peer-reviewed Journals:
Dental Health1) Di Pierro F, Zanvit A, Nobili P, Risso P, Fornaini C. (2015) Cariogram outcome after 90 days of oral treatment with Streptococcus salivarius M18 in children at high risk for dental caries: results of a randomized, controlled study. Clinical, Cosmetic and Investigational Dentistry, 7:107-113.
2) Scariya L, Nagarathna D and Varghese M. (2015) Probiotics in Periodontal Therapy. International Journal of Pharma and Bio Sciences, 6(1):242-250.
3) Burton J, Drummond B, Chilcott C, Tagg J, Thomson W, Hale J and Wescombe P. (2013) Influence of the probiotic Streptococcus salivarius strain M18 on indices of dental health in children: a randomized double-blind, placebo-controlled trial. Journal of Medical Microbiology, 62(6):875-884.
Patent Portfolio:
EP 1 483 366 “Antimicrobial Composition” (2010). US 7,226,590 “Antimicrobial Composition” (2007). This invention provides novel Streptococcus salivarius, compositions containing same, and use of S. salivarius strains as antimicrobial agents. The strains are bacterial inhibitors with respect to at least S. mutans and/or MS and therefore have a number of therapeutic applications. The applications include but are not limited to forming part of therapeutic formulations for use in controlling, treating, or preventing dental caries.
Abstract. – OBJECTIVE: Streptococcus sali-varius K12 (BLIS K12) is a probiotic strainstrongly antagonistic to the growth of Strepto-coccus pyogenes, the most important bacterialcause of pharyngeal infections in humans.Shown to colonize the oral cavity and to be safefor human use, BLIS K12 has previously been re-ported to reduce pharyngo-tonsillitis episodes inchildren or adults known to have experienced re-current streptococcal infection. The presentstudy was focussed upon evaluating the role ofBLIS K12 in the control of streptococcal diseaseand acute otitis media in children attending thefirst year of kindergarten.
PATIENTS AND METHODS: By randomization,222 enrolled children attending the first year ofkindergarten were divided into a treated group(N = 111) receiving for 6 months a daily treat-ment with BLIS K12 (Bactoblis®) and a controlgroup (N = 111) who were monitored as untreat-ed controls. During the 6 months of treatmentand 3 months of follow-up, the children wereevaluated for treatment tolerance, and forepisodes of streptococcal pharyngo-tonsillitis,scarlet fever and acute otitis media.
RESULTS: During the 6-month trial (N = 111per group) the incidence of streptococcalpharyngo-tonsillitis, scarlet fever and acute oti-tis media was approximately 16%, 9% and 44%respectively in the treated group and 48%, 4%and 80% in the control group. During the 3-months follow-up (N = 29 per group) the corre-sponding rates of infection were 15%, 0% and12% in the treated group and 26%, 6% and 36%in the controls. No apparent side effects weredetected in the treated group either during treat-
European Review for Medical and Pharmacological Sciences
Effect of administration of Streptococcussalivarius K12 on the occurrence ofstreptococcal pharyngo-tonsillitis, scarlet feverand acute otitis media in 3 years old children
F. DI PIERRO1, M. COLOMBO2, M.G. GIULIANI3, M.L. DANZA4, I. BASILE4,T. BOLLANI5, A.M. CONTI6, A. ZANVIT7, A.S. ROTTOLI8
1Scientific Department, Velleja Research, Milan, Italy2ATS, District 5, Milan, Italy3ATS District 8, Milan, Italy4ATS, District 6, Milan, Italy5ATS, District 4, Milan, Italy6ATS, District 7, Milan, Italy7Biological Dentistry Department, Italian Stomatology Institute, Milan, Italy8Pediatric Department, “Uboldo Hospital”, Cernusco S/N, Milan, Italy
Corresponding Author: Francesco Di Pierro, Ph.D; e-mail [email protected] 4601
ment or follow-up. All of the enrolled childrencompleted the study.
CONCLUSIONS: The daily administration ofBLIS K12 to children attending their first year ofkindergarten was associated with a significantreduction in episodes of streptococcal pharyngi-tis and acute otitis media. No protection againstscarlet fever was detected.
Key Words:Paediatric infections, Pharyngo-tonsillitis, Scarlet
fever, Acute otitis media, Blis K12, Bactoblis®.
Introduction
Streptococcus salivarius K12 (hereafter BLISK12) is a probiotic strain shown to strongly in-hibit the in vitro growth of Streptococcus pyo-genes, Streptococcus pneumonia, Haemophilusinfluenza, and Moraxella catarrhalis principaletiological agents respectively of bacterialpharyngo-tonsillitis and acute otitis media1,2. Thisantagonism seems to be due to the release of thelantibiotics salivaricin A2 and salivaricin B3. Af-ter oral administration, BLIS K12 colonizes theoral cavity, nasopharynx and adenoids4 persistingthere for up to one month after the last dose5. It isantibiotic-sensitive6 and has a thoroughly-investi-gated safety profile7. From a clinical perspective,administration of BLIS K12 has been shown to
2016; 20: 4601-4606
F. Di Pierro, M. Colombo, M.G. Giuliani, M.L. Danza, I. Basile, et al.
Exclusion CriteriaChildren were excluded from the study if they
were immunocompromised, had undergone ton-sillectomy or had an indication for adeno-tonsil-lectomy. Other exclusion criteria included a his-tory of rheumatic disorders, bronchospasmand/or a diagnosis of asthma and/or allergy; a di-agnosed respiratory or significant systemic disor-der. Also excluded were children who were eitherundergoing current pharmacological therapies toprevent recurrent respiratory infections or whopresented with conditions that could favour thedevelopment of acute otitis media, including se-vere atopy, acquired or congenital immunodefi-ciency, cleft palate, a chronically rupturedeardrum, craniofacial abnormalities or obstruc-tive adenoids, sleep apnoea syndrome or place-ment of tympanostomy tubes.
Study PatternAll individuals enrolled were first subjected to
a general medical examination and pharyngealswab (Test Strep-A, Gima, Gessate, Italy) andthen were randomized by tossed coin in twogroups: a treated group daily administered, for 6months, with BLIS K12 in the form of Bactoblis®
tablets and an untreated group not receiving anytreatment and simply monitored as control group.The parents of the children in the BLIS K12group were instructed on how to use the product.The tablets were to be administered for 180 con-secutive days. The children had to let one tabletdissolve slowly in the mouth immediately beforegoing to sleep, after brushing their teeth. The chil-dren were to be carefully instructed not to chewthe tablets or to swallow them whole. Further-more, they should not drink or swallow anythingelse just following the use of the product. For thetrial period, it was requested that at the first signof any oropharyngeal symptoms of infection thechildren should be brought to the clinic for an im-mediate medical examination and pharyngealswab test. In the case of a positive result, treat-ment was prescribed. The prescribed therapy forstreptococcal infection was a combination ofamoxicillin and clavulanic acid to be adminis-tered for 10 days. Following antibiotic therapy,treatment with BLIS K12 was resumed and con-tinued until the scheduled 180th day of the study.Infections accompanied by pharyngo-laryngealpain and/or a fever were treated with aceta-minophen or ibuprofen. Diagnosis of scarletfever13 and acute otitis14 media was done on thebasis of the microbial and clinical evidence and
reduce re-occurrences of streptococcal and viralpharyngo-tonsillitis as well as acute and secreto-ry otitis media8-12. A common denominator ofthese previous studies has been the enrolment ofsubjects having a history of recurrent streptococ-cal, infection, with no less than three episodesper year as demonstrated by culture growth ofStreptococcus pyogenes. By contrast, in the pre-sent study, we have evaluated whether BLIS K12could provide protection for children who wereattending their first year of kindergarten and whohad no recent history of recurrent streptococcalpharyngo-tonsillitis or acute otitis media.
Patients and Methods
ProductBLIS K12 was formulated as slowly-dissolv-
ing oral tablets by SIIT (Trezzano S/N, Milan,Italy) and notified to the Italian Ministry ofHealth as Bactoblis® by Omeopiacenza (Pon-tenure, Italy), according to the provisions of lawNo 169 of 2004, on July 5th, 2011 (notificationnumber: 53435). The preparation Bactoblis® usedin the clinical trial contained no less than 1 bil-lion CFU/tablet of Streptococcus salivarius K12(Blis Technologies Ltd., New Zealand).
Clinical TrialThis multicentre, open-label, randomized, con-
trolled clinical trial was conducted on 222 chil-dren (116 females and 106 males aged 33-45months) enrolled in the area of Milan (Italy). Thechildren were treated (N = 111) or untreated (N =111), between September 2015 and March 2016with Bactoblis®. Between April and July 2016 (N= 29 per group) a 90-days follow-up was per-formed. The trial was conducted according to thecriteria set by the Declaration of Helsinki andwith the approval of the local (Milan, Italy)Ethics Committee. The parents of all the partici-pants in the study were informed of the trialmethods and signed the appropriate consent andprivacy policy documents.
Inclusion CriteriaAt the time of enrolment, all of the children
were around 3 years of age and were soon to at-tend the first year of kindergarten. All partici-pants were free of streptococcal disease, as estab-lished by a rapid throat swab test for group Astreptococcus. None were clinically ill on enrol-ment.
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Treated Untreated
Total number 111 111Males 50 56Age of males (months ± SD) 36 ± 3.2 35 ± 3.0Females 61 55Age of females (months ± SD) 34 ± 3.0 35 ± 3.6Previous streptococcal episodes 16 14Previous AOM episodes 4 3Italians 75 69Arabs/Africans 30 33Asians 6 9Pneumococcal vaccine^ 9 10Naturally delivered 72 75Caesarean delivered 39 36Weight at birth (kg ± SD) 3.2 ± 0.7 3.3 ± 0.8Breastfed 39 42With older brothers 51 56Nursery attenders 45 51
Table I. Features* of the children (222) enrolled and ending the study.
*None of the features is significantly different between the two groups; ^PCV13; SD: standard deviation.
performed by trained investigators. Any otherpathologies possibly occurring during the studywere treated according to the recommendations ofthe Italian Paediatric guidelines.
Study AimsThe present study aimed to evaluate the fol-
lowing: (1) the onset of side effects or symptomsof toxicity while the product was being adminis-tered; (2) the efficacy of BLIS K12 in the preven-tion of Streptococcus pyogenes infections(pharyngo-tonsillitis and scarlet fever) during 6-months of treatment and a 3-month follow-up pe-riod; 3) the efficacy of BLIS K12 in reducing theoccurrence of acute otitis media.
Statistical AnalysisThe equivalence of the two subject groups was
determined using Fisher’s exact test and the two-tailed Wilcoxon-Mann-Whitney test respectively.The difference in terms of numbers of streptococ-cal pharyngo-tonsillitis, scarlet fever and acute oti-tis media episodes was determined using the two-tailed Wilcoxon-Mann-Whitney test. Statisticalsoftware used was JMP 10 for Mac OsX and thethreshold for statistical significance was 95%.
Results
Children (N = 222) attending the first year ofkindergarten in the area of Milan (Italy) were en-
rolled and randomized into two groups of 111children, one of which was treated, and the othernot treated for 6-months by daily administrationof Bactoblis® tablets to effect slow release intothe oral cavity of the anti-streptococcal probioticstrain BLIS K12. After this period some of thechildren (N = 29 per group) continued to bemonitored for a further three months (follow-up).None of the enrolled children presented charac-teristics of streptococcal recurrence and/or wereassessed as otitis media-prone. Compliancethroughout the 180 days of Bactoblis® treatmentwas assessed as very good; no side effects werereported and none of the children were with-drawn from the study (data not shown). Since, asshown in Table I, the two groups did not exhibitsignificantly different characteristics in terms ofage, sex, previous streptococcal or acute otitismedia episodes, ethnicity, pneumococcal vac-cine, type of delivery, weight at birth, type offeeding, presence of older brothers and previousattendance at nursery school, their backgroundswere considered to be comparable. Table IIshows the number of children diagnosed withstreptococcal pharyngo-tonsillitis, scarlet feverand acute otitis media during the 6-month treat-ment period. Eighteen of 111 (16.2%) of thetreated group and 54 of 111 (48.6%) in the con-trol group were diagnosed with streptococcalthroat infections. On the other hand, no statisticaldifference was found for episodes of scarlet fever(10 of the treated group versus 7 of the controls).
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BLIS K12 in 3 years old children
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A diagnosis of acute otitis media was made for49 (44.1%) of the 111 Bactoblis®-treated chil-dren and this was significantly fewer than the 89(80.2%) of 111 control group children having apositive diagnosis. Further analysis of the diag-nosed episodes of streptococcal throat infection(Table III) shows that in the treated group 16children experienced a single infection, one childhad two infections and another child had threeepisodes during the 6-month treatment period,for a total of 21 episodes. By comparison, in thecontrol group 67 streptococcal throat infectionswere diagnosed, with 45 children each having asingle episode, five with two episodes and fourchildren having three episodes. A similar situa-tion was observed for total episodes of acute oti-tis media: in the treated group 46 children had asingle episode, two children each experiencedtwo episodes and another child had threeepisodes giving a total of 53 episodes versus 101episodes in the control group, with 80 childrenhaving one episode, six having two episodes andthree with three episodes reported. During the 3-month follow-up period (Table IV) five (17.2%)of the 29 children in the treated group experi-enced streptococcal pharyngo-tonsillitis and four(13.8%) were reported to have single episodes ofacute otitis media. None of these children devel-oped scarlet fever. No significant difference inthe occurrence of streptococcal infections duringthe follow-up period was observed for the controlgroup, with eight (27.6) of the 29 children expe-riencing infection and two cases of scarlet feverreported. Nevertheless, a significant difference
was found for reported cases of acute otitis me-dia with 12 (41.3%) of the 29 children infected inthe control group.
Discussion
Achieving oral health benefits from probiotictherapy has recently become possible with thedevelopment of novel probiotics such as BLISK12 selected from the oral cavity commensalspecies Streptococcus salivarius. The BLIS K12strain was originally isolated from the oral cavityof a young child who had no recent experience ofS. pyogenes infection. The lantibiotic bacteri-ocins produced by this strain have subsequentlybeen shown to be inhibitory not only to S. pyo-genes but also to other oral cavity bacterialpathogens associated with acute otitis media2 andhalitosis15,16. Previous investigations have demon-strated that the oral administration to healthy vol-unteers of BLIS K12 reduces IL-8 plasma con-
F. Di Pierro, M. Colombo, M.G. Giuliani, M.L. Danza, I. Basile, et al.
PT % SF % AOM %
Treated 18* 16.2 10 9.0 49* 44.1Control 54 48.6 7 6.3 89 80.2
Table II. Number of children with pharyngo-tonsillitis (PT), scarlet fever (SF) and acute otitis media (AOM) during the 6-months treatment period in the two study groups (N=111/group).
*p < 0.01 vs. control.
PT AOM(1, 2, 3 episodes) (1, 2, 3 episodes)
Treated 21* (16, 1, 1) 53* (46, 2, 1)Control 67 (45, 5, 4) 101 (80, 6, 3)
Table III. Total episodes of pharyngo-tonsillitis (PT) andacute otitis media (AOM) during the 6-months treatment pe-riod in the two study groups (N=111/group). Into bracketsnumber of children with 1, 2 or 3 episodes.
*p < 0.01 vs. control.
PT % SF % AOM %
Treated 5 17.2 0 0 4* 13.8Control 8 27.6 2 6.9 12 41.3
Table IV. Number of children with pharyngo-tonsillitis (PT), scarlet fever (SF) and acute otitis media (AOM) during the 3-months follow-up in the two study groups (N=29/group).
*p < 0.051 vs. control.
centrations and increases salivary γ-interferon.2
These modulations may also rationally accountfor the anti-inflammatory, immuno-modulatingand anti-viral activity recently observed by ourgroup17,18. All of the clinical trials performed todate on BLIS K12 have aimed to reduce strepto-coccal pharyngo-tonsillitis or acute otitis mediaepisodes in subjects already having a clear histo-ry of recurrent streptococcal disease or otitis me-dia. It has not however yet been establishedwhether the prophylactic administration of BLISK12 can help provide a clinical benefit to indi-viduals not known to have a predilection to strep-tococcal infection or otitis media. The results ofthe current study provide some preliminary sup-port for this proposition. BLIS K12 prophylaxisgiven to 3 years old children attending the firstyear of kindergarten and who did not yet seem tobe either streptococcal pharyngitis or acute otitismedia prone, appeared to highlight a reduction inepisodes of both of these infections, the treatedchildren being protected by about 60% and 50%respectively by comparison with the children inthe control group.Also, the consideration of subjects who expe-
rienced infection recurrences appears to show abeneficial effect from the use of BLIS K12. In-deed, only two subjects of the treated group ver-sus nine of the controls were found to have recur-rent streptococcal throat infections. Also, onlythree of the treated group had recurrences ofacute otitis media by comparison with nine of thecontrol subjects. Perhaps anomalously the BLISK12 treatment did not seem to create any benefitin terms of prevention of scarlet fever episodes.Although this pathology occurs as a result ofStreptococcus pyogenes infection, its main char-acteristic, skin rash, is due to the release of ery-throgenic toxins19 that the bacterium produceswhen is infected by a specific phage20. One pos-sible hypothesis is that Streptococcus salivariusK12 more effectively antagonizes the replicationof Streptococcus pyogenes strains that are notphage-infected. Another possible theory is thatalthough strain K12 may have killed the phage-infected streptococci, the latter may still have re-leased rash-inducing concentrations of toxins in-to the throat tissues. The prophylactic roleagainst streptococcal throat infections andepisodes of otitis media played by BLIS K12during the 6-month treatment period seems alsoto have been maintained for acute otitis mediaduring the 3-month washout period. Even in thiscase, there is a possible bias, since the relatively
small number of subjects (29 per group), who ac-cepted to be monitored during the follow-upcould have led to an absence of significant resultsin terms of episodes of streptococcal throat infec-tions.
Conclusions
Limitations of the present study include theabsence of blind randomisation and a totally un-treated control group. Nevertheless, the resultsappear to demonstrate for the first time that theprophylactic administration of BLIS K12 couldgenerate positive clinical outcomes even in veryyoung children who have not been pre-selectedas recurrent streptococcal infection or acute otitismedia-prone. Follow-up studies should be con-ducted to establish whether BLIS K12 is forsome reason specifically non-protective againstdevelopment of scarlet fever. These studiesshould include in vitro tests of the action of BLISK12 against scarlet fever toxin-positive and -neg-ative strains of Streptococcus pyogenes and alsoa double-blind, placebo-controlled trial contain-ing a large number of young scarlet fever vulner-able subjects.
–––––––––––––––––-––––Conflict of InterestFDP is the main formulator of the tested product and he isinvolved in the Scientific Council of the Company (Omeopi-acenza®) trading the tested product. The other authors do notreport any conflict of interest.
References
1) WESCOMBE PA, BURTON JP, CADIEUX PA, KLESSE NA,HYINK O, HENG NC, CHILCOTT CN, REID G, TAGG JR.Megaplasmids encode differing combinations oflantibiotics in Streptococcus salivarius. AntonieVan Leeuwenhoek 2006; 90: 269-280.
2) WESCOMBE PA, HALE JD, HENG NC, TAGG JR. Devel-oping oral probiotics from Streptococcus salivar-ius. Future Microbiol 2012; 7: 1355-1371.
3) HYINK O, WESCOMBE PA, UPTON M, RAGLAND N, BUR-TON JP, TAGG. Salivaricin A2 and the novel lantibiot-ic salivaricin B are encoded at adjacent loci on a190-kilobase transmissible megaplasmid in theoral probiotic strain Streptococcus salivarius K12.Appl Environ Microbiol 2007; 73: 1107-1113.
4) POWER DA, BURTON JP, CHILCOTT CN, DAWES PJ, TAGGJR. Preliminary investigations of the colonisationof upper respiratory tract tissues of infants using
4605
BLIS K12 in 3 years old children
4606
a paediatric formulation of the oral probioticStreptococcus salivarius K12. Eur J Clin MicrobiolInfect Dis 2008; 27: 1261-1263.
5) HORZ HP, MEINELT A, HOUBEN B, CONRADS G. Distribu-tion and persistence of probiotic Streptococcus sali-varius K12 in the human oral cavity as determinedby real-time quantitative polymerase chain reaction.Oral Microbiol Immunol 2007; 22: 126-130.
6) BURTON JP, WESCOMBE PA, MOORE CJ, CHILCOTT CN,TAGG JR. Safety assessment of the oral cavity pro-biotic Streptococcus salivarius K12. Appl EnvironMicrobiol 2006; 72: 3050-3053.
7) BURTON JP, COWLEY S, SIMON RR, MCKINNEY J,WESCOMBE PA, TAGG JR. Evaluation of safety andhuman tolerance of the oral probiotic Streptococ-cus salivarius K12: a randomized, placebo-con-trolled, double-blind study. Food Chem Toxicol2011; 49: 2356-2364.
8) DI PIERRO F, ADAMI T, RAPACIOLI G, GIARDINI N, STREIT-BERGER C. Clinical evaluation of the oral probioticStreptococcus salivarius K12 in the prevention ofrecurrent pharyngitis and/or tonsillitis caused byStreptococcus pyogenes in adults. Expert OpinBiol Ther 2013; 13: 339-343.
9) DI PIERRO F, DONATO G, FOMIA F, ADAMI T, CAREDDUD, CASSANDRO C, ALBERA R. Preliminary pediatricclinical evaluation of the oral probiotic Streptococ-cus salivarius K12 in preventing recurrent pharyn-gitis and/or tonsillitis caused by Streptococcuspyogenes and recurrent acute otitis media. Int JGen Med 2012; 5: 991-997.
10) GREGORI G, RIGHI O, RISSO P, BOIARDI G, DEMURU G,FERZETTI A, GALLI A, GHISONI M, LENZINI S, MARENGHIC, MURA C, SACCHETTI R, SUZZANI L. Reduction ofgroup A beta-hemolytic streptococcus pharyngo-tonsillar infections associated with use of the oralprobiotic Streptococcus salivarius K12: a retro-spective observational study. Ther Clin Risk Man-ag 2016; 12: 87-92.
11) DI PIERRO F, DI PASQUALE D, DI CICCO M. Oral use ofStreptococcus salivarius K12 in children with secre-tory otitis media: preliminary results of a pilot, un-controlled study. Int J Gen Med 2015; 8: 303-308.
12) DI PIERRO F, COLOMBO M, ZANVIT A, RISSO P, ROTTOLIAS. Use of Streptococcus salivarius K12 in theprevention of streptococcal and viral pharyngo-tonsillitis in children. Drug Healthc Patient Saf2014; 6: 15-20.
13) WESSELS MR. Pharyngitis and Scarlet Fever. In: Fer-retti JJ, Stevens DL, Fischetti VA, editors. Strepto-coccus pyogenes: Basic Biology to Clinical Mani-festations. Oklahoma City (OK): University of Ok-lahoma Health Sciences Center, 2016.
14) LIEBERTHAL AS, CARROLL AE, CHONMAITREE T, GANIATSTG, HOBERMAN A, JACKSON MA, JOFFE MD, MILLERDT, ROSENFELD RM, SEVILLA XD. The diagnosis andmanagement of acute otitis media. Pediatrics2013; 131: e964-999.
15) BURTON JP, CHILCOTT CN, MOORE CJ, SPEISER G,TAGG JR. A preliminary study of the effect of pro-biotic Streptococcus salivarius K12 on oral mal-odour parameters. J Appl Microbiol 2006; 100:754-764.
16) MASDEA L, KULIK EM, HAUSER-GERSPACH I, RAMSEIER AM,FILIPPI A, WALTIMO T. Antimicrobial activity of Strepto-coccus salivarius K12 on bacteria involved in oralmalodour. Arch Oral Biol 2012; 57: 1041-1047.
17) DI PIERRO F, CAMPANA A, PANATTA ML, ANTENUCCI V,DE VINCENTIIS G. Possible use of Streptococcussalivarius K12 in counteracting PFAPA Syn-drome. Ped Intern. Alter Integr Med 2016. Sub-mitted.
18) DI PIERRO F, COLOMBO M, ZANVIT A, ROTTOLI AS. Posi-tive clinical outcomes derived from using Strepto-coccus salivarius K12 to prevent streptococcalpharyngo-tonsillitis in children. Drug, HealthcarePat Saf. Submitted July 2016.
19) RALPH AP, CARAPETIS JR. Group A streptococcal dis-eases and their global burden. Curr Top MicrobiolImmunol 2013; 368: 1-27.
20) SMITH NL, TAYLOR EJ, LINDSAY AM, CHARNOCK SJ,TURKENBURG JP, DODSON EJ, DAVIES GJ, BLACK GW.Structure of a group A streptococcal phage-en-coded virulence factor reveals a catalytically ac-tive triple-stranded beta-helix. Proc Natl Acad SciU S A 2005; 102: 17652-17657.
F. Di Pierro, M. Colombo, M.G. Giuliani, M.L. Danza, I. Basile, et al.
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Open Access Full Text Article
http://dx.doi.org/10.2147/DHPS.S117214
Positive clinical outcomes derived from using Streptococcus salivarius K12 to prevent streptococcal pharyngotonsillitis in children: a pilot investigation
Francesco Di Pierro1
Maria Colombo2
Alberto Zanvit3
Amilcare S Rottoli4
1Scientific Department, Velleja Research, Milan, 2Pediatric Department, University of Parma, Parma, 3Stomatology Institute, 4Pediatric Department, Uboldo Hospital, Cernusco S/N, Milan, Italy
Background: Streptococcus salivarius K12 (BLIS K12®) is a probiotic strain producing the
bacteriocins salivaricin A2 and salivaricin B, both of which strongly antagonize the growth of
Streptococcus pyogenes, the most important bacterial cause of pharyngeal infections in humans. It
successfully colonizes and exhibits persistence in the oral cavity and is endowed with an excellent
safety profile. Previous observations of a small group of children indicated that the use of BLIS
K12 could also reduce the occurrence of viral pharyngitis. The present study focused on a further
evaluation of the role of BLIS K12 in the control of pediatric streptococcal disease and moreover
whether its use could also help provide protection against various nonstreptococcal infections.
Methods: In total, 48 children with a recent history of recurrent pharyngeal streptococcal
disease were enrolled in the treated group. The control group comprised 76 children known to
have had a very low recent occurrence of oral streptococcal disease. The treated children were
given BLIS K12 daily for 90 days. The number of episodes of streptococcal pharyngotonsillitis,
tracheitis, viral pharyngitis, rhinitis, flu, laryngitis, acute otitis media, enteritis, and stomatitis
was recorded during probiotic treatment and for a follow-up period of 9 months, and this was
compared with the episodes of the control group over the corresponding period.
Results: Compared with the pretreatment time period, 2013, a 90% reduction of streptococcal
pharyngeal disease was observed in 2014; compared with untreated children, a statistically
significant reduction of all of the other disease conditions assessed, other than stomatitis, was
detected in the probiotic-treated children.
Conclusion: In agreement with previous findings, in the present study, it was found that the
daily use of BLIS K12 has been associated with a concurrent and persisting reduction in the
occurrence of pharyngeal, recurrent, streptococcal disease. Moreover, the benefits to children
may also extend to a reduction of nonstreptococcal diseases, including tracheitis, viral pharyn-
gitis, rhinitis, flu, laryngitis, acute otitis media, and enteritis.
Keywords: Streptococcus salivarius K12, pediatric infections, pharyngotonsillitis, rhinitis, flu,
tracheitis, laryngitis, stomatitis, enteritis, otitis
IntroductionThe oral probiotic Streptococcus salivarius strain K12 (also referred to here as BLIS
K12®) is known to produce the megaplasmid-encoded class I lantibiotics, salivaricin
A2, and salivaricin B.1 Expression of these two salivaricins enables BLIS K12 to coun-
teract the growth of Streptococcus pyogenes2 and, also to a lesser extent, Haemopilus
influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis, all of which are
involved in the etiopathogenesis of acute otitis media.3 BLIS K12 colonizes the oral
Correspondence: Francesco Di PierroScientific Department, Velleja Research, Viale Lunigiana 23, 20125 Milano, ItalyTel +39 349 552 7663Fax + 39 0523 511894Email [email protected]
Journal name: Drug, Healthcare and Patient SafetyArticle Designation: ORIGINAL RESEARCHYear: 2016Volume: 8Running head verso: Di Pierro et alRunning head recto: Clinical outcomes derived from using K12 in childrenDOI: http://dx.doi.org/10.2147/DHPS.S117214
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Di Pierro et al
cavity and, to a lesser extent, the nasopharynx and adenoids,4
remaining present in the tissues for up to 1 month after the
last administration.5 Characterized by excellent antibiotic
sensitivity6 and safety profiles7 and consistent with the known
activity spectrum of its salivaricin products, the regular use
of BLIS K12 has been shown to reduce reoccurrences of
streptococcal pharyngotonsillitis and acute otitis media.8–10
In addition, however, some preliminary studies have indi-
cated that BLIS K12 may also help prevent episodes of
oropharyngeal infections of viral origin.11 A potential basis
for this effect has been shown, whereby the administration of
BLIS K12 in adults can increase salivary γ-interferon levels
without modifying the levels of either interleukin-1β (IL-
1β) or tumor necrosis factor-α (TNF-α), but considerably
reducing IL-8 release.3 In the present study, whether there
is any associated reduction in the occurrence of episodes of
tracheitis, viral pharyngitis, rhinitis, flu, laryngitis, acute otitis
media, enteritis, and stomatitis in children having a history of
recurrent streptococcal pharyngotonsillitis who are receiving
a 3-month course of BLIS K12 was evaluated.
Materials and methodsProductBLIS K12 was formulated in the form of slowly dissolving
oral tablets by SIIT (Trezzano S/N, Milan, Italy) and notified to
the Italian Ministry of Health as Bactoblis® by Omeopiacenza
(Pontenure, Italy), according to the provisions of law number
169 of 2004, on July 5, 2011 (notification number: 53435).
The preparation strain K12 used in the clinical trial contained
>1 billion colony forming unit (CFU)/tablet of S. salivarius
K12 (BLIS Technologies Ltd., Otago, New Zealand).
Clinical trialThe multicenter, open, nonrandomized, controlled clinical
trial was conducted on 124 pediatric individuals enrolled in
the area of Milan (Italy) during 2014. The trial population
consisted of 65 boys and 59 girls. The first 90 days repre-
sented the treatment period. The following 9 months was
the follow-up period. The trial was conducted according to
the criteria set by the Declaration of Helsinki and with the
approval of the Local Ethics Committee (Milan, Italy). The
parents of all the participants in the study were informed
of the trial methods and signed the appropriate consent and
privacy policy documents.
Inclusion criteriaAll the individuals enrolled in this study were of 3–10 years
of age and attended preschool or school in the Milan area.
In terms of recurrent streptococcal pharyngotonsillitis,
the individuals enrolled for treatment (n=48) exhibited an
average of >3 episodes in the previous year (2013). The epi-
sodes were confirmed by a rapid swab positive for Group A
streptococcus (Test Strep-A; Gima, Gessate, Italy). None of
the individuals were clinically ill on enrolment. None of the
control group subjects had experienced recurrent streptococ-
cal pharyngotonsillitis in the previous year, and only 9 of the
76 children having single episode of streptococcal infection
had been reported.
Exclusion criteriaPotential subjects were excluded from the study if they were
immunocompromised, had undergone tonsillectomy or had an
indication for adenotonsillectomy, had a history of rheumatic
disorders, bronchospasm and/or a diagnosis of asthma and/
or allergy, had diagnosed respiratory or significant systemic
disorders, or were undergoing current pharmacological
therapies to prevent recurrent respiratory infections. Also
individuals presenting with conditions that could favor the
development of acute otitis media, including severe atopy,
acquired or congenital immunodeficiency, cleft palate, a
chronically ruptured eardrum, craniofacial abnormalities or
obstructive adenoids, sleep apnea syndrome, or placement
of tympanostomy tubes, were excluded.
Study patternAll the individuals enrolled in this study were first subjected
to a general medical examination and pharyngeal swab (Test
Strep-A) and then were subdivided, according to a previous
diagnosis of recurrent streptococcal pharyngotonsillitis, into
two groups: one group was treated with BLIS K12 in the form
of strain K12 tablets, whereas the other did not receive any
treatment and served as the control group. The individuals
in the BLIS K12-treated group were instructed on how to
use the product. The tablets were to be administered for 90
consecutive days. The children had to let one tablet dissolve
slowly in the mouth immediately before going to sleep, after
brushing their teeth. They were carefully instructed not to
chew the tablet or to swallow it whole. They were asked not
to drink or swallow anything else following the use of the
product. Before administration of the first tablet, the use of a
0.2% chlorhexidine mouthwash was recommended in order
to enhance the colonization process of the strain, by reducing
competition from endogenous S. salivarius already inhabiting
the mouth. For the trial period, it was requested that at the first
sign of any oropharyngeal symptoms of infection, the sub-
jects should be brought to the clinic for an immediate medical
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Clinical outcomes derived from using K12 in children
examination and pharyngeal rapid test. In case of a positive
result, treatment was prescribed. The prescribed therapy for
streptococcal infection was a combination of amoxicillin and
clavulanic acid to be administered for 10 days. Following the
antibiotic therapy, treatment with BLIS K12 was resumed
and continued until the scheduled 90th day of the study. Viral
infections accompanied by pharyngolaryngeal pain and/or
a fever were treated with acetaminophen or ibuprofen. Any
other pathologies present were treated according to the rec-
ommendations of the Italian Pediatric Guidelines.
Diagnosed pathologiesDiagnosis of viral infection was according to the follow-
ing criteria: negative rapid swab for streptococcal disease,
absence of submandibular lymphadenopathy, absence of
petechiae on the palate, mild dysphagia, absence of headache,
absence of abdominal pain, and absence of hyperpyrexia.
From a clinical standpoint, patients with viral pharyngitis pre-
sented with modest pharyngeal hyperemia, low-grade fever,
mild dysphagia, presence of rhinitis with serous secretion,
and spontaneous resolution of symptoms without medication
in ~48–72 hours. With regard to enteritis, according to the
Italian Pediatrics Guidelines, high fever (>40°C), live blood in
the stool, abdominal pain and involvement of the central ner-
vous system may suggest the presence of pathogenic bacteria
in the gut. Differently, vomiting and respiratory symptoms are
more frequently associated with a viral etiology. Therefore,
the diagnosis was clinical, and microbiological examination
of stool was indicated only if diarrhea is prolonged for >7–8
days or is relapsing. The diagnosis of stomatitis was also
clinical and according to the presence of ulcerations local-
ized preferentially in the fornix or in gingival–labial mucosa
of the lips. In all the cases, the etiology was considered to
be of herpetic origin, being further and more extensively
investigated only in the case of patients who were severely
debilitated. With regard to rhinitis, diagnosis was done on the
basis of absence of mucoid nasal secretion, absence of fever,
absence adenomegaly, no signs of retropharyngeal exudate,
and symptoms resolution within 72 hours. Diagnosis of
tracheitis was done clinically in the presence of mild fever,
mild/moderate pharyngeal redness, no submandibular lymph-
adenopathy, and crowing sound when inhaling. With regard
to laryngitis, diagnosis was done with moderate or no fever
resolving within 72 hours, pharyngeal redness with negative
streptococcal rapid swab, and clear signs of dysphonia along
with dysphagia. Diagnosis of acute respiratory infection (flu)
was also on a clinical basis with onset with fever, headache,
malaise, and myalgia followed by predominantly respiratory
symptoms (eg, cough, nasal congestion, and sore throat).
Virus isolation from throat swabs or sputum to identify the
causative agent is only recommended in carefully selected
cases, and this was not done in the present study. Acute otitis
media were diagnosed by pneumatic otoscopy performed by
a trained investigator.
Aims of the studyThe present study aimed to evaluate the following: 1) the
efficacy of the BLIS K12-containing product strain K12 in
the prevention of S. pyogenes pharyngotonsillitis in young
children during 3 months of treatment and a further 9-month
follow-up; 2) the efficacy of strain K12 in reducing tracheitis,
viral pharyngitis, rhinitis, flu, laryngitis, acute otitis media,
enteritis, and stomatitis in these same subjects; and 3) the
onset of side effects or toxicity while the product was being
administered.
Statistical analysisThe equivalence of the two subject groups in terms of sex
and age was determined by using Fisher’s exact test and the
two-tailed Wilcoxon–Mann–Whitney test, respectively. The
difference between the two groups in terms of numbers of
streptococcal pharyngotonsillitis, tracheitis, viral pharyngi-
tis, rhinitis, flu, laryngitis, acute otitis media, enteritis, and
stomatitis episodes was determined by using the two-tailed
Wilcoxon–Mann–Whitney test. Statistical software JMP
Version 10 for Mac OS X was used, and the threshold for
statistical significance was 95%.
ResultsForty-eight children having a diagnosis of recurrent strepto-
coccal pharyngotonsillitis were enrolled as subjects to assess
the preventive role associated with daily use of slowly dis-
solving oral tablets containing the oral probiotic S. salivarius
K12 (>1 billion CFU/tablet), against S. pyogenes infection.
The children were treated with 1 tablet of strain K12 each
day for 90 consecutive days and then continued to be moni-
tored for a further 9 months. The other group of 76 children,
not previously diagnosed with recurrent streptococcal pha-
ryngotonsillitis, served as controls for the same periods.
Compliance assessed throughout the 90 days of strain K12
treatment was very good, and no child withdrew from the
study. As shown in Table 1, the two groups did not exhibit
significantly different characteristics. The only significant
difference was in the diagnosis of recurrent streptococcal
pharyngotonsillitis, the distinctive feature of their enrolment
in the treatment group. Table 2 shows the total episodes per
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Di Pierro et al
child of diagnosed streptococcal pharyngotonsillitis occur-
ring during the study year 2014. When these findings are
compared to those obtained in 2013, the treated children can
be found to have experienced ~90% reduction in the diag-
nosed streptococcal pharyngitis episodes. By comparison, in
the control group, a nonsignificant increase in streptococcal
infections of ~30% was observed, when compared to 2013.
