Occurrence of Periodontopathic andSuperinfecting Bacteria in Chronic andAggressive Periodontitis Subjects in aColombian PopulationJavier Enrique Botero,*† Adolfo Contreras,*† Gloria Lafaurie,‡ Adriana Jaramillo,*†

Marisol Betancourt,*§ and Roger Mauricio Arce*

Background: Differences in the composition of subgingival micro-biota in periodontitis have been observed in different geographiclocations. Some of these microbial differences could have clinical sig-nificance. This study describes clinical parameters and the composi-tion of the subgingival microbiota in chronic periodontitis (CP) andaggressive periodontitis (AgP) patients in a Colombian population.

Methods: Clinical parameters (probing depth, clinical attachmentlevel, bleeding on probing, and plaque index) and plaque samplesfrom 68 CP, 12 AgP, and 30 periodontally healthy subjects were ana-lyzed. Subgingival samples were processed using culture, biochemicaltests, and polymerase chain reaction for the detection of periodon-tal and superinfecting pathogens. The analysis of variance, x2, andKruskal-Wallis tests were used to evaluate differences between groupsfor clinical parameters and microbiologic composition (P £0.05).

Results: Clinical parameters were significantly increased in CP andAgP patients compared to healthy subjects (P <0.001), but no differ-ences were found between periodontitis groups. Porphyromonas gin-givalis, Tannerella forsythensis, and Eikenella corrodens showedhigher frequencies in AgP compared to CP and healthy subjects(P <0.05). Extension (localized and generalized) of the periodontaldestruction had no effect on the composition of the subgingival micro-biota. Gram-negative enteric rods were more frequent in AgP patients(P <0.01).

Conclusions: This study demonstrated a high prevalence of P. gin-givalis, T. forsythensis, and E. corrodens in AgP patients. Gram-nega-tive enteric rods were frequent in AgP and CP patients. Differences inthe composition of subgingival microbiota in periodontitis patientsneed to be taken into account when considering the best therapeuticapproach for each individual, including the use of antibiotics. J Peri-odontol 2007;78:696-704.

KEY WORDS

Antimicrobial resistance; bacteria; chronic periodontitis; Gram-negative rods; Porphyromonas gingivalis.

Periodontal disease is an in-fectious clinical entity char-acterized by the destruction

of supporting tissues of the teeth(periodontal ligament and alveo-lar bone) as the result of an inflam-matory response in a susceptiblehost triggered by specific bacte-rial products from a dental bio-film. This response is alwaysevidenced clinically as gingivitisor periodontitis.1,2

In the United States, ;25%to 30% of subjects >30 years ofage have suffered to some extentfrom periodontal disease, fromslight inflammation with bleed-ing on probing to severe attach-ment loss, including 3.1% withsevere periodontitis.3 In generalterms, in Central and SouthAmerica there is a lack of studiesaddressing the prevalence ofperiodontal disease. Data fromBrazil, Argentina, and Chile showa prevalence of severe periodon-titis of 5.5%, 10% to 49%, and50%, respectively.4 Accordingto Axelsson et al.,5 periodon-tal disease also affects develop-ing countries in which ethnic,genetic, cultural, and individualaspects seem to influence theclinical features of this disease.

* Periodontal Medicine Group, University of Valle, Cali, Colombia.† Department of Periodontology, School of Dentistry, University of Valle.‡ School of Dentistry, El Bosque University, Bogota, Colombia.§ School of Bacteriology, University of Valle.

doi: 10.1902/jop.2007.060129

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Little data on periodontal disease have been pub-lished in Latin America, especially in Colombia. Alarge-scale study, conducted by the National HealthMinistry of Colombia in 1998 (ENSAB III), showed thatgeneralized periodontal diseases affect 12% of people<35 years of age, and 42% of people >60 years of age.With regard to clinical attachment level (CAL), 14%of the population around 35 years of age has moder-ate CAL, and by the age of 60 years, 40% of the pop-ulation has moderate to severe CAL. These findingsshow that periodontal disease is a common healthproblem in adult Colombians.6

