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TRANSCRIPT
6/21/2014
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Oral Health Applications for Probiotics
Andrew McBainBiofilm Research GroupManchester Pharmacy SchoolUniversity of Manchester
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Overview
• Oral microbiology introduction
• Dental probiotics and “replacement therapy”
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Distribution
Accumulation at: • Gingival margins
(gingivitis / periodontal disease)
• Tooth fissures (dental caries)
•Tongue and other mucosal surfaces
Density
•Saliva: c. 108 per ml
•Plaque c. 1011 per gram
The Oral Microbiota
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Taxonomy
Aas JA et al. (2005) Defining the normal bacterial flora of the oral cavity. J Clin Microbiol. 2005 Nov;43(11):5721-32.
c. 700* oral species. 50% currently uncultivated
Firmicutes (e.g. streptococci)Actinobacteria (e.g. Actinomyces)Proteobacteria (e.g. Neisseria)Bacteroidetes (e.g. Porphyromonas)Fusobacteria (e.g. Fusobacterium)TM7 phylum (so far uncultivated)
c. 50% culturability
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Planktonic bacteria
3. Biofilm dispersion
2.Plaque development1. Colonisation
Coaggregation
Coadhesion
Aggregation
, Plaque matrix; , Tooth surface , Bacteria.
How does plaque form?
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The Oral Microbiota and Disease Processes
•The specific plaque hypothesis- specific pathogens
•The non-specific plaque hypothesis- all plaque is bad
•The ecological plaque hypothesis- change in environment -> adverse change
in microbiota -> disease
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The ecological plaque hypothesisfor periodontal disease
GIN
GIV
AL
HE
ALT
HG
ING
IVIT
IS
INFLAMMATORYRESPONSE
ENVIRONMENTALCHANGE
ECOLOGICAL SHIFT
Plaquereduction
Plaqueaccumulation
Reduced inflammation
Increasedinflammation
Low GCF flowHigher oxygen
High GCF flowLower oxygen
PredominantlyGram +ve microflora
Facultative anaerobes
(StreprococciActinomycetes
etc.
PredominantlyGram -ve microfloraObligate
anaerobes
(Eubacterium Porthyromonas
Fusobacteriaetc.)
Philip Marsh
The ecological plaque hypothesisfor dental caries
HE
ALT
HC
arie
s
ENVIRONMENTAL SHIFT
DISEASE
Sugar intake
Acid production
Neutral pH
Low pH
High:Strep. Gordonii
Strep. OralisStrep. Mitis
A. naeslundii
Increased:
Strep. MutansLactobacilli
Bifidobacteria
Enamel health
Increased caries risk
Stress ECOLOGICAL SHIFT
Philip Marsh
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The Oral Systemic Interface
• Purported relationships between periodontal disease and heart disease
• Maternal oral health and premature birth
• Oral health and bacteraemia
• Oral bacterial involvement in device-related infections.
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Traditional managementDental hygiene
(Cleaning and antimicrobials)
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Plaque and antimicrobials
Biofilm bacteria are 50-1000 x less susceptible to antibiotics and biocides than are planktonic bacteria
McBain, 2009
[Antimicrobials] [Oxygen][Nutrients]
“Bulk” phase
, Persister cells
Taxonomically distinct cell
clusters
Phenotypically distinct cell
clusters
Plaque recalitrance…
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Viability Profiling of Streptococcus mutans Biofilms
Green= liveRed= dead
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“Replacement therapy”applied directly to a site with the intention of combating or preventing an infection.
“Probiotics”delivered orally and have been associated with a wide range of claimed localised and systemic beneficial effects, proven or otherwise.
(prebiotic equivalents include urea and arginine)
Alternative approaches
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Replacement therapy for the prevention of caries- a caries “vaccine”
Jeffrey Hillman originally at the Forsyth Dental Center, Boston
A key concept in this is the involvement of Streptococcus mutans in cariogenesis.
Replacement therapy for caries involves the use of mutants that do not produce lactic acid
From the press, “Oragenics, Jeff’s company, has patented a simple swab of bacteria that when wiped across a set of teeth will grant a lifetime of protection from tooth decay…”
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Replacement TherapyEvidence?
Evidence from rat studies for both caries and periodontal disease.
Anecdotal evidence that Prof. Hillman and four colleagues have remained colonised by the ‘effector strains’ for over 20 years…
Regulatory problems with using engineered mutans strains in humans, Now focusing on natural mutans and S. rattus.
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Dental Probiotics
Mainly lactobacilli (esp. L. reuteri)– Classed as safe (GRAS) by the FDA
Sporadic reports in the literature back to around 1954
Lactobacilli are lactic acid bacteria and lactic acid causes caries? Is there a conflict?
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Caglar et al. 2006 Administered Lactobacillus reuteri ATCC 55730 or placebo once
daily to healthy young adults (120 in total) in four groups of 30 volunteers.