As shown in Table 3, during 2013, the number of episodes
of tracheitis, flu, acute otitis media, enteritis, and stomatitis
overlapped between the two groups. A difference was pres-
ent only in the episodes of viral pharyngitis and rhinitis, the
incidences of which seemed to be higher and significant in
the treated group versus the control group. The differences
observed for laryngitis did not seem to be statistically sig-
nificant. As shown in Table 4, during 2014 when strain K12
treatment was performed, the episodes per child of tracheitis,
viral pharyngitis, rhinitis, flu, laryngitis, acute otitis media,
and enteritis, but not of stomatitis, seemed to be significantly
lower, showing an apparent protective effect associated with
the use of probiotic BLIS K12. Multivariate analysis (data not
shown) demonstrates the absence of any dependency between
these findings and sex and/or age variables.
DiscussionSome intestinal probiotics have been shown to be capable of
helping the consumer to counteract constipation, diarrhea,
irritable bowel syndrome, and a number of other gastrointesti-
nal disorders.12 Recently, the development of novel probiotics
such as BLIS K12 from the oral cavity commensal species S.
salivarius has introduced the prospect of specifically achiev-
ing oral health benefits from probiotic therapy. The BLIS K12
strain was originally isolated from the oral cavity of a young
child who had no recent experience of S. pyogenes infection.
The salivaricins produced by BLIS K12 have subsequently
been shown to be inhibitory not only to S. pyogenes but also
to oral cavity bacterial pathogens associated with acute otitis
media and halitosis. Some more recent studies demonstrated
that oral administration of BLIS K12, through a still not per-
fectly understood molecular mechanism, also reduces IL-8
plasma concentrations and increases salivary γ-interferon.3
These modulations may also rationally account for an anti-
inflammatory, immunomodulating and anti-viral activity,
which would augment the already-described beneficial
antibacterial action of BLIS K12. The results of the current
study provide further support for this proposition. BLIS
K12 prophylaxis of children who appeared historically to
be at an increased risk of streptococcal pharyngitis reduced
streptococcal infections by ~90%, a finding consistent with
those of previous studies but – and this is something new –
also demonstrated an apparent reduction of tracheitis, viral
pharyngitis, rhinitis, flu, laryngitis, acute otitis media, and
enteritis. Only the incidence of stomatitis seemed unchanged
in the treated group. This protection should not be due to a
Table 4 Episodes per child of tracheitis, viral pharyngitis, rhinitis, flu, laryngitis, acute otitis media, enteritis and stomatitis highlighted in 2014
Treated Untreated P-values Odds ratio (95% CI)
Tracheitis 0.08±0.29 1.17±1.00 <0.01 0.04 (0.01, 0.12)Pharyngitis (viral) 0.13±0.37 0.55±0.77 <0.01 0.19 (0.06, 0.52)Rhinitis 0.17±0.43 0.54±0.84 <0.05 0.36 (0.13, 0.96)Flu 0.02±0.17 0.44±0.58 <0.01 0.04 (0.01, 0.19)Laryngitis 0.02±0.17 0.28±0.55 <0.01 0.08 (0.01, 0.45)Acute otitis media 0.00±0.00 0.17±0.43 <0.01 0.01 (0.00, 0.24)Enteritis 0.05±0.24 0.30±0.56 <0.01 0.16 (0.03, 0.16)Stomatitis 0.00±0.00 0.08±0.30 ns 0.01 (0.00, 0.70)
Abbreviations: CI, confidence interval; ns, no significant differences between groups.
Table 1 Characteristicsa of the children enrolled and ending the study
Treated Untreated P-values
Total number 48 76Boys 25 40 0.43Age of boys 5.4±2.9 4.9±2.4 0.25Girls 23 36 0.52Age of girls 5.6±2.0 5.1±2.4 0.37Episodes/childb 3.208 0.118 0.0001
Notes: aNonsignificant differences between groups; age expressed as years ± standard deviation; bparameter expressed with reference to 12 months of 2013.
Table 2 Episodes of pharyngotonsillitis caused in 2013 and 2014 by Streptococcus pyogenes in the two study groups
2013 A/Ca 2014 A/Ca ∆% P-values
Treated (n=48) 154 3.208 16 0.333 −89.6 <0.01Control (n=76) 9 0.118 12 0.157 +33.3 ns
Note: aAverage/child.Abbreviation: ns, not significant.
Table 3 Episodes per child of tracheitis, viral pharyngitis, rhinitis, flu, laryngitis, acute otitis media, enteritis, and stomatitis highlighted in 2013
Treated Untreated P-values
Tracheitis 1.35±1.35 1.14±1.36 nsPharyngitis (viral) 1.40±1.61 0.71±1.40 <0.01Rhinitis 1.60±1.61 0.87±1.23 <0.01Flu 0.15±0.40 0.33±0.61 nsLaryngitis 0.90±1.74 0.49±1.32 nsAcute otitis media 0.25±0.60 0.22±0.61 nsEnteritis 0.20±0.67 0.30±0.62 nsStomatitis 0.08±0.29 0.11±0.40 ns
Abbreviation: ns, no significant differences between the groups.
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Clinical outcomes derived from using K12 in children
different use of antibiotics, vaccines, or immune stimulant
supplements or else occurred along the year of study, being
the two groups comparable in this perspective, too (data not
shown). Moreover, the slight difference in terms of antibi-
otic administration linked to the episodes (average/child) of
pharyngeal streptococcal disease between the treated and the
untreated group, 0.333 and 0.157, respectively (Table 2), is
not significant. Last, children resulted to be protected mostly
against viral diseases, where the use of antibiotics was not
effective. These results, together with the excellent tolerabil-
ity and compliance found in this study, as well as the absence
of side effects, show that prophylactic BLIS K12 administra-
tion could provide a safe, simple, and cost-effective preventa-
tive for a broad variety of pediatric infections and microbial
dysequilibria. The authors recognize that this observational
study has less validity than a double-blind, controlled, pro-
spective, and randomized investigation and also that it may
contain significant bias due to the relatively small number
of treated subjects and the absence of a control, placebo,
or alternative probiotic treatment. In any case, the findings
of this study confirm the anti-streptococcal action of BLIS
K12 and further demonstrate that its use can generate other
positive outcomes. Further studies are ongoing to highlight
why an oral colonizing probiotic strongly recognized as able
to antagonize streptococci and also to counteract pathologies
of viral etiology and/or disorders in nonoral tissues such as
enteritis.
DisclosureFDP is the main formulator of the tested product, and he is
involved in the Scientific Council of the Company (Omeopia-
cenza®) trading the tested product. The other authors report
no other conflicts of interest in this work.
References 1. Hyink O, Wescombe PA, Upton M, Ragland N, Burton JP, Tagg JR. Sali-
varicin A2 and the novel lantibiotic salivaricin B are encoded at adjacent loci on a 190-kilobase transmissible megaplasmid in the oral probiotic strain Streptococcus salivarius K12. Appl Environ Microbiol. 2007;73(4): 1107–1113.
2. Wescombe PA, Burton JP, Cadieux PA, et al. Megaplasmids encode dif-fering combinations of lantibiotics in Streptococcus salivarius. Antonie Van Leeuwenhoek. 2006;90(3):269–280.
3. Wescombe PA, Hale JD, Heng NC, Tagg JR. Developing oral probi-otics from Streptococcus salivarius. Future Microbiol. 2012;7(12): 1355–1371.
4. Power DA, Burton JP, Chilcott CN, Dawes PJ, Tagg JR. Preliminary investigations of the colonisation of upper respiratory tract tissues of infants using a paediatric formulation of the oral probiotic Strep-tococcus salivarius K12. Eur J Clin Microbiol Infect Dis. 2008; 27(12):1261–1263.
5. Horz HP, Meinelt A, Houben B, Conrads G. Distribution and persistence of probiotic Streptococcus salivarius K12 in the human oral cavity as determined by real-time quantitative polymerase chain reaction. Oral Microbiol Immunol. 2007;22(2):126–130.
6. Burton JP, Wescombe PA, Moore CJ, Chilcott CN, Tagg JR. Safety assessment of the oral cavity probiotic Streptococcus salivarius K12. Appl Environ Microbiol. 2006;72(4):3050–3053.
7. Burton JP, Cowley S, Simon RR, McKinney J, Wescombe PA, Tagg JR. Evaluation of safety and human tolerance of the oral probiotic Strepto-coccus salivarius K12: a randomized, placebo-controlled, double-blind study. Food Chem Toxicol. 2011;49(9):2356–2364.
8. Di Pierro F, Adami T, Rapacioli G, Giardini N, Streitberger C. Clinical evaluation of the oral probiotic Streptococcus salivarius K12 in the prevention of recurrent pharyngitis and/or tonsillitis caused by Strepto-coccus pyogenes in adults. Expert Opin Biol Ther. 2013;13(3):339–343.
9. Di Pierro F, Donato G, Fomia F, Adami T, Careddu D, Cassandro C, Albera R. Preliminary pediatric clinical evaluation of the oral probiotic Streptococcus salivarius K12 in preventing recurrent pharyngitis and/or tonsillitis caused by Streptococcus pyogenes and recurrent acute otitis media. Int J Gen Med. 2012;5:991–997.
10. Di Pierro F, Di Pasquale D, Di Cicco M. Oral use of Streptococcus salivarius K12 in children with secretory otitis media: preliminary results of a pilot, uncontrolled study. Int J Gen Med. 2015;8:303–308.
11. Di Pierro F, Colombo M, Zanvit A, Risso P, Rottoli AS. Use of Strep-tococcus salivarius K12 in the prevention of streptococcal and viral pharyngotonsillitis in children. Drug Healthc Patient Saf. 2014;6:15–20.
12. Hod K, Ringel Y. Probiotics in functional bowel disorders. Best Pract Res Clin Gastroenterol. 2016;30(1):89–97.
© 2016 Gregori et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you
hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).
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O R i g i n a l R e s e a R C h
open access to scientific and medical research
Open access Full Text article
http://dx.doi.org/10.2147/TCRM.S96134
Reduction of group a beta-hemolytic streptococcus pharyngo-tonsillar infections associated with use of the oral probiotic Streptococcus salivarius K12: a retrospective observational study
giuseppe gregori1
Ornella Righi1
Paolo Risso2
goffreda Boiardi1
giovanni Demuru1
anna Ferzetti1
antonio galli1
Marco ghisoni1
sonia lenzini1
Claudio Marenghi1
Caterina Mura1
Roberto sacchetti1
lucia suzzani1
1Primary Care Department, local health Unit (asl), Piacenza, 2Department of health science (Dissal), University of genoa, genoa, italy
Abstract: Recurrent pharyngo-tonsillar infections caused by group A beta-hemolytic streptococci
(GABHS) occur frequently in young children, and the treatment of these infections contributes
substantially to the total current requirement for antibiotic prescribing. Our study goal was to assess
through a retrospective observational analysis whether the administration of the oral probiotic,
Streptococcus salivarius K12 (SsK12), could reduce the occurrence of GABHS pharyngo-tonsillar
infections in children who had a recent history of recurrent episodes of these infections. Twelve
primary care pediatricians identified, through their databases, a total of 130 children who had experi-
enced recurrent GABHS pharyngo-tonsillar infections over a period of at least 6–12 months prior to
their inclusion in the study. Of these children, 76 then undertook a 90-day program requiring once-
a-day dosing with a commercially available (Bactoblis) lozenge containing SsK12. No probiotic
supplement was given to the remaining 54 (control) children. Each subject was monitored for the
occurrence of GABHS pharyngo-tonsillitis and also for acute otitis media, bronchitis, sinusitis, and
bronchopneumonia for at least 12 months following their entry to the study. Even 9 months after the
use of SsK12 had been stopped, the probability of new GABHS infections was significantly lower
(P.0.001) when compared to the period before dosing commenced. When compared to the untreated
children, those taking SsK12 appear to have had significantly fewer GABHS infections both during
the 90-day period of prophylaxis and during the following 9 months (P,0.001). These observations
are supportive of the use of probiotic SsK12 for the control of recurrent GABHS pharyngo-tonsillar
infections in children, and as an associated benefit, the use of this probiotic could lead to reduced
antibiotic consumption. Follow-up controlled prospective studies should now be initiated in order
to further establish the efficacy of this newly emerging prophylactic strategy.
Keywords: recurrent pharyngo-tonsillar infections, group A beta-hemolytic streptococcus,
Streptococcus salivarius K12
BackgroundGroup A beta-hemolytic streptococci (GABHS) are a frequent cause of recurrent
pharyngo-tonsillar infections (RPTIs) in young children, and this is associated with the
further requirements for recurrent clinical examinations, pharmacological treatments,
specialist consultations, and sometimes surgical intervention.
In Italy, oral penicillin is not available, and penicillin G is only provided through
the National Health Service for patients with rheumatic disease. Therefore, amoxicillin
is the drug of choice for treatment of single acute episodes of GABHS.1,2
Correspondence: giuseppe gregoriPrimary Care Department, local health Unit (asl), Via Conciliazione 45/a, 29121 Piacenza, italyemail [email protected]
Journal name: Therapeutics and Clinical Risk ManagementArticle Designation: Original ResearchYear: 2016Volume: 12Running head verso: Gregori et alRunning head recto: Reduction of GABHS pharyngo-tonsillar infectionsDOI: http://dx.doi.org/10.2147/TCRM.S96134
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gregori et al
For those experiencing recurrent GABHS infections,
cycles of antibiotic therapy and tonsillectomy are given con-
sideration. Recently, an orally administered probiotic product
(Bactoblis), based on Streptococcus salivarius K12 (SsK12),
became available in Italy. SsK12, a normal inhabitant of the
human oral cavity, produces two bacteriocins, salivaricin A2
and salivaricin B, both of which interfere with the growth of
GABHS.3,4 Bacteriocins are antimicrobials having relatively
specific killing activity. Their action leads to suppression
of the growth of bacteria that are phylogenetically closely
related to the bacteriocin-producing strain. Unlike the classi-
cal antibiotics used to treat infections, the action of bacterio-
cins does not extend to microbial species that are distanced
phylogenetically from the producer strain.
As is often the case for nutraceutical products, reports of
its efficacy are still quite limited.5
The product is provided as tablets to be sucked slowly
in the evening before bedtime with a recommended dosing
program of one tablet daily for 90 days.
We asked the primary care pediatricians of the Local
Health Unit (LHU) of Piacenza to analyze their databases
retrospectively for children experiencing GABHS RPTIs,
and then to compare the subsequent clinical sequelae in the
children who had been treated with the recommended pro-
gram of SsK12 with those who had not taken this product.
The primary objective of the study was to assess retro-
spectively if SsK12 use in pediatric patients with GABHS
RPTIs could:
(a) significantly reduce the occurrence of GABHS relapses
during the treatment period itself and over the following
9 months, when compared to the 6- to 12-month
period immediately prior to the start of their probiotic
treatment; and
(b) significantly reduce the occurrence of GABHS relapses
during the treatment period and over the following
9 months, when compared with a control group of children
experiencing GABHS RPTIs but nontreated with SsK12.
A secondary study objective was to assess whether the
subjects treated with SsK12 had experienced any significant
differences in the occurrence of bronchitis, otitis, sinusitis,
or bronchopneumonia.
Materials and methodsThis study was performed according to the criteria contained
in the Declaration of Helsinki and was approved by the Eth-
ics Committee of the Local Health Authority of Piacenza.
A written consent was obtained from parents of children
enrolled in the study.
Methods and selection of patientsTwelve of the 33 primary care pediatricians of the LHU of
Piacenza participated in the study. Each pediatrician collected
the retrospective data of patients ranging from 3 to 7 years of
age who had received a diagnosis of GABHS RPTIs during
the period January 1, 2011 to December 31, 2013.
Since 2010, the primary care pediatricians of LHU have
used standardized clinical and microbiological criteria for the
diagnosis of GABHS infections based on the McIsaac clinical
score6 and the rapid throat swab (RAD), as a requirement of
the ProBA (Project Children-Antibiotics) regional project.7
According to the McIsaac clinical score and flow chart, the
diagnosis of GABHS infection could, in probability terms,
be excluded, confirmed, or remain questionable: in the case
of a questionable clinical score, the availability of RAD
enables a diagnosis to be made with reduced error rates.8
Therefore, the following definitions were adopted for the
present study.
Definition of pharyngo-tonsillar infectionMcIsaac score with clinical score $2 (adenopathy,
fever .38°C, absence of cough, pharyngo-tonsillar exudate,
age, season) + confirmation of GABHS presence with RAD
method or McIsaac score =5.
Definition of RPTIRPTI is defined as three or more episodes of pharyngo-
tonsillitis over a period of 6 months, or four or more episodes
over a period of 12 months.
For each patient, a form was completed listing infec-
tious events observed over the 12 months following his/her
RPTI diagnosis, and/or any antibiotic therapy used. Patients
diagnosed as having RPTIs who then received a standard
3-month treatment with SsK12 were then observed for a fur-
ther 9 months so that all patients in the study were monitored
for at least 12 months after RPTI diagnosis.
administration of ssK12SsK12 was administered as tablets sucked slowly in the
evening just before bedtime, one tablet each day for 90 days.
Every tablet (Bactoblis commercial lozenges) contains one
billion units forming colonies of SsK12/dose (based on the
product expiry date).
statistical analysisFor a suitable statistical assessment, it was necessary to have
a sample size of at least 100 cases of RPTIs. Nonparametric
tests were used. To compare the results of the 12-month
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89
Reduction of gaBhs pharyngo-tonsillar infections
observations of the two groups of patients with RPTIs
(ie, those first treated for 3 months with SsK12 vs those
not treated with SsK12), Fisher’s exact test was used for
contingency analysis, while the Mann–Whitney test was
applied for ordinal variables analysis.
ResultsIncluded in the present study were 130 children who were
established to be affected by RPTIs and whose clinical
records could be followed up for a subsequent period of
12 months: 76 children were first treated for 90 days with
SsK12, while 54 did not receive SsK12 and were considered
to be the control group. The group of treated children con-
sisted of 38 males, mean age 5.0±1.3 years, and 38 females,
mean age 4.9±1.6 years. The group of nontreated children
consisted of 25 males, mean age 5.3±1.7 years, and 29
females, mean age 5.3±1.5 years. The two groups had no
statistically significant differences in age and sex.
The children treated with SsK12 had a significantly
lower number of pharyngo-tonsillar infections than in
the period before treatment (P,0.001). Interestingly,
the control group also experienced a significantly lower
occurrence of GABHS infections during the observation
period of 1 year following RPTI diagnosis. However, the
reduction of infections obtained in the treated group was
statistically higher in the SsK12-treated children (P,0.001)
(Table 1).
By comparison with the control group, the group of
children treated with SsK12 experienced significantly fewer
GABHS infections both during the initial 90 days of inclu-
sion in the study, during which the treatment group received
SsK12 (nine relapses vs 42; P,0.001, odds ratio 0.03), and
in the following 9 months (eleven relapses vs 39; P,0.001,
odds ratio 0.07) (Tables 2 and 3).
Multivaried analysis has also demonstrated the absence
of any dependency on sex and age variables. Regarding
documented episodes of acute otitis media, bronchitis,
sinusitis, and bronchopneumonia, no significant differences
were found between the group treated with SsK12 and the
nontreated group.
DiscussionGABHS pharyngo-tonsillitis is one of the most frequently
occurring infectious diseases in the pediatric age. Although
RPTIs are a well-recognized problem in geographical
areas such as the North of Italy, there are relatively little
epidemiological data on this topic. Cardiac and rheumatic
complications, although still present in developing countries,
have been greatly reduced in recent decades in most western
countries, and so their prevention is no longer the primary
goal of pharyngo-tonsillitis therapy.9 Appropriate antibiotic
therapy typically effects a rapid healing of acute pharyngo-
tonsillitis, and this, in practice, is the most pressing need for
parents, as a consequence of current social and economic
patterns differing from those of the past.
The secondary prevention of GABHS RPTIs typically
focuses upon tonsillectomy as a therapeutic option, although
this is less frequently applied than it was 20–30 years ago.
In Italy, the use of penicillin G has been gradually neglected
because of increased concern about the risk of adverse ana-
phylactic reactions, and it is not granted by National Health
Service, with a high cost per vial. At present, there are no
validated alternative pharmacological options for the second-
ary prevention of GABHS RPTIs.
Studies of a group of school children in New Zealand10
demonstrated that some of the children had bacteriocin-
producing S. salivarius present in their saliva, which had strong
inhibitory action against Streptococcus pyogenes. S. salivarius
are known to be harmless, frequently occurring inhabitants of
the human oral cavity, and one isolate, named SsK12, was
shown to produce two bacteriocins, salivaricin A2 and salivari-
cin B, both having strong inhibitory action against GABHS.3,4
The ability of SsK12 to colonize the upper respiratory
tract when taken as a probiotic preparation in tablet form has
been established both in adults and in children.11,12 The pres-
ence of SsK12 and of its released bacteriocins is detectable,
through use of bacterial culture analysis and polymerase
chain reaction methodologies for at least 32 days after its
last administration.12
The safety profile of SsK12 has been assessed in several
studies, and according to the Ministry of Health directives,
Table 1 incidence of gaBhs infections in treated group and control group with RPTis before enrollment and in the next 12 months
Monthly GABHS frequency on enrollment
Monthly GABHS in the next 12 months
Wilcoxon signed-rank test
ssK12 group 0.38±0.08 0.03±0.07 Significant at P,0.001Control group 0.39±0.08 0.17±0.1 Significant at P,0.001Wilcoxon signed-rank test Not significant Significant at P,0.001
Abbreviations: gaBhs, group a beta-hemolytic streptococci; RPTis, recurrent pharyngo-tonsillar infections; ssK12, Streptococcus salivarius K12.
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90
gregori et al
Tab
le 3
sta
tistic
al a
naly
sis
of g
aBh
s in
fect
ions
in g
roup
tre
ated
with
ssK
12 a
nd in
con
trol
gro
up d
urin
g 9
mon
ths
of o
bser
vatio
n af
ter
ther
apy
Subj
ects
gro
up s
sK12
76C
ontr
ol54
App
roac
h A
App
roac
h B
Odd
s ra
tio
Gen
eral
dat
aA
naly
sis
wit
hout
mul
tipl
icit
yA
naly
sis
wit
h m
ulti
plic
ity
Ana
lysi
s pe
rfor
med
on
the
num
ber
of e
vent
s w
ith
cont
inge
ncy
anal
ysis
Fis
her’
s ex
act
test
Ana
lysi
s pe
rfor
med
on
the
num
ber
of e
vent
s w
ith
cont
inge
ncy
anal
ysis
Fis
her’
s ex
act
test
One
-way
mea
n M
ann–
Whi
tney
no
npar
amet
ric
test
Gro
upN
o ev
ent
1 ev
ent
2 or
mor
e ev
ents
Gro
upE
vent
sN
o ev
ent
Gro
upN
umbe
rM
edia
ssK
12 1
2 m
onth
s65
92
ssK
12 1
2 m
onth
s11
65ss
K12
12
mon
ths
760.
220.
07 (0
.03±
0.16
)C
ontr
ol 1
2 m
onth
s15
2316
Con
trol
12
mon
ths
3915
Con
trol
12
mon
ths
541.
1P,
0.00
1P,
0.00
1P,
0.00
1
Abb
revi
atio
ns: g
aBh
s, g
roup
a b
eta-
hem
olyt
ic s
trep
toco
cci;
ssK
12, S
trept
ococ
cus
saliv
ariu
s K
12.
Tab
le 2
sta
tistic
al a
naly
sis
of g
aBh
s in
fect
ions
in g
roup
tre
ated
with
ssK
12 a
nd in
con
trol
gro
up d
urin
g 90
-day
the
rapy
Subj
ects
gro
up s
sK12
76C
ontr
ol54
App
roac
h A
App
roac
h B
Odd
s ra
tio
Gen
eral
dat
aA
naly
sis
wit
hout
mul
tipl
icit
yA
naly
sis
wit
h m
ulti
plic
ity
Ana
lysi
s pe
rfor
med
on
the
num
ber
of e
vent
s w
ith
cont
inge
ncy
anal
ysis
Fis
her’
s ex
act
test
Ana
lysi
s pe
rfor
med
on
the
num
ber
of e
vent
s w
ith
cont
inge
ncy
anal
ysis
Fis
her’
s ex
act
test
One
-way
mea
n M
ann–
Whi
tney
no
npar
amet
ric
test
Gro
upN
o ev
ent
1 ev
ent
2 or
mor
e ev
ents
Gro
up1
or m
ore
even
tsN
o ev
ent
Gro
upN
umbe
rM
edia
ssK
12 1
2 m
onth
s67
63
ssK
12 1
2 m
onth
s9
67ss
K12
12
mon
ths
760.
140.
03 (0
.02±
0.11
)C
ontr
ol 1
2 m
onth
s12
366
Con
trol
12
mon
ths
4212
Con
trol
12
mon
ths
540.
88P,
0.00
1P,
0.00
1P,
0.00
1
Abb
revi
atio
ns: g
aBh
s, g
roup
a b
eta-
hem
olyt
ic s
trep
toco
cci;
ssK
12, S
trept
ococ
cus
saliv
ariu
s K
12.
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91
Reduction of gaBhs pharyngo-tonsillar infections
it is considered safe for human use in probiotic formulations
intended to achieve oral colonization.13,14
During our study, compliance rate assessed throughout
the 3-month period on SsK12 was very good: no child has
stopped therapy earlier than established.
The present retrospective observational study has indi-
cated that the use of SsK12 has significantly reduced the
occurrence of GABHS pharyngo-tonsillitis in a group of
children established to have GABHS RPTIs, by comparison
to the occurrence of these infections both in this group of
children in the period prior to their use of SsK12 and in a
control group of children characterized by the same clinical
history of RPTIs, but untreated with SsK12.
Control group also showed reduced rate of GABHS
infections over a period of 12 months: this is more likely
due to immune competence that physiologically increases in
childhood; nevertheless, there remained a very high statistical
difference when compared with group treated with SsK12.
Ours is a retrospective observational study, and hence, the
different number of subjects in the two groups. Anyway, we
are not concerned about the effects of numerical imbalance
between the two groups because, according to Ruvuna,15
we calculated the statistical impact on the power of our test
discovering it as minimal.
According to our data, SsK12 assumption makes four
times less likely the need for antibiotic therapy against
GABHS infections providing the chance of reducing antibi-
otic pressure in the era of multi-resistant germs.
It is recognized that this retrospective, observational study
has less validity than a double-blind, controlled, prospective,
and randomized investigation and also that it may contain
significant bias: for example, no account has been taken
of poorly compliant subjects who may either have stopped
taking inconsistently used SsK12 during the recommended
90-day course of treatment. Also, no account has been made
of the use by the test or control subjects of any other nutri-
tional and/or probiotic products. Moreover, entry to the study
occurred in a voluntary way, and the criteria established by the
pediatricians for the recommendation for SsK12 use by each
patient with an RPTI diagnosis were purely subjective. On the
other hand, the treatment and control groups were uniform
for age and sex, and the diagnostic–therapeutic management
protocols for pharyngo-tonsillitis (ie, clinical assessment with
McIsaac score and the use of RAD) have been well estab-
lished for all of the pediatricians participating in the study.
Few studies are available about clinical evaluation of the oral
probiotic SsK12 in preventing recurrent pharyngitis and/or
tonsillitis in childhood caused by S. pyogenes, all of them not
randomized or placebo-controlled and also not blinded and
with fewer children enrolled.5 Up to now, our study is the
one with the largest number of children enrolled.
ConclusionOn the basis of the results of this observational and retrospec-
tive study, it appears that oral preparations containing SsK12
may provide a beneficial option for the prevention of pedi-
atric GABHS RPTIs: their use may be particularly useful in
patients who would otherwise be forced to undergo frequent
cycles of antibiotic therapy. Hopefully, further investigations
of this new approach to prophylaxis against GABHS infection
will follow, also bearing in mind the ever-increasing need to
reduce our antibiotic usage in patients of all ages in order to
reduce the risk of antibiotic resistance development.
Author contributionsGG and OR designed the study and have made substantial
contributions in drafting manuscript. PR performed statistical
analysis. GB, GD, AF, AG, SL, CMa, and CMu performed
data acquisition and validation and contributed to interpreta-
tion of data. MG and LS verified data analysis and revised
the manuscript critically. All authors contributed toward data
analysis, drafting and critically revising the paper and agree
to be accountable for all aspects of the work.
DisclosureThe authors report no conflicts of interest in this work.
References1. Van Driel ML, De Sutter AIM, Keber N, Habraken H, Christiaens T.
Different antibiotic treatments for group A streptococcal pharyngitis. Cochrane Database Syst Rev. 2013;(4):CD004406.
2. Spinks A, Glasziou PP, DelMar CB. Antibiotics for sore throat. Cochrane Database Syst Rev. 2013;(11):CD000023.
3. Tagg JR. Streptococci as effector organism for probiotic and replacement therapy. In: Versalovic J, Wilson M, editors. Therapeutic Microbiology: Probiotics and Related Strategies. Washington, DC: ASM Press; 2008: 61–81.
4. Tagg JR, Dierksen KP. Bacterial replacement therapy: adapting ‘germ war-fare’ to infection prevention. Trends Biotechnol. 2003;21(5):217–223.
5. Di Pierro F, Donato G, Fornia F, et al. Preliminary pediatric clinical evaluation of the oral probiotic Streptococcus salivarius K12 in preventing recurrent pharyngitis and/or tonsillitis caused by Streptococcus pyogenes and recurrent acute otitis media. Int J Gen Med. 2012;5:991–997.
6. McIsaac WJ, Kellner JD, Aufricht P, Vanjaka A, Low DE. Empirical validation of guidelines for the management of pharyngitis in children and adults. JAMA. 2004;291(13):1587–1595.
7. E-R Agenzia sanitaria e sociale regionale. Dossier n.153/2007 – Farin-gotonsillite in età pediatrica. Linea Guida regionale [update February 15, 2013]. Available from: http://assr.regione.emilia-romagna.it/it/servizi/pubblicazioni/dossier/doss153. Accessed March 24, 2015.
8. Di Mario S, Gagliotti C, Moro ML. Nuovelinee Guidafaringotonsil-lite – Regione Emilia – Romagna. June 2015. Available from: http://assr.regione.emilia-romagna.it/it/servizi/pubblicazioni/rapporti-documenti/faringotonsillite-guida-rapida-2015
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92
gregori et al
9. National Institute for Health and Clinical Excellence. Centre for Clinical Practice. Respiratory tract infections – antibiotic prescribing. Prescrib-ing of antibiotics for selflimiting respiratory tract infections in adults and children in primary care (Clinical guideline; no. 69). London, UK: NICE; 2008.
10. Tagg JR. Prevention of streptococcal pharyngitis by anti-Streptococcus pyogenes bacteriocin-like inhibitory substances (BLIS) produced by Streptococcus salivarius. Indian J Med Res. 2004;119(Suppl):13–16.
11. Power DA, Burton JP, Chilcott CN, Dawes PJ, Tagg JR. Preliminary investigations of the colonization of upper respiratory tract tissues of infants using a paediatric formulation of the oral probiotic Streptococ-cus salivarius K12. Eur J Clin Microbiol Infect Dis. 2008;27(12): 1261–1263.
12. Horz HP, Meinelt A, Houben B, Conrads G. Distribution and persistence of probiotic Streptococcus salivarius K12 in the human oral cavity as determined by real-time quantitative polymerase chain reaction. Oral Microbiol Immunol. 2007;22(2):126–130.
13. Burton JP, Wescombe PA, Moore CJ, Chilcott CN, Tagg JR. Safety assessment of the oral cavity probiotic Streptococcus salivarius K12. Appl Environ Microbiol. 2006;72(4):3050–3053.
14. Burton J, Chilcott C, Wescombe P, Tagg J. Extended safety data for the oral cavity probiotic Streptococcus salivarius K12. Probiot Antimicrob Proteins. 2010;2:135–144.
15. Ruvuna F. Unequal center sizes, sample size, and power in multicenter clinical trials. Drug Inf J. 2004;38(4):387–394.
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Open access Full Text article
http://dx.doi.org/10.2147/IJGM.S92488
Oral use of Streptococcus salivarius K12 in children with secretory otitis media: preliminary results of a pilot, uncontrolled study
Francesco Di Pierro1
Daniele Di Pasquale2
Maurizio Di cicco2
1Velleja research, Milan, Italy; 2Orl Department, Ospedale Maggiore Policlinico ca’ Grande Irccs, Milan, Italy
correspondence: Francesco Di Pierro Velleja research, Viale lunigiana 23, 20125 Milan, Italy Tel +39 349 552 7663 email [email protected]
Abstract: Secretory otitis media (SOM) remains a common disease among children. Although
its cause is not yet perfectly established, the pathology, often a sequel of acute otitis media
(AOM), is mainly characterized by persistent fluid in the middle ear cavity. Twenty-two children
with a diagnosis of SOM were treated daily for 90 days with an oral formulation containing
the oral probiotic Streptococcus salivarius K12 (Bactoblis®). After treatment, the children
were evaluated for AOM episodes and subjected to tone audiometry, tympanometry, endonasal
endoscopy, otoscopy, and tonsillar examination. Subject compliance and probiotic tolerability
and side effects have also been evaluated. Our results indicate a good safety profile, a substantial
reduction of AOM episodes, and a positive outcome from the treatment for all of the clinical
outcomes tested. We conclude that strain K12 may have a role in reducing the occurrence and/or
severity of SOM in children. From our perspective, this study constitutes a starting point toward
the organization of a more extensive placebo-controlled study aimed at critically appraising our
preliminary observations.
Keywords: BLIS K12, Bactoblis®, acute otitis media, exudative otitis media
IntroductionOtitis media is one of the most frequent problems that must be addressed by physicians
dealing with pediatric patients. Approximately 80% of children have at least one episode
of acute otitis media (AOM), and between 80% and 90% of preschool children have
at least one episode of secretory otitis media (SOM).1,2 In AOM, bacterial pathogens
such as Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis,
and Streptococcus pyogenes ascend through the eustachian tube from the nasopharynx
to the middle ear, causing an inflammatory response.3 SOM, asymptomatic persistence
of effusion in the middle ear cavity, is a possible sequel of AOM. SOM often resolves
spontaneously but in some cases, especially when bilateral exudate persists for more
than 3 months, the insertion of a tympanostomy tube may be required for drain-
age to avoid hearing difficulties and/or chronic anatomical damage of the tympanic
membrane.4 Antibiotics, decongestants, and corticosteroids fail to eliminate middle
ear exudation and therefore are not recommended.5,6 It has been observed that otitis-
prone children carry more bacterial pathogens in their nasopharyngeal microbiota and
fewer potentially-interfering microorganisms such as alpha-hemolytic streptococci,
nonhemolytic streptococci, Prevotella and Peptostreptococcus species.7,8 Some strains
of alpha-hemolytic streptococci depress the growth of pathogenic bacteria in vitro9 and
nasal spraying with alpha-hemolytic streptococci in otitis-prone children reduces the
risk of recurrence of AOM and the development of SOM.10 Also, spray treatment with
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Di Pierro et al
60 days of clinical observationof the status of asymptomatic SOM
Enrollmentphysical examination
bilateral tone audiometric testingbilateral tympanometry
bilateral otoscopyendonasal endoscopytonsillar examination
T=−60 T=−30 T=0 T=30 T=60 T=90
Physical examinationbilateral tone audiometric testing
bilateral tympanometrybilateral otoscopy
endonasal endoscopytonsillar examinationstatistical analysis
90 days of treatment withStreptococcus salivarius K12
Figure 1 scheme of the study.Abbreviation: sOM, secretory otitis media.
alpha-hemolytic streptococci in children with established
SOM can diminish the amount of fluid in the middle ear, thus
reducing hearing problems and rendering surgery unneces-
sary in many cases.11 Many oral commensal streptococci
have been recently investigated for their ability to interfere
with the growth of pathogens inhabiting the oral cavity and/
or nasopharynx.12 Of these, the oral probiotic Streptococcus
salivarius K12 has been most thoroughly studied. Originally
isolated from the throat of a New Zealand child, strain K12
produces two distinct megaplasmid-encoded lantibiotics,
named salivaricin A2 and salivaricin B, that inhibit the
growth of S. pyogenes, S. pneumoniae, and M. catarrhalis,
all of which are involved in the pathogenesis of AOM and
bacterial pharyngotonsillitis in children and adults.13–17 Strain
K12 typically colonizes the oral cavity of more than 30% of
children after 3 days of administration, with clear coloniza-
tion of even the nasopharynx and adenoids detected for up to
32 days after the last administration.18,19 The K12 strain is also
endowed with an excellent antibiotic-sensitivity profile and
high safety-assurance characteristics, as demonstrated in tests
in laboratory animals and humans.20,21 Recent clinical trials22–24
conducted both in adults and children demonstrated that treat-
ment with the strain K12 reduces recurrences of bacterial
pharyngotonsillitis and AOM. The present preliminary and
uncontrolled study, performed in children diagnosed with
recurrent AOM and also affected by asymptomatic SOM, was
therefore designed first to evaluate the safety and tolerability
profile of strain K12 when administered in children with
clear presence of a middle ear exudate, second to establish
the possible protective effect in terms of reduction of AOM
recurrences, and third to follow the progression of SOM by
using tone audiometry, tympanometry, endonasal endoscopy,
otoscopy, and tonsillar examination.
Materials and methodssubjectsTwenty-two children (3–9 years old) having a recent his-
tory of recurrent AOM and with unilateral or bilateral fluid
in the middle ear for at least 2 months were included in this
preliminary, uncontrolled study after informed consent was
obtained from their parents. Exclusion criteria were severe
underlying disease, immune deficiency, heart disease or
congenital heart defects, sore mucosae, antibiotic use within
the last month, and upper respiratory tract infection in the
10 days preceding the enrollment.
study schemeThis pilot, uncontrolled study was conducted in the field of
routine clinical practice in the area of Milan (Italy) between
November 2013 and September 2014, in agreement with the
criteria set by the Declaration of Helsinki. As the product
being tested is a nutraceutical, the approval from the ethical
board was not required. The parents of all the participants
in the study were informed of the trial methods and signed
the consent and privacy policy documents giving the autho-
rization to publish the results. The subjects were followed
for 2 months before entering the study. At the enrollment
(T=0) and after 90 days, all the subjects underwent to physi-
cal examination, pharyngeal buffer (Test Strep-A; Gima,
Gessate, Italy), bilateral tone audiometric testing, bilateral
tympanometry, bilateral otoscopy, endonasal endoscopy,
and tonsillar examination (Figure 1). Every 15 days, for
the whole duration of the study, all of the enrolled subjects
were in contact with the physician responsible for the study
to report their medical condition and specific study param-
eters such as probiotic tolerability and dosing compliance
as well as to enable documentation of the occurrence of any
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role of K12 in sOM
side effects possibly linked to the treatment. The subjects
were also provided with the possibility of daily access to
the physician responsible for the study. The treatment was
for 3 months from T=0 to T=90. All 22 enrolled subjects
completed the study, and statistical analysis was performed
on all subjects.