Because specific microorganisms initiate peri-odontal disease, the study of the subgingival micro-biota is an important aspect of periodontal research.The interaction between the subgingival microbiotaand the host results in an inflammatory processresponsible for the etiopathogenesis of periodontaldisease. Microorganisms, such as Actinobacillus ac-tinomycetemcomitans, Porphyromonas gingivalis,Tannerella forsythensis, Fusobacterium spp., Campy-lobacter rectus, Eikenella corrodens, Micromonasmicros, Eubacterium spp., and recently, Dialisterpneumosintes, have been associated as importantperiodontal pathogens.7-9 Other opportunistic spe-cies have been described in periodontitis patients,such as enteric rods, Pseudomonas spp., Acinetobac-ter spp., and Alcaligenes spp.10 Studies conducted inthe United States, Latin America, and Europe haveshown that the composition of the microbiota andprevalence of certain periodontopathic microorgan-isms in periodontitis patients could be related to eth-nicity and geographic locations.11-13

The aim of this study was to describe the clinicalparameters, subgingival microbiota, and antibioticresistance of enteric rods in the localized and gen-eralized forms of chronic periodontitis (LCP and GCP,respectively) and aggressive periodontitis (LAgPand GAgP, respectively) in a Colombian population.

MATERIALS AND METHODS

SubjectsPatients attending the dental clinics of the Universityof Valle were invited to participate in the study be-tween January and December of 2004. Informedand written consent was obtained from each partici-pant. In the case of underage patients, parents signedthe consent and approved their participation in thestudy. The study design was approved by the EthicsCommittee on Human Research of the School ofHealth of the University of Valle according to the Dec-laration of Helsinki on experimentation involvinghuman subjects.

Patients with a diagnosis of CP and AgP were con-sidered candidates for the study. Exclusion criteriaincluded diabetes, cardiovascular disease, human

immunodeficiency virus (HIV) infection, pulmonarydisease, or any other systemic disease that could alterthe course of periodontal disease. Pregnant women,heavy smokers (>15 cigarettes per day), previous(6 months) consumption of systemic antimicrobialsor anti-inflammatory drugs, and periodontal treat-ment also served as exclusion criteria.

Periodontal ExaminationOne of the authors (JEB) carried out a completeperiodontal examination. Clinical parameters wererecorded as follows: plaque index, percentage ofbleeding on probing sites, probing depth in millime-ters, and CAL in millimeters. A marked probei wasused in all instances. Periodontal diagnosis was estab-lished based on the consensus report of the AmericanAcademy of Periodontology.14,15 Extension of peri-odontal destruction was classified as localized (£30%of sites affected) and generalized (>30% of sites af-fected). The following groups of patients were formed:68 subjects in the CP group consisting of 41 subjectsin the LCP group and 27 subjects in the GCP group.Twelve patients were assigned to the AgP group andsubsequently stratified as five in the LAgP groupand seven in the GAgP group. Periodontal bone loss(‡50%) was assessed on individual radiographic films.Thirty periodontally healthy subjects were studied todetermine differences with the periodontitis groups.

Microbial AnalysisSubgingival microbial samples were taken from thesix deepest proximal sites using sterile paper pointsinserted into the bottom of the periodontal pocket-sulcus and kept in place for 15 seconds. The paperpoints were pooled in screw cap vials containing Via-bility Medium Gotenborg Anaerobical (VGMA) III trans-port medium. The same process was repeated andpaper points were stored in eppendorf vials (-70�C)for polymerase chain reaction (PCR) detection.

Before sampling, supragingival plaque was re-moved from the sampled site with sterile gauze andisolated with cotton rolls. The samples were analyzedusing microbial culture techniques for the presenceof periodontopathic bacteria according to Slots.16

Briefly, all samples were processed in £24 hours atroom temperature (25�C) and immediately incubatedin CO2 and anaerobic culture systems. Brucella bloodagar medium was incubated at 35�C in an anaerobicjar for 7 days. The trypticase-soy with serum, bacitra-cin, and vancomycin medium was incubated in 10%CO2 in air at 37�C for 4 days. Presumptive identifica-tion was performed according to methods describedby Slots and Reynolds17 and Slots et al.18 to identifyby colony morphology and Gram stains for Campylo-bacter spp., Eubacterium spp., Fusobacterium spp.,

i UNC-15, Hu-Friedy, Chicago, IL.