Group A drank 200ml of water containing a probiotic through a straw; Group B 200ml placebo water;
Group C sucked probiotic tablet and Group D sucked placebo tables.
Consumption of the probiotics via the tablet route resulted in highly significant decreases in S. mutans counts.
Significant decreases in mutans also occurred for individuals who used the straw.
The authors postulated that the lowering of S. mutans might occur by a systemic mechanism rather than direct interaction between probiotic and S. mutans.
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Krasse et al. 2006
Administered two different Lactobacillus reuteri strains of human origin, or a placebo to 59 patients with moderate to severe gingivitis in Sweden.
Improvements (decreases in gingival indexes)were reported for the probiotic and control groups which were more significant for the probiotic group (both Lactobacillus strains). Interestingly, one of the lactobacillus strains appeared to be better clinically than the others.
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Iniesta et al. 2012• Forty gingivitis subjects over 8 weeks administration of a daily tablet,
containing Lactobacillus reuteri or placebo.
• There were no significant changes between and within the groups in the clinical variables.
• In saliva, total anaerobic counts after 4 weeks and counts of Prevotella intermedia after 8 weeks, showed significant reductions in the test group.
• In subgingival samples, significant reductions were observed for P. gingivalis counts. L. reuteri ATCC-PTA-5289 was more frequently detected than L. reuteri DSM-17938 by PCR.
• The effect of L. reuteri administered in tablets resulted in a reduction in the number of selected periodontal pathogens in the subgingival microbiota, but without an associated clinical impact.
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Sunstar GUM
• The lozenges are stated to contain at least 200 million active viable Lactobacillus reuteri “ProdentisTM” (a patented mixture of equal amounts of L. reuteri ATCC 55730 and L. reuteri ATCC PTA 5289)
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• GRAS (generally regarded as safe)- FDA
• Easy to scale up
• Palatable
• Antagonistic to pathogens?
• Immunomodulation?
• Efficacy?
• But is acidogenesis an issue?
Why lactobacilli as dentalprobiotics?
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Facultative anaerobes
Pre-dosing Dosing Post-dosing
2
3
4
5
6
7
8Total anaerobes
Pre-dosing Dosing Post-dosing
2
3
4
5
6
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8Gram -negative anaerobes
Pre-dosing Dosing Post-dosing
Log1
0 cf
u.m
m-2
2
3
4
5
6
7
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Total streptococci
Pre-dosing Dosing Post-dosing
2
3
4
5
6
7
8Total lactobacilli
Pre-dosing Dosing Post-dosing
2
3
4
5
6
7
8Lactobacillus reuteri
Pre-dosing Dosing Post-dosing
2
3
4
5
6
7
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Log1
0 cf
u.m
m-2
Mature plaques
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Nascent plaques
Total anaerobes
6 5 5 6 6
2
3
4
5
6
7
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Log
10C
FU
.mm
-2L
og1
0C
FU
.mm
-2
* *
Gram-negative anaerobes
2
3
4
5
6
7
8
**
Total streptococci
2
3
4
5
6
7
8
*
Total aerobes
2
3
4
5
6
7
8
*
Total lactobacilli
2
3
4
5
6
7
8
*
*
Control PT
LRPT
LRBF
ControlBF
Control PT
LRPT
LRBF
ControlBF
Control PT
LRPT
LRBF
ControlBF
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Figure 3 Box plots showing a comparison of L. reuteri tracked by colony morphology (backward diagonals) with major bacterial groups i.e. total anaerobes (white), aerobes (forward diagonals) and total lactobacilli (crossed pattern). The vertical dotted lines enclose period of probiotic treatment. The horizontal bar inside the box represents the median while * indicates significant change observed (p < 0.05).
Total eubacteria
Pre-dosing Dosing Post-dosing
copi
es.m
m-2
2
4
6
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Lactobacillus group qPCR
Pre-dosing Dosing Post-dosing
2
4
6
8
10
Lactobacillus reuteri qPCR
Pre-dosing Dosing Post-dosing
copi
es.
mm
-2
2
4
6
8
10
Persistence of L. reuteri in plaques
Up to three weeks after dosing
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Direct antagonism?
No inhibition detected against actinomyces, Veillonella dispar, Lactobacillus rhamnosus, Prevotella oralis, Streptococcus mutans, Neisseria subflavaand Streptococcus oralis.
Even at glycerol concentrations of 200mM both Lactobacillus reuteri strains L1 and L2 did not produce any zones of inhibition.
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Conclusions
• Oral biofilms- taxonomically complex but relatively culturable.
• Specific pathogens implicated in disease but also, considerable functional redundancy in the oral microbiome
• Physical cleaning and antimicrobial- based control regimes remain the clinically proven option.
• Considerable interest in probiotics which are currently (perhaps counter-intuitively) dominated by organisms of intestinal origin. More good quality clinical trials are needed. Mechanisms poorly understood. Considerable potential.