Tested productS. salivarius K12, also known as BLIS K12 (BLIS Technolo-
gies, Dunedin, New Zealand), was formulated as slowly-
dissolving oral tablets by SIIT (Trezzano, Milan, Italy) and
notified as nutritional supplement to the Italian Ministry of
Health as Bactoblis® by Omeopiacenza (Pontenure, Italy),
according to the provisions of law 169 of 2004, on July 5,
2011 (notification number 53435). The preparation of Bac-
toblis® used in the clinical trial contained no less than 1 billion
colony-forming units/tablet of S. salivarius K12.
Treatment protocolStarting from T=0 to T=90, 1 tablet of Bactoblis® was
administered to each subject every night, just before sleep.
The tablet was allowed to slowly dissolve in the oral cavity,
without biting or swallowing. Saliva production is typically
reduced in the evening hours and this improves the effective-
ness of oral colonization. Only for the very first treatment,
the administration of the tablet was preceded, approximately
30 minutes before, by the use of a chlorhexidine-based (0.2%)
mouthwash. This procedure improves the efficacy of oral
colonization by BLIS K12 by creating bacteria-depleted
niches in the oral tissues. In order to evaluate the level of
subject adherence to the established protocol, the subjects
were asked to return any unused product boxes and tablets.
Acceptable adherence was considered to be the administra-
tion of not less than 95% of the allocated tablets.
study objectivesThe study aims were to evaluate: 1) the safety profile of
S. salivarius K12 when administered for 90 days to children
with a previous diagnosis of recurrent AOM and in presence
of middle ear exudate for at least 2 months; 2) the capabil-
ity of S. salivarius K12 to protect against AOM recurrence;
3) to follow the possible evolution of SOM by using tone
audiometry, tympanometry, endonasal endoscopy, otoscopy,
and tonsillar examination.
aOM incidenceThe incidence of AOM has been calculated as episodes of
AOM per month per child. The value of AOM incidence
described at T=0 corresponds to the one calculated according
to the episodes occurred the previous 12 months. The value
of AOM incidence described at T=90 corresponds to the
one calculated according to the episodes occurred during
the 90 days of treatment.
audiometryAM13 FreeQuency (Tecnomed) audiometer, TDH 39
(Telephonics) headphones, and a soundproof booth (Mitaso)
were used. Pure tone audiometry (air conduction) was per-
formed at frequencies ranging from 250 to 8,000 Hz for
the hearing threshold test. When necessary the bone route
(frequencies of 250 to 4,000 Hz) was applied. The technique
used was sound-to-silence, and the threshold was considered
to be the lowest intensity at which the child responded 100%
of the times to the presence of sound. To classify the degree,
we used the mean tonal thresholds per airway at the frequen-
cies of 250–2,000 Hz and values proposed by Northern and
Downs25 for children classifying as normal (score =0; up to
15 dB); mild transmissive hypoacusis (score =1; from 16
to 70 dB); severe transmissive hypoacusis (score =2; when
more than 71 dB).
TympanometryA Zodiac 901 tympanometer (Madsen Electronics, Taastrup,
Denmark) was used to assess the condition of the middle ear.
The tympanogram was evaluated according to three types
of conditions: type A (normal); type B (presence of middle
ear exudate); type C (tubaric dysfunction).
OtoscopyThe data obtained from otoscopy were categorized into
four grades of classification: normal tympanic membrane
(score =0); matt-like tympanic membrane (score =1); matt-
like and retracted tympanic membrane (score =2); and adher-
ent (glue-ear) tympanic membrane (score =3).
endonasal endoscopyEndonasal endoscopic examination was done using an
Olympus pediatric fiberscope with a 2.2 mm flexible nasal
endoscope. The other equipment used for assessing slow
movements were video camera attached to the endoscope,
a colored television, and an image recorder. Data have been
evaluated as percentage of obstruction.
Tonsillar examinationTonsil volume was classified according to validated criteria26
as follows: tonsils in the tonsillar fossa barely seen behind the
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Di Pierro et al
Table 1 Demographic characteristics of the enrolled subjects
Sex Number Age (yr) Weight (kg) Height (m) BMI (kg/m2) Allergy Breastfed PCV13 SBEA
Female 5 4.8±1.0 18.4±3.3 1.05±0.07 16.7±2.9 1 4 5 0Male 17 5.8±2.2 24.2±6.3 1.14±0.11 18.5±2.8 7 16 15 4Total 22 5.6±2.1 22.9±6.2 1.12±0.11 18.1±2.8 8 20 20 4
Abbreviations: BMI, body mass index; PcV13, 13-valent pneumococcal vaccine; sBea, Streptococcus beta-hemolytic group-a (Streptococcus pyogenes); yr, year.
anterior pillar (score =0); tonsils visible behind the anterior
pillar (score =1); hypertrophic tonsils extended three-quarters
of the way to middle line (score =2); and tonsils completely
obstructing the airway, known as kissing tonsils (score =3).
statistical analysisThe difference between the two groups of clinical variables,
pre- and posttreatment with S. salivarius K12, was deter-
mined using the two-tailed Wilcoxon–Mann–Whitney test.
Only for endonasal endoscopy, since it is a ratio variable,
we transformed the data with arcsin function before applying
the Wilcoxon–Mann–Whitney test. The difference between
pre- and posttympanometry data was determined using the
Fisher’s exact test. Statistical software used was JMP® 10 for
Mac OS X (SAS Institute, Cary, NC, USA), and the threshold
for statistical significance was 95%.
ResultsIn our study, 22 children aged between 3 and 9 years were
enrolled. At the end of the study, all were considered eligible
for statistical analysis, having completed the study according
to the protocol. In Table 1, sex, age, weight, height, body
mass index, and other clinical characteristics of the enrolled
subjects are reported. Among the 22 enrolled subjects,
eight reported a history of allergy, 20 had been breastfed
for at least 4 months since delivery, and 20 had been vac-
cinated with 13-valent pneumococcal conjugate vaccine. At
enrollment, four of the 22 subjects were swab-test positive
but asymptomatic for S. pyogenes. The same four subjects
were also swab-test positive and asymptomatic at the end
of the study, indicating that they were healthy carriers. At
the end of the study, no other subjects were swab-test posi-
tive for S. pyogenes. As shown in Table 2, the oral use of
S. salivarius K12 appears to have modified some clinically
relevant outcomes. AOM incidence, calculated as the number
of episodes per month per child, was reduced by approxi-
mately 40% during the 3 months of treatment as compared
with the corresponding incidence calculated according to the
number of episodes that occurred in the previous year. Pure
tone audiometry improved by more than 50% (left ear) and
60% (right ear), and, otoscopy, bilaterally, by approximately
40%. Eustachian tube obstructions due to adenoid vegetation
hypertrophy decreased by approximately 30%. Collectively,
the sizes of palatine tonsils were reduced by 40%, and only
two cases of kissing tonsils were detected at T=90 by com-
parison to ten cases at T=0 (data not shown). As shown in
Table 3, tympanometry was substantially improved, particu-
larly concerning the presence of middle ear exudate which
occurred at T=90 in only two cases, bilaterally. No relevant
differences were observed concerning tubaric dysfunction.
Finally, the probiotic treatment demonstrated a very good
safety profile with no treatment-related side effects occurring
and no subject drop out. As shown in Table 4, tolerability
was assessed as “good” and “very good” in 20 of the 22
subjects and overlapping results were obtained as regards
to compliance.
DiscussionIn a previous study, a nasal spray bacterial treatment was
shown to reduce the development of SOM as a sequel to
AOM in otitis-prone children.10 In a later study, it was shown
that a spray treatment with alpha-hemolytic streptococci led
to complete or almost complete resorption of middle ear
effusion in one-third of treated patients having long-standing
SOM.11 In our preliminary uncontrolled study, performed
in children diagnosed with recurrent AOM and affected by
asymptomatic SOM, we have used the well-established probi-
otic strain S. salivarius K12. Strain K12 was administered to
children having well-documented middle ear exudate and/or
rhinotubaric dysfunction. Our results demonstrate that strain
K12: 1) is endowed with a good safety profile when used in
otitis-prone children affected by asymptomatic SOM; 2) has
a protective effect against AOM recurrence; and 3) seems to
improve some of the clinical outcomes and features relevant
in children with SOM. Our study can be criticized for being
uncontrolled and for utilizing subjective clinical examination.
Nevertheless, 1) this study principally proposed to reevaluate
the already well-documented general safety profile of strain
K12, but now, specifically with respect to children with SOM;
2) this was the first time that strain K12 has been administered
to evaluate its possible benefit in subjects experiencing middle
ear asymptomatic exudate and/or tubaric dysfunction; 3) most
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role of K12 in sOM
Table 2 clinical outcome in children with secretory otitis media treated by oral route with Streptococcus salivarius K12
T=0 T=90 Δ (%)a P
aOM incidenceb 0.40 0.23 42.5 ,0.01Tone audiometry (right) 1.2±0.6 0.4±0.5 66 ,0.01Tone audiometry (left) 1.1±0.7 0.4±0.6 54.6 ,0.01Otoscopy (right) 1.5±0.9 0.9±1.0 40 ,0.05Otoscopy (left) 1.3±0.8 0.8±0.3 38.5 ,0.05endonasal endoscopyc 70 50 28.6 ,0.01Tonsillar examination 1.9±1.1 1.2±0.8 36.8 ,0.01
Notes: aT=90 versus T=0; bepisodes/month/child; creported as average value of the % of obstruction, statistical analysis has been performed by using arcsin value.Abbreviation: aOM, acute otitis media.
Table 3 left and right tympanometry in children with secretory otitis media treated by oral route with Streptococcus salivarius K12
Left T=0 T=90 P Right T=0 T=90 P
a 6 14 ,0.05 a 7 13 ,0.05B 9 2 ,0.05 B 10 2 ,0.05c 7 6 ns c 5 7 ns
Notes: a = normal; B = presence of middle ear exudate; c = tubaric dysfunction.Abbreviation: ns, not significant.
children are not likely to improve spontaneously after having
experienced chronic SOM. If the encouraging preliminary
results that we have obtained are confirmed in a subsequent
double-blind, placebo-controlled study, two hypothesis can
be formulated. The first is that the oral treatment with strain
K12 may have reduced populations of relevant pathogens in
the nasopharyngeal microbiota. Indeed, S. salivarius K12 has
been shown to be endowed with strong in vitro inhibitory activ-
ity against S. pyogenes, S. pneumoniae, and M. catarrhalis.
Moreover, children with recurrent AOM seem to have a rela-
tive absence of interfering commensal streptococci in their
nasopharyngeal flora.7,8 In this study, we did not establish
whether alterations in the populations of pathogens occurred.
Further trials should include this as an important endpoint.
The pathogenesis of SOM is still poorly understood. Analyzed
exudates from children with SOM have demonstrated the pres-
ence of pathogens in approximately 35% of cases, by culture
techniques, and in approximately 75% of cases, by polymerase
chain reaction.11,27 The presence of bacterial pathogens in the
exudate could lead to inflammation. Indeed, high levels of
proinflammatory cytokines were found in exudates of children
with SOM.28 S. salivarius K12 has been shown to lower IL-8,
a well-known proinflammatory cytokine, in the oral mucosa
of colonized subjects.12 A possible second hypothesis is then
that treatment with strain K12 might have stimulated innate
immunity, promoting the clearance of bacteria from the middle
ear. As a support for this hypothesis, we know that S. salivarius
K12 is able to reduce colonization by Candida in the oral
mucosa and that this effect is not mediated by any type of
chemical interference.29 This could suggest that stimulation
of antibacterial immune effector mechanisms, rather than (or
in addition to) bacterial interference, might be responsible
for the observed clinical benefits. Both of these hypotheses
should now be tested. Originally we also thought that a third
possible hypothesis could be drawn to explain the obtained
results. It could be that the children with a spontaneous, or
caused by the administration of the strain K12, improvement
of the adenoid condition also had a clear improvement in
terms of exudate presence in the middle ear. We have then
decided to statistically analyze the impact of the improvements
observed by endonasal endoscopy to possibly correlate them
with a parallel and proportional improvement in audiometry,
otoscopy, and tympanometry. Statistical results of this analysis
(data not shown) demonstrated that no correlation like that
could be established. To conclude, our preliminary results
have reconfirmed the good safety profile of strain K12 and
also seem to indicate that oral treatment with the strain K12 in
children with SOM could diminish the amount of fluid in the
middle ear and improve some possible hearing problems. This
study has to be considered only a starting point to evaluating
the role of strain K12 in children with AOM and/or SOM.
AcknowledgmentsThe authors wish to thank Dr Risso P for the statistical analy-
sis of the results and Dr Tagg J for the kind suggestions and
review of the paper.
DisclosureDi Pierro F is the Scientific Director of Velleja Research,
the company that developed the finished product tested in
this study. The authors report no other conflicts of interest
in this work.
References1. Tos M. Epidemiology and natural history of secretory otitis. Am J Otol.
1984;5(6):459–462.
Table 4 Tolerability, compliance, and side effects in children (n=22) with secretory otitis media treated for 90 days by oral route with Streptococcus salivarius K12 as reported by parents and established by clinician
Tolerability Compliance Side effects
Very good n=13 n=16 noneGood n=7 n=4 noneacceptable n=2 n=2 noneUnacceptable n=0 n=0 none
Note: n = number of children.
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2. Burrows HL, Blackwood RA, Cooke JM, Cooke JM, Harrison RV, Passamani PP; Otitis Media Guideline Team. University of Michigan Health System: Otitis Media Guideline 2013.
3. Loos BG, Bernstein JM, Dryja DM, Murphy TF, Dickinson DP. Determination of the epidemiology and transmission of non-typable Haemophilus influenza in children with otitis media by comparison of total genomic DNA restriction fingerprints. Infect Immun. 1989; 57(9):2751–2757.
4. Rosenfeld RM, Schwartz SR, Pynnonen MA, et al. Clinical practice guideline: tympanostomy tubes in children. Otolaryngol Head Neck Surg. 2013;149(Suppl 1):S1–S35.
5. Gluth MB, McDonald DR, Weaver AL, Bauch CD, Beatty CW, Orvidas LJ. Management of eustachian tube dysfunction with nasal steroid spray: a prospective, randomized, placebo-controlled trial. Arch Otolaryngol Head Neck Surg. 2011;137(5):449–455.
6. American Academy of Family Physicians; American Academy of Otolaryngology-Head and Neck Surgery; American Academy of Pediatrics Subcommittee on Otitis Media with Effusion. Otitis media with effusion. Pediatrics. 2004;113(5):1412–1429.
7. Brook I, Gober AE. In vitro bacterial interference in the nasopharynx of otitis media-prone and non-otitis media-prone children. Arch Oto-laryngol Head Neck Surg. 2000;126:1011–1013.
8. Bernstein JM, Faden HF, Dryja DM, Wactawski-Wende J. Micro-ecology of the nasopharyngeal bacterial flora in otitis-prone and non-otitis-prone children. Acta Otolaryngol. 1993;113:88–92.
9. Tano K, Olofsson C, Grahn-Hakansson E, Holm SE. In vitro inhibi-tion of S. pneumoniae, non-typable H. influenzae and M. catarrhalis by alpha-hemolytic streptococci from healthy children. Int J Pediatr Otorhinolaryngol. 1999;47:49–56.
10. Roos K, Hakansson EG, Holm S. Effect of re-colonization with “inter-fering” alpha streptococci on recurrences of acute and secretory otitis media in children: randomized placebo controlled trial. BMJ. 2001;322: 210–212.
11. Skovbjerg S, Roos K, Holm SE, et al. Spray bacteriotherapy decreases middle ear fluid in children with secretory otitis media. Arch Dis Child. 2009;94(2):92–98.
12. Wescombe PA, Hale JD, Heng NC, Tagg JR. Developing oral probi-otics from Streptococcus salivarius. Future Microbiol. 2012;7(12): 1355–1371.
13. Tagg JR. Prevention of streptococcal pharyngitis by anti-Streptococcus pyogenes bacteriocin-like inhibitory substances (BLIS) produced by Streptococcus salivarius. Indian J Med. 2004;119(Suppl):13–16.
14. Hyink O, Wescombe PA, Upton M, Ragland N, Burton JP, Tagg JR. Salivaricin A2 and the novel lantibiotic salivaricin B are encoded at adjacent loci on a 190-kilobase transmissible megaplasmid in the oral probiotic strain Streptococcus salivarius K12. Appl Environ Microbiol. 2007;73(4):1107–1113.
15. Sharma S, Verma KK. Skin and soft tissue infection. Indian J Pediatr. 2001;68(Suppl 3):S46–S50.
16. Wescombe PA, Burton JP, Cadieux PA, et al. Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius. Antonie Van Leeuwenhoek. 2006;90(3):269–280.
17. van Zon A, van der Heijden GJ, van Dongen TM, Burton MJ, Schilder AG. Antibiotics for otitis media with effusion in children. Cochrane Database Syst Rev. 2012;9:CD009163.
18. Power DA, Burton JP, Chilcott CN, Dawes PJ, Tagg JR. Preliminary investigations of the colonisation of upper respiratory tract tissues of infants using a paediatric formulation of the oral probiotic Strep-tococcus salivarius K12. Eur J Clin Microbiol Infect Dis. 2008; 27(12):1261–1263.
19. Horz HP, Meinelt A, Houben B, Conrads G. Distribution and persistence of probiotic Streptococcus salivarius K12 in the human oral cavity as determined by real-time quantitative polymerase chain reaction. Oral Microbiol Immunol. 2007;22(2):126–130.
20. Burton JP, Wescombe PA, Moore CJ, Chilcott CN, Tagg JR. Safety assessment of the oral cavity probiotic Streptococcus salivarius K12. Appl Environ Microbiol. 2006;72(4):3050–3053.
21. Burton JP, Cowley S, Simon RR, McKinney J, Wescombe PA, Tagg JR. Evaluation of safety and human tolerance of the oral probiotic Strepto-coccus salivarius K12: a randomized, placebo-controlled, double-blind study. Food Chem Toxicol. 2011;49(9):2356–2364.
22. Di Pierro F, Adami T, Rapacioli G, Giardini N, Streitberger C. Clinical evaluation of the oral probiotic Streptococcus salivarius K12 in the pre-vention of recurrent pharyngitis and/or tonsillitis caused by Streptococ-cus pyogenes in adults. Expert Opin Biol Ther. 2013;13(3):339–343.
23. Di Pierro F, Donato G, Fomia F, et al. Preliminary pediatric clinical evaluation of the oral probiotic Streptococcus salivarius K12 in pre-venting recurrent pharyngitis and/or tonsillitis caused by Streptococcus pyogenes and recurrent acute otitis media. Int J Gen Med. 2012;5: 991–997.
24. Di Pierro F, Colombo M, Zanvit A, Risso P, Rottoli AS. Use of Strepto-coccus salivarius K12 in the prevention of streptococcal and viral phar-yngotonsillitis in children. Drug Healthc Patient Saf. 2014;6:15–20.
25. Northern JL, Downs MP. Hearing in Children. 3rd ed. Philadelphia, PA: Williams and Wilkins; 1984.
26. Friedman M, Tanyeri H, La Rosa M, et al. Clinical predictors of obstruc-tive sleep apnea. Laryngoscope. 1999;109:1901–1907.
27. Matar GM, Sidani N, Fayad M, Hadi U. Two-step PCR-based assay for identification of bacterial etiology of otitis media with effusion in infected Lebanese children. J Clin Microbiol. 1998;36:1185–1188.
28. Schousboe LP, Ovesen T, Eckhardt L, Rasmussen LM, Pedersen CB. How does endotoxin trigger inflammation in otitis media with effusion? Laryngoscope. 2001;111(2):297–300.
29. Ishijima SA, Hayama K, Burton JP, et al. Effect of Streptococcus salivarius K12 on the in vitro growth of Candida albicans and its protective effect in an oral candidiasis model. Appl Environ Microbiol. 2012;78(7):2190–2199.
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59665
Use of Streptococcus salivarius K12 in the prevention of streptococcal and viral pharyngotonsillitis in children
Francesco Di Pierro1
Maria colombo2
alberto Zanvit3
Paolo risso4
amilcare S rottoli5
1Scientific Department, Velleja research, Milan, 2Pediatric Department, University of Parma, Parma, 3Stomatology institute, Milan, 4laboratory of epidemiology and Social Psychiatry, Mario negri institute, Milan, 5Pediatric Department, Uboldo Hospital, cernusco sul naviglio, italy
correspondence: Francesco Di Pierro Scientific Department, Velleja Research, 23 Viale lunigiana, Milan 20125, italy Tel +39 349 552 7663 Fax +39 0523 511 894 email [email protected]
Background: Streptococcus salivarius K12 is an oral probiotic strain releasing two lantibiotics
(salivaricin A2 and salivaricin B) that antagonize the growth of S. pyogenes, the most important
bacterial cause of pharyngeal infections in humans also affected by episodes of acute otitis media.
S. salivarius K12 successfully colonizes the oral cavity, and is endowed with an excellent safety
profile. We tested its preventive role in reducing the incidence of both streptococcal and viral
pharyngitis and/or tonsillitis in children.
Materials and methods: We enrolled 61 children with a diagnosis of recurrent oral strep-
tococcal disorders. Thirty-one of them were enrolled to be treated daily for 90 days with a
slow-release tablet for oral use, containing no less than 1 billion colony-forming units/tablet
of S. salivarius K12 (Bactoblis®), and the remaining 30 served as the untreated control group.
During treatment, they were all examined for streptococcal infection. Twenty children (ten per
group) were also assessed in terms of viral infection. Secondary end points in both groups were
the number of days under antibiotic and antipyretic therapy and the number of days off school
(children) and off work (parents).
Results: The 30 children who completed the 90-day trial with Bactoblis® showed a significant
reduction in their episodes of streptococcal pharyngeal infection (.90%), as calculated by
comparing the infection rates of the previous year. No difference was observed in the control
group. The treated group showed a significant decrease in the incidence (80%) of oral viral
infections. Again, there was no difference in the control group. With regard to secondary end
points, the number of days under antibiotic treatment of the treated and control groups were
30 and 900 respectively, days under antipyretic treatment 16 and 228, days of absence from
school 16 and 228, and days of absence from work 16 and 228. The product was well tolerated
by the subjects, with no side effects, and only one individual reported bad product palatability
and dropped out.
Conclusion: Prophylactic administration of S. salivarius K12 to children with a history of
recurrent oral streptococcal disease resulted in a considerable reduction of episodes of both
streptococcal and viral infections and reduced the number of days under antibiotic and/or anti-
pyretic therapy and days of absence from school or work.
Keywords: Blis K12, pediatric trial, Bactoblis, S. pyogenes, antibiotic therapy
IntroductionStreptococcus salivarius K12 is a strain isolated from the throat of a New Zealand
child,1 and capable of producing two distinct lantibiotics – salivaricin A2 and salivaricin
B – encoded on two adjacent loci on a 190-kilobase megaplasmid.2 Owing to these
two salivaricins, the K12 strain successfully counteracts the growth of S. pyogenes
in vitro.3 In contrast the K12 P– strain (negative plasmid) is totally ineffective.4
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Di Pierro et al
The K12 strain is not only effective against S. pyogenes but
also inhibits the growth of such pathogens as Haemophilus
influenzae, S. pneumoniae, and Moraxella catarrhalis, all
of which are involved in the etiopathogenesis of acute otitis
media.5 The four pathogens are responsible for almost all
bacterial pharyngotonsillitis cases in children and adults.6
The results of K12 administration in children demonstrate
that the strain can colonize the oral cavity in some 30% of
children as early as on the third administration day, with clear
colonization even in the nasopharynx and adenoids,7 and
can remain in tissue for up to 32 days after the last admin-
istration.8 The K12 strain is also endowed with an excellent
antibiotic-sensitivity profile and high safety characteristics,
as demonstrated in tests in animals9 and humans.10 Recent
clinical trials, conducted both in adults11 and children,12
demonstrated that treatment with the K12 strain reduces
recurrences of bacterial pharyngotonsillitis by approxi-
mately 80% and 90%, respectively. Even if a treatment over
at least 90 days is followed by a 6-month washout period,
the protection rates against recurrence remain high (about
60% in either case). Preliminary data indicate that treatment
with the K12 strain also seems to reduce acute otitis media
recurrences by 40% in children.12 As the K12 strain can
also inhibit proliferation of Micrococcus luteus, S. angi-
nosus, Eubacterium saburreum, and Micromonas micros,
it has been successfully used in the treatment of halitosis
by eliminating volatile sulfur compounds in 14 days.13,14 A
recent study highlighted the action of the K12 strain against
Candida albicans, though in this case the authors did not
demonstrate a cytotoxic action referable to the release of
the two salivaricins (A2 and B), but reported a mechani-
cal antihyphae action.15 In addition to the aforementioned
biological actions, the K12 strain also seems to possess
preventive properties against oropharyngeal infections of
viral origin and gingivitis. This possibility seems to be
confirmed by the fact that administration of the K12 strain
in adults may increase salivary interferon-γ levels without
modifying the levels of either interleukin (IL)-1β or tumor
necrosis factor-α, but considerably reducing IL-8 release.5
The incidence of pharyngotonsillitis is very high in children,
and is caused by bacteria in about 35% of cases (with 80%
of these being caused by S. pyogenes) and by respiratory
viruses in about 65% of cases.16,17 We resolved to verify
the prophylactic action of the K12 strain administered in
children with a diagnosis of recurrent pharyngotonsillitis
caused by β-hemolytic streptococcus, while assessing the
aspects of potential prevention of either S. pyogenes infec-
tions or possible viral infections.
Materials and methodsThe productThe K12 strain was formulated in the form of slowly dis-
solving oral tablets by SIIT (Trezzano, Milan, Italy) and
notified to the Italian Ministry of Health as Bactoblis® by
Omeopiacenza (Pontenure, Italy), according to the provisions
of law 169 of 2004, on July 5, 2011 (notification number
53435). The preparation of Bactoblis® used in the clinical trial
contained no less than 1 billion colony-forming units (CFU)/
tablet of S. salivarius K12 (BLIS Technologies, Dunedin,
New Zealand).
The clinical trialThe multicenter, open, nonrandomized, controlled clinical
trial was conducted on 61 pediatric individuals enrolled in
the area of Milan, Italy, and treated between January 31 and
April 30, 2013. The trial population consisted of 32 males
(53.4%) and 29 females (46.6%). The trial was conducted
according to the criteria set by the Declaration of Helsinki
and with the approval of the local ethics committee. The
parents of all the participants in the study were informed of
the trial methods and signed the consent and privacy-policy
documents.
inclusion criteriaAll the enrolled individuals were 3–13 years of age and
attended school in the Milan area. In terms of recurrent
pharyngotonsillitis, the enrolled individuals exhibited an
average of not less than three episodes in the same quarter
as that of the study (January 31 to April 30) of the previous
year (2012). The episodes were confirmed by a rapid swab
positive for group A and group B streptococci. None of the
individuals were affected by infectious diseases of any nature
on enrolment.
exclusion criteriaThe following exclusion criteria were used: immunocom-
promised individuals, individuals who had undergone
tonsillectomy or with an indication for adenotonsillectomy,
individuals with rheumatic disorders, individuals with
bronchospasm and/or a diagnosis of asthma and/or allergy,
individuals with respiratory disorders or important systemic
disorders, and individuals undergoing therapies to prevent
any recurrent respiratory infections.
Study patternThe individuals enrolled following their parents’ approval
were first subjected to a general medical examination and
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S. salivarius K12 for pharyngotonsillitis prevention
pharyngeal buffer (Test Strep-A; Gima, Gessate, Italy) and
then subdivided, according to a simple 1+1 enrolment key,
into two groups: one group was treated with Bactoblis®,
while the other did not receive any treatment and served as
the control group. The individuals of the Bactoblis® group
were instructed on how to use the product. The product was
to be administered for 90 consecutive days. The children
had to let it dissolve slowly in the mouth immediately before
going to sleep, after brushing their teeth. They were care-
fully instructed not to chew the product or swallow it whole.
They were asked not to drink or swallow any substance fol-
lowing the administration of the product. During the whole
trial period, the individuals were invited to come to the
clinic as soon as any oropharyngeal symptoms suggested
an infection. In this case, the individual was immediately
subjected to a medical examination and pharyngeal buffer
test. In the case of a positive result, treatment was prescribed
to the individual. The prescribed therapy was antibiotics in
the case of streptococcal infection. The selected antibiotic
in the study was the combination of amoxicillin and clavu-
lanic acid to be administered for 10 days. At the end of the
prescribed antibiotic therapy, treatment with Bactoblis® was
resumed and continued until the scheduled 90th day. In the
case of a viral infection accompanied by pharyngolaryngeal
pain and/or a fever, treatment was based on acetaminophen
or ibuprofen.
Diagnosis of viral infectionDiagnosis of viral infection was done according to the fol-
lowing criteria: negative rapid swab for streptococci, absence
of submandibular lymphadenopathy, absence of petechiae on
the palate, mild dysphagia, absence of headache, absence
of abdominal pain, and absence of hyperpyrexia. From a
clinical standpoint, patients with viral pharyngitis presented
with modest pharyngeal hyperemia, low-grade fever, mild
dysphagia, presence of rhinitis with serous secretion, and
spontaneous resolution of symptoms without medication in
48–72 hours. Differential diagnosis with mononucleosis, ie,
absence of adenomegaly, absence of splenomegaly, absence
of plaque exudates on the tonsils, and absence of hyperpy-
rexia, was also made.
Study objectivesThe study aimed to verify the following parameters: the
efficacy of Bactoblis® in the prevention of pharyngotonsil-
litis from group A S. pyogenes during the study period, the
efficacy of Bactoblis® in reducing viral pharyngotonsillar
infections in the same period, the onset of side effects or
toxicity while the product was being administered, and the
compliance of the individuals. Secondary objectives were the
collection of information concerning resorting to antibiotic
therapy, treatment with antipyretics, working days lost by
parents, and days of absence from school (or preschool for
children under 6 years of age).
Statistical analysisThe equivalence of the two treatment groups in terms of
sex and age was determined using Fisher’s exact test and
the two-tailed Wilcoxon–Mann–Whitney test, respectively.
The difference between the two treatment groups in terms of
number of pharyngotonsillitis episodes was determined using
the two-tailed Wilcoxon–Mann–Whitney test. Statistical soft-
ware used was JMP® 10 for Mac OS X (SAS Institute, Cary,
NC, USA), and the threshold for statistical significance was
95%. The sample size was determined in the following way.
We estimated the prevalence of pharyngotonsillitis episodes
in the untreated population to be 90% of the whole popula-
tion, while the application of the product could determine
a reduction to 50%. For the hypothesis of 95% specificity
and 90% test power, in consideration of Fisher’s exact test,
the resulting sample size amounted to 58 units, to be equally
divided into the two treatment branches. It was thus decided
to include at least 60 individuals to obtain the required sta-
tistical effectiveness.
ResultsSixty children with a diagnosis of recurrent pharyngotonsil-
litis were enrolled to highlight the preventive role played by
slowly dissolving oral tablets (Bactoblis®) containing a strain
of S. salivarius K12 (no less than 1 billion CFU/tablet),
against β-hemolytic streptococcal or viral pharyngotonsillitis.
Thirty children were treated with one tablet of product a day
for 90 consecutive days. The other 30 children served as
the control group in the same period. As shown in Table 1,
the two groups did not exhibit such important characteris-
tics as to make them significantly different. The statistical
Table 1 characteristics* of the children who completed the study
Treated Untreated P-value
Total number 30 30Males 19 13 0.20age† of males 6.7±2.5 6.1±2.8 0.14Females 11 17 0.20age† of females 5.7±1.9 5.2±1.8 0.14episodes/child‡ 3.1 3.0 0.70
Notes: *Nonsignificant differences between groups; †years ± standard deviation; ‡for the quarter considered for enrolment (January 31–april 30, 2012).
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Di Pierro et al
equivalence in sex, age, and number of past episodes between
the treatment groups was verified using Fisher’s exact test
(P=0.20), the Wilcoxon–Mann–Whitney test (P=0.14), and
the Wilcoxon–Mann–Whitney test (P=0.70), respectively.
Table 2 shows the data on the prevalence of streptococcal
pharyngotonsillitis episodes (diagnosed with a rapid pharyn-
geal swab). During the 90-day treatment in the 2013 quarter,
the 30 treated children who completed the study were affected
by three episodes of streptococcal etiology: pharyngotonsil-
litis was diagnosed in two children and scarlet fever in a third
child. These same 30 children had suffered from 94 episodes
of oral streptococcal infection in the same quarter of the
previous year. The children in the control group, who had
suffered from 90 episodes of oral streptococcal infection in
the same quarter of 2012, suffered from 84 episodes in the
same quarter of 2013. Prophylaxis with Bactoblis® reduced
the incidence of β-hemolytic streptococcal infections by some
96% (about 7% reduction observed in the control group).
The statistical analysis also shows that in 2012, the two study
groups did not exhibit statistically significant differences in
the onset of tonsillitis (Wilcoxon–Mann–Whitney P=0.16).
On the contrary, in 2013 the two groups were found to differ
in a statistically significant manner with regard to the onset of
tonsillitis (Wilcoxon–Mann–Whitney P,0.001). The result is
that the odds ratio of suffering from tonsillitis following the
administration of Bactoblis® was equal to 0.003, with a 95%
confidence interval amounting to 0.001–0.026. The assess-
ment of viral oropharyngeal infections was performed only
in one of the medical centers involved in the study. For this
reason, the data reported in Table 3 refer to only ten children
of either group. As the table shows, the ten children of the
treated group had had 25 infections of viral etiology in the
corresponding quarter of 2012, with a prevalence of 100%.
In the same quarter of 2013, viral infections diagnosed in
the same ten children during treatment were only five, with
a prevalence of 30% (two children with two episodes each
and one with one episode). In the control group, with the
assessment being referred again to ten children, the 28 viral
infections diagnosed in the quarter of 2012, with a prevalence
of 100%, were reduced to 24 in the corresponding quarter of
2013 (prevalence 100%). Treatment with Bactoblis® reduced
the incidence of viral oropharyngeal infections by 80%
(about 14% reduction observed in the control group). From
a statistical perspective, in 2013 we observed a prevalence of
viral episodes in the untreated group of 100%, while in the
treated one we observed viral infection only in 30%; by the
hypothesis of 95% specificity, this leads to a test power of
70%. Therefore these results are significant at P,0.01. The
treatment (Table 4), referable only to the 30 children who had
received Bactoblis®, was well tolerated and without any side
effects worth mentioning. Compliance was very good, with
only one child complaining of the bad taste of the product.
Because of the perceived poor palatability, the individual
dropped out of the study immediately on the first enrolment
day. As shown in Table 5, the number of days on which the
children were treated with antibiotics or antipyretics were 30
and 16, respectively, in the treated group. Antipyretics were
administered for 6 days following pain/fever due to a strep-
tococcal infection and for 10 days for the same reasons, but
due to a viral infection; this caused the loss of 16 school days
(or preschool days, depending on the child’s age) and the loss
of 16 working days by the children’s parents. In the control
group, there were instead 900 antibiotic therapy days and 228
antipyretic therapy days, 180 of which owing to streptococcal
infections and 48 owing to viral infections. This caused the
loss of 228 school (or preschool) days and the loss of 228 Table 2 episodes of pharyngotonsillitis caused by Streptococcus pyogenes in the two study groups (n=30/group)
Treated A/C Untreated A/C
January 31–april 30, 2012 94 3.1 90 3.0January 31–april 30, 2013 3*,† 0.1 84‡ 2.8% reduction of episodes 96.79 6.79
Notes: *P,0.001 versus episodes relatable to the same quarter of 2012 and versus episodes relatable to the control group in the same quarter of 2013; †one child with scarlet fever, two children suffering from one pharyngotonsillitis episode each; ‡15 children suffering from four pharyngotonsillitis episodes each, ten children suffering from one pharyngotonsillitis episode each and one from scarlet fever, two children suffering from two pharyngotonsillitis episodes each, three children without any episodes.Abbreviation: a/c, average/child.
Table 3 rhino-pharyngotonsillar episodes of viral etiopathogenesis in two subgroups of individuals (n=10/group)
Treatment 2012 quarter
A/C 2013 quarter
A/C Delta %
Treated 25 2.5 5* 0.5 80control 28 2.8 24 2.4 14.3
Note: *P,0.01 versus the 2012 quarter and versus the control group result relatable to the 2013 quarter.Abbreviation: a/c, average/child.
Table 4 Tolerability, compliance, and side effects during the 90-day treatment in 31 individuals enrolled in the Bactoblis® group
Tolerability Compliance Side effects
excellent n=30 n=30 nonegood n=0 n=0 noneacceptable n=0 n=0 noneUnacceptable n=1* n=1* none
Note: *Refused to continue on the very first treatment day due to an obvious distaste for the product.
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S. salivarius K12 for pharyngotonsillitis prevention
working days. In terms of costs (see Table 6), the treated
group reported a total expenditure of slightly over €2,600.
The overall expenditure of the control group amounted to
approximately €1,530. In particular, the treated group spent
€2,558.25 to acquire the product Bactoblis®, €44.79 for anti-
biotics (about €11 borne by the families and the remainder
by the public health service) and €36.80 to buy antipyretics.
The control group spent €1,343.70 in antibiotics (€335.70
borne by the families and the remainder by the public health
service) and €184 in antipyretics.
DiscussionThe chief objective of treatment with probiotics – that is,
with strains isolated from human feces (or from the feces of
mice, pigs, or other animals) – is the achievement of intes-
tinal health. Probiotics are described as capable of counter-
acting constipation, diarrhea, or irritable bowel syndrome.