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Capnocytophaga spp., and D. pneumosintes. Colonymorphology characterized by the presence of an innerstar and a catalase-positive test were used to identifyA. actinomycetemcomitans. P. gingivalis, Prevotellaintermedia/nigrescens, T. forsythensis, M. micros,and E. corrodens were identified by the use of a com-mercial micromethod system.¶ Gram-negative en-teric rods were subcultured and colony purified onMacConkey and cetrimide agar plates and identifiedusing a standardized biochemical test.# Total viablecounts were defined as the total number of colony-forming units (CFU) obtained on non-selective mediaplates. Species found on selective media were enu-merated and presented as counts · 105.

Antimicrobial Susceptibility TestingSelected colonies of Gram-negative enteric rods frompure cultures were used to test susceptibility to amox-icillin, tetracycline, and ciprofloxacin.** Briefly, via-ble colonies were homogenized in 0.85% saline, andthe turbidity was adjusted to MacFarland 0.5 standard(1.5 · 108 CFU/ml). Using a sterile swab, inoculumwas spread over Mueller-Hinton agar plates,†† anddried for 15 minutes at room temperature. Strips weregently placed onto the agar surface and incubated at37�C for 18 to 24 hours.‡‡ The elliptic zone of inhibi-

tion was examined after incubation. The reading atthe intersection of the bacterial zone of inhibitionand the strip represented the minimum inhibitory con-centration of the organism. The minimum inhibitoryconcentration breakpoints were interpreted accord-ing to the Clinical Laboratory Standards Institute (CLSIM100-S15) guidelines.19 Specifically, antimicrobialsusceptibility of Enterobacteriaceae was tested againstamoxicillin, tetracycline, and ciprofloxacin. In the caseof non-fermentative microorganisms, amoxicillin wasnot tested because of its poor effect on these bacteria.

PCR DetectionPCR detection was conducted for selected periodontalpathogens to determine how the presence of entericrods affects the detection of periodontopathic bacte-ria in culture plates. Primers sequence and a PCR pro-cedure were done according to Ashimoto et al.20 andContreras et al.21 Briefly, paper points were diluted in0.5 ml double-distilled water and homogenized in vor-tex for 1 minute. After removal of the paper points,samples were boiled for 10 minutes, chilled on ice

Table 1.

Demographic and Clinical Description of Study Sample

Parameter

CP Group AgP GroupControl

GroupGCP LCP Total GAgP LAgP Total

Subjects (N) 27 41 68 7 5 12 30

Age (years – SD) 42.81 – 9.31 41.88 – 12.57 42.25 – 11.32 22.29 – 5.73 30.20 – 7.25 25.58 – 7.32 26.90 – 7.17

Gender (N) (F/M) 14/13 31/10 45/23 6/1 4/1 10/2 18/12

Race (N)

Black 5 11 16 3 2 5 0

Mixed race 22 30 52 4 3 7 30

Cigarettesmokers (N)

1 2 3 0 0 0 4

PI (% – SD) 48.49 – 3.72 45.72 – 11.51 46.82 – 16.05 43.03 – 20.15 32.54 – 19.03 38.66 – 19.56 8.26 – 7.15

BOP (% – SD) 54.41 – 22.13 42.27 – 15.23 47.09 – 19.09 44.91 – 19.42 41.44 – 11.76 43.47 – 15.44 10.01 – 13.09

PD site sampled(mm – SD)

7.6 – 1.02 7.58 – 1.20 7.62 – 1.12 7.82 – 1.06 8.76 – 1.76 8.21 – 1.39 1.99 – 0.59*

CAL site sampled(mm – SD)

8.24 – 1.50 7.75 – 1.70 7.94 – 1.63 7.40 – 1.00 8.80 – 1.03 7.98 – 1.20 1.17 – 0.92*

PD (mm – SD) 4.11 – 0.55 3.62 – 0.49 3.81 – 0.57 4.42 – 0.95 4.12 – 0.47 4.30 – 0.77 1.92 – 0.51*

CAL (mm – SD) 4.54 – 1.45 3.71 – 0.86 4.04 – 1.19 3.90 – 0.63 3.86 – 0.72 3.88 – 0.64 1.17 – 0.89*

F/M = female/male; PI = plaque index; BOP = bleeding on probing; PD = probing depth at sample site and subject level; CAL = clinical attachment level atsample site and subject level. One-way analysis of variance test was used to compare groups (*P £0.01).

¶ RapID ANA II, Remel, Norcross, GA.# API 20E, BioMerieux, Marcy l’Etoile, France.** E-tests, AB Biodisk, Solna, Sweden.†† BD, Sparks, MD.‡‡ E-tests, AB Biodisk.