These effects can be achieved, but are closely linked to their
intestinal colonization capability. Probiotic therapy may
be also aimed at a nonintestinal benefit. Examples of these
applications are attempts to prevent or treat such gynecologi-
cal and/or urological conditions as vaginitis, vaginosis, or
cystitis, particularly in patients with recurrent forms. Even
in these cases, however, the effect seems to be directly con-
nected to the ability of the probiotic to colonize the intestine.
Once the strain has reached and colonized the intestine, it
can transmigrate to the surrounding, anatomically connected
tissues, like the vagina or bladder, and exert its biological
action by counteracting the growth of pathogens.18 In the case
of the K12 strain, the issue of this study, things are different.
This strain, isolated from the oral cavity through a pharyngeal
swab, can colonize the oropharynx rather than the intestine
and locally release the two lantibiotics (salivaricin A2 and B),
which have been described as able to counteract the action of
β-hemolytic streptococcus. Its oral colonization reduces the
risk of oral colonization by S. pyogenes. As a matter of fact,
the same antagonism described for β-hemolytic streptococ-
cus has also been observed for other strains, which are also
sensitive to the same two lantibiotics and responsible for acute
otitis media and halitosis. More recent information5 seems
to demonstrate that oral administration of the K12 strain
can not only colonize the oropharynx with the release of the
two salivaricins acting as antagonists for certain pathogenic
strains but also, through a still not perfectly clear molecular
mechanism, reduce IL-8 plasma concentrations and increase
salivary interferon-γ. These modulations may also rationally
account for anti-inflammatory and antiviral activity, which
would then be added to the antibacterial action of the K12
strain already described. The results of the trial described in
this work go exactly in this direction. Prophylaxis with the
K12 strain reduced streptococcal infections by over 90%,
thus confirming the results of other works,11,12 but – and this
is something new – also demonstrated an 80% reduction of
viral infections. Use of the K12 strain also contributed to an
important reduction in the children’s and their parents’ days
off school and work. In consideration of the need to rest at
home during a high-grade fever or high infectivity hazard,
use of the K12 strain resulted in absence from school, pre-
school, or work for 32 days in all, corresponding to about
1 day per family. On the contrary, not using it caused absence
from school/preschool/work for 456 days, corresponding
to about 15 days per family. With regard to the expenditure
parameter, the advantage of not having lost 14 school/ working
days per family cost the group treated with the K12 strain
about €900 more than the expenditure borne by the control
group. To put this in individual family terms, this €900 delta
corresponds to a cost of €30 per family, and €30 spread
over 90 treatment days corresponds to 33 a day per family.
The same calculation can be demonstrated by arguing on
the basis of not losing school days and working days. In this
case, as stated earlier, use of the K12 strain prevented the
loss of 14 days off school or work per family. As each fam-
ily spent €30 more than those who did not resort to the K12
strain, it can be stated that each “saved” day cost about €2
per family. Hiring a babysitter to avoid losing a working day
will certainly cost much more than that. The most impor-
tant benefit offered by the use of the K12 strain in children
suffering from recurrent oral infections of a streptococcal
nature is not the undoubted economic advantage, but rather
the dramatic reduction in the number of days under antibi-
otic therapy. As shown in Table 5, in the group treated with
Table 5 Days under treatment with antibiotics and/or antipyretics or days off preschool/school or working days lost by parents in the study groups (n=30/group)
Group Antibiotics Antipyretics* School/preschool Work
Treated 30 6+10 16 16control 900 180+48 228 228
Note: *The first number indicates the antipyretic administered because of a streptococcal disease, and the second the antipyretic administered because of a viral disease.
Table 6 expenses (€) borne by the two groups to buy Bactoblis®, antibiotics, and antipyretics (n=30/group)
Group Bactoblis® Antibiotics Antipyretics Total
Treated 2,558.25 44.79 36.80 2,639.84control 0 1,343.70 184.00 1,527.70
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Di Pierro et al
the K12 strain, resorting to antibiotic therapy was 30 times
lower. Similar data can be evinced from those resorting to
antipyretic/anti-inflammatory/analgesic therapy. In this case,
use of the K12 strain reduced the occurrence of resorting to
acetaminophen or ibuprofen 14-fold. These results, together
with the excellent tolerability and compliance, as well as the
absence of side effects, demonstrate that the K12 strain can
be a valid therapeutic solution in the prevention of infective
diseases of the oral cavity, whether of streptococcal or viral
etiology, and in particular those of a recurrent nature. This
study confirms, at least with regard to streptococcal infection
prevention, the data observed and published in two previ-
ous works,11,12 and adds another piece of information: the
prevention of viral infections, which will obviously require
further clinical confirmation before it can be validated with
greater certainty. This study certainly exhibits some limits:
the absence of a placebo group, the absence of blind condi-
tions, the small size of the sample, only one center involved
in checking viral infection, and an inability to follow up the
enrolled children in the 6–9 subsequent months to assess
further trends in infective oropharyngeal events. These limits
will be overcome in a subsequent multicenter study presently
in progress in the Emilia Romagna (Italy) territory.
DisclosureFDP is the main formulator of the tested product and involved
in the scientific council of the company (Omeopiacenza) trad-
ing the tested product. The other authors report no conflicts
of interest in this work.
References1. Tagg JR. Prevention of streptococcal pharyngitis by anti-Streptococcus
pyogenes bacteriocin-like inhibitory substances (BLIS) produced by Streptococcus salivarius. Indian J Med. 2004;119 Suppl:13–16.
2. Hyink O, Wescombe PA, Upton M, Ragland N, Burton JP, Tagg. Salivaricin A2 and the novel lantibiotic salivaricin B are encoded at adjacent loci on a 190-kilobase transmissible megaplasmid in the oral probiotic strain Streptococcus salivarius K12. Appl Environ Microbiol. 2007;73(4):1107–1113.
3. Sharma S, Verma KK. Skin and soft tissue infection. Indian J Pediatr. 2001;68 Suppl 3:S46–S50.
4. Wescombe PA, Burton JP, Cadieux PA, et al. Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius. Antonie Van Leeuwenhoek. 2006;90(3):269–280.
5. Wescombe PA, Hale JD, Heng NC, Tagg JR. Developing oral probi-otics from Streptococcus salivarius. Future Microbiol. 2012;7(12): 1355–1371.
6. van Zon A, van der Heijden GJ, van Dongen TM, Burton MJ, Schilder AG. Antibiotics for otitis media with effusion in children. Cochrane Database Syst Rev. 2012;9:CD009163.
7. Power DA, Burton JP, Chilcott CN, Dawes PJ, Tagg JR. Preliminary investigations of the colonisation of upper respiratory tract tissues of infants using a paediatric formulation of the oral probiotic Streptococ-cus salivarius K12. Eur J Clin Microbiol Infect Dis. 2008;27(12): 1261–1263.
8. Horz HP, Meinelt A, Houben B, Conrads G. Distribution and persistence of probiotic Streptococcus salivarius K12 in the human oral cavity as determined by real-time quantitative polymerase chain reaction. Oral Microbiol Immunol. 2007;22(2):126–130.
9. Burton JP, Wescombe PA, Moore CJ, Chilcott CN, Tagg JR. Safety assessment of the oral cavity probiotic Streptococcus salivarius K12. Appl Environ Microbiol. 2006;72(4):3050–3053.
10. Burton JP, Cowley S, Simon RR, McKinney J, Wescombe PA, Tagg JR. Evaluation of safety and human tolerance of the oral probiotic Strepto-coccus salivarius K12: a randomized, placebo-controlled, double-blind study. Food Chem Toxicol. 2011;49(9):2356–2364.
11. Di Pierro F, Adami T, Rapacioli G, Giardini N, Streitberger C. Clinical evaluation of the oral probiotic Streptococcus salivarius K12 in the prevention of recurrent pharyngitis and/or tonsillitis caused by Streptococcus pyogenes in adults. Expert Opin Biol Ther. 2013;13(3): 339–343.
12. Di Pierro F, Donato G, Fomia F, et al. Preliminary pediatric clinical evaluation of the oral probiotic Streptococcus salivarius K12 in preventing recurrent pharyngitis and/or tonsillitis caused by Streptococcus pyogenes and recurrent acute otitis media. Int J Gen Med. 2012;5:991–997.
13. Burton JP, Chilcott CN, Tagg JR. The rationale and potential for the reduction of oral malodour using Streptococcus salivarius probiotics. Oral Dis. 2005;11 Suppl 1:29–31.
14. Burton JP, Chilcott CN, Moore CJ, Speiser G, Tagg JR. A preliminary study of the effect of probiotic Streptococcus salivarius K12 on oral malodour parameters. J Appl Microbiol. 2006;100(4):754–764.
15. Ishijima SA, Hayama K, Burton JP, et al. Effect of Streptococcus salivarius K12 on the in vitro growth of Candida albicans and its protective effect in an oral candidiasis model. Appl Environ Microbiol. 2012;78(7):2190–2199.
16. Murray RC, Chennupati SK. Chronic streptococcal and non- streptococcal pharyngitis. Infect Disord Drug Targets. 2012;12(4):281–285.
17. Bonsignori F, Chiappini E, De Martino M. The infections of the upper respiratory tract in children. Int J Immunopathol Pharmacol. 2010;23(Suppl 1):16–19.
18. Singh VP, Sharma J, Babu S, Rizwanulla MT, Singla A. Role of probiotics in health and disease: a review. J Pak Med Assoc. 2013;63(2): 253–257.
1. Introduction
2. Materials and methods
3. Results
4. Discussion
Original Research
Clinical evaluation of the oralprobiotic Streptococcus salivariusK12 in the prevention of recurrentpharyngitis and/or tonsillitiscaused by Streptococcuspyogenes in adultsFrancesco Di Pierro†, Teresa Adami, Giuliana Rapacioli, Nadia Giardini &Christian Streitberger†Velleja Research, Scientific Department, Milano, Italy
Background: Streptococcus salivarius K12 has been shown to inhibit the
growth of Streptococcus pyogenes due to bacteriocins release. Because of
its ability to colonize the oral cavity, we have tested the strain K12 for its
efficacy in preventing streptococcal pharyngitis and/or tonsillitis in adults.
Methods: Forty adults with a diagnosis of recurrent oral streptococcal pharyn-
gitis were enrolled in the study. Twenty of these subjects took for 90 days a
tablet containing Streptococcus salivarius K12 (Bactoblis�). The other
20 subjects served as untreated controls. A 6-month follow-up was included
to evaluate any persistent protective role.
Results: The 20 adults who completed the 90-day course of Bactoblis� showed
a reduction in their episodes of streptococcal pharyngeal infection (about
80%). The 90 days treatment was also associated with an approximately
60% reduction in the incidence of reported pharyngitis in the 6-month period
following use of the product. The product was well tolerated by the subjects
with no treatment-related side effects or drop-outs reported.
Conclusion: Prophylactic administration of Streptococcus salivarius K12 to
adults having a history of recurrent oral streptococcal pathology reduced
the number of episodes of streptococcal pharyngeal infections and/
or tonsillitis.
Keywords: bactoblis�, BLIS, pharyngitis, Streptococcus salivarius K12, tonsillitis
Expert Opin. Biol. Ther. [Early Online]
1. Introduction
Most probiotics currently in use by humans have been derived from intestinal sour-ces and they have targeted the improvement of intestinal tract maladies [1].However, more recently it has been recognized that there is potential for probioticintervention in non-intestinal body sites using effector strains of species that areindigenous to alternative target tissues, in order to possibly obtain more specificand enduring health benefits [2]. This kind of approach could be referred to as“bacterial therapy” or “bioprotic” therapy, where bio indicates “living bacteria,” prostands for “in favor of” and tic refers to “a well identified and precise pharmaceuticalor nutraceutical application.” To our knowledge, Streptococcus salivarius K12 couldbe one of the first examples of the “bioprotic” approach. One probiotic on the mar-ket, called BLIS K12 (where BLIS is the acronym for Bacteriocin-Like InhibitorySubstances) uses the S. salivarius strain K12 which was isolated from the mouth
10.1517/14712598.2013.758711 © 2013 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 1All rights reserved: reproduction in whole or in part not permitted
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of an healthy child [3]. It is known to inhibit the growth of alltested strains of b-hemolytic (Lancefield group A) S. pyogenes,the most common cause of bacterial pharyngitis and tonsillitisand also sometimes implicated in acute otitis media(AOM) [4,5]. This inhibitory activity is known to be due tothe action of the lantibiotics salivaricin A2 and salivaricin B,both of which are encoded by a 190 kb megaplasmid presentin strain K12 [6]. Derivatives of strain K12 lacking the mega-plasmid DNA fail to show any BLIS activity againstS. pyogenes [7]. The inhibitory spectrum of BLIS K12 alsoincludes strains of Micrococcus luteus, Streptococcus anginosus,Eubacterium saburreum andMicromonas micros some of whichcan contribute to the symptoms of halitosis [8,9]. Pilot studieshave shown that BLIS K12 can achieve persistent colonizationof various tissues within the upper respiratory tract of infants(oral cavity, nasopharynx, and adenoid tissues) [10,11]. On thebasis of its i) good oral cavity colonization capabilities,ii) excellent safety record (recently having achieved self-affirmed GRAS status) [12,13] and iii) reputed ability to pre-vent or treat certain oral infections [14], we decided to evaluatethe efficacy of BLIS K12 when administered to adults havinga history of recurrent streptococcal pharyngitis and/or tonsillitis with the principal endpoint being the numberof episodes of oral streptococcal infections.
2. Materials and methods
The study has been carried out in a routine clinical practicesetting, following international guidelines and in line with theprinciples outlined in the Declaration of Helsinki. This studywas carried out in out-patient department (Merano Hospital,Merano, Italy) and in clinics (Verona and Piacenza, Italy) whereit is not mandatory to obtain ethical approval in order toperform experimental protocols on nutraceutical products.Inclusion criteria were informed signed consent; age between18 and 65 years; total absence of symptoms of infective diseaseat the time of enrolment; diagnosis of recurrent, streptococcal(group A hemolytic Streptococcus) pharyngitis and/or tonsillitisin the previous year. Exclusion criteria were absence of informedconsent; age below 18 or above 65; severe respiratory and/or systemic pathologies; asthma; healthy carriage of Streptococcuspyogenes. In the study, fromOctober 2011 to August 2012, therewere 40 subjects enrolled with a diagnosis of not less thanfour episodes of recurrent pharyngitis and/or tonsillitis in theprevious year confirmed by throat swab (S. pyogenes positive).Selection was done on the basis of the information available inthe files of the out-patient department and clinics involved inthe study. These files contained information either comingdirectly from the department and clinics or coming from a visitreport filled by the medical doctors visiting the patient in thefirst aid of an hospital. Twenty subjects were selected for thetreatment group and 20 for the not-treated (control) group.These subjects were followed for 90 days. After this period, allsubjects in the treated group were considered valid for theaims of the study, having declared a total adherence to the
treatment protocol. In a second part of the study, 16 subjectsof treated group and 17 of the not-treated group were followedfor a further 6 months, during which time the product was notadministered to them. The product, Bactoblis� in agreementwith the Italian law number 169/2004, has been notified tothe Minister of Health on July the 5th, 2011 (Registrationnumber still pending) and registered as a food supplement.Bactoblis� contains at the time of manufacturing, 5 billionCFU/tablet of S. salivariusK12 ATCCBAA-1 024 (BLIS Tech-nologies Ltd., New Zealand) and has been manufactured bySIIT (Italy). According to the treatment protocol, the round-shaped, vanilla-flavored, slowly-dissolving tablet, had to beadministered just before bedtime (after teeth-washing and/or mouthwash use) once daily for 90 days. The correct adminis-tration of the product requires that the tablet is not to be chewedor directly swallowed, but that it has to be sucked for about4 -- 5 min. Before administration of the first tablet, a chlorhexi-dine (0.2%)mouthwash was given to decrease the population ofendogenous S. salivarius inhabiting the mouth in order toenhance the colonization process. Data were collected on adocument filled out by the physician during each subject visit.The visits occurred every 15 days regardless of the presence ofany apparent pathology. When oral pathology (sore throat)was apparent, the subjects reported straight away to the medicalcenter prior to the 15th day appointment. The following infor-mation was entered into the document: name of physician,inclusion and exclusion criteria, beginning and end date of ther-apy, name of patient, age, weight, height, sex, other concomitanttherapy, clinical history during the 90 days of trial, number ofepisodes of oral S. pyogenes infection in the previous year, typesof vaccines given to subject, results of oral swab at the time ofenrollment to exclude healthy carriers, results of oral swab per-formed at every visit, types of side effects observed during the90 days of treatment, tolerance to the course of tablets and com-pliance at the end of the therapy. Primary study endpoints werethe evaluation, throughmedical visits and throat swabs, of num-ber of episodes of pharyngitis and/or tonsillitis in the treated andin the not-treated groups during the 90 days of treatment withthe product and during the 6-month follow-up period whenthe product was not administered. Secondary study endpointswere the evaluation of parameters like tolerability, complianceand side effects during the 90 days of treatment. With respectto the assessment of tolerability and compliance we have definedfour terms to describe the different reactions: very good, good,acceptable, and unacceptable. Compliance, as adherence to ther-apy, was bothmonitored by a document to be filled every day bythe subjects, to be delivered every 30 days to the physician, andby a check of the returned boxes of the product every 30 days.The follow-up data were collected using procedures identicalto those reported above with the exception that the plannedvisits occurred every 30 days and the parameters of compliance,tolerability and side effects were not evaluated. Out of the20 subjects in the treated group, one was experiencing chronicgingivitis with bleeding and two had recurrent aphthous stoma-titis previously demonstrated not to be responsive to treatment
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with hyaluronic acid, chlorhexidine, topical antibiotics andcorticosteroids. Unscheduled endpoints were for evaluation ofthese two localized mouth complaints. The statistical analysiswas performed using the Standardized Incidence Ratio (SIR)and its confidence interval 100(1-)% as proposed by Vanden-broucke [15]. If the range includes 100% it is highly likely thatthe difference between observed and expected is due to chance(random fluctuations in the data).On the other hand, if the con-fidence interval does not include 100%, it is very likely that thedifference is not due to chance. The statistical comparisonsbetween treatment and past controls are shown in the tableswhere the real number of episodes is reported along with, intable notes, the same value/4 to allow a real statistical compari-son otherwise not possible using values obtained in 12 monthswith values obtained in 3 months.
3. Results
In this study, we have attempted to evaluate the preventiverole played by BLIS K12 when administered to adults havinga history of recurrent pharyngitis and/or tonsillitis of strepto-coccal origin. The main endpoint was the number of episodesof streptococcal oral pathology. The 40 adults enrolled inthe study were assigned either to the treated (20 subjects)or not-treated (20 subjects) group. As shown in Table 1
the demographic characteristics of the 40 enrolled, andtherapy-adherent, adults do not differ statistically.
Statistically-significant results have however been seenduring the 90 days of treatment with BLIS K12 (Table 2) interms of episodes of streptococcal pharyngitis and/or tonsilli-tis in the 20 adults enrolled on the basis that they had experi-enced not less than four episodes of streptococcal pharyngitis
and/or tonsillitis in the previous year. These 20 adults had98 streptococcal episodes in the previous 12 months, but dur-ing the 90 days of treatment the subjects experienced onlyfour diagnosed episodes of oral streptococcal infection, theincidence per month per subject dropping from 0.410 to0.067.
The control group, adults enrolled with prior diagnosis ofrecurrent streptococcal oral disease but not treated withBLIS K12, showed an increase in episodes of streptococcalpharyngitis and/or tonsillitis in comparison with the previousyear, as shown by the calculation of the incidence per monthper subject (Table 3). This increase, from 0.421 to 0.48, islikely due to seasonal variations, since the previous year valuealso includes a period of warm months when the incidence ofstreptococcal infections normally drops, while the secondvalue includes infections acquired only during 3 wintermonths.
Sixteen and 17, respectively of the adults in the treated andnon-treated groups consented to continue with a 6-monthfollow-up component of the study to evaluate possible ongo-ing protection against streptococcal pathology following the90 days of treatment with BLIS K12. As shown in Table 5,in the 6-month period the 17 adults from the not-treated group had 14 episodes of oral streptococcal pathologywhereas the 16 adults coming from the treated group experi-enced only five oral streptococcal infections. This representsa reduction in incidence of approximately 60%.
4. Discussion
Pharyngitis is a common medical problem in the outpatientmedical setting, resulting in millions of patient visits eachyear in Italy. Most episodes of pharyngitis are caused by infec-tious etiologies, especially viruses. However, some of the moreserious types of pharyngitis are bacterial in origin, and themost common agent is S. pyogenes. Complications ofuntreated S. pyogenes pharyngitis include rheumatic fever,deep space abscesses, and toxic shock. Although most episodesof pharyngitis are acute in nature, a small percentage becomerecurrent or chronic. Antibiotic therapy is typically prescribedto treat both acute and recurrent infections. However, whenpatients present with sore throat, physicians must also con-sider a wider range of potential pathogens including virusesand other bacteria. Aside from a few rare streptococcal infec-tions due to species other than S. pyogenes, antimicrobial ther-apy is of no proven benefit to treat any other causes ofpharyngitis other than those provoked by Lancefield groupA S. pyogenes. Inappropriate antibiotic therapy imposesunnecessary expense and also contributes to the emergenceof antibiotic-resistant bacteria, which are being reportedwith increasing frequency. Consequently a conservativeapproach to managing sore throats is increasingly promoted,with antibiotic therapy held in reserve until S. pyogenes infec-tion is confirmed. Prevention of infection by non-antibiotictherapy is a preferable approach to the use of repeated
Table 1. Demographic parameters of enrolled adults.
Group N M F Age*
Treated 20 7 13 33.0 ± 6.4Not-treated 20 8 12 35.7 ± 7.0
*Expressed as median ± standard deviation.
F: Females; N: Number of subjects; M: Males.
Table 2. Episodes of streptococcal oral pathology
during the 90 days of treatment with BLIS K12 in
adults (n = 20) with recurrent streptococcal pharyngitis
and/or tonsillitis.
Pharyngitis/
tonsillitis in the
previous year
Pharyngitis/
tonsillitis while
taking BLIS K12
Number of episodes 98 (1 year) 4 (90 days)Incidence/month/subject 0.410 0.067*Delta (%) -83.7
*p < 0.001 considering 98 episodes and p < 0.01 considering 25 episodes (98/4).
Preventive role of BLIS K12 in adult
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treatment doses of antibiotics. The ability of the normal oralcavity bacterial microflora of some individuals to inhibit thegrowth of S. pyogenes has been previously established. Mostof this inhibitory activity has been attributed to BLIS-producing S. salivarius [16]. Since S. salivarius has extremelylow pathogenic potential and it is a prominent member ofthe normal oral microbiota it is regarded as an excellent can-didate for bacterial interference-mediated prevention of recur-rent pharyngitis and tonsillitis. Certain S. salivarius caninterfere with the growth of S. pyogenes due to their produc-tion of BLIS. For example, S. salivarius K12 has been shownto reduce acquisitions of S. pyogenes by school-aged children
and also the prevalence of sore throats [17-20]. These datahave been recently confirmed in a pediatric clinical investiga-tion performed in Italy [21] in which children with a history ofrecurrent streptococcal oral pathology were for 90 days, givena nutritional supplement (Bactoblis�) containing as its uniqueactive ingredient 5 billion CFU/tablet of S. salivarius K12. Inthat same study, the influence on acute otitis media (AOM)incidence in the children was also assessed. Since that investi-gation included some risk of the incidence values potentiallybeing affected by normal fluctuations of oral and ear pathol-ogy, the incidence values in the treated group were comparedwith those of two untreated groups. The first untreated grouphad the same characteristic as the treated one, being consti-tuted of children enrolled because of recurrent streptococcaloral pathology. However, for an additional control of possibleincidence fluctuations, disease episodes were also monitoredin a second group of children in whom previous recurrentoral streptococcal pathology had not been demonstrated.This approach to the study showed that the approximately90% reduction in the incidence of S. pyogenes pathology dur-ing the 90 days of treatment with BLIS K12 was not due tofortuitous fluctuations in exposure of the subjects to S. pyo-genes during the study period. In the second part of that study,an assessment was made of whether the administration ofBLIS K12 resulted in persistent protection in the 6 monthsfollowing treatment. This evaluation demonstrated that theprior use of the product provided enduring protection againstoral streptococcal pathology and AOM, with about 65%reduction compared to the control. In spite of several limita-tions inherent in that study design; it was not randomized,placebo-controlled or blinded, and it was based on use of arelatively small number of children -- the results demonstratedfor the first time that the use of the oral probiotic S. salivariusK12 by children having a history of recurrent oral streptococ-cal disease, could substantially reduce the incidence ofbacterial infections of the throat and ear. In the present study,we have decided to evaluate the same clinical endpoints in acohort of adult people enrolled on the basis of their recurrentstreptococcal oral pathology. Of the 40 enrolled subjects,20 were treated for 90 days with 5 billion CFU/tablet ofS. salivarius K12. The other 20 subjects served as untreatedcontrols. Supporting the outcome of our parallel pediatricstudy, for the treated group the protection rate was about80% with a very high profile in terms of tolerability, compli-ance and side effects (Table 4). Following the 90 days oftreatment, the protocol included an assessment of whetherthe prior administration of BLIS K12 resulted in persistentprotection in the following 6 months. As was also foundin the pediatric study enduring protection against oralstreptococcal pathology was observed with about 60%reduction found by comparison to the control group. Alsoof note, the treated group included one subject sufferingchronic gingivitis with bleeding and two having recurrentaphthous stomatitis, previously demonstrated not to beresponsive to treatment with B-vitamins supplementation or
Table 3. Episodes of streptococcal oral pathology
during 90 days in adults (n = 20) with recurrent
streptococcal pharyngitis and/or tonsillitis not treated
with BLIS K12.
Pharyngitis/
tonsillitis in the
previous year
Pharyngitis/
tonsillitis in
90 days
Number of episodes 101 29Incidence/month/subject 0.421 0.48*Delta (%) +38.5
*p < 0.001 considering 101 episodes and p > 0.05 considering 25 episodes
(101/4).
Table 4. Tolerability, compliance and side effects
during the 90 days of treatment with BLIS K12 in
adults (n = 20) having a history of recurrent
streptococcal pharyngitis and/or tonsillitis.
Tolerability Compliance Side effects
Very Good n = 19 n = 19 NoneGood n = 1 n = 1 NoneAcceptableUnacceptable
N: Number of adults.
Table 5. Episodes of oral streptococcal pathology
during the course of a 6-month follow-up of adults
who had been previously treated or not-treated with
BLIS K12.
Group Number of
episodes
Incidence/
month/
subject
% vs
control
Control (n = 17)(from not-treated group)
14 0.137
Tested (n = 16)(from treated group)
5 0.052* -62.1
*p = 0.0389 (Pearson chi-squared test for difference in proportion. Chi^2 = 4.26).
N: Number of children.
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with hyaluronic acid, chlorhexidine, antibiotic and corticoste-roids. Clinical assessments of these three patients have shownthat a total resolution of these conditions occurred in associa-tion with their exposure to BLIS K12. Whether theseresponses are directly related to the use of BLIS K12 remainsan open question. In conclusion, the regular use of BLISK12 appears to have effected a substantial reduction in theincidence of recurrent oral streptococcal pathology, reducingthe requirement for these BLIS K12-treated individuals tobe exposed to therapeutic courses of antibiotics. On the basis
of the encouraging outcome from our tests of BLIS K12efficacy in both children and adults, our group is currentlyorganizing a much larger randomized, blinded, placebo-controlled study to more thoroughly evaluate the findings ofthese preliminary investigations.
Declaration of interest
FDP is the principal formulator of the product. There are noother conflicts of interest.
Bibliography
1. Sathyabama S, Vijayabharathi R,
Bruntha Devi P, et al. Screening for
probiotic properties of strains isolated
from feces of various human groups.
J Microbiol 2012;50:603-12
2. Tagg JR, Dierksen KP. Bacterial
replacement therapy: adapting ’germ
warfare’ to infection prevention.
Trends Biotechnol 2003;21:217-23
3. Tagg JR. Prevention of streptococcal
pharyngitis by anti-Streptococcus
pyogenes bacteriocin-like inhibitory
substances (BLIS) produced by
Streptococcus salivarius. Indian J
Med Res 2004;119(Suppl):13-16
4. Jack RW, Tagg JR, RAY B. Bacteriocins
of gram-positive bacteria. Microbiol Rev
1995;59:171-200
5. Sharma S, Verma KK. Skin and soft
tissue infection. Indian J Pediatr
2001;68(Suppl 3):S46-50
6. Hyink O, Wescombe PA, Upton M, et al.
Salivaricin A2 and the novel lantibiotic
salivaricin B are encoded at adjacent loci on
a 190-kilobase transmissible megaplasmid
in the oral probiotic strain Streptococcus
salivarius K12. Appl Environ Microbiol
2007;74:1107-13
7. Wescombe PA, Burton JP, Cadieux PA,
et al. Megaplasmids encode differing
combinations of lantibiotics in
Streptococcus salivarius. Antonie van
Leeuwenhoek 2006;90:269-80
8. Burton JP, Chilcott CN, Tagg JR. The
rationale and potential for the reduction
of oral malodour using Streptococcus
salivarius probiotics. Oral Dis
2005;11(Suppl 1):29-31
9. Burton JP, Chilcott CN, Moore CJ,
et al. A preliminary study of the effect of
probiotic Streptococcus salivarius K12 on
oral malodour parameters.
J Appl Microbiol 2006;100:754-64
10. Power DA, Burton JP, Chilcott CN,
et al. Preliminary investigations of the
colonisation of upper respiratory tract
tissues of infants using a paediatric
formulation of the oral probiotic
Streptococcus salivarius K12. Eur J Clin
Microbiol Infect Dis 2008;27:1261-3
11. Horz HP, Meinelt A, Houben B, et al.
Distribution and persistence of probiotic
Streptococcus salivarius K12 in the human
oral cavity as determined by real-time
quantitative polymerase chain reaction.
Oral Microbiol Immunol 2007;22:126-30
12. Burton JP, Wescombe PA, Moore CJ,
et al. Safety assessment of the oral cavity
probiotic Streptococcus salivarius K12.
Appl Environ Microbiol 2006;72:3050-3
13. Burton JP, Cowley S, Simon RR, et al.
Evaluation of safety and human tolerance
of the oral probiotic Streptococcus
salivarius K12: a randomized,
placebo-controlled, double-blind study.
Food Chem Toxicol 2011;49:2356-64
14. Tagg J, Wescombe P, Burton J. Oral
streptococcal BLIS: Heterogeneity of the
effector molecules and potential role in
the prevention of streptococcal infections.
Int Congress Series 2006;1289:347-50
15. Vandenbroucke JP. A shortcut method
for calculating the 95 per cent confidence
interval of the standardised mortality
ratio. Am J Epidemol 1982;115:303
16. Tagg JR. A longitudinal study of
Lancefield group A streptococcus
acquisitions by a group of young
Dunedin school children. NZ Med J
1990;103:429-31
17. Fantinato VC, Shimizu MT. Production
of bacteriocin-like inhibitory substances
(BLIS) by Streptococcus salivarius strains
isolated from the tongue and throat of
children with and without sore throat.
Rev Microbiol 1999;30:332-4
18. Ragland N, Tagg JR. Applications of
bacteriocin-like inhibitory substance (BLIS)
typing in a longitudinal study of the oral
carriage of beta-haemolytic streptococci by
a group of Dunedin schoolchildren.
Zentralbl Bakteriol 1990;274:100-8
19. Tagg JR. Significance of bacteriocin
production by oral streptococci. In:
Lutticken R, editor. Proceedings of the
Xth Lancefield Symposium. Gustav Fischer
Verlag; Stuttgart and New York; 1990
20. Dierksen KP, Tagg JR. The influence of
indigenous bacteriocin-producing
Streptococcus salivarius on the acquisition
of Streptococcus pyogenes by primary
school children in Dunedin. In:
Martin DR, Tagg JR, editors. Streptococci
and streptococcal diseases entering the new
millennium. Securacopy; Auckland, New
Zealand: 2000. p. 81-5
21. Di Pierro F, Donato G, Fomia F, et al.
Preliminary pediatric clinical evaluation of
the oral probiotic Streptococcus salivarius
K12 in preventing recurrent pharyngitis
and/or tonsillitis caused by Streptococcus
pyogenes and recurrent acute otitis media.
Int J Gen Med 2012;5:991-7
AffiliationFrancesco Di Pierro†1, Teresa Adami2,
Giuliana Rapacioli3, Nadia Giardini4 &
Christian Streitberger4
†Author for correspondence1Velleja Research, Scientific Department, Viale
Lunigiana 23, 20125, Milano, Italy
Tel: +39 3495527663; Fax: +39 0523 511894;
E-mail: [email protected] Diseases, Verona, Italy3A.I.O.R., Piacenza, Italy4Merano Hospital, ORL Department, Merano,
Italy
Preventive role of BLIS K12 in adult
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© 2012 Di Pierro et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.
International Journal of General Medicine 2012:5 991–997
International Journal of General Medicine
Preliminary pediatric clinical evaluation of the oral probiotic Streptococcus salivarius K12 in preventing recurrent pharyngitis and/or tonsillitis caused by Streptococcus pyogenes and recurrent acute otitis media
Francesco Di Pierro1
Guido Donato2
Federico Fomia3
Teresa Adami4
Domenico Careddu5
Claudia Cassandro6
Roberto Albera6
1Scientific Department, Velleja Research, Milano, 2ASL 1, Cuneo, 3ASL 3, Brescia, 4Infective Diseases, Verona, 5ASL 13, Novara, 6Surgical Science Department, Università degli Studi, Torino, Italy
Correspondence: Francesco Di Pierro Velleja Research, Viale Lunigiana 23, 20125, Milano, Italy Tel +39 34 9552 7663 Fax +39 05 2351 1894 Email [email protected]
Background: The oral probiotic Streptococcus salivarius K12 has been shown clearly to antago-
nize the growth of Streptococcus pyogenes, the most important bacterial cause of pharyngeal
infections in humans, by releasing two bacteriocins named salivaricin A2 and salivaricin B.
Unpublished observations indicate that it can also antagonize the growth of other bacteria
involved in acute otitis media. Because of its ability to colonize the oral cavity and its safety
profile, we have tested its efficacy in reducing the incidence of streptococcal pharyngitis and/or
tonsillitis and episodes of acute otitis media.
Methods: We enrolled 82 children, including 65 with and 17 without a recent diagnosis of
recurrent oral streptococcal pathology. Of those with recurrent pathology, 45 were treated daily
for 90 days with an oral slow-release tablet containing five billion colony-forming units of
S. salivarius K12 (Bactoblis®), and the remaining 20 served as an untreated control group. The
17 children without a recent diagnosis of recurrent oral pathology were used as an additional
control group. After 90 days of treatment, a 6-month follow-up period without treatment
was included to evaluate a possible persistent protective role for the previously administered
product.
Results: The 41 children who completed the 90-day course of Bactoblis showed a reduction
in their episodes of streptococcal pharyngeal infection (about 90%) and/or acute otitis media
(about 40%), calculated by comparing infection rates in the previous year. The 90-day treatment
also reduced the reported incidence of pharyngeal and ear infections by about 65% in the 6-month
follow-up period during which the product was not administered. Subjects tolerated the product
well, with no side effects or dropouts reported.
Conclusion: Prophylactic administration of S. salivarius K12 to children with a history of
recurrent oral streptococcal pathology reduced episodes of streptococcal pharyngeal infections
and/or tonsillitis as well as episodes of acute otitis media.
Keywords: BLIS K12, bacteriocin-like inhibitory substance K12, Streptococcus salivarius
K12, Bactoblis®, pharyngitis, tonsillitis, acute otitis media
IntroductionTo date, the use of probiotic strains has almost exclusively focused on the gastrointestinal
benefits of ingestion of selected bacteria obtained from intestinal sources.1 However,
the potential for probiotic intervention at nonintestinal body sites suggests possible
application of effector strains of species selected from alternative target tissues in order
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International Journal of General Medicine 2012:5
to obtain more specific and durable benefits.2 Streptococcus
salivarius K12, also known as BLIS (bacteriocin-like
inhibitory substance) K12, was isolated in New Zealand
from the mouth of a healthy child.3 It is known to release two
lantibiotic bacteriocins named salivaricin A2 and salivaricin
B, with high efficiency.4 Via these two lantibiotics, encoded by
a 190 kb megaplasmid,5 BLIS K12 can effectively counteract
the growth of β-hemolytic (group A) Streptococcus pyogenes,
a common cause of pharyngitis, tonsillitis, and acute otitis
media.6 This inhibitory action is strongly linked to the release
of lantibiotics because BLIS K12 P(-), the same strain without
the 190 kb plasmid, does not show any antagonism of growth
of Streptococcus pyogenes.7
In addition to its action against S. pyogenes, BLIS K12 can
also inhibit growth of Haemophilus influenzae, Streptococcus
pneumoniae, Moraxella catarrhalis, Micrococcus luteus,
Streptococcus anginosus, Eubacterium saburreum, and
Micromonas micros.8 Many of these are potential pathogens
in the ear and oral cavity, causing acute otitis media9 and
halitosis.10 Preliminary investigations have shown that
BLIS K12 colonizes the upper respiratory tract of infants
(oral cavity, nasopharyngeal and adenoid tissues)11 and with
good persistence, given that after only 3 days of administration,
it can still be detected 32 days later.12 Therefore, because
of its good colonization capability and very high safety
profile,13,14 combined with its reputed ability to counteract
oral pathology,15 we decided to evaluate the preventive role
of BLIS K12 when administered to children having a history
of recurrent streptococcal pharyngitis and/or tonsillitis. Our
main endpoint was the number of episodes of streptococcal
infections and acute otitis media.