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for 2 minutes, and centrifuged at 10.000 rpm · 1 min-ute. Samples were stored at -70�C until the PCR pro-cedure. A 45-ml reaction mixture containing 5 ml 10 ·PCR buffer,§§ 1.25 unit Taq DNA polymerase, 0.2 mMof each deoxyribonucleotide, 1 to 1.5 mM MgCl2, and1 ml primer mix was prepared. A final volume of 50 mlwas completed with 5 ml DNA sample. PCR amplifica-tion was conducted on a DNA thermal cycler.ii Tem-perature conditions were as follows for T. forsythensis,C rectus, E. corrodens, and P. gingivalis: 95�C · 2 min-utes, followed by 36 cycles at 95�C · 30 seconds,60�C · 1 minute, 72�C · 1 minute, and a final stepat 72�C · 2 minutes. Temperature conditions wereas follows for A. actinomycetemcomitans, P. interme-dia, P. nigrescens, and D. pneumosintes: 95�C · 2minutes; 36 cycles at 94�C · 30 seconds; 55�C ·1 minute; 72�C · 2 minutes; and a final step at 72�C ·10 minutes. PCR products were analyzed in a 2% ag-arose gel stained with ethidium bromide (0.5 mg/ml),and observed under ultraviolet light (300 nm).

Statistical AnalysisA descriptive analysis was conducted (mean, stan-dard error, and frequency detection) for collecteddata. Differences in clinical parameters betweengroups were established with the one-way analysisof variance test. The frequency detection of importantperiodontopathic bacteria was analyzed with the x2

test. The Kruskal-Wallis test was used to assess thecultivable subgingival microbiota composition. Sta-tistical significance was assumed when P £0.05. Datawere analyzed with statistical software.¶¶

RESULTS

Table 1 describes the clinical and demographic char-acteristics of study subjects. A total of 41 subjects inthe LCP group (age: 41.88 – 12.57 years), 27 subjectsin the GCP group (age: 42.81 – 9.31 years), five sub-jects in the LAgP group (age: 30.20 – 7.25 years),seven subjects in the GAgP group (age: 22.29 – 5.73years), and 30 subjects in the control group (age:26.90 – 7.17 years) were studied. Some differencesin probing depth and CAL were observed betweenthe groups (P £0.01). Periodontitis patients presentedincreased probing depths and CAL compared to theperiodontally healthy group, but no significant differ-ences were noted between AgP and CP groups.

The frequency detection of periodontopathic andsuperinfecting bacteria in localized and generalizedformsofperiodontitis isdepicted inTable2.P.gingivalis,T. forsythensis, P. intermedia/P. nigrescens, andFusobacterium spp. were detected in between 50%

Table 2.

Number and Percentage of Positive Subjects for Each Microorganism According toPeriodontal Status

CP AgPControl Group

(N = 30)GCP (N = 27) LCP (N = 41) GAgP (N = 7) LAgP (N = 5)

Microorganism N % N % N % N % N %

P. gingivalis 19 70.40 33 80.50 6 85.70 5 100 3 10

T. forsythensis 13 48.10 21 51.20 3 42.90 3 60.00 2 6.70

P. nigrescens/P. intermedia 18 66.70 33 80.50 6 85.70 3 60.00 7 23.30

Fusobacterium spp. 22 81.50 37 90.20 7 100 5 100 15 50

M. micros 0 0.0 2 4.90 0 0.0 1 20.00 2 6.70

Campylobacter spp. 4 14.80 10 24.40 0 0.0 0 0.0 1 3.30

Eubacterium spp. 4 14.80 14 34.10 2 28.60 1 20.00 2 6.70

A. actinomycetemcomitans 5 18.50 5 12.20 0 0.0 1 20.00 2 6.70

E. corrodens 7 25.90 11 26.80 3 42.90 3 60.00 7 23.30

D. pneumosintes 2 7.40 2 4.90 2 28.60 0 0.0 0 0.0

Enteric rods 9 33.30 10 24.40 1 14.30 3 60.00 4 13.33

§§ Promega, Madison, WI.ii PTC-100, MJ Research, Boston, MA.¶¶ GraphPad Prism version 4.00 for Windows, GraphPad Software, San

Diego, CA.