Materials and methodsThis research was carried out in the field of routine clinical
practice, following international guidelines and in line with
the principles outlined in the Declaration of Helsinki, such that
approval from local ethics boards was not required. The study
was carried out in five Italian day care centers, located in Cuneo,
Brescia, Verona, Novara, and Torino, where it is not mandatory
to obtain ethical approval in order to perform experimental
protocols on nutraceutical products. Inclusion criteria were:
informed signed consent from parents; age 3–12 years; total
absence of symptoms of infective disease at the time of
enrollment; and diagnosis of recurrent streptococcal (group A
hemolytic Streptococcus) pharyngitis and/or tonsillitis in the
previous year. Exclusion criteria were: lack of parental signature
of informed consent; age below 3 years or above 12 years;
diagnosis of obstructive sleep apnea syndrome, respiratory,
and/or systemic severe pathologies; asthma; and being a
healthy carrier of S. pyogenes. Eighty-two children were
enrolled in the study, which was conducted from October 2011
to August 2012. Sixty-five were with and 17 were without a
diagnosis of not less than three episodes of recurrent pharyngitis
and/or tonsillitis in the previous year confirmed by throat swab
(positive for group A hemolytic Streptococcus). In agreement
with their parents, 45 of the 65 children with recurrent oral
pathology were selected as the treated group and 20 as the not-
treated group. The 17 children enrolled without a diagnosis of
recurrent pathology were selected as a not-treated group. The
recurrent-treated (n = 45), recurrent-not-treated (n = 20), and
not-recurrent-not-treated (n = 17) subjects were followed for
90 days. After this period, 41 of the 45 children in the recurrent-
treated group were considered appropriate for the aim of the
study, their parents having declared total adherence to the
treatment protocol.
In a second part of the study, in accordance with parental
consent, 16 of the 41 children in the recurrent-treated group
and 14 of the 20 children in the recurrent-not-treated group
were enrolled for follow-up lasting a further 6 months,
during which the product was not administered. The product,
Bactoblis®, in agreement with Italian law (169/2004),
was notified to the Minister of Health on July 5, 2011 and
registered as a food supplement. At the time of manufacturing,
Bactoblis contains five billion colony-forming units per tablet
of S. salivarius K12 ATCC BAA-1 024 (BLIS Technologies
Ltd, North Dunedin, New Zealand) and is manufactured by
SIIT, (Trezzano S/N, Milan,Italy). In accordance with the
treatment protocol, the product is administered as one tablet
daily for 90 days. The product, an oral, round-shaped, vanilla-
tasting, slow-release tablet (dissolving in about 5 minutes)
is administered just before bedtime (ie, after teeth brushing
and/or mouthwashing). Correct administration of the product
requires that the tablet is not chewed or directly swallowed, but
is sucked for about 4–5 minutes. Before administration of the
first tablet, a chlorhexidine 0.2% mouthwash is recommended
in order to enhance the colonization process of the strain,
reducing extreme competition from endogenous S. salivarius
inhabiting the mouth.
The primary study endpoints were evaluation by
medical visits, results of a throat swab, and otoscopic
signs of acute otitis media, and episodes of pharyngitis,
tonsillitis, and/or acute otitis media in the recurrent-treated,
recurrent-not-treated, and not-recurrent-not-treated groups
during 90 days of treatment with the product and during
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Di Pierro et al
International Journal of General Medicine 2012:5
the 6-month follow-up period in which the product was not
administered. Secondary study endpoints were tolerability,
compliance, and side effects during the 90 days of treatment.
As regards to tolerability and compliance, we defined four
terms able to describe the different conditions, ie, very good,
good, acceptable, and unacceptable.
Statistical analysisThe statistical analysis was performed using the standardized
incidence ratio and its confidence interval 100 (1 - α)% as
proposed by Vandenbroucke.16 If the range includes 100%,
it is highly likely that the difference between observed and
expected values is due to chance (random fluctuations in
the data). On the other hand, if the confidence interval does
not include 100%, it is very likely that the difference is not
due to chance. The statistical comparisons between treatment
and past controls are shown in Tables 2–7, where the real
number of episodes is reported along with, in table notes, the
same value/4 to allow a real statistical comparison, otherwise
not possible comparing values obtained in 12 months with
values obtained in 3 months.
ResultsIn this study, we attempted to establish the preventive
role played by BLIS K12 when administered to children
with a history of recurrent pharyngitis and/or tonsillitis of
streptococcal origin. The main endpoint was the number of
episodes of oral streptococcal pathology and/or acute otitis
media. The 82 children enrolled in this study were assigned to
one of three groups, ie, recurrent-treated, recurrent-not-treated,
or not-recurrent-not-treated. Four children were eliminated
because of failure to adhere strictly to therapy. As shown in
Table 1, the demographic characteristics of the 78 therapy-
adherent enrolled children did not differ statistically.
Statistically significant results were seen during the 90 days
of treatment with BLIS K12 (Table 2) in terms of episodes of
streptococcal pharyngitis and/or tonsillitis in the 41 children
having had more than three episodes of streptococcal pharyngitis
and/or tonsillitis in the previous year. These 41 children had
Table 1 Demographic parameters of enrolled children
Group n M F Age, years*
Recurrent-treated 41 19 22 4.5 ± 1.4Recurrent-not-treated 20 9 11 4.2 ± 1.3Not-recurrent-not-treated 17 9 8 5.1 ± 1.5
Note: *Expressed as the median ± standard deviation. Abbreviations: n, number of children; M, males; F, females.
Table 2 Episodes of streptococcal oral pathology during 90 days of treatment with BLIS K12 in children (n = 41) with recurrent streptococcal pharyngitis and/or tonsillitis
Pharyngitis/ tonsillitis in the previous year
Pharyngitis/ tonsillitis during BLIS K12
Number of episodes 152 (1 year) 3 (90 days)Incidence/month/child 0.309 0.024*Delta (%) -92.2
Notes: *P , 0.0001 considering 152 episodes and P , 0.01 considering 38 episodes (152/4). Abbreviation: BLIS, bacteriocin-like inhibitory substance.
Table 4 Episodes of streptococcal oral pathology during 90 days in children (n = 17) without recurrent streptococcal pharyngitis and/or tonsillitis and not treated with BLIS K12
Pharyngitis/ tonsillitis in previous year
Pharyngitis/ tonsillitis in 90 days
Number of episodes 4 4Incidence/month/child 0.020 0.078*Delta (%) +290
Note: *Not significant considering four episodes and P , 0.05 considering one episode (4/4). Abbreviation: BLIS, bacteriocin-like inhibitory substance.
Table 3 Episodes of streptococcal oral pathology during 90 days in children (n = 20) with recurrent streptococcal pharyngitis and/or tonsillitis not treated with BLIS K12
Pharyngitis/ tonsillitis in the previous year
Pharyngitis/ tonsillitis in 90 days
Number of episodes 78 27Incidence/month/child 0.325 0.45*Delta (%) +38.5
Note: *P , 0.001 considering 78 episodes and not significant considering 19.5 episodes (78/4). Abbreviation: BLIS, bacteriocin-like inhibitory substance.
Table 5 Episodes of acute otitis media during the 90 days of treatment with BLIS K12 in children (n = 41) with recurrent streptococcal pharyngitis and/or tonsillitis
AOM in previous year
AOM during BLIS K12
Number of episodes 27 4 (90 days)Incidence/month/child 0.055 0.033*Delta (%) -40
Note: *P , 0.01 considering 27 episodes and not significant considering 6.75 episodes (27/4). Abbreviations: AOM, acute otitis media; BLIS, bacteriocin-like inhibitory substance.
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Table 7 Episodes of acute otitis media during 90 days in children (n = 17) without recurrent streptococcal pharyngitis and/or tonsillitis and not treated with BLIS K12
AOM in previous year
AOM in 90 days
Number of episodes 4 2Incidence/month/child 0.020 0.039*Delta (%) +95
Note: *P , 0.05 considering four episodes and not significant considering one episode (4/4). Abbreviations: AOM, acute otitis media; BLIS, bacteriocin-like inhibitory substance.
had 152 episodes in 12 months, and during the 90 days of
treatment, only three episodes of oral streptococcal infection
were diagnosed, with the calculated incidence per month per
child dropping from 0.3109 to 0.024.
The control group, (children enrolled with a diagnosis of
recurrent oral streptococcal disease but not-treated) showed an
increase in terms of episodes of streptococcal pharyngitis and/
or tonsillitis in comparison with the previous year, as shown
by the incidence per month per child (Table 3). This increase,
from 0.325 to 0.45, is likely due to seasonal reasons being the
first value calculated, considering also warm months where the
incidence normally decreases, while the second value calcu-
lated is only considered during the three winter months.
The other controls, (not-recurrent not-treated children
enrolled without a diagnosis of recurrent oral streptococcal
disease) also showed an increase in terms of episodes of
streptococcal pharyngitis and/or tonsillitis in comparison
with the previous year, as shown by the incidence per month
per child (Table 4). This value increased from 0.020 to 0.078.
This is again likely due to seasonal reasons being the first
value calculated considering also warm months where the
incidence normally drops down, while the second value is
calculated only considering the 3 winter months.
Relevant (-40%) results were seen during 90 days of
treatment with BLIS K12 (Table 5) in terms of episodes of
acute otitis media in the 41 children enrolled for having had no
fewer than three episodes of streptococcal pharyngitis and/or
tonsillitis in the previous year. In fact, these 41 children had
had 27 episodes in 12 months and four episodes during the
90 days of treatment, with the incidence per month per child
dropping from 0.055 to 0.033.
In the control (not-treated) children enrolled with a
diagnosis of recurrent oral streptococcal disease, there was
an increase in terms of episodes of acute otitis media in
comparison with the previous year, as shown by the incidence
per month per child (Table 6). This increase, from 0.054 to
0.117, is again likely due the seasonal reasons explained earlier.
Table 6 Episodes of AOM during 90 days in children (n = 20) with recurrent streptococcal pharyngitis and/or tonsillitis not treated with BLIS K12
AOM in previous year
AOM in 90 days
Number of episodes 13 7Incidence/month/child 0.054 0.117*Delta (%) +116
Note: *P , 0.05 considering 13 episodes and not significant considering 3.25 episodes (13/4). Abbreviations: AOM, acute otitis media; BLIS, bacteriocin-like inhibitory substance.
In the other control (not-recurrent-not-treated) children
enrolled without a diagnosis of recurrent streptococcal oral
disease, there was an increase in terms of episodes of acute
otitis media in comparison with the previous year, as shown
by the incidence per month per child (Table 7). This value
increased from 0.020 to 0.039, again likely for seasonal
reasons.
The evaluation of tolerability, compliance, and side
effects is reported only for the recurrent-treated group as
enrolled in terms of number (n = 45). As shown in Table 8,
treatment with BLIS K12 seemed to be well tolerated and
devoid of side effects. The four children who were excluded
from the study were removed because they did not adhere
strictly to the study therapy, not because of side effects.
According to their parents, they have missed more than
20 days of treatment.
With parental consent, only 16 of the 41 recurrent-treated
children and 14 of the 20 recurrent-not-treated children,
respectively, continued into the 6-month follow-up period
to determine if BLIS K12 had a protective role. As shown
in Table 9, the 14 children in the recurrent-not-treated
group had eight episodes of oral streptococcal pathology
and 10 episodes of acute otitis media over 6 months. The
16 children in the recurrent-treated group were confirmed to
be protected, having had four oral streptococcal infections,
two episodes of acute otitis media, and one case of scarlet
fever, with a reduction by about 65% of incidence.
DiscussionAcute pharyngitis and/or tonsillitis in children are among
the most frequent recurrent illnesses presenting to general
practitioners and pediatricians. Group A beta-hemolytic
streptococci is the most common bacterial cause of acute
pharyngitis and tonsillitis. Antibiotic therapy is typically pre-
scribed to treat the acute infection and to prevent development
of sequelae, such as rheumatic fever. However, when patients
present with sore throat, physicians must also consider a
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Table 8 Tolerability, compliance and side effects during the 90 days of treatment with BLIS K12 in children (n = 45) with recurrent streptococcal pharyngitis and/or tonsillitis as reported by parents and established by clinician
Tolerability Compliance Side effects
Very good n = 42 n = 42 NoneGood n = 3 n = 1 NoneAcceptable n = 2 NoneUnacceptable
Abbreviations: n, number of children; BLIS, bacteriocin-like inhibitory substance.
Table 9 Episodes of oral streptococcal pathology and acute otitis media in a 6-month follow-up period in children coming from the recurrent-treated and from the recurrent-not-treated groups
Group Number of episodes
Incidence/ month/child
% versus control
Control (n = 14) (from recurrent-not-treated)
18° 0.214
Tested (n = 16) (from recurrent-treated)
7°° 0.073* -65.9
Notes: *P = 0.0278 (Pearson Chi-squared test for difference in proportions, Chi-squared test = 4.84); °oral streptococcal pathology (n = 8) and acute otitis media (n = 10); °°oral streptococcal pathology (n = 4), acute otitis media (n = 2), scarlet fever (n = 1).
wider range of potential pathogens, including viruses and
other bacteria. Apart from a few rare non-group A streptococ-
cal infections, antimicrobial therapy is of no proven benefit to
treat causes of pharyngitis other than those provoked by
group A beta-hemolytic streptococci. Inappropriate antibiotic
therapy imposes unnecessary expense and also contributes
to the emergence of antibiotic-resistant bacteria, which are
being reported with increasing frequency. Consequently, a
conservative approach to managing sore throats is being
promoted increasingly, with antibiotic therapy held in reserve
until group A beta-hemolytic streptococcal infection is con-
firmed. Prevention of recurrent infection by nonantibiotic
therapy is preferable than having repeated doses of antibiot-
ics. The ability of the normal bacterial microflora in the oral
cavity to inhibit the growth of group A streptococci has been
established previously. Most of this inhibitory activity has
been attributed to BLIS-producing S. salivarius.17 Because S.
salivarius is a member of the normal bacterial flora found in
the oral cavity and is considered to be essentially nonpatho-
genic, it is regarded as an excellent candidate for bacterial
interference-mediated prevention of recurrent pharyngitis
and tonsillitis.
Our study is based upon the observation that certain
strains of S. salivarius are capable of preventing the growth
of bacteria associated with sore throat due to their production
of BLIS. In particular, it has been shown that S. salivarius
K12 reduces group A streptococcus acquisition and also
the prevalence of sore throat.18–21 Therefore, the feasibility
of using this harmless strain as a prophylactic agent was
investigated in this preliminary study. Ear infections are
also common in children, and in severe cases can lead to
deafness. Acute otitis media is the most common bacterial
infection in young children. It is thought that the bacteria
from the nasopharyngeal tissue that infect the middle ear do
so via the eustachian tubes. In Italy, over 90% of cases of
acute otitis media result in antibiotic treatment. Relapses are
common, and repeat treatment may contribute to a reservoir of
resistant microorganisms. Published studies have shown that
at least 50% of patients acquire a new otitis media infection
within 3 months of a previous episode.22 The ability of the
normal microflora of the upper airways to inhibit growth of
potential pathogens in vitro has been well described.23 Most of
this inhibitory activity has been attributed to alpha-hemolytic
streptococci. One as yet unpublished study, (data on file at
BLIS Ltd, Dunedin, New Zealand) has shown that S. salivarius
K12 produces BLIS with activity against S. pneumoniae, S.
pyogenes, H. influenzae, and M. catarrhalis, ie, the principal
agents known to be causative of acute otitis media. On this
basis, we decided to test the role played by S. salivarius K12 in
the prevention of streptococcal oral pathology and acute otitis
media. Sixty-five children with a history of recurrent oral
streptococcal pathology were given Bactoblis, a nutritional
supplement containing as its unique active ingredient five
billion colony-forming units per tablet of S. salivarius K12
for 90 days.
We also checked the incidence of acute otitis media
during treatment. This preliminary investigation was not
placebo-controlled, so risks determining incidence values
potentially affected by normal fluctuations of mouth and
ear pathology. To minimize such a risk, we compared the
incidence values in the treated group with those of two
untreated groups. The first untreated group had the same
characteristics as the treated one, being comprised of children
enrolled because of recurrent oral streptococcal pathology.
For further control of possible fluctuations in incidence, we
monitored disease episodes in a second group of children in
whom oral streptococcal pathology had not been recurrent.
This methodological approach allowed us to demonstrate that
the approximately 90% reduction in incidence observed by
administering BLIS K12 for 90 days was not due to random
fluctuations during the study period in 2012.
In the second part of the study, we investigated whether
administration of BLIS K12 resulted in durable protection
in the months following treatment. This evaluation lasted
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Preventive role of BLIS K12 in oral and ear pathologies
International Journal of General Medicine 2012:5
from March to August 2012, and demonstrated that prior
use of the product provided durable protection against oral
streptococcal pathology and acute otitis media, with about
65% reduction compared with controls.
This study has several limitations, in that it is not ran-
domized nor placebo-controlled, and was also not blinded.
Furthermore, it was carried out in a relatively small number
of children with recurrent oral streptococcal pathology but
not specifically with recurrent acute otitis media, with epi-
sodes of the latter only being an endpoint. However, in spite
of these limitations, the results demonstrate for the first time
that use of S. salivarius K12, an oral probiotic, can reduce
the incidence of bacterial throat and ear infections in children
with a history of recurrent oral streptococcal infection. Our
research group is currently organizing a larger, randomized,
blinded, placebo-controlled study in children with recur-
rent oral streptococcal pathology to confirm more precisely
what has been observed in this preliminary investigation.
This study will be performed with approval and a reference
number from our local ethics committee.
DisclosureFDP is the main formulator of the study product. Otherwise,
the authors report no conflicts of interest in this work.
References1. Sathyabama S, Vijayabharathi R, Bruntha Devi P, Ranjith Kumar M,
Priyadarisini VB. Screening for probiotic properties of strains isolated from feces of various human groups. J Microbiol. 2012;50:603–612.
2. Tagg JR, Dierksen KP. Bacterial replacement therapy: adapting ‘germ warfare’ to infection prevention. Trends Biotechnol. 2003;21: 217–223.
3. Tagg JR. Prevention of streptococcal pharyngitis by anti-Streptococcus pyogenes bacteriocin-like inhibitory substances (BLIS) produced by Streptococcus salivarius. Indian J Med Res. 2004;119 Suppl:13–16.
4. Jack RW, Tagg JR, Ray B. Bacteriocins of Gram-positive bacteria. Microbiol Rev. 1995;59:171–200.
5. Hyink O, Wescombe PA, Upton M, Ragland N, Burton JP, Tagg JR. Salivaricin A2 and the novel lantibiotic salivaricin B are encoded at adjacent loci on a 190-kilobase transmissible megaplasmid in the oral probiotic strain Streptococcus salivarius K12. Appl Environ Microbiol. 2007;73:1107–1113.
6. Sharma S, Verma KK. Skin and soft tissue infection. Indian J Pediatr. 2001;68 Suppl 3:S46–S50.
7. Wescombe PA, Burton JP, Cadieux PA, et al. Megaplasmids encode dif-fering combinations of lantibiotics in Streptococcus salivarius. Antonie Van Leeuwenhoek. 2006;90:269–280.
8. Burton JP, Chilcott CN, Tagg JR. The rationale and potential for the reduction of oral malodour using Streptococcus salivarius probiotics. Oral Dis. 2005;11 Suppl 1:29–31.
9. van Zon A, van der Heijden GJ, van Dongen TM, Burton MJ, Schilder AG. Antibiotics for otitis media with effusion in children. Cochrane Database Syst Rev. 2012;9:CD009163.
10. Burton JP, Chilcott CN, Moore CJ, Speiser G, Tagg JR. A preliminary study of the effect of probiotic Streptococcus salivarius K12 on oral malodour parameters. J Appl Microbiol. 2006;100:754–764.
11. Power DA, Burton JP, Chilcott CN, Dawes PJ, Tagg JR. Preliminary investigations of the colonisation of upper respiratory tract tissues of infants using a paediatric formulation of the oral probiotic Streptococcus salivarius K12. Eur J Clin Microbiol Infect Dis. 2008;27:1261–1263.
12. Horz HP, Meinelt A, Houben B, Conrads G. Distribution and persistence of probiotic Streptococcus salivarius K12 in the human oral cavity as determined by real-time quantitative polymerase chain reaction. Oral Microbiol Immunol. 2007;22:126–130.
13. Burton JP, Wescombe PA, Moore CJ, Chilcott CN, Tagg JR. Safety assessment of the oral cavity probiotic Streptococcus salivarius K12. Appl Environ Microbiol. 2006;72:3050–3053.
14. Burton JP, Cowley S, Simon RR, McKinney J, Wescombe PA, Tagg JR. Evaluation of safety and human tolerance of the oral probiotic Strepto-coccus salivarius K12: a randomized, placebo-controlled, double-blind study. Food Chem Toxicol. 2011;49:2356–2364.
15. Tagg J, Wescombe P, Burton J. Oral streptococcal BLIS: heterogeneity of the effector molecules and potential role in the prevention of streptococ-cal infections. International Congress Series 1289. 2006:347–350.
16. Vandenbroucke JP. A shortcut method for calculating the 95 per cent confidence interval of the standardised mortality ratio. Am J Epidemiol. 1982;115:303–304.
17. Tagg JR. A longitudinal study of Lancefield group A streptococcus acquisitions by a group of young Dunedin school children. N Z Med J. 1990;103:429–431.
18. Fantinato VC, Shimizu MT. Production of bacteriocin-like inhibitory substances (BLIS) by Streptococcus salivarius strains isolated from the tongue and throat of children with and without sore throat. Revista de Microbiologia. 1999;30:332–334.
19. Ragland N, Tagg JR. Applications of bacteriocin-like inhibitory sub-stance (BLIS) typing in a longitudinal study of the oral carriage of beta-haemolytic streptococci by a group of Dunedin schoolchildren. Zentralbl Bakteriol. 1990;274:100–108.
20. Tagg JR. Significance of bacteriocin production by oral streptococci. In: R Lutticken R, editor. Proceedings of the Xth Lancefield Symposium. New York, NY: Gustav Fischer Verlag; 1990.
21. Dierksen KP, Tagg JR. The influence of indigenous bacteriocin-producing Streptococcus salivarius on the acquisition of Streptococcus pyogenes by primary school children in Dunedin, New Zealand, In: Martin DR, Tagg JR, editors. Streptococci and Streptococcal Diseases Entering the New Millenium. Auckland, New Zealand: Securacopy; 2000.
22. Roos K, Hakansson EG, Holm S. Effect of recolonisation with “interfering” alpha streptococci on recurrences of acute and secretory otitis media in children: randomized placebo controlled trial. BMJ. 2001;322:210–212.
23. Tano K, Olofsson C, Grahn-Hakansson E, Holm SE. In vitro inhibi-tion of S. pneumoniae, nontypable H. influenzae and M. catharralis by alpha-hemolytic streptococci from healthy children. Int J Pediatr Otorhinolaryngol. 1999;47:49–56.
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Preventive role of BLIS K12 in oral and ear pathologies
AOB-2765; No. of Pages 7
Antimicrobial activity of Streptococcus salivarius K12 onbacteria involved in oral malodour
L. Masdea a, E.M. Kulik a, I. Hauser-Gerspach a,*, A.M. Ramseier a, A. Filippi b, T. Waltimo a
a Institute of Preventive Dentistry and Oral Microbiology, School of Dental Medicine, University of Basel, SwitzerlandbDepartment of Oral Surgery, Oral Radiology and Oral Medicine and the Centre of Dental Traumatology, School of Dental Medicine, University
of Basel, Switzerland
a r c h i v e s o f o r a l b i o l o g y x x x ( 2 0 1 2 ) x x x – x x x
a r t i c l e i n f o
Article history:
Accepted 11 February 2012
Keywords:
Streptococcus salivarius K12
Halitosis
Solobacterium moorei
Deferred antagonism test
Bacteriocin
a b s t r a c t
Objective: To investigate the antimicrobial activity of the bacteriocin-producing strain
Streptococcus salivarius K12 against several bacteria involved in halitosis.
Design: The inhibitory activity of S. salivarius K12 against Solobacterium moorei CCUG39336,
four clinical S. moorei isolates, Atopobium parvulum ATCC33793 and Eubacterium sulci
ATCC35585 was examined by a deferred antagonism test. Eubacterium saburreum ATCC33271
and Parvimonas micra ATCC33270, which have been tested in previous studies, served as
positive controls, and the Gram-negative strain Bacteroides fragilis ZIB2800 served as a
negative control. Additionally, the occurrence of resistance in S. moorei CCUG39336 to S.
salivarius K12 was analysed by either direct plating or by passage of S. moorei CCUG39336 on
chloroform-inactived S. salivarius K12-containing agar plates.
Results: S. salivarius K12 suppressed the growth of all Gram-positive bacteria tested, but the
extent to which the bacteria were inhibited varied. E. sulci ATCC35585 was the most sensitive
strain, while all five S. moorei isolates were inhibited to a lesser extent. Natural resistance
seems to be very low in S. moorei CCUG39336, and there was only a slight decrease in
sensitivity after exposure to S. salivarius K12 over 10 passages.
Conclusion: Our studies demonstrate that S. salivarius K12 has antimicrobial activity against
bacteria involved in halitosis. This strain might be an interesting and valuable candidate for
the development of an antimicrobial therapy for halitosis.
# 2012 Elsevier Ltd. All rights reserved.
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
1. Introduction
Oral malodour, also called halitosis, afflicts a significant
proportion of the adult population and is of common interest
due to its compromising influence in social and working
environments. Most halitosis oral malodour compounds are
by-products of the metabolism of certain species of oral
bacteria, mainly those on the dorsum of the tongue.1,2 These
* Corresponding author at: Institute of Preventive Dentistry and OraHebelstrasse 3, 4056 Basel, Switzerland. Tel.: +41 061 267 25 98; fax: +
E-mail address: [email protected] (I. Hauser-Gerspach
Please cite this article in press as: Masdea L, et al. Antimicrobial activityArchives of Oral Biology (2012), doi:10.1016/j.archoralbio.2012.02.011
0003–9969/$ – see front matter # 2012 Elsevier Ltd. All rights reservedoi:10.1016/j.archoralbio.2012.02.011
compounds consist of VSC (volatile sulphur compounds),
valeric acid, butyric acid and putrescine.2 A diverse group of
Gram-negative and Gram-positive bacteria has been found to
contribute to the problem. By contrast, certain bacterial
species that predominate in the mouths of ‘‘healthy’’ subjects
are noticeably absent in subjects with halitosis.3
Current treatments focus on the use of chemical or physical
antibacterial regimens to reduce the numbers of these bacteria.
The treatments typically provide only short-term relief because
l Microbiology, School of Dental Medicine, University of Basel,41 061 267 26 58.).
of Streptococcus salivarius K12 on bacteria involved in oral malodour.
d.
a r c h i v e s o f o r a l b i o l o g y x x x ( 2 0 1 2 ) x x x – x x x2
AOB-2765; No. of Pages 7
the offensive bacteria quickly recover after treatment is
stopped.4
The use of probiotics has long been popular in the food
industry. The World Health Organisation defines probiotics as
a ‘live organism which when administered in adequate
amounts confers a health benefit on the host’. Their use in
clinical practice has previously been discussed.5 One potential
and clinically important use of probiotics is in the prevention
of dental caries.6–10
Preventing the re-growth of odour-causing organisms
through the pre-emptive colonisation of the oral cavity with
non-odorous, commensal microorganism may be a reason-
able alternative to chemical or physical antibacterial regi-
mens. Given that the dorsum of the tongue is the origin of
most halitosis problems, a candidate probiotic to counter this
condition should be able to persist in this particular ecosys-
tem. The production of anti-competitor molecules such as
bacteriocins also appears to confer an ecological advantage to
some bacteria. A probiotic strain that efficiently colonises the
tongue surface and does not produce odours metabolic by-
products would be highly advantageous.
Streptococcus salivarius is known to be a pioneer coloniser of
oral surfaces and is found predominant in ‘healthy’ humans
not affected by halitosis.3 BLIS K12 Throat Guard lozenges
(BLIS Technologies, Centre for Innovation, Dunedin, New
Zealand) contain S. salivarius K12, which has been shown to
help maintain throat health by supporting the defence against
undesirable bacteria.11 The bacterium is not genetically
modified or engineered, and the product is available in three
flavours (vanilla, strawberry and peppermint). The particular
strain used produces two natural antibacterial peptides,
salivaricin A212,13 and salivaricin B,14 which are lantibiotic-
type bacteriocins. In deferred antagonism studies, S. salivarius
K12 inhibited the Gram-positive bacteria Streptococcus anginosis
T29, Eubacterium saburreum and Micromonas micros, which are
implicated in halitosis, and significantly inhibited black-
pigmented colony types present in saliva samples.4
Based on these investigations and other promising results,
S. salivarius K12 has an excellent potential for use as a probiotic
targeting halitosis producing bacteria.
The aim of this study was to evaluate the extent of the
inhibitory spectrum of S. salivarius K12 against three additional
bacterial species recently found to be implicated in halitosis
and to investigate the development of bacterial resistance
against S. salivarius K12.
2. Materials and methods
2.1. Bacterial strains and growth conditions
The bacteriocin-producing strain S. salivarius K12 and the
nonproducer S. salivarius MU, were kindly provided by Prof. J.
Tagg (Department of Microbiology and Immunology, Univer-
sity of Otago, Dunedin, New Zealand).4
The indicator strains used in this study included the
following: E. saburreum ATCC 33271; Parvimonas micra (previ-
ously known as Micromonas micros or Peptostreptococcus micros)
ATCC 33270, which served as a positive control4 and Bacteroides
fragilis ZIB 2800 (School of Dental Medicine, University of Basel,
Please cite this article in press as: Masdea L, et al. Antimicrobial activityArchives of Oral Biology (2012), doi:10.1016/j.archoralbio.2012.02.011
Switzerland), which served as a negative control. The test
strains included Atopobium parvulum ATCC 33793, Eubacterium
sulci ATCC 35585, Solobacterium moorei CCUG 39336 and four
clinical S. moorei isolates, CH1#23, CH3A#109A, CH3#63 and
CH8#20,15 which had, to date, not yet been tested for
susceptibility against S. salivarius K12 in vitro.
All bacteria were grown on Columbia agar (Columbia Agar
Base [BBL Becton Dickinson, Allschwil, Switzerland]) supple-
mented with 4 mg/l hemin (Fluka, Buchs, Switzerland), 1 mg/l
menadione (VWR International, Dietikon, Switzerland) and
50 ml/l human blood (Blutspendezentrum, Basel, Switzerland)
under anaerobic conditions (Oxoid AnaeroGen Compact,
Oxoid, Pratteln, Switzerland) at 37 8C for 2–4 days.
2.2. Antimicrobial activity of S. salivarius K12
Inhibitory activities of S. salivarius K12 and the salivaricin non-
producer S. salivarius MU were analysed using a modified
deferred antagonism test.16 Sterile blotting paper (Inapa
Schweiz AG, Regensdorf, Switzerland) was cut to the size of
9 cm � 1 cm and carefully immersed in a S. salivarius culture
with a density of 4-5 McFarland standard. After removing excess
fluid, the blotting paper was placed in the middle of a plate of
Columbia agar containing 5% human blood and 0.1% calcium
carbonate (CaCO3) (E. Merck, Darmstadt) left in place for 2 s and
then removed. The plates were incubated at 37 8C under
anaerobic conditions for 24 h. After incubation, the growth
was removed with a sterile cotton swab. To kill any residual
bacterial cells on the mediums surface, the plate was exposed to
chloroform (E. Merck, Darmstadt) vapours for 30 min at room
temperature. The plate was then aired for 30 min.
Several colonies of each indicator strain grown on Colum-
bia blood agar-calcium carbonate medium were suspended in
3 ml Todd-Hewitt broth and streaked at right angles to the
original S. salivarius culture zone with a sterile cotton swab.
The plates were incubated under anaerobic conditions at 37 8C
for at least 48 h, and the extent of inhibition was recorded in
mm (the distance between the original producer line and the
inhibition line of indicator strains). Each test was performed at
least three times.
2.3. Test for resistance of S. moorei CCUG 39336 againstS. salivarius K12
S. salivarius K12 or S. salivarius MU cells were each suspended
in 3 ml Todd Hewitt broth and swabbed onto Columbia blood
agar-calcium carbonate medium. Afterwards, the plates were
incubated at 37 8C under anaerobic conditions for 24 h until
confluent growth was observed. Bacterial cells were removed
from the plates with sterile cotton swabs, and the agar
surfaces exposed to chloroform vapour for 30 min and aired
for another 30 min. Control plates without S. salivarius were
also exposed to the same conditions.
To detect bacteriocin-resistant S. moorei isolates, several
colonies of S. moorei CCUG 39336 were inoculated in 2 ml Todd-
Hewitt broth. After incubation at 37 8C under anaerobic
conditions for 24 h, 1 ml of this suspension was centrifuged
at 10,000 rpm for 15 min at 15 8C and resuspended in 300 ml
Todd-Hewitt broth. The exact cell density was determined
by plating appropriate dilutions onto Columbia blood
of Streptococcus salivarius K12 on bacteria involved in oral malodour.
Antimicrobial Activity of S. salivarius K12
inh
ibitio
n z
on
e ±
SD
[m
m]
0
5
10
15
20
25
A. parv
ulum
E. sab
urreu
m
E. sulc
i
P. micr
a
S. moo
rei C
CUG 3
9336
S. moo
rei C
H1#
23
S. moo
rei C
H3A
#109
A
S. moo
rei C
H3#
63
S. moo
rei C
H8#
20
Fig. 1 – Mean inhibition zone W standard deviation of S.
salivarius K12 against nine Gram-positive indicator strains
(n = 3).
a r c h i v e s o f o r a l b i o l o g y x x x ( 2 0 1 2 ) x x x – x x x 3
AOB-2765; No. of Pages 7
agar-calcium carbonate medium. One-hundred microlitres of
this S. moorei suspension was streaked onto the agar plate
pretreated with S. salivarius K12 and 100 ml onto the agar plate
pretreated with S. salivarius MU.
2.4. Test for induction of resistance in S. moorei CCUG39336 against the bacteriocins from S. salivarius K12
Bacteriocin-producing S. salivarius K12 and the indicator strain
S. moorei CCUG 39336 were grown, streaked onto Columbia
blood agar-calcium carbonate medium and incubated as
described above for the modified deferred antagonism test.
The S. moorei colonies closest to the inhibition zone were
subcultivated onto Columbia blood agar-calcium carbonate
medium and again tested against S. salivarius K12. This
procedure was repeated for 10 passages.
2.5. Statistical analysis
The inhibitory activity of S. salivarius K12 against the indicator
strains was tested using a linear model. The dependent
Fig. 2 – Inhibitory effect of S. salivarius K12 (a and b) compared to S. salivarius MU (c and d). A clear inhibition zone was
produced against S. moorei CCUG 39336 culture (a), whereas growth of B. fragilis was not inhibited by S. salivarius K12 (b).
The non-producer strain S. salivarius MU did not inhibit the growth of either S. moorei CCUG 39336 (c) or B. fragilis (d).
Please cite this article in press as: Masdea L, et al. Antimicrobial activity of Streptococcus salivarius K12 on bacteria involved in oral malodour.Archives of Oral Biology (2012), doi:10.1016/j.archoralbio.2012.02.011
Table 1 – Results of the statistical analysis of theinhibition of the Gram-positive indicator bacteria by S.salivarius K12 compared to the bacteriocin-nonproducingstrain S. salivarius MU. Shown are the respective indi-cator strain, the estimated mean differences (est. meandifference) in mm, the upper and lower 95% confidenceintervals (95% confint) in mm and the corresponding P-values.
Indicator strain Est. meandifference
95% confint P-Value
Lower Upper
A. parvulum 14.38 12.40 16.35 <0.001
E. saburreum 8.59 6.52 10.66 <0.001
E. sulci 16.67 12.71 20.63 <0.001
P. micra 9.28 6.99 11.56 <0.001
S. moorei CCUG 39336 9.75 7.33 12.17 <0.001
S. moorei CH1#23 5.29 3.31 7.27 <0.001
S. moorei CH3A#109A 9.17 6.88 11.45 <0.001
S. moorei CH3# 63 9.04 7.06 11.02 <0.001
S. moorei CH8#20 5.54 3.56 7.52 <0.001
Table 2 – Results of the statistical analysis comparing thesize of the inhibition zones of the first passage withthose of the following passages. Shown are the respec-tive passage number, the estimated mean differences(est. mean difference) in mm, the upper and lower 95%confidence intervals (95% confint) in mm and thecorresponding P-value.
Passagenumber
Est. meandifference
95% confint P-Value
Lower Upper
2 �0.67 �1.20 �0.14 0.017
3 �0.83 �1.36 �0.30 0.004
4 �0.67 �1.20 �0.14 0.017
5 �1.83 �2.36 �1.30 <0.001
6 �2.00 �2.53 �1.47 <0.001
7 �2.33 �2.86 �1.80 <0.001
8 �1.67 �2.20 �1.14 <0.001
9 �2.00 �2.53 �1.47 <0.001
10 �2.00 �2.53 �1.47 <0.001
a r c h i v e s o f o r a l b i o l o g y x x x ( 2 0 1 2 ) x x x – x x x4
AOB-2765; No. of Pages 7
variable was the size of the inhibition zone, the independent
variable was the indicator strain. To compare the inhibition
zones of each indicator strain against the salivaricin non-
producer S. salivarius MU, a model with no intercept term was
used. Means were estimated with 95% confidence intervals
with corresponding P-values.
To analyse the induction of resistance in S. moorei CCUG
39336 against the salivaricins from S. salivarius K12, a linear
mixed effects model (LME) was used because data structures
with serial dependency had to be described. The dependent
variable was the size of the inhibition zones and the
independent variable was the passage number. The experi-
mental unit was treated as a random factor.
The results are presented as differences of means, with 95%
confidence intervals and corresponding P-values. P-
values < 0.05 were considered statistically significant.
All statistical evaluations and graphs were done with the
publicly available R software v. 2.14.0 for Windows1.17 The
linear model was calculated using function lm( ) and corre-
sponding confidence intervals were calculated using the
function confint( ) (package stats). The linear mixed effects
model was calculated using the function lme( ) and confidence
intervals were estimated using the function intervals( ) (pack-
age nlme). Tests for normality of distribution were conducted
using the function qqPlot( ) fom the package car. No systematic
deviations from normal distribution were observed. For crea-
tion of graphs, packages plotrix and gplots were used.
3. Results
3.1. Antimicrobial activity of S. salivarius K12
All Gram-positive indicator strains were inhibited by S. salivarius
K12 (Fig. 1), while B. fragilis, a Gram-negative bacterium, was not
inhibited (Fig. 2b). The mean size of the inhibition zones for the
five S. moorei isolates were between 5.3 mm for S. moorei CH1#23
and 9.8 mm for the type strain S. moorei CCUG 39336. The zones
of inhibition for E. saburreum and P. micra were in the same
range, whereas A. parvulum and E. sulci where more susceptible
to S. salivarius K12 with inhibition zones of 14.4 mm and
16.7 mm, respectively. Variability was evident within most
species. E. saburreum, E. sulci and P. micra showed the biggest
variation in the results, whereas S. moorei CCUG 39336 and S.
moorei CH1#23 had the smallest variation.
The bacteriocin-nonproducing strain S. salivarius MU did
not inhibit any of the indicator strains (Fig. 2c and d). The
inhibition of all Gram-positive indicator bacteria by S.
salivarius K12 was statistically significant (P < 0.001) when
compared to the bacteriocin-nonproducing strain S. salivarius
MU (Table 1).
3.2. Test for resistance of S. moorei CCUG 39336 againstS. salivarius K12
To test for an intrinsic resistance of S. moorei against the
bacteriocins produced by S. salivarius K12, up to 9.1 � 107 S.
moorei CCUG 39336 cells were streaked on plates previously
seeded with S. salivarius K12 or S. salivarius MU. No growth
could be detected on the plates pretreated with S. salivarius K12
Please cite this article in press as: Masdea L, et al. Antimicrobial activityArchives of Oral Biology (2012), doi:10.1016/j.archoralbio.2012.02.011
even after prolonged incubation, while there was confluent
growth of S. moorei CCUG 39336 on plates pretreated with S.
salivarius MU.
3.3. Test for induction of resistance in S. moorei CCUG39336 against the bacteriocins from S. salivarius K12
The inhibition zones of S. moorei CCUG 39336 decreased
slightly with each passage, from 8.2 � 0.6 mm at the beginning
to 6.2 � 0.3 mm after 10 passages (Fig. 3). Comparing inhibition
zones of successive passages with those of the first passage,
the linear mixed-effect model indicated that this reduction
was statistically significant (Table 2). From the 5th passage on,
all differences were highly significant (P < 0.001).
4. Discussion
To compete with other species for nutrients in the same
ecological niche, many different bacterial species produce
of Streptococcus salivarius K12 on bacteria involved in oral malodour.
1 98765432 10
6
7
8
9
Induction of Solobacterium moorei CCUG 39336
passages
Inh
ibitio
n z
on
e ±
SD
[m
m]
Fig. 3 – Test for induction of resistance in S. moorei CCUG
39336 against the bacteriocins from S. salivarius K12.
Shown are levels of inhibition (mm) in different passages.
a r c h i v e s o f o r a l b i o l o g y x x x ( 2 0 1 2 ) x x x – x x x 5
AOB-2765; No. of Pages 7
bacteriocins. These ribosomally synthesised peptides or
proteins have antibacterial activity.18,19 Due to their potential
as food preservatives and their antagonistic effect against
important food pathogens, the bacteriocins that have been
studied most extensively are derived from lactic acid bacteria.
The lantibiotic nisin, which is produced by different Lactococcus
lactis spp., is the best studied bacteriocin and, so far, the only
one that is used as a food additive.20,21 However, bacteriocin-
producing starter cultures are commonly used in food
fermentations, and probiotic bacteria have recently gained
increased interest and acceptance due to their potential health
benefit. Production of antimicrobial substances against patho-
gens has been proposed as an important mechanism by which
probiotic bacteria may improve human health.22,23
There have been few attempts to examine the effects of
probiotic bacteria in the oral cavity.24 This complex ecosystem
is inhabited by more than 700 bacterial species,25 some of
which have been shown to produce antimicrobial substances,
including bacteriocins. The caries pathogen Streptococcus
mutans produces several kinds of bacteriocins called mutacins.
The efficient replacement of indigenous cariogenic mutans
streptococci by a genetically modified S. mutans strain is based
on the production of the broad-spectrum lantibiotic mutacin
1140. Animal testing indicates that an avirulent S. mutans
strain producing ethanol instead of lactic acid and harbouring
mutacin 1140 can successfully displace other S. mutans strains
and lead to significantly reduced level of caries.26 Phase I
safety trials using an auxotrophic strain are planned to
determine the level of transmission of this bacterium.27
A few other studies have examined the effect of probiotic
bacteria, mainly lactic acid bacteria, on salivary bacterial
counts and caries prevention. These initial studies yielded
promising results; a reduction of salivary S. mutans counts and
a reduced caries risk was found in most studies.6,7
S. salivarius, one of the predominant commensal bacteria
of the oral cavity, is known to produce bacteriocins and
bacteriocin-like inhibitory substances, which makes
S. salivarius strains promising candidates for the development
of oral probiotics against oral infectious diseases. It has
already been shown that S. salivarius can antagonise the action
of Streptococcus pyogenes, the main etiological agent of bacterial
Please cite this article in press as: Masdea L, et al. Antimicrobial activityArchives of Oral Biology (2012), doi:10.1016/j.archoralbio.2012.02.011
pharyngitis in children; indeed, lozenges containing
S. salivarius K12 are sold in some countries as an oral probiotic
to maintain throat health.11,28–31
Therefore, the possibility of screening probiotics against
several bacteria implicated in halitosis seems very promising.
The experiments in the present study were performed to
elucidate the inhibitory effect of the probiotic S. salivarius K12,
which produces at least two lantibiotic bacteriocins, on strains
of several species of Gram-positive bacteria.
Recently, the use of S. salivarius K12 as a probiotic in clinical
practice has been tested.5 In a deferred antagonism test, Burton
et al. reported strong inhibition by S. salivarius K12 against Gram-
positive halitosis associated species, including E. saburreum and P.
micra (M. micros).4 The procedures followed in our study were
related to the test used by Tagg and Bannister.16 The results
demonstrated an inhibition of E. saburreum and P. micra,
indicating adequate culture conditions for the bacteriocin-
production of S. salivarius. In addition, Gram-negative bacteria
showed no inhibition, which was consistent with the corre-
sponding literature. Thus, the applied test arrangement could be
routinely used to study further bacterial species implicated in
halitosis.
S. moorei has recently been identified in specimens from
patients suffering from halitosis.3,15 Inhibition by S. salivarius
K12 was demonstrated against type strain CCUG 39336 and four
clinical isolates of S. moorei (CH1#23, CH3A#109A, CH3#63,
CH8#20)15 that originated from samples taken from the human
oral cavity. A. parvulum ATCC 33793 and E. sulci ATCC 35585 were
used for this study because they are known to be implicated in
halitosis and because they had not been tested with the
deferred antagonism test. The variation in inhibition zones of E.
saburreum, E. sulci and P. micra could be attributed to their
demanding growth conditions and challenging cultivation.
The development of strain resistance is of major concern for
the in vivo application of probiotic strains, and the emergence of
resistance against bacteriocins has been best documented for
nisin. In laboratory settings, nisin-resistant bacteria can be
obtained by repeatedly exposing sensitive strains to increasing
amounts of nisin. Gram-positive and Gram-negative bacteria
can exhibit resistance against nisin. The molecular mechanisms
leading to nonsusceptibility have been shown to involve changes
in the bacterial cell membrane or cell wall, although the precise
nature of the factors involved in resistance development
remains elusive, and bacteria may employ several strategies
simultaneously to acquire nisin resistance (reviewed in Ref. 21).
A possible mechanism leading to the acquisition of resis-
tance is horizontal gene transfer wherein genes are transferred
between bacteria. This mode of gene transfer was demonstrat-
ed in S. salivarius K12 where the large plasmid harbouring the
loci for bacteriocins production could be transferred in vivo into
a plasmid-negative S. salivarius strain by oral transmission.32,33
So far, no studies have been conducted to determine the host
range of this plasmid or whether there is transmission to other
oral streptococci or even to potential pathogens.
No resistance against the bacteriocins produced by orally
administrated S. salivarius K12 have been reported so far. In
our study, no intrinsic resistance of S. moorei CCUG 39336
against S. salivarius K12 could be detected, although there was
a decrease in sensitivity when S. moorei CCUG 39336 was
repeatedly exposed to S. salivarius K12 over 10 passages.
of Streptococcus salivarius K12 on bacteria involved in oral malodour.
a r c h i v e s o f o r a l b i o l o g y x x x ( 2 0 1 2 ) x x x – x x x6
AOB-2765; No. of Pages 7
Further studies are needed to determine if resistances might
also occur in vivo.
In contrast to the situation with antibiotics in which there
is currently no antibiotic in clinical use to which resistance has
not developed, bacteriocin resistance does not yet pose a
serious problem. However, cross-resistance between bacter-
iocins have been observed and thought to represent a general
mechanism of resistance, and this emphasises the need for
efficient and safe probiotics.34,35
5. Conclusions
In conclusion, our study demonstrated that the bacteriocin-
producing strain S. salivarius K12 displayed antimicrobial
activities against several halitosis bacteria including S. moorei,
which has recently been found to be a major contributor to oral
malodour. Additionally, the type strain S. moorei CCUG 39336 did
not seem to have a natural resistance against S. salivarius K12,
and there was only a slight decrease in sensitivity after repeated
exposure to S. salivarius K12. Based on these results, S. salivarius
K12 might be an interesting and valuable candidate for the
development of an antimicrobial therapy to treat oral malodour.
Funding
None.
Competing interests
None declared.
Ethical approval
Not required.
Acknowledgments
The authors would like to thank E. Filipuzzi and I. Schweizer
for their skilful technical assistance, V. Haraszthy (University
at Buffalo, School of Dental Medicine, Buffalo, N.Y.) for the
clinical isolates of Solobacterium moorei, J. Tagg (Department of
Microbiology and Immunology, University of Otago, Dunedin,
New Zealand) for the strains S. salivarius K12 and S. salivarius
MU and dipl. math A. Schotzau (Basel, Switzerland) for his
expert help with statistical analysis. The authors declare no
potential conflicts of interest with respect to the authorship
and/or publication of this article.
r e f e r e n c e s
1. Scully C, el-Maaytah M, Porter SR, Greenman J. Breath odor:etiopathogenesis, assessment and management. Eur J OralSci 1997;105:287–93.
Please cite this article in press as: Masdea L, et al. Antimicrobial activityArchives of Oral Biology (2012), doi:10.1016/j.archoralbio.2012.02.011
2. Loesche WJ, Kazor C. Microbiology and treatment ofhalitosis. Periodontol 2000 2002;28:256–79.
3. Kazor CE, Mitchell PM, Lee AM, Stokes LN, Loesche WJ,Dewhirst FE, et al. Diversity of bacterial populations on thetongue dorsa of patients with halitosis and healthy patients.J Clin Microbiol 2003;41:558–63.
4. Burton JP, Chilcott CN, Moore CJ, Speiser G, Tagg JR. Apreliminary study of the effect of probiotic Streptococcussalivarius K12 on oral malodour parameters. J Appl Microbiol2006;100:754–64.
5. Reid G, Jass J, Sebulsky MT, McCormick JK. Potential uses ofprobiotics in clinical practice. Clin Microbiol Rev 2003;16:658–72.
6. Nase L, Hatakka K, Savilahti E, Saxelin M, Ponka A, Poussa T,et al. Effect of long-term consumption of a probioticbacterium, Lactobacillus rhamnosus GG, in milk on dentalcaries and caries risk in children. Caries Res 2001;35:412–20.
7. Ahola AJ, Yli-Knuuttila H, Suomalainen T, Poussa T,Ahlstrom A, Meurman JH, et al. Short-term consumption ofprobiotic-containing cheese and its effect on dental cariesrisk factors. Arch Oral Biol 2002;47:799–804.
8. Comelli EM, Guggenheim B, Stingele F, Neeser JR. Selectionof dairy bacterial strains as probiotics for oral health. Eur JOral Sci 2002;110:218–24.
9. Montalto M, Vastola M, Marigo L, Covino M, Graziosetto R,Curigliano V, et al. Probiotic treatment increases salivarycounts of lactobacilli: a double-blind, randomized,controlled study. Digestion 2004;69:53–6.
10. Twetman S, Stecksen-Blicks C. Probiotics and oral healtheffects in children. Int J Paediatr Dent 2008;18:3–10.
11. Horz HP, Meinelt A, Houben B, Conrads G. Distribution andpersistence of probiotic Streptococcus salivarius K12 in thehuman oral cavity as determined by real-time quantitativepolymerase chain reaction. Oral Microbiol Immunol2007;22:126–30.
12. Ross KF, Ronson CW, Tagg JR. Isolation and characterizationof the lantibiotic salivaricin A and its structural gene salAfrom Streptococcus salivarius 20P3. Appl Environ Microbiol1993;59:2014–21.
13. Upton M, Tagg JR, Wescombe P, Jenkinson HF. Intra- andinterspecies signaling between Streptococcus salivarius andStreptococcus pyogenes mediated by SalA and SalA1 lantibioticpeptides. J Bacteriol 2001;183:3931–8.
14. Tagg JR, Dierksen KP. Bacterial replacement therapy:adapting ‘germ warfare’ to infection prevention. TrendsBiotechnol 2003;21:217–23.
15. Haraszthy VI, Gerber D, Clark B, Moses P, Parker C,Sreenivasan PK, et al. Characterization and prevalence ofSolobacterium moorei associated with oral halitosis. J BreathRes 2008;2:017002.
16. Tagg JR, Bannister LV. Fingerprinting beta-haemolyticstreptococci by their production of and sensitivity tobacteriocine-like inhibitors. J Med Microbiol 1979;12:397–411.
17. R Development Core Team. R: a language and environment forstatistical computing. Vienna, Austria: R Foundation forStatistical Computing; 2011.
18. Jack RW, Tagg JR, Ray B. Bacteriocins of Gram-positivebacteria. Microbiol Rev 1995;59:171–200.
19. Cotter PD, Hill C, Ross RP. Bacteriocins: developing innateimmunity for food. Nat Rev Microbiol 2005;3:777–88.
20. Cleveland J, Montville TJ, Nes IF, Chikindas ML. Bacteriocins:safe, natural antimicrobials for food preservation. Int J FoodMicrobiol 2001;71:1–20.
21. Chatterjee C, Paul M, Xie L, van der Donk WA. Biosynthesisand mode of action of lantibiotics. Chem Rev 2005;105:633–84.
22. De Vuyst L, Leroy F. Bacteriocins from lactic acid bacteria:production, purification, and food applications. J MolMicrobiol Biotechnol 2007;13:194–9.
of Streptococcus salivarius K12 on bacteria involved in oral malodour.
a r c h i v e s o f o r a l b i o l o g y x x x ( 2 0 1 2 ) x x x – x x x 7
AOB-2765; No. of Pages 7
23. Gillor O, Etzion A, Riley MA. The dual role of bacteriocinsas anti- and probiotics. Appl Microbiol Biotechnol 2008;81:591–606.
24. Meurman JH. Probiotics: do they have a role in oral medicineand dentistry? Eur J Oral Sci 2005;113:188–96.
25. Paster BJ, Olsen I, Aas JA, Dewhirst FE. The breadth ofbacterial diversity in the human periodontal pocket andother oral sites. Periodontol 2000 2006;42:80–7.
26. Hillman JD, Brooks TA, Michalek SM, Harmon CC, Snoep JL,van Der Weijden CC. Construction and characterization ofan effector strain of Streptococcus mutans for replacementtherapy of dental caries. Infect Immun 2000;68:543–9.
27. Hillman JD, Mo J, McDonell E, Cvitkovitch D, Hillman CH.Modification of an effector strain for replacement therapy ofdental caries to enable clinical safety trials. J Appl Microbiol2007;102:1209–19.
28. Tagg JR. Prevention of streptococcal pharyngitis by anti-Streptococcus pyogenes bacteriocin-like inhibitory substances(BLIS) produced by Streptococcus salivarius. Indian J Med Res2004;119 Suppl.:13–6.
29. Wescombe PA, Heng NC, Burton JP, Chilcott CN, Tagg JR.Streptococcal bacteriocins and the case for Streptococcussalivarius as model oral probiotics. Future Microbiol2009;4:819–35.
Please cite this article in press as: Masdea L, et al. Antimicrobial activityArchives of Oral Biology (2012), doi:10.1016/j.archoralbio.2012.02.011
30. Wescombe PA, Upton M, Renault P, Wirawan RE, Power D,Burton JP, et al. Salivaricin 9, a new lantibiotic produced byStreptococcus salivarius. Microbiology 2011;157:1290–9.
31. Guglielmetti S, Taverniti V, Minuzzo M, Arioli S, StuknyteM, Karp M, et al. Oral bacteria as potential probiotics forthe pharyngeal mucosa. Appl Environ Microbiol2010;76:3948–58.
32. Wescombe PA, Burton JP, Cadieux PA, Klesse NA, Hyink O,Heng NC, et al. Megaplasmids encode differingcombinations of lantibiotics in Streptococcus salivarius. AntonVan Leeuw 2006;90:269–80.
33. Hyink O, Wescombe PA, Upton M, Ragland N, Burton JP,Tagg JR. Salivaricin A2 and the novel lantibiotic salivaricinB are encoded at adjacent loci on a 190-kilobasetransmissible megaplasmid in the oral probiotic strainStreptococcus salivarius K12. Appl Environ Microbiol2007;73:1107–13.
34. Gravesen A, Ramnath M, Rechinger KB, Andersen N, JanschL, Hechard Y, et al. High-level resistance to class IIabacteriocins is associated with one general mechanism inListeria monocytogenes. Microbiology 2002;148:2361–9.
35. Naghmouchi K, Kheadr E, Lacroix C, Fliss I. Class I/Class IIabacteriocin cross-resistance phenomenon in Listeriamonocytogenes. Food Microbiol 2007;24:718–27.
of Streptococcus salivarius K12 on bacteria involved in oral malodour.
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Open access Full Text article
http://dx.doi.org/10.2147/CCIDE.S93066
Cariogram outcome after 90 days of oral treatment with Streptococcus salivarius M18 in children at high risk for dental caries: results of a randomized, controlled study
Francesco Di Pierro1
alberto Zanvit2
Piero nobili2
Paolo risso3
Carlo Fornaini4
1scientific Department, Velleja research, 2stomatology Institute, Milan, Italy; 3Department of health science, University of genoa, genoa, Italy; 4Teleo laboratory, Faculty of Dentistry, University of nice, nice, France
Correspondence: Francesco Di Pierro Scientific Department, Velleja Research, Viale lunigiana 23, 20125 Milan, Italy Tel +39 34 9552 7663 email [email protected]
Abstract: Dental caries is the most common chronic disease of childhood. Cariogram is a
well-recognized algorithm-based software program based on different caries-related risk fac-
tors and intended to aid clinicians in performing more objective and consistent dental caries
risk assessments. This type of approach precedes the diagnosis of caries and allows the dentist
to identify at-risk patients and then take appropriate preventive measures before caries develop
further. One of the etiological factors favoring the development of dental caries is the mutans
streptococci. These acidogenic dental plaque inhabitants can be effectively antagonized by
the activity of bacteriocins released by the probiotic Streptococcus salivarius M18 (salivarius
M18). Moreover, salivarius M18 after colonizing the human oral mucosa produces the enzymes
dextranase and urease that are able to counteract plaque formation and saliva acidity, respectively.
Seventy-six subjects at high risk of dental caries were randomized and then either treated or
not treated for 90 days with an oral formulation containing the oral probiotic salivarius M18
(Carioblis®). The results indicate that the use of salivarius M18 increases the chances of avoid-
ing new dental caries development in children, and its application could be proposed as a new
tool in the dentist’s armory to be adopted in subjects considered at high risk on the basis of
their Cariogram outcome.
Keywords: BLIS M18, caries prediction, dextranase, urease, Streptococcus mutans, Streptococcus
sobrinus, plaque, salivary pH, bacteriocins
IntroductionDental caries is the most common chronic disease of childhood and its prevalence
continues to increase in many populations worldwide.1,2 It is a multifactorial disease
mainly caused by interactions between mutans streptococci, especially Streptococcus
mutans and Streptococcus sobrinus, and individual caries risk factors, such as saliva
composition, fluoride exposure, and dietary habits.3 Despite dental caries being pre-
ventable and the many major technological advances in dentistry in recent years,4
dental caries remains a very diffuse and unsolved medical problem. Being a pathology
sustained by microbial pathogens, treatments using conventional antistreptococcal
antibiotics can be effective in the short term to reduce dental plaque levels and to
decrease counts of the mutans streptococci. However, as most antibiotics have relatively
broad-spectrum antimicrobial activity, they indiscriminately destroy both commensal
and potentially harmful bacteria and thereby create population imbalances within the
microflora.5 This outcome could be a consequence of using well-known natural or
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Di Pierro et al
synthetic antibiotics and also of using new herbal medicines
endowed with antibiotic activity.6,7 It is now becoming clear
that the severity of some oral pathologies, including dental
caries, otitis media, halitosis, and streptococcal pharyngoton-
sillitis, can be related to the development of oral microbiota
disequilibria. The application of oral probiotics to help restore
a balanced microbiota and thereby improve oral health is a
relatively new concept.8 Some putative commensal bacteria
have been assessed for their ability to help prevent dental
caries. Some initial studies based on the use of intestinal
probiotics have reported a reduction in levels of S. mutans
and apparently fewer dental caries.9,10 However, because these
strains have limitations in terms of their colonization of oral
tissues, a new generation of probiotic strains sourced from
the human oral cavity and belonging to commensal species
known to have extremely low pathogenic potential has more
recently been developed. In this regard, a key species is
Streptococcus salivarius and the oral probiotic identified as
strain K12 has been the most thoroughly studied in terms of
its bacteriocin production, oral colonization, and oral per-
sistence and also its efficacy in counteracting halitosis, oral
candidosis, pharyngotonsillitis, and acute otitis media.11–22
Streptococcus salivarius M18 (salivarius M18) (IDA clas-
sification: DSM 14865),23 a strain originally isolated from a
healthy female adult subject during a specific search for an
oral commensal strain capable of inhibiting mutans strep-
tococci, has subsequently been shown to have relatively
broad spectrum bacteriocin-like inhibitory substance (BLIS)
activity against S. mutans and S. sobrinus and to produce
both dextranase and urease enzymes, the activities of which
could potentially help limit the progression of dental caries
by reducing plaque accumulation and plaque acidification,
respectively.24–26 The whole genome of strain salivarius M18
has been published recently, and its bacteriocin repertoire
includes the megaplasmid-encoded salivaricin A2, salivaricin
MPS, and salivaricin 9, and the chromosomally encoded
salivaricin M.23,24,27 Recent trials have revealed, along with
its safety and tolerability profiles, the capability of salivarius
M18 to colonize and persist in the human oral cavity,28 to
reduce plaque formation and to lower S. mutans counts in
colonized primary-school-aged children,29 and to reduce both
moderate and severe gingivitis and periodontitis in adults.30
On the basis of these biochemical, microbiological, and clini-
cal findings, we determine whether the oral and daily use of
the strain salivarius M18 affects or modifies the Cariogram
outcome after 90 days of treatment in children at high risk
of developing new dental caries. Cariogram is an algorithm-
based software program developed in Sweden in 1997 by the
University of Malmö, based on nine different caries-related
risk factors, along with physician judgment, intended to aid
clinicians in performing more objective and consistent dental
caries risk assessments.31 The performance of the program
has been validated in preschool children, schoolchildren,
young adults, and the elderly.32–38
Materials and methodssubjects and criteriaSeventy-six children (aged 6–17 years) classified as high
risk on the basis of the Cariogram results performed at
day 0 (chance to avoid new cavities ,25) were included in
this randomized, controlled study after informed consent
was obtained from their parents. Exclusion criteria were
diagnosis of heart, respiratory, renal, liver, or intestinal dis-
ease, or undergoing current therapy with antibiotics and/or
corticosteroids for the prevention/treatment of recurrent
bacterial pathologies, such as cystitis, pharyngotonsillitis,
and acute otitis media, or to counteract allergic reactions
and/or asthma. According to the protocol, occasional use of
acetaminophen or ibuprofen for fever and/or pain control and
of physician-prescribed antibiotics was allowed. During the
use of antibiotics, treated children were asked to stop using
the salivarius M18-based product. As there were no drop-
outs, all the 76 children (38 children in the treated group and
38 in the control, untreated group) attended the follow-up
examination performed after 90 days and were included in
the statistical analysis.
study schemeThis randomized, controlled study was conducted in the
field of routine clinical practice in the area of Milan (Italy)
between March and September 2014, in agreement with the
criteria set by the Declaration of Helsinki and the Milan Ethi-
cal Board gave the approval for this study. The parents of all
the participants in the study were informed of the trial meth-
ods and signed the consent and privacy-policy documents
giving the authorization to publish the results. As shown in
Figure 1, 76 of the 100 children analyzed were considered
eligible for enrollment and were randomly assigned to be
supplemented once a day for 3 months with the test product
(treated group; n=38) or not to receive any treatment (con-
trol group; n=38). Randomization was carried out using the
sealed envelope system. After 90 days, 76 children attended
the follow-up examination and were subjected to their second
Cariogram test. Every 15 days, during the study, all of the
enrolled subjects were in contact with the dentists responsible
for the study to report their medical condition and specific
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role of BlIs M18 in Cariogram
N=38
N=38
Cariogram testn=38
Day 0:
1. Cariogram testperformed on n=100
No treatment
Day 30
2. Enrolled subjectsat high risk (n=76)
3. Randomizationprocedure (n=76)
Cariogram testn=38
Streptococcus salivarius M18
Day 60Day 90
Day 30 Day 60Day 90
Figure 1 scheme of the study.
study parameters such as probiotic tolerability and dosing
compliance, as well as to enable documentation of the occur-
rence of any side effects possibly linked to the treatment.
The subjects were also provided with the possibility of daily
access to the physicians responsible for the study.
Tested productSalivarius M18 (IDA classification: DSM 14865), also
named by the manufacturer as BLIS M18 (BLIS Technolo-
gies, Dunedin, New Zealand), was formulated as slowly
dissolving oral tablets by SIIT (Trezzano S/N, Italy) and
notified as nutritional supplement to the Italian Ministry
of Health as Carioblis® by Omeopiacenza (Pontenure,
Italy), according to the provisions of law 169 of 2004, on
July 19, 2013 (notification number 69163). The prepara-
tion of Carioblis® used in our research contained no less
than 1 billion colony-forming units (CFU)/tablet of strain
salivarius M18.
Treatment protocolStarting from day 0 to 90, one tablet of Carioblis® was
administered to each subject every night, just before sleep.
The tablet was allowed to slowly dissolve in the oral cavity,
without biting or swallowing. Saliva production is typically
reduced in the evening hours and this improves the effective-
ness of oral colonization. Only for the very first treatment,
the administration of the tablet was preceded, approximately
30 minutes before, by the use of a chlorhexidine-based (0.2%)
mouthwash. This procedure improves the efficacy of oral
colonization by BLIS M18 by creating bacteria-depleted
niches in the oral tissues. In order to evaluate the level of
subject adherence to the established protocol, the subjects
were asked to return any unused product boxes and tablets.
Acceptable adherence was considered to be the administration
of not less than 95% of the allocated tablets.
Mutans, saliva, and plaqueTo evaluate the presence of S. mutans, the GC Saliva-Check
Mutans test (monoclonal antibody-based) was used. As
regards saliva, to evaluate pH and quantity, the GC Saliva-
Check Buffer test was used. In order to obtain the samples of
saliva for the analysis of mutans streptococci, the saliva secre-
tion rate (mL/min), and buffer capability, paraffin-stimulated
whole saliva was collected from all children. The presence of
dental plaque was assessed by using the GC Plaque Indicator
test. All kits are supplied by GC Europe, Leuven, Belgium.
study objectivesThe principal objectives for the study were 1) to establish the
safety and tolerability profiles of the salivarius M18-based
product in children at high risk of developing new dental
caries and 2) to evaluate in the same children whether any
Cariogram modifications occurred after 90 days of treatment
with the salivarius M18-based product.
statistical analysisTo study the null hypothesis of no effect of treatment on
Carioblis® for each clinical variable, and for a global summa-
tion, we applied the two-tailed Wilcoxon test for matched pairs
with signed ranks. To study the effect of Carioblis® therapy on
Cariogram scores, using averaged clinical variables, we used
the two-tailed Fisher’s exact test. Statistical software JMP®
10 for Mac OS X (SAS Institute Inc., Cary, NC, USA) was
used, and the threshold for statistical significance was 95%.
To calculate the caries risk of a group of subjects (results are
shown in Table 1 and Figure 2), we used the average value of
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Di Pierro et al
Table 1 Cariogram values (at day 0 and day 90) calculated using the average value of the items listed
Treatment, time period
Actual chance to avoid new cavities (%)
Diet (%) Bacteria (%) Susceptibility (%) Circumstances (%)
Salivarius M18, day 0 20 17 29 23 11Salivarius M18*, day 90 70 7 7 9 7Untreated, day 0 20 17 29 23 11Untreated, day 90 37 11 26 16 10
Note: *All values of M18, day 90 are significant (P,0.01) vs treatment M18, day 0.Abbreviation: Salivarius M18, Streptococcus salivarius M18.
20
17
29
23
11
Salivarius M18 treated group (day 0) Salivarius M18 treated group (day 90)
20
17
29
23
11
Untreated group (day 0)
37
1126
16
10
Untreated group (day 90)
Chance to avoid new cavities
Circumstances
Susceptibility
Bacteria
Diet
70
7
77
9
7
Figure 2 graphical representation of Cariogram values (%) calculated using the average value of the items listed.Abbreviation: Salivarius M18, Streptococcus salivarius M18.
any single items of the Cariogram and considered equal to 0 if
the decimal values stands between 0.1 and 0.4 and equal to 1 if
the decimal values were between 0.5 and 0.9. The Cariogram
software used the Java Internet 2004 version.
ResultsThis randomized and controlled study has been carried out
on 76 children at high risk of new dental caries development.
Thirty-eight of these subjects were treated for 90 days with
Carioblis® (a S. salivarius M18-based product) and the others
served as controls (untreated group). There was no dropout,
therefore all of the children were considered eligible for the
statistical analysis. As shown in Table 2, no statistical dif-
ferences existed between the two groups in terms of sex and
age. Ninety days of treatment with strain M18 produced in
the treated group a statistically significant reduction, by more
than 30%, in the global Cariogram outcome. No statistical
difference was observed in the control group (Table 3). By
analyzing every individual parameter of the Cariogram results
in the salivarius M18 treated group (Table 4) one observes
that, other than for “caries experience”, “related diseases”,
and “clinical judgment”, all of the parameters are improved.
Some improvements are probably due to a better control of
the aspects of diet (diet content and frequency) or changes
in oral hygiene and/or in prophylaxis (fluoride program) and
cannot be linked to the treatment, with clear evidence. Others,
such as “plaque amount”, “mutans streptococci”, and “buf-
fer capacity” could be a direct consequence of the treatment
because salivarius M18 releases bacteriocins able to kill
mutans streptococci, and dextranase and urease enzymes,
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role of BlIs M18 in Cariogram
which are capable of counteracting plaque formation and
increasing saliva pH, respectively. Noteworthy, “plaque
control” and “mutans streptococci” were reduced by approxi-
mately 50% and 75%, respectively. By contrast, as shown in
Table 5, the untreated group did not show the same type of
improvement and the only statistically significant changes are
due to a better control of diet, oral hygiene, and prophylactic
approach. The Cariogram software was uniquely imple-
mented to calculate the caries risk of individual subjects.
Nevertheless, we have exploited the algorithm of the Cario-
gram software and used the average value of any single items
of the Cariogram to calculate the caries risk of a group of
subjects. This nonvalidated procedure allows construction
of an image representative of the likely impact that a treat-
ment can have on a group of patients. As shown in Table 1
and Figure 2, treatment with the strain M18-based product
significantly improves the “chances of avoiding new dental
caries”, from 20 to 70, reduces the parameter “bacteria”,
from 29 to 7, and reduces the “susceptibility”, from 23 to 9.
No relevant variations were evident in the untreated group.
Finally, in Table 6, the M18-based treatment demonstrated a
very good safety profile with no treatment-related side effects
and no subject dropout. Tolerability was assessed as “good”
and “very good” in 35 of the 38 subjects and overlapping
results were obtained with regard to compliance.
DiscussionCaries risk assessment is an important tool assisting the dentist
in obtaining a better understanding of the dental profile of a
patient. The Cariogram software has been clinically proven to
be effective in evaluating such a risk.39 Cariogram is based on
a set of nine pathological and protective factors, in addition
to the professional judgment of the expert dentist. Among
these factors, the likely most relevant variable in caries risk
prediction is “caries experience” and, as a matter of fact, a
strong relationship has been shown between caries experience
and caries risk profile.40,41 Apparently, microbial tests, aimed
at evaluating the presence of mutans streptococci, do not seem
to be equally relevant. This could be because, in the presence
of fluoride, along with an appropriate diet in terms of quality
and quantity, a high number of mutans streptococci may be
tolerated without causing significant harm to the teeth.42
Fluoride is not the only potentially protective factor in the
presence of an abundance of deleterious streptococci. Within
the oral microbiota, populations of mutans streptococci can
indeed be balanced by the presence of antagonizing bacteria.
Among these, a particularly important role is thought to be
played by S. salivarius, one of the most prevalent of the com-
mensal oral bacteria. Different strains of S. salivarius have
Table 3 global Cariogram outcome at day 0 and day 90
Salivarius M18-treated group (n=38)
Untreated group (n=38)
Day 0** 15.9±2.6 (16) 16.3±2.9 (16)Day 90** 11.1±2.0 (11)* 14.4±3.2 (14)Δ % vs day 0 30.2 11.7
Notes: *P,0.01 vs day 0; **data expressed as mean ± standard deviation (median).Abbreviation: Salivarius M18, Streptococcus salivarius M18.
Table 2 Characteristics* of the enrolled children
Salivarius M18-treated group (n=38)
Untreated group (n=38)
Males, n 25 21age° of males 11.2±3.2 12.1±2.9Females, n 13 17age° of females 11.5±3.6 11.8±3.8
Notes: *Nonsignificant differences between groups; °age expressed in years ± standard deviation.Abbreviation: Salivarius M18, Streptococcus salivarius M18.
Table 4 Cariogram: outcome of individual parameters in the salivarius M18-treated group (n=38)
Day 0* Day 90* P
Caries experience 2.7±0.5 (3) 2.7±0.5 (3) nsrelated diseases 0.0±0.2 (0) 0.1±0.2 (0) nsDiet, content 1.9±1.0 (2) 1.4±0.7 (1) ,0.05Diet, frequency 1.4±0.8 (1) 1.2±0.5 (1) ,0.05Plaque amount 2.0±0.8 (2) 1.0±0.6 (1) ,0.01Mutans streptococci 2.7±0.5 (3) 0.7±0.8 (0) ,0.01Fluoride program 2.4±0.7 (2) 1.8±0.5 (2) ,0.01saliva secretion 1.7±0.9 (2) 1.2±1.1 (1) ,0.05Buffer capacity 0.0±0.2 (0) 0.0±0.0 (0) ,0.05Clinical judgment 1.1±0.4 (1) 1.0±0.2 (1) ns
Note: *Data expressed as mean ± standard deviation (median).Abbreviations: ns, not significant; salivarius M18, Streptococcus salivarius M18.
Table 5 Cariogram: outcome of individual parameters in the untreated group (n=38)
Day 0* Day 90* P
Caries experience 2.8±0.5 (3) 2.8±0.6 (3) nsrelated diseases 0.1±0.2 (0) 0.2±0.2 (0) nsDiet, content 2.0±1.0 (2) 1.3±0.4 (1) ,0.01Diet, frequency 1.3±0.8 (1) 1.1±0.7 (1) ,0.05Plaque amount 2.0±0.9 (2) 2.1±0.7 (2) nsMutans streptococci 2.6±0.5 (3) 2.5±0.6 (3) nsFluoride program 2.3±0.7 (2) 1.6±0.7 (1) ,0.01saliva secretion 1.8±0.9 (2) 1.4±1.2 (1) ,0.05Buffer capacity 0.2±0.2 (0) 0.2±0.2 (0) nsClinical judgment 1.2±0.4 (1) 1.2±0.1 (1) ns
Note: *Data expressed as mean ± standard deviation (median).Abbreviation: ns, not significant.
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Di Pierro et al
been shown capable of counteracting the growth of mutans
streptococci8 and, of these, the strongest clinical potential has
been shown by strain M18.29,30 On this basis, we decided to
test the capability of salivarius M18 to modify the Cariogram
outcome. According to our results, 90 days treatment with
this oral probiotic has increased the chances of avoiding new
cavities in children. This outcome is considered attributable
to the specific anticariogenic characteristics of strain M18
that, after colonizing the oral mucosa, is able to release
bacteriocins, limiting the growth of S. mutans and S. sobri-
nus, and the enzymes dextranase and urease, catalyzing the
breakdown of dextran (aiding solubilization of plaque) and the
hydrolysis of urea (increasing saliva pH). The present study
does contain some bias: 1) it is not a blinded study; 2) there
is no placebo group; 3) the control group comprises untreated
subjects; and 4) the number of enrolled subjects is rather
small. Nevertheless, this study represents one of the pioneer
attempts to analyze the significance of salivarius M18 in
dental practice. If these preliminary results can be confirmed
with a larger number of subjects and in double-blind clinical
conditions, the practical application of strain M18 could be
proposed in the future as a new tool in the dentist’s armory,
along with the already available strategies (eg, anticaries diets,
fluoride, and oral hygiene) to be adopted in subjects consid-
ered at high risk on the basis of their Cariogram outcome. On
the basis of the calculated risk to develop new dental caries,
subjects are divided into three groups: low, medium, and
high. Depending on these groups, the fundamental aspects
of primary prevention are applied to different extents for
protocol and rigor: light in those at low risk, moderate in
those at intermediate risk, and close and manifold in high-
risk individuals. Certainly, in patients defined at high risk, but
possibly also in those of intermediate risk, the addition of a
protocol incorporating the administration of the salivarius
M18 could be crucial to addressing and further reducing the
risk of tooth-decay receptivity. The caries risk is configured as
the predisposition of an individual to be affected by the carious
pathology, regardless of the fact of presenting caries at the time
of the dental examination. This type of diagnosis precedes
then the diagnosis of caries, allowing dentist to intercept the
at-risk patient and take the appropriate preventive measures
to intercept the development of tooth decay.