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and 90% of LCP cases in contrast to the GCP groups,where they accounted for 47% to 81% of cases.The frequency detection of P. gingivalis (100%) andT. forsythensis (60%) was higher in the LAgP groupcompared to the GAgP group (85.70% versus42.60%, respectively). Detection of periodontopathicbacteria in the control group was lower (<25%) com-pared to localized and generalized forms of periodon-titis. The detection of enteric rods was higher in the

AgP group (33.3%), followed by the CP group(27.9%) and periodontally healthy group (13.33%).

When the microbiota in chronic, aggressive, andhealthy subjects was compared (Table 3), P. gingiva-lis, T. forsythensis, P. nigrescens/P. intermedia, andenteric rods were most frequently detected in peri-odontitis patients (P £0.05). P. gingivalis and E. corro-dens were more frequent in AgP subjects comparedto CP subjects (P £0.05).

The proportions of peri-odontopathic microorganismsin GCP, LCP, GAgP, and LAgPsubjects are presented in Fig-ures 1 and 2. Levels of P. gingi-valis, T. forsythensis, and P.intermedia/P. nigrescens wereincreased inCPpatients incon-trast to periodontally healthysubjects (P £0.01). The samefindings were observed forAgP (Fig. 2). The levels of P.gingivalis were higher in AgPcompared to CP patients.

To assess the effect of en-teric rods on the detection ofperiodontopathic microorgan-isms in culture plates, PCRdetection was carried out forP. gingivalis, T. forsythensis,Campylobacter spp., A. acti-nomycetemcomitans, E. cor-rodens, and D. pneumosintesin 21 CP patients (Fig. 3).PCR detection of P. gingivalis,C. rectus, and A. actinomyce-temcomitans yielded a higherfrequency than culture tech-niques (P £0.05).

Table 3.

Number and Percentage of Subjects Positive forEach Microorganism in the CP and AgP andNon-Periodontitis Subjects

Microorganism

CP (N = 68) AgP (N = 12)

Control Group

(N = 30)

N % N % N %

P. gingivalis 52 76.47* 11 91.6*† 3 10

T. forsythensis 34 50* 6 50* 2 6.70

P. nigrescens/P. intermedia 51 75* 9 75* 7 23.30

Fusobacterium spp. 59 86.76 12 100 15 50

M. micros 2 2.94 1 8.30 2 6.70

Campylobacter spp. 14 20.58 0 0.0 1 3.30

Eubacterium spp. 18 26.47 3 25 2 6.70

A. actinomycetemcomitans 10 14.70 1 8.30 2 6.70

E. corrodens 18 26.47 6 50*† 7 23.30

D. pneumosintes 4 5.88 2 16.60 0 0.0

Enteric rods 19 27.94* 4 33.33* 4 13.33

* Compared to control group (x2; P £0.05).

† Compared to CP (x2; P £0.05).

Figure 1.Proportions of microorganisms in GCP and LCP subjects compared to non-periodontitis subjects. Subgingival microbiota was assessed by cultureand biochemical tests (see Materials and Methods). The Kruskal-Wallis test was used to compare groups (*P £0.01; †P £0.001).A. actinomyc = A. actinomycetemcomitans. Data are presented as counts · 105 – SEM.

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Table 4 presents the frequency of Gram-negativeenteric rods in positive subjects according to peri-odontal status. Species of the Enterobacteriaceaefamily were the most prevalent in all groups, followedby non-fermentative Gram-negative rods. Klebsiellapneumoniae and Enterobacter cloacae were the mostfrequent species in the CP group.

The antimicrobial susceptibility of superinfectingorganisms is shown in Table 5. Enterobacteriaceaespecies were very resistant to amoxicillin, whereastetracycline and ciprofloxacin showed the best activ-ity. Non-fermentative Gram-negative rods displayedsensitivity to ciprofloxacin; however, these were mod-erately resistant to tetracycline.

DISCUSSION

Patients with untreated CP and AgP showed increasedclinical parameters compared to subjects with ahealthy or minimally inflamed periodontium (Table1). Differences in the subgingival microbiota wereobserved for some species between AgP and CP

Figure 2.Proportions of microorganisms in generalized and localized aggressive periodontitis compared to non-periodontitis subjects. Subgingival microbiota wasassessed by culture and biochemical tests (see Materials and Methods). Kruskal-Wallis test was used to compare groups (*P £0.01; †P £0.001).A. actinomyc = A. actinomycetemcomitans. Data are presented as counts · 105 – SEM.

Figure 3.Frequency detection (%) of selected periodontopathic microorganisms inCP subjects (N = 21) as detected by culture and PCR. PCR detectionwas conducted for selected periodontopathic bacteria in CP subjectswho presented enteric rods in culture plates (see Materials andMethods). The x2 test was used to assess differences between PCRand culture detection (‡P £0.05).

Table 4.

Frequency of Gram-Negative EntericRods in Positive Subjects Accordingto Periodontal Status

Microorganism

CP

(N = 19)*

AgP

(N = 4)

Control

Group

(N = 4)

N % N % N %

K. pneumoniae 8 42.10 1 25 0 0.0

E. cloacae 5 26.31 2 50 2 50

Enterobacter gergoviae 2 10.53 0 0.0 1 25

Enterobacter aerogenes 0 0.0 1 25 0 0.0

Serratia liquefaciens 1 5.26 0 0.0 0 0.0

Citrobacter freundii 1 5.26 0 0.0 0 0.0

Pseudomonas aeruginosa 3 15.79 0 0.0 0 0.0

Pseudomonas putida 0 0.0 0 0.0 1 25

Pseudomonas spp. 1 5.26 0 0.0 0 0.0

Acinetobacter baumannii 1 5.26 0 0.0 0 0.0

Acinetobacter lwoffi 1 5.26 0 0.0 0 0.0

Stenotrophomonas maltophilia 3 15.79 0 0.0 0 0.0

Gram-negative enteric rods were subcultured on MacConkey and cetrimideagar plates and identified using a biochemical test (see Materials and Methods).Microorganisms from the Enterobacteriaceae family were the most prevalentin all groups. K. pneumoniae was found to be the most prevalent in CP patients.* Number of subjects positive for one or more enteric rods.

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groups (P £0.05). For example, P. gingivalis, T. for-sythensis, and E. corrodens showed higher frequencyin the AgP group followed by the CP and healthy group,respectively (Fig. 2). A similar finding for P. gingivalisand E. corrodens was reported in Chile,22 and for P.gingivalis in Jamaica23 and Brazil,24 in patients withAgP. In contrast, the levels of colonization were similarbetween periodontitis groups, but still higher com-pared to healthy subjects. This explains, in part,why some patients develop periodontitis comparedto healthy carriers, but fails to discriminate betweenAgP and CP.25 Therefore, it can be stated that otherfactors besides mere qualitative microbiologic differ-ences between the clinical forms of periodontitis areinvolved in periodontal pathogenesis.

This study observed that periodontopathic bacteriaheavily colonize the microbiota in periodontitis pa-tients in contrast to healthy or gingivitis patients. Thisis in agreement with other studies on healthy popula-tions, where P. gingivalis, T. forsythensis, and P. inter-media/nigrescens were minimally detected or almostabsent.26,27 More plaque accumulation and the patho-logic deepening of the gingival sulcus in the peri-odontitis patients facilitate more counts of thesepathogenic bacteria. To what extent the presence ofa specific microorganism predicts the progressionfrom a healthy or gingivitis site to a periodontitis siteis still unknown. It is likely that periodontitis patientscan harbor more bacteria or more virulent strains thatinduce disease progression.

P. gingivalis, E. corrodens, and T. forsythensis wereprevalent microorganisms in AgP, in contrast to re-sults from European and North American popula-tions, where A. actinomycetemcomitans has beenproposed as a frequently recovered microorganismfrom AgP patients, especially in the LAgP patients.In this study, A. actinomycetemcomitans frequencywas low (8.3%) in AgP, compared to previous28-30

and recent studies.31,32 Ethnicity and geographicand cultural factors could influence the colonizationof A. actinomycetemcomitans in Western, African,and Asian populations in contrast to Latin Americans.