AcknowledgmentThe authors thank Dr J Tagg for suggestions and review of
the paper.
DisclosureF Di Pierro is the Scientific Director of Velleja Research, the
company that developed the finished product tested in this
study. The other authors report no other conflicts of interest
in this work.
References 1. Bagramian RA, Garcia-Godoy F, Volpe AR. The global increase in dental
caries. A pending public health crisis. Am J Dent. 2009;22:3–8. 2. Vachirarojpisan T, Shinada K, Kawaguchi Y, Laungwechakan P,
Somkote T, Detsomboonrat P. Early childhood caries in children aged 6–19 months. Community Dent Oral Epidemiol. 2004;32(2): 133–142.
3. Selwitz RH, Ismail AI, Pitts NB. Dental caries. Lancet. 2007;369: 51–59.
4. Shah N, Bansal N, Logani A. Recent advances in imaging technologies in dentistry. World J Radiol. 2014; 6(10):794–807.
5. ten Cate JM, Zaura E. The numerous microbial species in oral bio-films: how could antibacterial therapy be effective? Adv Dent Res. 2012;24(2):108–111.
6. Silva JP, Castilho AL, Saraceni CH, Díaz IE, Paciencia ML, Suffredini IB. Anti-Streptococcal activity of Brazilian Amazon Rain Forest plant extracts presents potential for preventive strategies against dental caries. J Appl Oral Sci. 2014;22(2):91–97.
7. Brighenti FL, Salvador MJ, Delbem AC, et al. Systematic screen-ing of plant extracts from the Brazilian Pantanal with antimicrobial activity against bacteria with cariogenic relevance. Caries Res. 2014;48(5):353–360.
8. Wescombe PA, Hale JD, Heng NC, Tagg JR. Developing oral probi-otics from Streptococcus salivarius. Future Microbiol. 2012;7(12): 1355–1371.
9. Caglar E, Sandalli N, Twetman S, Kavaloglu S, Ergeneli S, Selvi S. Effect of yogurt with Bifidobacterium DN-173 010 on salivary mutans streptococci and lactobacilli in young adults. Acta Odontol Scand. 2005;63:317–320.
10. Nase L, Hatakka K, Savilahti E, et al. Effect of long-term consumption of a probiotic bacterium, Lactobacillus rhamnosus GG, in milk on dental caries and caries risk in children. Caries Res. 2001;35:412–420.
11. Tagg JR. Prevention of streptococcal pharyngitis by anti-Streptococcus pyogenes bacteriocin-like inhibitory substances (BLIS) produced by Streptococcus salivarius. Indian J Med. 2004;119:13–16.
12. Hyink O, Wescombe PA, Upton M, Ragland N, Burton JP, Tagg JR. Salivaricin A2 and the novel lantibiotic salivaricin B are encoded at adjacent loci on a 190-kilobase transmissible megaplasmid in the oral probiotic strain Streptococcus salivarius K12. Appl Environ Microbiol. 2007;73(4):1107–1113.
13. Sharma S, Verma KK. Skin and soft tissue infection. Indian J Pediatr. 2001;68(Suppl 3):S46–S50.
14. Wescombe PA, Burton JP, Cadieux PA, et al. Megaplasmids encode dif-fering combinations of lantibiotics in Streptococcus salivarius. Antonie Van Leeuwenhoek. 2006;90(3):269–280.
Table 6 Tolerability, compliance, and side effects in children (n=38) treated for 90 days by oral route with Streptococcus salivarius M18 as reported by themselves and/or parents and established by dentists responsible for the study
Tolerability Compliance Side effects
Very good n=30 n=32 nonegood n=5 n=6 noneacceptable n=3 n=0 noneUnacceptable n=0 n=0 none
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role of BlIs M18 in Cariogram
15. van Zon A, van der Heijden GJ, van Dongen TM, Burton MJ, Schilder AG. Antibiotics for otitis media with effusion in children. Cochrane Database Syst Rev. 2012;9:CD009163.
16. Power DA, Burton JP, Chilcott CN, Dawes PJ, Tagg JR. Preliminary investigations of the colonisation of upper respiratory tract tissues of infants using a paediatric formulation of the oral probiotic Streptococcus salivarius K12. Eur J Clin Microbiol Infect Dis. 2008;27(12): 1261–1263.
17. Horz HP, Meinelt A, Houben B, Conrads G. Distribution and persistence of probiotic Streptococcus salivarius K12 in the human oral cavity as determined by real-time quantitative polymerase chain reaction. Oral Microbiol Immunol. 2007;22(2):126–130.
18. Burton JP, Wescombe PA, Moore CJ, Chilcott CN, Tagg JR. Safety assessment of the oral cavity probiotic Streptococcus salivarius K12. Appl Environ Microbiol. 2006;72(4):3050–3053.
19. Burton JP, Cowley S, Simon RR, McKinney J, Wescombe PA, Tagg JR. Evaluation of safety and human tolerance of the oral probiotic Strepto-coccus salivarius K12: a randomized, placebo-controlled, double-blind study. Food Chem Toxicol. 2011;49(9):2356–2364.
20. Di Pierro F, Adami T, Rapacioli G, Giardini N, Streitberger C. Clinical evaluation of the oral probiotic Streptococcus salivarius K12 in the prevention of recurrent pharyngitis and/or tonsillitis caused by Streptococcus pyogenes in adults. Expert Opin Biol Ther. 2013;13(3):339–343.
21. Di Pierro F, Donato G, Fomia F, et al. Preliminary pediatric clinical evaluation of the oral probiotic Streptococcus salivarius K12 in preventing recurrent pharyngitis and/or tonsillitis caused by Streptococcus pyogenes and recurrent acute otitis media. Int J Gen Med. 2012;5:991–997.
22. Di Pierro F, Colombo M, Zanvit A, Risso P, Rottoli AS. Use of Streptococcus salivarius K12 in the prevention of streptococcal and viral pharyngotonsillitis in children. Drug Healthc Patient Saf. 2014;6:15–20.
23. Chilcott CN, Tagg JR. Antimicrobial composition. United States patent US 7226590. 2007.
24. Heng NC, Haji-Ishak NS, Kalyan A, et al. Genome sequence of the bacteriocin producing oral probiotic Streptococcus salivarius strain M18. J Bacteriol. 2011;193:6402–6403.
25. Chen YY, Clancy KA, Burne, RA. Streptococcus salivarius urease: genetic and biochemical characterization and expression in a dental plaque streptococcus. Infect Immun. 1996;64:585–592.
26. Ohnishi Y, Kubo S, Ono Y, et al. Cloning and sequencing of the gene coding for dextranase from Streptococcus salivarius. Gene. 1995;156: 93–96.
27. Wescombe PA, Upton M, Renault P, et al. Salivaricin 9, a new lantibiotic produced by Streptococcus salivarius. Microbiology. 2011;157: 1290–1299.
28. Burton JP, Wescombe PA, Macklaim JM, et al. Persistence of the oral probiotic Streptococcus salivarius M18 is dose dependent and mega-plasmid transfer can augment their bacteriocin production and adhesion characteristics. PLoS One. 2013;8(6):e65991.
29. Burton JP, Drummond BK, Chilcott CN, et al. Influence of the probiotic Streptococcus salivarius strain M18 on indices of dental health in children: a randomized double-blind, placebo-controlled trial. J Med Microbiol. 2013;62(Pt 6):875–884.
30. Litty S, Nagarathna D, Merline V. Probiotics in periodontal therapy. Int J Pharm Bio Sci. 2015;6(1):242–250.
31. Bratthall D, Hänsel PG. Cariogram: a multifactorial risk assessment model for a multifactorial disease. Community Dent Oral Epidemiol. 2005;33:256–264.
32. Holgerson PL, Twetman S, Stecksèn-Blicks C. Validation of an age-modified caries risk assessment program (Cariogram) in preschool children. Acta Odontol Scand. 2009;67:106–112.
33. Hänsel Petersson G, Twetman S, Bratthall D. Evaluation of a computer program for caries risk assessment in schoolchildren. Caries Res. 2002;36:327–340.
34. Campus G, Cagetti MG, Sale S, Carta G, Lingström P. Cariogram validity in schoolchildren: a two-year follow-up study. Caries Res. 2012;46:16–22.
35. Petersson GH, Isberg PE, Twetman S. Caries risk assessment in school children using a reduced Cariogram model without saliva tests. BMC Oral Health. 2010;19(10):5.
36. Zukanović A. Caries risk assessment models in caries prediction. Acta Med Acad. 2013;42:198–208.
37. Alian AY, McNally ME, Fure S, Birkhed D. Assessment of caries risk in elderly patients using the Cariogram model. J Can Dent Assoc. 2006;72:459–463.
38. Celik EU, Gokay N, Ates M. Efficiency of caries risk assessment in young adults using Cariogram. Eur J Dent. 2012;6:270–279.
39. Tellez M, Gomez J, Ellwood R, Ismail AI. Evidence on existing caries risk assessment systems: are they predictive of future caries? Community Dent Oral Epidemiol. 2013;41(1):67–78.
40. Campus G, Cagetti MG, Sacco G, Benedetti G, Strohmenger L, Lingström P. Caries risk profiles in Sardinian schoolchildren using Cariogram. Acta Odontol Scand. 2009;67(3):146–152.
41. Hänsel Petersson G, Twetman S, Bratthall D. Evaluation of a computer program for caries risk assessment in schoolchildren. Caries Res. 2002;36(5):327–340.
42. Baehni PC, Guggenheim B. Potential of diagnostic microbiology for treatment and prognosis of dental caries and periodontal diseases. Crit Rev Oral Biol Med. 1996;7(3):259–277.
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Research Article Pharmaceutics
International Journal of Pharma and Bio Sciences ISSN
0975-6299
PROBIOTICS IN PERIODONTAL THERAPY
LITTY SCARIYA*1, NAGARATHNA D.V2 AND MERLINE VARGHESE1
1 Post Graduate Student, Department of Periodontics, A.J Institute of Dental Sciences, Mangalore, India.
2 Professor, Department of Periodontics, A. J Institute of Dental Sciences, Mangalore, India.
ABSTRACT
To partially assess the efficacy of probiotic lozenges, in the treatment of periodontal disease. Material and methods: Twenty eight subjects, of both sexes, were selected and divided into 4 groups (2 test groups and 2 control groups).The test group was instructed to consume probiotic lozenges whereas the control group did not receive any probiotic product. Clinical parameters such as plaque index, gingival index, modified sulcular bleeding index and probing pocket depth were recorded and assessed at baseline, day 15, 30, 45 and day 60. The Test group showed significant reduction in all parameters when compared to that of Control group. After stopping probiotic administration on day 30, the test group showed a significant increase in all the clinical parameters except probing pocket depth on day 45 and day 60. Conclusions: The results show that probiotic lozenges were efficacious in reducing both moderate to severe gingivitis and moderate periodontitis. KEY WORDS: Probiotics, Gingivitis, Periodontitis
*Corresponding author
LITTY SCARIYA
Post Graduate Student, Department of Periodontics,
A.J Institute of Dental Sciences, Mangalore, India.
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INTRODUCTION
The World Health Organization defines probiotics as “living organisms, principally bacteria that are safe for human consumption and when ingested in sufficient quantities, have beneficial effects on human health, beyond the basic nutrition”1. The concept of probiotics dates back to the 20th century when Ukrainian bacteriologist and Nobel laureate, Elie Metchnikoff laid down the scientific foundation of probiotic. He proposed that Bulgarian people had longer longevity due to fermented milk containing viable bacteria. The term ‘probiotics’, the antonym for the term antibiotics, was introduced in 1965 by Lilly and Stillwell as substances produced by microorganisms which promote the growth of other microorganisms1. Probiotic therapy has been studied extensively in a variety of systemic indications and medical disorders and have also been introduced in the field of periodontal healthcare. The discovery of the role of free radicals in cancer, diabetes, cardiovascular diseases, and other chronic diseases, including periodontal disease has led to the emergence of antioxidants as prophylactic and therapeutic agents2. The development of resistance to antibiotics has raised the possibility of a return to the pre-antibiotic dark ages. Here, probiotics provide an effective alternative way, which is economical and natural to combat periodontal disease3. The aim of this study was to evaluate the efficacy of orally administered probiotic lozenges in the treatment of chronic gingival and periodontal disease by evaluating changes in monitored clinical parameters. Lozenges containing Streptococcus salivarius were selected because of their innate capacity to bind and persist on the tongue dorsum. Some strains of Streptococcus salivarius release into saliva, copious quantities of bacteriocins that could provide a targeted way of removing deleterious bacteria making them a more effective probiotic organism. They also regularly produce the enzymes dextranase and urease, which could help reduce dental plaque accumulation and acidification, respectively4.
MATERIALS AND METHODS
(i) Materials used
Lozenges containing not less than 100 million Streptococcus salivarius bacteria per tablet were imported from BLIS Technologies Ltd, Dunedin New Zealand. (ii) Methods of Randomisation of Subjects This study was conducted on 28 subjects between the age of 20 and 60 years of age. Subjects were selected from those attending the Department of Periodontics, A.J Dental College and Hospital, Mangalore. Subjects were selected on the basis of the following criteria by examining the periodontium. Inclusion criteria 1. Good general health and age ranges
between 20 to 60years 2. Not participated in any clinical trial
during the previous 4 weeks 3. No ongoing antibiotic treatment 4. Only individuals with moderate and
severe gingivitis, and moderate periodontitis
Exclusion criteria 1. Individuals with systemic disease
predisposing to periodontitis. 2. Individuals with probing pocket depth more
than 6mm. 3. Presence of tooth with grade II or grade III
mobility or abscess formation. 4. Pregnancy or breastfeeding 5. Physical or mental handicaps that may
interfere with an adequate oral hygiene. 6. History of drug abuse 7. Allergies Groups The selected subjects were divided into four groups, with 7 subjects in each group.
Group1:Test Seven male subjects with gingival index score 3 or 2 with periodontal pocket less than 6 mm treated with probiotic lozenges.
Group2:Test Seven female subjects with gingival index
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scores 3 or 2 with periodontal pocket less than 6 mm treated with probiotic lozenges.
Group3:Control Seven male subjects with gingival index scores 3 or 2 with periodontal pocket less than 6mm treated without probiotic lozenges.
Group4:Control Seven female subjects with gingival index scores 3 or 2 with periodontal pocket less than 6mm treated without probiotic lozenges. The participants were briefed in detail regarding the study. The proposed study was reviewed by the ethical committee of the institution and clearance was obtained. An informed consent was obtained from each subject before conducting the trial. Preselected participants were scheduled for a dental examination. The subjects were allotted into groups by a second post graduate student, while the clinician conducting the clinical examinations was not informed whether subjects were actively taking the lozenges or not. The test group subjects were instructed to store the lozenges in a refrigerator, as recommended by the manufacturer. Study protocol The study period was 60 days. Subjects in Group1 and Group2, after initial scaling and root planning, were instructed to consume 2 lozenges containing Streptococcus salivarius M18 every day for the next 30 days. Subjects in Group 3 and Group 4 were not instructed to consume any lozenges but underwent scaling and root planning. Participants in Group1 and Group2 were directed to place one lozenge in their oral cavity for few minutes after brushing their teeth once in the morning and in the evening, allowing the tablet to dissolve.
The patients were also instructed on how to brush and floss effectively. Participants in the test group were instructed to bring the remaining lozenges during their visits to the hospital. A count of the remaining lozenges was taken to monitor whether the subjects were regularly consuming the lozenges. Clinical parameters were obtained for all the subjects on day 0 (Baseline); day 15, day 30, day 45, and day 60. All 7 subjects in each group were analysed. At the end of the study period, 6 M18 tablets were returned to BLIS Technologies Ltd New Zealand for quality assurance testing.
RESULTS
The following clinical parameters were assessed in all subjects during each visit. 1. Supragingival plaque was scored by
Plaque Index (P.I) (Silness and Loe1964).
2. The Gingival Index (G.I) (Loe and Silness 1963) was scored.
3. Bleeding on probing by The Modified Sulcular Bleeding Index (mSBI) by Mombelli et al 1987.
4. Probing pocket depth (PPD) measured using Williams Periodontal Probe.
Data Analysis The data was tabulated in Microsoft excel and analysed using SPSS (Statistical Product and Service Solutions)version-16. The comparison between test and control group in each category (PI-plague index, GI-gingival index, mSBI-modified sulcular bleeding index, PD-probing pocket depth) at each interval was done using an independent T test. The level of significance was set to p< 0.05 (where ‘p’ is the probability value).
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Plaque Index Comparision Between Test And Control Group
Graph 1 Plaque index Comparison between Control and Test Group
Graph 1 shows the plaque index (PI) scores for both test and control group at baseline, day 15, day 30 day, 45 and day 60. The mean score in Test and Control groups were almost similar at base line. On day 15 the mean score was reduced in both groups. At day 30 the mean plaque index score of the Test group was significantly lower when compared to that of the control group for which there was an increase in the score compared to day 15. There was an increase in the mean
score of PI in the test group on day 45 and day 60, whereas in the control group there was a continuous increase in the mean score from day 15 to day 30, day 45 and day 60 respectively. There was no statistically significant difference between the plaque index scores of test and control group at baseline and day 15 (p>0.05). The difference between two group on day 30 day 45 and 60 was statistically significant (p<0.001).
Gingival Index Comparison Between Test And Control Group
Graph 2 Gingival Index Comparison between Control and Test Group
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Graph 2 shows the gingival index scores for both test and control groups at baseline, day 15 day 30 day 45 and day 60. There was no statistically significant difference between the two groups at baseline and day 15. The difference between the two groups on day 30, day 45, and day 60 was significantly higher (p<0.001). The mean score of GI in the test and control group at baseline were similar. There was a reduction in the mean score of GI for both groups on day
15. The Test group showed a significant reduction in GI mean score on day 30 when compared to that of the control group which showed an increased GI score when compared to day 15. Similarly to the plaque index results, the mean GI score showed an increase on day 45 and day 60 in the Test group after stopping the administration of probiotic lozenges. The control group showed an increase in mean score from day 15 to day 30, day 45 and day 60 respectively.
Modified Sulcular Bleeding Index Comparion Between Test And Control Group.
Graph 3 Modified Sulcular Bleeding Index Comparison between
Control Group and Test Group.
Graph 3 shows the modified sulcular bleeding index (mSBI) scores for both the test and control group at baseline, day 15, day 30, day 45 and day 60. The mean mSBI score for the test group has continued to reduce from baseline to day 30 during the time the tablets were administered after which the mean mSBI scores are observed to increase. Although the control group’s mean mSBI score are
seen to decrease on day 15. The mean mSBI score of the control group shows a sharp increase on day 30, day 45 and day 60. There was no statistically significant difference between the two groups at baseline. On day 15 the difference between the 2 groups was significant (p=0.017). The difference between the two groups on day 30, day 45 and day 60 was significantly high (p<0.001).
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Probing Pocket Depth Comparison Between Test And Control
Graph 4 Probing Pocket Depth Comparison Between Test Group and Control Group
Graph 4 shows the Probing Pocket Depth (PPD) scores for both the test and control group at baseline, day 15, day 30, day 45 and day 60. The mean score PPD at baseline were similar in the Test and the Control groups and on day 15 both group showed a slight reduction in mean score which can be credited to scaling and root planning. On day 30, the Test group showed greater reduction in scores when compared to that of Control group which may be due to probiotic lozenges but on day 45 and day 60 there was no increase or decrease in mean score of both groups. There was no statistically significant difference between the PPD scores of test and control group at baseline and day 15 (p>0.05). The difference between the two groups on day 30, day 45 and day 60 was statistically significant (p<0.05).
DISCUSSION
Probiotic bacteria, generally regarded as safe, may favour periodontal health if they are able to establish themselves in oral biofilm and inhibit pathogen growth and metabolism5. Various studies on effects of probiotic therapy showed positive results for gastrointestinal disorders as well as for caries associated riskfactors6,7,8,9,10. However, there are only a few studies which have investigated the influence of probiotics on gingivitis or periodontitis. Twetman et al.
reported a reduction of clinical symptoms caused by gingivitis after the use of chewing gum containing Lactobacillus reuteri for two weeks.11Krasse et al. documented the effects of probiotic microorganism Lactobacillus reuteri for a 2 week period during which gingival inflammation was significantly reduced12. It was also demonstrated that probiotic bacteria accumulated in microbial biofilms thus replacing or reducing pathogenic bacteria13. Ishikawa et al14 and Matsuoka et al15 demonstrated that the use of probiotic pills containing L.salivarius significantly reduced the concentration of the periopathogenic bacterium P.gingivalis in saliva and subgingival plaque in healthy volunteers. Shimauchi et al16 documented a reduced concentration of periodontopathogenic bacteria after administration of probiotic Lactobacilli over a period of weeks, which was associated with improved periodontal conditions17. It is well known that the effect of professional cleaning of teeth is effective in short term treatment of gingivitis. In the present study there was no difference in mean scores between the Test and Control group at base line. On day 15 both the groups showed a reduction in PI, GI, mSBI, and PD. This may be because scaling and root planning was carried out in both groups. A patient’s ability to maintain oral hygiene may also be a factor. But on day 30, the
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test group exhibited a greater reduction in their PI score than did the control group. Similar results were obtained in a study conducted by Shimauchi et al16. The test subjects also showed a significant reduction in the GI score when compared to the control group. Similar results were obtained in a study conducted by Krasse et al12, Shimauchi et al16 , Della Riccia et al18. In the case of the mSBI, a significant reduction in the score on day 30 was observed in the test group when compared to the control. On day 45 and day 60, the score increased, i.e. the number of bleeding site increased as soon as the probiotic intake was stopped. Similar observations were seen in the study by Twetman et al11. On day 30, a significant beneficial effect of the probiotic treatment was observed for the PPD based on comparison with the control group. Similar findings were reported in the study conducted by Matsuoka et al15, Shimauchi et al16 In general, beneficial effects from a probiotic will only take place as long as the probiotic is applied. Therefore probiotic therapy should not be seen as a treatment that permanently alters the oral microbiota as evidence indicates they are not able to sustain a shift to a stable non-pathogenic microbiota19. This observation is supported by the present study with beneficial effects being most obvious during the actual dosing phase for all parameters monitored. The reduction in all clinical parameters in the Test group appear to be due to administration of the probiotic lozenges. Once the administration of probiotic lozenge was stopped all the clinical parameters (PI, GI, mSBI) showed increases in their mean scores although generally they remained lower than the corresponding scores for the control group with the exception of PPD, which almost remained constant. It was not possible to determine why mean PPD scores remained constant after an initial reduction. The mean score of all the clinical parameters (PI, GI, mSBI) except PPD increased in the control group from day
15 to day 30, 45 and 60. PPD remained constant on day 30, day 45 and day 60. Despite the effect of professional cleaning, the reduction in all indexes was stronger and significantly better than the control group in subjects supplemented with probiotics lozenges. QUALITY ASSURANCE TEST The test report concludes that the levels of Streptococcus salivarius M18 in the tablet may not have been optimal during the entire course of the trial, although it is likely that there should still be enough live probiotic bacteria to impact on the oral health of the test subjects.
CONCLUSION
Limitations of this study are as follows: 1. Microbial analysis not carried out. 2. All lozenges were not refrigerated. 3. Study was not restricted to any particular
social strata. 4. Cell count conducted on sample tablets
after the study period found that levels of S. salivarius M18 may not have been optimal during the entire course of the trial. Despite some of these limitations, the reduction in all indices monitored was seen to be stronger and significantly better in all subjects that were supplemented with probiotic M18 tablets than in the control group who were not administered with any probiotic. This study concludes that Streptococcus salivarius M18 may be potentially useful as an aid in improving the oral health of periodontal patients. Further studies including microbial analysis need to be performed to confirm the initial findings of this report. The effect of probiotics on different strata’s of Indian society and the survivability of Streptococcus salivarius M18 bacteria in different climatic conditions such as that found in India, also need to be further probed.
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ACKNOWLEDGEMENT The authors are thankful to BLIS Technologies Ltd, Dunedin, New Zealand, for providing the probiotic lozenges.
REFERENCES
1. Sandeep Lawande. Probiotics for
management of periodontal disease: a novel therapeutic strategy.J Pharmacol, 2 (4):41-46,(2012).
2. AditiMathur,LalitMathur, BalajiManohar, HemantMathur, Rajesh Shankarapillai, et al. Antioxidant therapy as monotherapy or as an adjunct to treatment of periodontal disease. J Indian Soc Periodontol, 17(1):21-24, (2013).
3. Anirban Chatterjee,Hirak Bhattacharya, AbhishekKandwal. Probiotics in periodontal health and disease-review.J Indian Soc Periodontol, 15(1): 23-28, (2011).
4. Jeremy P.Burton, Philip A.Wescombe, Jean M.Macklaim, Melissa H.C.Chai, Kyle MacDonald,et al. Persistance of the oral probiotic Streptococcus salivarius M18 is dose dependent and megaplasmid transfer can augment their bacteriocin production and adhesion characteristics. PLOS ONE ,8(6):e65991, (2013).
5. Iva Stamatova and Jukka H. Meurman. Probiotic: Health benefits in the mouth Am J Dent, 22(6):329-338, (2009).
6. Broekaert IJ and Walker WA. Probiotics and chronic disease. J ClinGastroenterol,2006; 40: 270-274, (2006).
7. Cremonini F, Di Caro S, Nista EC, Bartolozzi F, Capelli G, Gasbarrini G et al. Meta-analysis: the effect of probiotic administration on antibiotic-associated diarrhoea. Aliment PharmacolTher ,16:1461-1467,(2002).
8. D'Souza AL, Rajkumar C, Cooke J and Bulpitt CJ. Probiotics in prevention of antibiotic associated diarrhoea: meta-analysis. BMJ ,324:1361, (2002).
9. Kligler B and Cohrssen A. Probiotics. Am FamPhysician ,78:1073-1078, (2008).
10. Cildir SK, Germec D, Sandalli N, Ozdemir FI, Arun T, Twetman S et al. Reduction of salivary Mutans streptococci in
orthodontic patients during daily consumption of yoghurt containing probiotic bacteria. Eur J Orthod,31: 407-411,(2009).
11. Twetman S, Derawi B, Keller M, Ekstrand K, Yucel-Lindberg T and Stecksen-Blicks C. Short-term effect of chewing gums containing probiotic Lactobacillus reuteri on the levels of inflammatory mediators in gingival crevicular fluid. ActaOdontolScand67: 19-24, (2009).
12. Krasse P, Carlsson B, Dahl C, Paulsson A, Nilsson A and Sienkiewicz G. Decreased gum bleeding and reduced gingivitis by the probiotic Lactobaccilusreuteri. Swed Dent J ,30: 55-60, (2006).
13. Tsubura S, Mizunuma H, Ishikawa S, Oyake I, Okabayashi M, Katoh K et al. The effect of Bacillus subtilis mouth rinsing in patients with periodontitis. EurJClinMicrobiol Infect Dis ,28: 1353-1356, (2009).
14. Ishikawa H, Aiba Y, Nakanishi M, Ohhashi Y and Koga Y. Suppression of periodontal pathogenic bacteria in the saliva of humans by the Administration of Lactobacillus salivarius TI2711. Journal of the Japanese Society of Periodontology ,45: 105-112, (2003).
15. Matsuoka T, Sugano N, Takigawa S, Takane M, Yoshimura N, Ito K et al. Effect of oral Lactobacillus salivarius TI 2711 (LS1) administration on periodontopathic bacteria in subgingival plaque. Journal of the Japanese Society of Periodontology ,48: 315-324, (2006).
16. Shimauchi H, Mayanagi G, Nakaya S, Minamibuchi M, Ito Y, Yamaki K et al. Improvement of periodontal condition by probiotics with Lactobacillus salivarius WB21: a randomized, double-blind, placebo-controlled study. J ClinPeriodontol ,35: 897-905, (2008).
17. Slawik Sabina, Staufenbiel Ingmar, SchilkeReinhard, Nicksch Sonja, Weinspach Knut. Probiotics affect the
Int J Pharm Bio Sci 2015 Jan; 6(1): (P) 242 - 250
This article can be downloaded from www.ijpbs.net
P- 250
clinical parameters of experimental gingivitis in humans. European Journal of Clinical Nutrition, 65: 857-863, ( 2011).
18. Della Riccia DN, Bizzini F, Perilli MG, Polimeni A, Trinchieri V, Amicosante G, et al. Anti-inflammatory effects of Lactobacillus brevis (CD2) on periodontal disease. Oral Dis, 13:376–385, (2007).
19. TeughelsW,LoozenG,Quirynen M. Do probiotics offer opportunities to manipulate the periodontal oral microbiota?.J ClinPeriodontol ,38(11):159-177,(2011).
20. Perkrasse, BirgittaCarlsson, Carina Dahl, Annette Paulsson, Asa Nilsson, et al. Decreased gum bleeding and reduced gingivitis by the probiotic Lactobacillus reuteri. SWED DENT J , 30(2):55-56 (2005).
21. Vivek Gupta, Bhavana Gupta. Probiotics and periodontal disease: a current
update. J Oral Health Comm Dent ,4:35-37,(2010).
22. Shivkumar,gopinath. Probiotics in periodontology. Indian Journal of Multidisciplinary Dentistry ,1(6):315-320, (2011).
23. Dr.J.Bhuvaneswarri, Dr.V.Ramya, Dr.Manisundar, Dr.Preethi.Probiotics and Its Implications In Periodontal Therapy-A Review. Indian J MedSci , 2(5):11-15, (2012).
24. Jeremy P. Burton, Bernadette K. Drummond, Chris N. Chilcott, John R. Tagg, W. Murray Thomson et al .The influence of the probioticStreptococcussalivarius M18, on indices of dentalhealth in children: a randomised double-blind placebo-controlled trial. Journal of Medical Microbiology Papers in Press ( 2013).
Influence of the probiotic Streptococcus salivariusstrain M18 on indices of dental health in children: arandomized double-blind, placebo-controlled trial
Jeremy P. Burton,1,2 Bernadette K. Drummond,3 Chris N. Chilcott,1
John R. Tagg,1,4 W. Murray Thomson,5 John D. F. Hale1
and Philip A. Wescombe1
Correspondence
Philip A. Wescombe
Received 22 December 2012
Accepted 26 February 2013
1BLIS Technologies Ltd, Centre for Innovation, University of Otago, Dunedin, New Zealand
2Canadian Research and Development Centre for Probiotics, Lawson Health Research Institute,St Joseph’s Health Care, London, Ontario, Canada
3School of Dentistry, University of Otago, Dunedin, New Zealand
4Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
5Oral Sciences, Faculty of Dentistry, University of Otago, Dunedin, New Zealand
The prevalence of dental caries continues to increase, and novel strategies to reverse this trend
appear necessary. The probiotic Streptococcus salivarius strain M18 offers the potential to confer
oral health benefits as it produces bacteriocins targeting the important cariogenic species
Streptococcus mutans, as well as the enzymes dextranase and urease, which could help reduce
dental plaque accumulation and acidification, respectively. In a randomized double-blind, placebo-
controlled study of 100 dental caries-active children, treatment with M18 was administered for
3 months and the participants were assessed for changes to their plaque score and gingival and
soft-tissue health and to their salivary levels of S. salivarius, S. mutans, lactobacilli, b-haemolytic
streptococci and Candida species. At treatment end, the plaque scores were significantly
(P50.05) lower for children in the M18-treated group, especially in subjects having high initial
plaque scores. The absence of any significant adverse events supported the safety of the
probiotic treatment. Cell-culture analyses of sequential saliva samples showed no differences
between the probiotic and placebo groups in counts of the specifically enumerated oral micro-
organisms, with the exception of the subgroup of the M18-treated children who appeared to have
been colonized most effectively with M18. This subgroup exhibited reduced S. mutans counts,
indicating that the anti-caries activity of M18 probiotic treatments may be enhanced if the
efficiency of colonization is increased. It was concluded that S. salivarius M18 can provide oral
health benefits when taken regularly.
INTRODUCTION
Dental caries is the most common chronic disease ofchildhood and, despite major technological advances andthe introduction of many new initiatives by the dentalprofession, its prevalence continues to increase in manypopulations worldwide (Bagramian et al., 2009).Expression of the disease is characterized initially bydissolution of the mineral portion of the tooth (whitespot lesions), progressing to localized destruction of theenamel and dentine, and followed ultimately by inflam-mation of the pulp and periapical tissues if left untreated.Whilst recent research indicates a multi-species aetiology
for dental caries, the mutans streptococci (MS) – a clusterof acidogenic, dental plaque-inhabiting streptococcalspecies – are still recognized as major constituents of mostactive dental caries lesions. Of the various MS species, it isStreptococcus mutans and Streptococcus sobrinus that havebeen principally implicated in dental caries development inhumans. Dental caries imposes a major health andeconomic burden internationally, and a wide variety ofapproaches for its control has been developed and applied,with varying degrees of success. Treatments using conven-tional anti-streptococcal antimicrobials can be effective inthe short-term to reduce dental plaque levels and todecrease counts of MS, but, as most therapeutic antibioticshave relatively broad-spectrum antimicrobial activity, theyindiscriminately destroy both commensal and potentially
Abbreviations: BLIS, bacteriocin-like inhibitory substance; MS, mutansstreptococci; OHI-S, simplified Oral Hygiene Index.
Journal of Medical Microbiology (2013), 62, 875–884 DOI 10.1099/jmm.0.056663-0
056663 G 2013 SGM Printed in Great Britain 875
harmful bacteria and thereby create population imbalanceswithin the microflora. In addition, conventional antimi-crobial treatments are often unpalatable for youngchildren, resulting in poor compliance and therebycompromising the likelihood of demonstrating beneficialoutcomes.
The use of probiotics to effect an improvement in oralhealth without impacting negatively on the normal oralmicrobiota is a relatively new concept. Conventionally,probiotics – defined by the World Health Organization as‘live organisms which, when administered in adequateamounts confer a health benefit on the host’ – have almostexclusively been bacteria of intestinal origin, and theirapplication has largely been targeted at relieving maladiesof the gastrointestinal tract. However, because it is nowbecoming clear that many human illnesses are relatedeither directly (for example, dental caries and periodontaldisease) or indirectly (for example, cardiovascular disease)to the development of oral microbiota disequilibria; newways of reducing the disease burden imposed by thesedysfunctional microbial populations are being investigated(Zarco et al., 2012). A variety of putative commensalbacteria have been assessed for their potential to preventdental caries. Studies based on use of the intestinalprobiotics Lactobacillus rhamnosus GG (Nase et al., 2001),Lactobacillus reuteri ATCC 55739 and Bifidobacterium DN-173 010 (Caglar et al., 2005) have each reported achievingreduced levels of S. mutans and, moreover, the childrentaking L. rhamnosus GG developed fewer dental caries.Whilst these strains have shown some promise for theprevention of dental caries, a new generation of probioticstrains sourced from the human oral cavity and belongingto commensal species known to have extremely lowpathogenic potential are now being developed. In thisregard, a key species is Streptococcus salivarius, which haspreviously been investigated for its role in the prevention ofpharyngitis caused by Streptococcus pyogenes (Dierksen &Tagg, 2000), dental caries (Tanzer et al., 1985a, b),periodontal disease (Guglielmetti et al., 2010; Teughelset al., 2007) and halitosis (Burton et al., 2006a).
Many S. salivarius strains produce bacteriocins, which areribosomally synthesized antimicrobials that typically have anarrow inhibitory spectrum directed against relativelyclosely related bacteria (Wescombe et al., 2009). In orderto minimize widespread disruption within the oralmicroflora, bacteriocin-producing probiotics targetingMS are now under consideration as a replacement therapyapproach to the control of dental caries (Hillman et al.,1987). Key characteristics of an effector strain for use inreplacement therapy include: (i) the absence of virulencedeterminants, (ii) colonization capability, and (iii) theability to competitively displace the target bacterium(Burton et al., 2011a). Perhaps not unexpectedly, the mostcommon producers of anti-MS bacteriocins are otherstrains of MS. Indeed, a number of S. mutans producingstrong anti-S. mutans activity have been identified aspotential effectors of replacement therapy (Hillman et al.,
1987). However, unless stable non-cariogenic naturalvariants of these strains can be derived, their acceptanceand application for dental caries control seems unlikely togain either regulatory or public acceptance (Hillman et al.,1990, 1998).
S. salivarius is a numerically predominant, exclusivelyhuman, oral streptococcal species that is not known tohave any disease associations in healthy humans.Previously, S. salivarius TOVE-R (Tanzer et al., 1985a, b)has been shown capable of effecting a reduction in dentalcaries in animal models, but this strain had only relativelyweak bacteriocin activity in vitro (Wescombe et al., 2011,2006). Heightened interest in the probiotic potential of S.salivarius stems from the recognition that some strainsproduce a particularly diverse array of bacteriocins,together with the demonstration that the application ofstrain K12, the prototype of S. salivarius probiotics, canhelp control a variety of upper respiratory tract ailmentsincluding streptococcal sore throat, otitis media andhalitosis (Burton et al., 2006a, 2006b, 2011b; Wescombeet al., 2009). In addition, some S. salivarius strains havebeen shown to produce the exoenzymes dextranase andurease, the activities of which could potentially help limitthe progression of dental caries by reducing plaqueaccumulation and plaque acidification, respectively (Chenet al., 1996; Li et al., 2000; Nascimento et al., 2009; Ohnishiet al., 1995). S. salivarius M18 (also referred to as strainMia or DSM 14685; Chilcott & Tagg, 2007), a strainoriginally isolated from a healthy adult subject during aspecific search for S. salivarius capable of inhibiting MS,has subsequently been shown to have relatively broad-spectrum bacteriocin-like inhibitory substance (BLIS)activity against MS, and to produce both dextranase andurease enzymes (Heng et al., 2011). The genome of M18has recently been published, and its megaplasmid-encodedbacteriocin repertoire includes salivaricin A, salivaricin M,salivaricin MPS and salivaricin 9 (Chilcott & Tagg, 2007;Heng et al., 2011; Wescombe et al., 2006, 2011). Theseidiosyncratic characteristics of M18 make it an attractivecandidate with potential application to the prevention andtreatment of dental disease.
The present study provides a preliminary evaluation of S.salivarius M18 for its probiotic application to theprevention, or a reduction in the risk, of dental caries.The objective was to compare the influence on severalreadily measured indices of potential for dental cariesactivity in children who were given lozenges containingeither M18 or placebo over a 3-month study period.
METHODS
Screening for antimicrobial activity. The spectrum of antimicro-
bial activity of M18 was detected using the deferred antagonism
method, essentially as originally described by Tagg & Bannister
(1979). Briefly, an agar plate, comprising tryptic soy broth (BBL)
supplemented with 2 % yeast extract (Difco), 1 % CaCO3 and 0.7 %
Bacto agar and adjusted to pH 6.5 before autoclaving (TSYCa), was
J. P. Burton and others
876 Journal of Medical Microbiology 62
seeded diametrically with a 1 cm wide inoculum of the test strain
from an 18 h culture of the producer strain grown on Columbia
blood agar base (Difco) supplemented with 5 % human blood using a
cotton swab. Following incubation for 18 h at 37 uC in 5 % CO2 in
air, the culture growth was removed using a glass slide and the agar
surface sterilized by exposure to chloroform vapour for 30 min,
followed by airing for a further 30 min. Indicator bacteria, from 18 h
Todd–Hewitt broth (Difco) cultures, were then inoculated with a
swab at right angles across the line of the original diametric streak
culture of the test strain, and the plate was reincubated for 18 h at
37 uC in 5 % CO2 in air. Zones of inhibition were scored as ‘2’ for no
inhibition or ‘+’ where definite interference with the growth of the
indicator was evident.
Preparation of test material. Freeze-dried preparations of S.
salivarius M18 cells were produced by the Microbial Fermentation
Unit (Fonterra, Palmerston North, New Zealand), an ISO 9001
quality-accredited facility. The cell powder was blended with
flavouring agents and the food-grade sugar substitutes trehalose and
maltodextrin, prior to forming into lozenges by Good Manufacturing
Practice-certified Alaron Products (Nelson, New Zealand). Cell
counts were obtained just prior to commencement and at completion
of the study, and each lozenge was determined to contain 3.66109
c.f.u. S. salivarius M18, a level that was maintained throughout the
3 months of the active dosing period. The placebo differed only in
containing additional sugar substitutes in place of the M18 cell
powder. The active and placebo preparations were identical in
appearance and taste.
Participants. The study was approved by the Otago Ethics
Committee (approval no. 02/09/099). Six schools with dental clinics
on site were randomly selected. One hundred Dunedin schoolchildren
aged 5–10 years who had previously experienced dental caries (and
had at least three dental restorations, including one placed within the
previous 12 months) were recruited into the study through their
school dental clinics. Two saliva specimens were procured from each
child within a 2-week period and tested for their content of S. mutans
and BLIS-producing S. salivarius. The inclusion criteria for the
treatment phase of the study were: (i) no natural strong BLIS-
producing S. salivarius detected, and (ii) .104 c.f.u. S. mutans ml21
in at least one of the two pre-screen saliva samples. During the
recruitment phase, 18 children were excluded who were either lactose
intolerant, allergic to dairy products, immunologically compromised
or taking antibiotics. One hundred children meeting the selection
criteria were assigned randomly into two groups of 50 and
commenced the dosing programme; however, after exclusions due
to non-compliance or incomplete data collection, 40 (80 %) and 43
(86 %) children, respectively, from each group completed the
probiotic- and placebo-dosing courses. The probiotic group com-
prised 12 males and 28 females (mean age 8.5 years) and the placebo
group had 21 males and 22 females (mean age 8.5 years). A
combination of count back of the number of lozenges returned and a
sticker chart administered by the parents was used for the monitoring
of subject compliance. Parents/guardians were asked via telephone
interview each month whether the children had experienced any ill
effects and these were recorded.
Clinical examinations. At the first visit, a dental therapist (whose
assessment regimen had previously been calibrated) carried out a
clinical examination and recorded the numbers of decayed, missing or
filled teeth and any new caries lesions that had occurred since the
child’s most recent school dental care visit. Also recorded at this visit
(and at the 1-, 3- and 7-month visits) were data on soft-tissue health,
gingival health and dental plaque.
Evaluation of soft-tissue health was based on the appearance of the
oral mucosa and involved recording the presence of any tissue
abnormalities such as ulceration, redness/inflammation, abscesses orwhite patches. The surfaces examined were the lips, sulci, buccalmucosa, floor of the mouth, tongue, hard palate, soft palate andgingiva/alveolar processes. For analysis purposes, all abnormalitiesdetected were treated as isolated events and the sum of abnormalitiesat each time point for each group was compared.
For the gingival health assessment, the Gingival Index of Loe & Silness(1963) was utilized. The appearance of the gingival tissues (colour,inflammation, swelling or signs of bleeding) was recorded for six teeth(the buccal aspects of the upper right second primary molar, upperleft second primary molar, upper right central incisor and lower leftcentral incisor, and the lingual aspects of the lower left secondprimary molar and lower right second primary molar). The categoriesand codes used were: normal gingivae, scoring 0; mild inflammation,slight difference in colour or slight oedema, 1; moderate inflam-mation, redness, oedema and glazing, 2; and severe inflammation,manifesting as redness and oedema with signs of bleeding, 3.
Plaque was scored using an adaptation of the simplified Oral HygieneIndex (OHI-S) of Greene & Vermillion (1964). The OHI-S has twocomponents, the Debris Index and the Calculus Index. Each of theseis based on numerical determinations representing the amount ofdebris or calculus on index tooth surfaces. The distribution of dentalplaque was assessed following the use of a plaque-disclosing solution.The teeth scored were the same as those selected for the gingivalhealth assessment.
Treatment protocol. On the first day of the study protocol, thechildren used a fluoride-containing toothpaste not containing anysupplementary antibacterial agents and, under the supervision of adental therapist, brushed their teeth until all of their disclosed plaquehad been removed. The dental therapist then flossed the children’steeth. With a second brush, the teeth were then brushed for 1 minusing a 2 % chlorhexidine gel. Two hours later, the children wereasked to suck two lozenges containing either S. salivarius M18 or theplacebo. Two of the corresponding lozenges were also given to thechildren at the end of that school day. On days 2 and 3, the teeth werebrushed for a timed 1 min with 2 % chlorhexidine gel. Two hourslater, the children were given two lozenges and then another twolozenges just before they went home. Beyond day 3, a supply of theappropriate lozenges (plus toothpaste and toothbrushes) wasprovided for home use, together with a sticker chart to encourageand record compliance. The protocol required the children to sucktwo lozenges each day for 3 months, one after brushing the teeth inthe morning and one after teeth brushing at night. At 1, 3 and7 months, the children’s mouths were examined and scored forplaque distribution and clinical health as described above. Salivasamples for analyses of dental caries indicator microbes (MS,lactobacilli and yeast) and S. salivarius (total count and probioticM18) were obtained pre-colonization and at 1, 2, 3 and 7 months.Approximately 1 ml saliva was collected at each assessment. If thechild had difficulty salivating, he/she was asked to chew on a plasticfilm (Parafilm) to stimulate saliva flow.
Bacteriological analysis of saliva samples. CandidaChromogenic agar (for Candida spp.) (Fort Richard Laboratories),Rogosa SL agar (BD Difco) (for lactobacilli) and Mitis Salivarius agar(BD Difco) (for S. salivarius) were used. The MS selective mediumwas TYCSB, as described by Van Palenstein Helderman et al. (1983).CNA-P is a blood agar medium formulated to enhance the detectionof haemolytic streptococci (Dierksen et al., 2000). Incubation was inair for 48 h (for Candida), in 5 % CO2 in air for 24 h (for S.salivarius) or was anaerobic (85 % N2, 10 % H2, 5 % CO2) for 48 h(for MS or haemolytic streptococci). Saliva samples were seriallydiluted in sterile PBS in duplicate, and appropriate dilutions wereplated in duplicate and cultivated under the appropriate conditions.CNA-P medium was used to determine the number of b-haemolytic
Impact of S. salivarius M18 on children’s oral health
http://jmm.sgmjournals.org 877
streptococci present in saliva samples; the numbers were graded from0 (no haemolytic colonies) to 4 (all colonies apparently b-haemolytic)(Dierksen et al., 2000). Streptex testing (Remel) established whetherthe haemolytic colonies on CNA-P were Lancefield group A (S.pyogenes). Data were log10 transformed for analysis. To determinewhether M18 had colonized in the child’s oral cavity, 80 represent-ative S. salivarius-like colonies from the Mitis Salivarius agar cultureswere sampled using toothpicks and tested for simultaneousantagonism inhibitory activity against S. mutans OMZ 175 andMicrococcus luteus I1, as described previously (Tagg & Bannister,1979). The characteristic activity of M18 against these two indicatorsprovides a specific presumptive identification of M18. In addition,total streptococcal populations (taken from Mitis Salivarius agar)were tested for their deferred antagonism inhibitory profile (producertype) against a set of nine standard indicators, as described previously(Tagg & Bannister, 1979), to further confirm the prevalence of M18 asa significant proportion of the oral salivary population.
Statistical analysis. Following the computation of univariatedescriptive statistics, bivariate associations were tested for statisticalsignificance using analysis of variance or Kruskal–Wallis tests (asappropriate, depending upon the distribution of the dependentvariable). Where the dependent variable was not normally distributed,it was log transformed prior to modelling. Linear regressionmodelling was used to examine the effect of the intervention whilecontrolling for baseline status and putative confounding variables.
RESULTS
Antimicrobial spectrum of S. salivarius M18
To determine the potential applications of S. salivariusM18, an in vitro screen against a variety of bacterial speciesof significance for human health was carried out using thedeferred antagonism test (Table 1). In particular, repres-entative strains of a number of species identified ascausative agents of either dental caries or periodontaldisease in humans were tested and those inhibited were: S.mutans (11/11), Actinomyces naeslundii (1/1), Actinomycesviscosus (2/2), Enterococcus faecalis (1/1), Lactobacillus spp.(3/3) and S. sobrinus (1/1). Other important upperrespiratory tract pathogens inhibited by M18 included allS. pyogenes (causative agent of streptococcal sore throat),all Streptococcus pneumoniae (associated with pneumonia,meningitis and otitis media), half of the Moraxellacatarrhalis (otitis media), both Streptococcus agalactiae(major cause of infant septicaemia) and half of theStaphylococcus aureus (regularly carried in the nasopharynxand a common source of community- and hospital-acquired infections) isolates.
Compliance and adverse reactions
Dosing with probiotics twice daily required a significantcommitment from the children, and therefore it wasessential to determine the compliance rates in both thetreatment and placebo arms of the trial. The chewablestrawberry lozenges proved to be a suitable delivery format,with compliance found to be .80 %. Compliance wasmonitored by the use of sticker charts and collecting andcounting unused lozenges at the end of treatment months 1
and 2, and the subjects were assessed as compliant if theyconsumed ¢75 % of the prescribed lozenges each month.There were no significant differences in compliance ratesbetween the treatment and placebo groups. Participantswere excluded from the analysis if they failed to be assessedas compliant for both of the months monitored. Data forsix participants were excluded from the treatment groupand two from the placebo group based on this compliancecriterion. Six participants dropped out of the study becausethey did not like the taste of the lozenges; one waseliminated because of protocol breaches, and four were lost
Table 1. Spectrum of antibacterial activity of strain M18 whentested by the deferred antagonism method on TSYCa agar
Indicator species No. strains inhibited/total tested
Actinomyces naeslundii 1/1
Actinomyces viscosus 2/2
Bacillus cereus 0/1
Candida albicans 0/3
Clostridium perfringens 1/1
Clostridium sporogenes 1/1
Corynebacterium diphtheriae 1/1
Enterobacter aerogenes 0/1
Enterococcus faecalis 1/3
Enterococcus hirae 1/2
Escherichia coli 0/1
Haemophilus influenzae 2/3
Klebsiella pneumoniae 0/1
Lactobacillus acidophilus 1/1
Lactobacillus brevis 1/1
Lactobacillus casei 1/1
Lactococcus lactis 1/1
Listeria greyii 3/5
Listeria monocytogenes 5/5
Micrococcus luteus 1/1
Moraxella catarrhalis 2/4
Moraxella lacunata 1/1
Moraxella osloensis 1/2
Porphyromonas gingivalis 0/2
Prevotella intermedia 0/2
Proteus vulgatus 0/1
Rothia mucilagenosa 1/1
Staphylococcus aureus 3/6
Staphylococcus cohnii 2/2
Staphylococcus hominus 1/1
Staphylococcus saprophyticus 2/2
Staphylococcus simulans 0/0
Streptococcus agalactiae 2/2
Streptococcus mitis 1/1
Streptococcus mutans 11/11
Streptococcus pneumoniae 8/8
Streptococcus pyogenes 8/8
Streptococcus rattus 0/1
Streptococcus salivarius 24/46
Streptococcus sanguis 0/1
Streptococcus sobrinus 1/1
Streptococcus uberis 2/2
J. P. Burton and others
878 Journal of Medical Microbiology 62
to follow-up due to moving away. There were four cases ofadverse reactions, as monitored by self-reporting using themonthly questionnaires: three were for the M18 group andone was in the placebo group. None of the adverse eventsresulted in the participants leaving the trial, and none wasof a serious nature.
Colonization with M18
One of the major aims of this trial was to determinewhether dosing with M18 resulted in its persistentcolonization of the oral cavity, with assessments beingmade at day 3 and after 1, 2 and 3 months. In the M18-treated group, nine subjects retained M18 populationscomprising at least 5 % of their total salivary S. salivariuspopulation at the 3-month time point. Furthermore, P-typing of the 3-month samples of the total S. salivariuspopulations of these nine subjects showed that five gaveinhibition profiles consistent with that given by purecultures of M18. In addition to having their data evaluatedtogether with that of the other members of the M18treatment group, the plaque scores and S. mutans countsfor these nine apparently more highly colonized individualswere also analysed separately to determine whether theremight be additional oral health benefits associated withcolonization efficacy and persistence.
Effect on plaque
Comparison of the total plaque scores of the two groups ofsubjects at the start of the study, after taking the lozengesfor 1 and 3 months, and 4 months after dosing wasterminated (i.e. at 7 months) demonstrated that, by theend of the treatment period, there was a significantdifference in mean plaque scores between the M18 groupand the placebo group (Table 2). As a consequence of thedental plaque treatment regimen given to all of theparticipants at the beginning of the study, there was alsoa decrease in plaque scores between the start of the studyand after 1 month for both groups.
Although the plaque scores were lowest for the grouptaking the M18 lozenges at all sampling points, it should benoted that a disproportionate number of participants in
this group also had relatively low preliminary plaque scores(Table 2). Closer analysis of the M18 group showed that itcontained fewer children falling into the high plaque scorecategory of ¢7 [n516 (40 %) for the M18 group versusn526 (60 %) for the placebo group]. However, controllingfor baseline plaque scores using linear regression estab-lished that the plaque scores after 3 months weresignificantly lower in the M18 group (Table 3).Moreover, when the participants in the placebo and M18groups who had high plaque scores (i.e. ¢7) at thebeginning of the trial were followed separately, there was astrong difference between the two groups throughout thetreatment phase of the study, with 87.5 % of children in theM18 treatment group maintaining lower plaque scoresthan their pre-treatment scores (defined as a decrease inscore of 3 or more), whilst only 44 % of those in theplacebo group had lower plaque scores at the same timepoint (Fig. 1).
While only small numbers of children were successfullycolonized with M18 during the study period, the plaquescores of the nine who were well colonized showed a greaterplaque reduction than both the entire M18- and placebo-treated groups. Indeed, the proportion of participants
Table 2. Summary data on plaque scores at each time point by group
Assessment time Mean (SD) P value
Treatment group (n540) Placebo (n543)
Baseline 6.0 (3.3) 6.9 (3.2) 0.182
1 month 3.4 (2.1) 4.1 (2.6) 0.175
3 months 5.3 (3.2) 7.0 (4.1) 0.022
7 months 4.7 (2.7) 4.4 (2.9) 0.852
Table 3. Regression models for plaque score
B (95 % CI)* P value
Plaque score at 1 month
Intercept 4.26 (2.12, 6.40) ,0.001
Baseline plaque score 0.03 (20.21, 0.27) 0.816
Treatment groupD 21.12 (22.91, 0.67) 0.212
Plaque score at 3 months
Intercept 5.86 (2.62, 9.10) 0.001
Baseline plaque score 0.27 (20.09, 0.64) 0.137
Treatment groupD 22.71 (25.4, 0.01) 0.050
Plaque score at 7 months
Intercept 5.45 (2.64, 8.26) 0.001
Baseline plaque score 20.03 (20.36, 0.30) 0.847
Treatment groupD 0.22 (22.27, 2.70) 0.859
*B, regression coefficient; CI, confidence interval.
DReference category5control group.
Impact of S. salivarius M18 on children’s oral health
http://jmm.sgmjournals.org 879
having reduced plaque scores was much greater in thecolonized group than for the placebo group or for those whowere not well colonized with M18 (Fig. 1).
Gingival and soft-tissue health
Analysis of the gingival health scores and soft-tissue scoresshowed no significant differences between the M18-treatedchildren and the controls (Table 4). Whilst both scoreswere low at the beginning of the trial, the soft-tissue scoreswere observed to decrease further for both groups over thecourse of the monitored period.
Salivary microbial levels
No significant differences between the treatment andplacebo groups were observed in the mean S. mutanscounts at any of the time points assessed (Table 5).However, the five children who were identified as havingbeen well colonized (by both the detection of M18-likecolonies by the use of the simultaneous antagonismmethod and by having a total streptococcal P-typeconsistent with that of M18; i.e. P-type 677) tended tohave lower S. mutans counts during the dosing phase whencompared with their baseline salivary S. mutans levels (Fig.2).
The presence of S. pyogenes was monitored at thepreliminary, 1-, 2- and 3-month time points by plating thesaliva onto CNA-P medium. Haemolytic colonies wereidentified as S. pyogenes if they were positive for Lancefieldserogroup A using a Streptex agglutination test. The rate ofS. pyogenes acquisition during the 3-month treatment phasefor the M18 group was 17.9 % (seven new acquisitions) andfor the placebo group was 25.6 % (ten new acquisitions).
Part
icip
ants
(%
)
1 3
80
70
60
50
40
30
20
10
90
100
Treatment month
M18 colonized (n=4)
Placebo (n=24)
Treatment (n=15)
Fig. 1. Percentage of children for each group who had high pre-treatment plaque scores (¢7) and who demonstrated plaquescore improvements of ¢3 at treatment months 1 and 3.
Ta
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J. P. Burton and others
880 Journal of Medical Microbiology 62
The salivary levels of S. salivarius, lactobacilli, haemolyticstreptococci and Candida spp. did not differ substantiallybetween the two groups (data not shown).
DISCUSSION
Previous studies of the applicability of probiotics to theprevention of dental caries have focused largely on thetesting of gastrointestinal tract strains that had initiallybeen developed as probiotics to help counter gut ailments.Here, we have provided evidence to support the use of S.salivarius strain M18 – a bacterium isolated from thehuman oral cavity and shown to have antibacterial activityagainst a number of clinically important human pathogens– to reduce dental plaque accumulation in schoolchildren.In addition, the findings of this preliminary double-blind,placebo-controlled trial, which included the longitudinalmonitoring of the individuals’ soft-tissue and periodontalclinical parameters, further attest to the safety of S.salivarius M18.
The treatment dosing regime of two lozenges per day,taken morning and night after tooth brushing, was welltolerated by the individuals (compliance .80 %) and this
indicates that this is a suitable format for use in primary-school-aged populations (5–12 years). Despite the high rateof compliance, only 22 % of the children in the M18 grouphad detectable probiotic in their saliva at the end of thetreatment period. This observation indicates that anyeffects directly attributable to the presence of the probioticare only likely to occur during the period of active dosing,which is in contrast to S. salivarius strain K12, a probioticthat has been shown to exhibit more persistent coloniza-tion of the human oral cavity (Horz et al., 2007). It is,however, noteworthy that, in the time period since thepresent trial was conducted, significant improvements havebeen introduced to the production process for M18,resulting in considerably improved colonization efficacy(J. P. Burton, unpublished data).
In the present study, the primary clinical measure ofclinical efficacy was the plaque score (as measured by theOHI-S), which has been ratified as an essentially validmethod for measuring oral hygiene in large epidemiolo-gical studies (Broadbent et al., 2011). The plaque score wasmonitored for each participant prior to the start of dosingand then at months 1, 3 and 7. The significant plaque scoredifference between the M18 group and the placebo groupobserved at the end of the treatment phase (month 3)indicated that the regimen implemented during this studywas efficacious for a reduction in dental plaque. However,no significant differences were observed at any of the othertime points between the two treatment groups, althoughboth the placebo and M18 groups experienced a largereduction at the 1-month time point compared with theirpre-treatment scores. The observed reduction in plaquescore at 1 month appeared to be due to the efficient plaqueremoval carried out under the supervision of the dentalhygienist as part of the initial pre-treatment regimen priorto beginning the trial. Both groups had similar plaquescores at the 7-month time point, indicating that thebenefit of M18 treatment did not extend to 4 months pastthe termination of the probiotic treatment. However, forfuture studies, it would be beneficial to have examinationtime points closer to the end of active treatment todetermine the extent of any persistent benefit occurringpost-treatment. An interesting observation was that, for thesubgroups with pre-treatment plaque scores of ¢7, 87.5 %
Table 5. Summary data on natural-logged S. mutans scores at each time point by group
Assessment time Mean (SD) P value
Treatment group (n540) Placebo (n543)
Baseline 1 10.7 (2.1) 10.6 (2.6) 0.868
Baseline 2 10.3 (2.4) 11.3 (2.4) 0.129
Day 3 8.9 (2.2) 9.8 (2.1) 0.061
1 Month 11.3 (2.5) 11.6 (2.8) 0.593
2 Months 11.4 (2.2) 11.7 (2.5) 0.490
3 Months 10.6 (2.3) 10.9 (2.7) 0.755
7 Months 10.4 (2.6) 11.4 (2.3) 0.156
0 50
0.20
0.30
0.40
0.
–0.20
–0.10
0.00
0.10
M18
Placebo
M18 colonized
–0.40
–0.30
Treatment time (days)
Lo
g1
0 d
iffe
rence in S
. mut
ans
sco
res
0 906030
Fig. 2. Change in S. mutans scores (log10) from the pre-treatmentvalues for the total M18 group (n540), placebo group (n543) andsubgroup (n55) that had detectable levels of M18 followingtreatment (M18 colonized).
Impact of S. salivarius M18 on children’s oral health
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of the M18 group and 44 % of the placebo group hadplaque score reductions of ¢3 from their pre-treatmentscore at the end of treatment. This large difference betweenthe two groups indicates that M18 treatment may providegreater benefit to individuals with existing high plaquelevels and may be most efficacious when used inconjunction with a preliminary plaque removal proceduresuch as was implemented in the present study. Anothergroup of individuals who may derive benefit fromprobiotic-mediated plaque reduction are adults experi-encing gingival inflammation in whom the development ofperiodontitis is closely linked to the level of plaqueaccumulation (Broadbent et al., 2011). Further evidencesupporting a potential role for M18 in the control ofgingivitis comes from recent experiments by Adam et al.(2011) who investigated whether M18 could potentiallyimpact on pathogen-induced pro-inflammatory cytokineexpression in gingival fibroblasts. A variety of pathogenshave been implicated in the development of both gingivitisand periodontitis, and the aetiology of these diseases is nowstrongly linked to the inflammatory response of the hostcells to the bacterial pathogens (Fisher et al., 2010;Seymour & Gemmell, 2001). In earlier work, M18 wasco-incubated with gingival fibroblasts both prior to andconcomitantly with exposure to periodontal pathogenssuch as Porphyromonas gingivalis, Aggregatibacter actino-mycetemcomitans and Fusobacterium nucleatum. StrainM18 significantly inhibited the expression of the pro-inflammatory cytokines IL-6 and IL-8 commonly asso-ciated with periodontitis, indicating that dosing with theseprobiotics may potentially be useful in the treatment ofinflammatory periodontal diseases (Adam et al., 2011).
The other clinical parameters examined in the present trialwere the soft-tissue and periodontal tissue scores. Theseoutcomes were monitored largely to establish whetherthere was evidence of any soft-tissue damage, ulcers or gumdisease developing as a result of the twice-daily probiotictreatment regimen, thereby further monitoring andevaluating the safety of M18 and the efficacy of thedelivery format for use in children. No significantdifference was observed between the placebo or M18treatment group for either of these clinical parameters;indeed, for the soft-tissue scores, both groups showed someimprovement over the course of the trial, a findingproviding some support for twice-daily oral dosing witha probiotic preparation having a positive rather than adetrimental general effect on oral health. This improve-ment may reflect either improved oral hygiene awarenessand/or be related to seasonal changes over the period of thetrial.
S. mutans has long been considered one of the principalaetiological agents of dental caries, and this has also beensupported by more modern molecular analyses (Kanasiet al., 2010). In the present study, the inter- and intra-individual salivary levels of cultivable S. mutans appearedquite variable, a finding in part due to the difficulty ofselective propagation of these bacteria. A direct comparison
of the mean numbers of S. mutans in those receiving M18
probiotic treatment with those in the placebo group showed
no significant difference. However, as the overall rates of
colonization were quite low, the subgroup of individualsknown to have been colonized with M18 were compared for
their mean log reduction in S. mutans counts against both
the placebo group and the entire M18 group. The M18
colonized subgroup showed a larger decrease in S. mutanscounts than both the placebo group and the entire M18
group over the treatment period, indicating that the longer-
term establishment of an M18 population may be required
to impact significantly on the salivary levels of S. mutans. Asthe number of children in this colonized group was low
(n55), this observation must be considered preliminary and
should now be supported by the conduct of further trials
using the improved fermentation and manufacturingprocedures that have been developed more recently for
M18. Whilst it has been established that the presence of S.
mutans is an important risk factor for the development of
dental caries, recent molecular studies have highlighted theimportance of microbial consortia in the aetiology and
development of caries, and indeed S. mutans may simply act
as a disease indicator organism, with its plaque predom-
inance signifying that the oral conditions are changing to a
more caries-active state (Marsh, 2003).
In previous studies, the impact of naturally occurring BLIS-producing S. salivarius on the acquisition of S. pyogenes hasbeen determined to be of the order of a 47 % reduction innew acquisitions (Dierksen & Tagg, 2000). In the presentstudy, 18 % of those in the M18 group experienced a newacquisition of S. pyogenes during the treatment phase and26 % of the children in the placebo group experienced newacquisitions. Unfortunately, this analysis suffers from type2 error, as the numbers are too small. In order todemonstrate statistical significance at this level of differ-ence, 100 individuals would have been required in eachgroup. Nevertheless, the apparent difference in acquisitionobserved (in spite of poor colonization efficacy occurring)may provide encouragement for the implementation of alarger trial investigating the protective effect of M18 againstS. pyogenes infections.
There were no significant differences in any of the othermicrobial indices that were measured [total Lactobacillus,Candida and S. salivarius c.f.u. (ml saliva)21]. This isnoteworthy, as large quantities of exogenous bacteria wereinstilled into the oral cavity twice daily for an extendedperiod of time and yet there were no detectable populationshifts in these non-targeted microbes. Whilst the micro-organisms quantified in the present study can constitute alarge proportion of the cultivatable population, it is clear thatthe use of traditional microbiological culture technologydoes not suffice to detect relatively minor fluctuations inmicrobiota composition. In addition, the culture methodo-logy employed did not support detection of the occurrence ofmore subtle intra-species (strain-specific) populationchanges. Further studies are now being undertaken using,
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882 Journal of Medical Microbiology 62
as a population analysis tool, higher-sensitivity, next-genera-tion sequencing.
In this study, we demonstrated that twice-daily dosing withthe probiotic strain S. salivarius M18 is a safe andefficacious way of effecting a significant reduction inplaque formation in primary-school-aged children. Inaddition, whilst there was no overall reduction in S.mutans carriage rates for the treatment group, the smallsubgroup of individuals who demonstrated persistentcolonization by the probiotic strain showed lower S.mutans counts than their baseline scores, indicating thatimproved efficacy occurs when the probiotic strain isestablished within the oral microbiota of the host.
ACKNOWLEDGEMENTS
We would like to acknowledge excellent technical support from
Hannah Clark, Vidya Kulkarni, Chris Moore, Erin Isdale, Nikolai
Klesse and Megan Inglis. All funding for this trial was provided by
BLIS Technologies Ltd. P. A. W. and J. D. F. H. are currently employed
by BLIS Technologies Ltd. J. R. T. was the founding scientist of the
company and currently is a consultant, whilst C. N. C. and J. P. B. are
former employees. W. M. T. performed all statistical analysis of the
data and has no affiliation (financial or otherwise) with BLIS
Technologies Ltd.
REFERENCES
Adam, E., Jindal, M., Seney, S., Summers, K., Hamilton, D. W.,Hatibovic-Kofman, S. & Cadieux, P. A. (2011). Streptococcus salivarius
K12 and M18 probiotics reduce periodontal pathogen-induced
inflammation. In IADR/AADR/CADR 89th General Session and
Exhibition, 16–19 March 2011, San Diego, CA, USA. Paper no. 150126.
Bagramian, R. A., Garcia-Godoy, F. & Volpe, A. R. (2009). The global
increase in dental caries. A pending public health crisis. Am J Dent 22,
3–8.
Broadbent, J. M., Thomson, W. M., Boyens, J. V. & Poulton, R. (2011).Dental plaque and oral health during the first 32 years of life. J Am
Dent Assoc 142, 415–426.
Burton, J. P., Chilcott, C. N., Moore, C. J., Speiser, G. & Tagg, J. R.(2006a). A preliminary study of the effect of probiotic Streptococcus
salivarius K12 on oral malodour parameters. J Appl Microbiol 100,
754–764.
Burton, J. P., Wescombe, P. A., Moore, C. J., Chilcott, C. N. & Tagg,J. R. (2006b). Safety assessment of the oral cavity probiotic
Streptococcus salivarius K12. Appl Environ Microbiol 72, 3050–3053.
Burton, J. P., Wescombe, P. A., Cadieux, P. A. & Tagg, J. R. (2011a).Beneficial microbes for the oral cavity: time to harness the oral
streptococci? Benef Microbes 2, 93–101.
Burton, J. P., Cowley, S., Simon, R. R., McKinney, J., Wescombe, P. A.& Tagg, J. R. (2011b). Evaluation of safety and human tolerance of the
oral probiotic Streptococcus salivarius K12: a randomized, placebo-
controlled, double-blind study. Food Chem Toxicol 49, 2356–2364.
Caglar, E., Sandalli, N., Twetman, S., Kavaloglu, S., Ergeneli, S. &Selvi, S. (2005). Effect of yogurt with Bifidobacterium DN-173 010
on salivary mutans streptococci and lactobacilli in young adults. Acta
Odontol Scand 63, 317–320.
Chen, Y. Y., Clancy, K. A. & Burne, R. A. (1996). Streptococcus
salivarius urease: genetic and biochemical characterization and
expression in a dental plaque streptococcus. Infect Immun 64, 585–
592.
Chilcott, C. N. & Tagg, J. R. (2007). Antimicrobial composition. US
Patent 7226590.
Dierksen, K. P. & Tagg, J. (2000). The influence of indigenous
bacteriocin-producing Streptococcus salivarius on the acquisition of
Streptococcus pyogenes by primary school children in Dunedin, NewZealand. In Streptococci and Streptococcal Diseases Entering the New
Millenium, pp. 81–85. Edited by D. R. Martin & J. R. Tagg. Auckland:
Securacopy.
Dierksen, K. P., Ragland, N. L. & Tagg, J. R. (2000). A new alkaline
pH-adjusted medium enhances detection of b-hemolytic streptococciby minimizing bacterial interference due to Streptococcus salivarius.
J Clin Microbiol 38, 643–650.
Fisher, M. A., Borgnakke, W. S. & Taylor, G. W. (2010). Periodontaldisease as a risk marker in coronary heart disease and chronic kidney
disease. Curr Opin Nephrol Hypertens 19, 519–526.
Greene, J. C. & Vermillion, J. R. (1964). The simplified oral hygiene
index. J Am Dent Assoc 68, 7–13.
Guglielmetti, S., Taverniti, V., Minuzzo, M., Arioli, S., Stuknyte, M.,Karp, M. & Mora, D. (2010). Oral bacteria as potential probiotics for
the pharyngeal mucosa. Appl Environ Microbiol 76, 3948–3958.
Heng, N. C., Haji-Ishak, N. S., Kalyan, A., Wong, A. Y., Lovric, M.,Bridson, J. M., Artamonova, J., Stanton, J. A., Wescombe, P. A. &other authors (2011). Genome sequence of the bacteriocin-
producing oral probiotic Streptococcus salivarius strain M18.J Bacteriol 193, 6402–6403.
Hillman, J. D., Dzuback, A. L. & Andrews, S. W. (1987). Colonizationof the human oral cavity by a Streptococcus mutans mutant producing
increased bacteriocin. J Dent Res 66, 1092–1094.
Hillman, J. D., Duncan, M. J. & Stashenko, K. P. (1990). Cloning andexpression of the gene encoding the fructose-1,6-diphosphate-
dependent L-(+)-lactate dehydrogenase of Streptococcus mutans.
Infect Immun 58, 1290–1295.
Hillman, J. D., Novak, J., Sagura, E., Gutierrez, J. A., Brooks, T. A.,Crowley, P. J., Hess, M., Azizi, A., Leung, K. & other authors (1998).Genetic and biochemical analysis of mutacin 1140, a lantibiotic from
Streptococcus mutans. Infect Immun 66, 2743–2749.
Horz, H. P., Meinelt, A., Houben, B. & Conrads, G. (2007).Distribution and persistence of probiotic Streptococcus salivarius
K12 in the human oral cavity as determined by real-time quantitative
polymerase chain reaction. Oral Microbiol Immunol 22, 126–130.
Kanasi, E., Dewhirst, F. E., Chalmers, N. I., Kent, R., Jr, Moore, A.,Hughes, C. V., Pradhan, N., Loo, C. Y. & Tanner, A. C. (2010). Clonalanalysis of the microbiota of severe early childhood caries. Caries Res
44, 485–497.
Li, Y. H., Chen, Y. Y. & Burne, R. A. (2000). Regulation of urease geneexpression by Streptococcus salivarius growing in biofilms. Environ
Microbiol 2, 169–177.
Loe, H. & Silness, J. (1963). Periodontal disease in pregnancy. I.
Prevalence and severity. Acta Odontol Scand 21, 533–551.
Marsh, P. D. (2003). Are dental diseases examples of ecologicalcatastrophes? Microbiology 149, 279–294.
Nascimento, M. M., Gordan, V. V., Garvan, C. W., Browngardt, C. M. &Burne, R. A. (2009). Correlations of oral bacterial arginine and urea
catabolism with caries experience. Oral Microbiol Immunol 24, 89–95.
Nase, L., Hatakka, K., Savilahti, E., Saxelin, M., Ponka, A., Poussa, T.,Korpela, R. & Meurman, J. H. (2001). Effect of long-term
consumption of a probiotic bacterium, Lactobacillus rhamnosus GG,
in milk on dental caries and caries risk in children. Caries Res 35, 412–420.
Impact of S. salivarius M18 on children’s oral health
http://jmm.sgmjournals.org 883
Ohnishi, Y., Kubo, S., Ono, Y., Nozaki, M., Gonda, Y., Okano, H.,Matsuya, T., Matsushiro, A. & Morita, T. (1995). Cloning andsequencing of the gene coding for dextranase from Streptococcussalivarius. Gene 156, 93–96.
Seymour, G. J. & Gemmell, E. (2001). Cytokines in periodontaldisease: where to from here? Acta Odontol Scand 59, 167–173.
Tagg, J. R. & Bannister, L. V. (1979). ‘‘Fingerprinting’’ b-haemolyticstreptococci by their production of and sensitivity to bacteriocine-likeinhibitors. J Med Microbiol 12, 397–411.
Tanzer, J. M., Kurasz, A. B. & Clive, J. (1985a). Inhibition of ecologicalemergence of mutans streptococci naturally transmitted between ratsand consequent caries inhibition by Streptococcus salivarius TOVE-Rinfection. Infect Immun 49, 76–83.
Tanzer, J. M., Kurasz, A. B. & Clive, J. (1985b). Competitivedisplacement of mutans streptococci and inhibition of tooth decayby Streptococcus salivarius TOVE-R. Infect Immun 48, 44–50.
Teughels, W., Newman, M. G., Coucke, W., Haffajee, A. D., Van DerMei, H. C., Haake, S. K., Schepers, E., Cassiman, J. J., Van Eldere, J.& other authors (2007). Guiding periodontal pocket recolonization: aproof of concept. J Dent Res 86, 1078–1082.
Van Palenstein Helderman, W. H., Ijsseldijk, M. & Huis in ’t Veld, J. H.(1983). A selective medium for the two major subgroups of the
bacterium Streptococcus mutans isolated from human dental plaque
and saliva. Arch Oral Biol 28, 599–603.
Wescombe, P. A., Burton, J. P., Cadieux, P. A., Klesse, N. A., Hyink,O., Heng, N. C., Chilcott, C. N., Reid, G. & Tagg, J. R. (2006).Megaplasmids encode differing combinations of lantibiotics in
Streptococcus salivarius. Antonie van Leeuwenhoek 90, 269–
280.
Wescombe, P. A., Heng, N. C., Burton, J. P., Chilcott, C. N. & Tagg, J. R.(2009). Streptococcal bacteriocins and the case for Streptococcus
salivarius as model oral probiotics. Future Microbiol 4, 819–
835.
Wescombe, P. A., Upton, M., Renault, P., Wirawan, R. E., Power, D.,Burton, J. P., Chilcott, C. N. & Tagg, J. R. (2011). Salivaricin 9, a new
lantibiotic produced by Streptococcus salivarius. Microbiology 157,
1290–1299.
Zarco, M. F., Vess, T. J. & Ginsburg, G. S. (2012). The oral
microbiome in health and disease and the potential impact on
personalized dental medicine. Oral Dis 18, 109–120.
J. P. Burton and others
884 Journal of Medical Microbiology 62