One important finding of this study was the pres-ence of relatively high levels of unusual microorgan-isms (Figs. 2 and 3; Table 2) showing statisticaldifferences between periodontal health and disease.Enterobacteriaceae organisms are mostly found inthe digestive system of humans and animals, wherethey are considered normal microbiota, and someof them have shown opportunistic roles in humaninfections.33,34 Few studies have reported differentprevalence among populations in the world, includinghealthy carriers of potential pathogenic organismsbelonging to the Enterobacteriaceae family.35-39 Ithas also been proposed that these superinfectingorganisms could be associated with halitosis in hu-mans,40 and they have also been found in AgP.41,42

Nevertheless, their prevalence varies amply withinstudied populations, ranging from 14% in the UnitedStates,43 61% in Romania,44 67% in the DominicanRepublic,45 and up to 92% in Sudan.46,47 Similar prev-alence to our study was reported in Brazil (31.2%).48

Factors, such as antibiotics misuse, could explainthe subgingival overgrowth of these superinfect-ing organisms.49 For example, Enterobacteriaceaeand Pseudomonadaceae organisms frequently showantibiotic resistance to common drug therapy forperiodontitis patients (amoxicillin, doxycycline,metronidazole, and tetracyclines).50-52 In the pasttwo decades, researchers have found different preva-lences of unusual microorganisms and have sug-gested that these organisms may have an impact onprogression and treatment of periodontal disease.At this moment, the role of superinfection is unknown,but it may be of therapeutic interest in periodontal dis-ease. Further studies evaluating risk factors, mecha-nisms of virulence, and treatment impact are needed.

To determine whether the presence of enteric rodscould have an impact on the detection of periodonto-pathic microorganisms in the culture, PCR detectionwas carried out for the indicated periodontopathicbacteria in 21 CP patients. It has been shown thatthe prolonged time (>72 hours) of samples in VMGAIII transport media could permit the overgrowth of

Table 5.

Number of Clinical Isolates of Gram-Negative Enteric Rods Susceptible andResistant to Three Antimicrobial Agents

Microorganisms

Amoxicillin Tetracycline Ciprofloxacin

N

Susceptible

N

Total

%

Resistant

N

Susceptible

N

Total

%

Resistant

N

Susceptible

N

Total

%

Resistant

Non-fermentativeGram-negative rods

NP NP NP 3 10 70 9 10 10

Enterobacteriaceae 3 24 87.5 23 24 4.17 24 24 0.0

NP = not performed.

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enteric rods.44 Samples in this study were processedimmediately or £24 hours to avoid overgrowth ofsuperinfecting microorganisms. However, higher fre-quencies of P. gingivalis, C rectus, A. actinomycetem-comitans, and D. pneumosintes were observed withPCR detection than culture. The colonies of entericrods are bigger in size, and this may have interferedwith the detection of periodontopathic bacteria.Nonetheless, when samples were processed for cul-ture, they were diluted before plating, and still highcounts of enteric rods were observed. This could indi-cate that these microorganisms could colonize theperiodontal pocket in high proportions. On the otherhand, PCR detection does not take into considerationwhether the sample is viable, and thus may yield to ahigher frequency. We report on the frequency detec-tion of periodontopathic microorganisms based pri-marily on culture techniques because it allows uslater to work with the cultured microorganisms. Thepresence of subgingival enteric rods should be con-sidered cautiously, because their role in destructiveperiodontal disease has not been elucidated.

Relatively high antimicrobial resistance of Gram-negative enteric rods to amoxicillin was determined(Table 5). These results may have important clinicalimplications in the selection of proper antimicrobialtherapy. Thus, antibiotics should always be prescribedafter microbial culture identification and antibiotic sen-sitivity determination in periodontitis patients.

CONCLUSIONS

This study found elevated proportions and frequencyof P. gingivalis, T. forsythensis, and E. corrodens inAgP patients compared to CP and healthy subjects.Periodontitis patients also harbored high levels ofGram-negative enteric rods. Subgingival microbiotacomposition in periodontitis patients should be takeninto account in the mechanical and antimicrobialtreatment of periodontal disease among Colombians.

ACKNOWLEDGMENTS

This study was partially supported by the ColombianInstitute for Science and Technology Development.‘‘Francisco Jose de Caldas’’ (grant 1308-04-13001),Bogota, Colombia; University of Bosque; and the Uni-versity of Valle. The authors thank Senobia Buritica,Periodontal Medicine Group, University of Valle, andNathaly Vasquez, School of Bacteriology, Universityof Valle, for their valuable assistance in the laboratory.

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Correspondence: Dr. Javier Enrique Botero, Departmentof Periodontology, School of Dentistry, University of Valle,Calle 4B #36-00 San Fernando, Cali, Columbia. E-mail:drjavo@yahoo.com.

Accepted for publication October 18, 2006.

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