Chairside testing for cariogenic bacteria: current ... · The biofilm which causes dental caries arises from the normally thin dental plaque biofilm which is present continuously

JournalOfMinimum Intervention In Dentistry

J Minim Interv Dent 2008; 1 (2)

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Abstract

Modern approaches to dentalcaries such as STEM (System forTotal EnvironmentalManagement) are based on awhole-of-patient and whole-of-biofilm approach to the disease.There are now multiple lines ofevidence which indicate thatdental caries is a multi-pathogendisease and that mutansstreptococci may participate butare not critically essential fordisease to occur. A number ofnew strategies for controllingcaries risk are currently beingexplored, based on the modernbiofilm concept. Clinicaldiagnostic kits which examineplaque or saliva for features ofthe bioilm that are surrogates(markers) of the disease processcan be useful in clinical patientmanagement since these can beundertaken at chairside. Firstpublished in Int Dent S Afric 2008; 10 50-65.

1 The University of Queensland School ofDentistry

Address of first author:School of DentistryThe University of QueenslandTurbot StreetBrisbane QLD 4000 Australia

Introduction

Dental caries can be defined as adiet- and saliva-modified bacterialdisease1. The key microbial featureof dental caries is a dietarycarbohydrate-induced enrichment ofthe normal oral flora with bacteriathat are both acidogenic (acidproducing) and aciduric (acidtolerant) located within a densebiofilm, rich in extra cellularpolysaccharides, which maintains afavorable environment for microbialcommunity and protects it fromphysical and chemical assaults.

The biofilm which causesdental caries arises from thenormally thin dental plaque biofilmwhich is present continuously onhard surfaces in the oral cavity,when this is subject to particularecological pressures related to intakeof fermentable carbohydrates andsubsequent production of organicacids by bacterial fermentation.Within the dental plaque biofilm,bacteria inhabit a diverse range ofecological niches (“habitats”), andexist not as isolated species but incomplex physical and metabolicallysynergistic relationships with otherspecies, which provides metabolicadvantages to each.

Within a cariogenic biofilm,factors affecting bacterial growth andmetabolism include:

Water – which is a source ofhydrogen and oxygen. This isdelivered throughout thebiofilm by water channels,which develop as part of thebiofilm structure.

Chairside testing for cariogenic bacteria: currentconcepts and clinical strategies

Walsh L J1 , Tsang A K1



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Carbon - from carbon dioxideand from carbohydrates.Organic nutrients -Carbohydrates, proteins(peptides), and amino acids.Bacteria require amino acidsfor the protein synthesis thatis essential for theirreplication by binary fission.The breakdown andmetabolism of certain aminoacids such as arginine canalter the local pH, as will bediscussed further below.Various peptides (termedbacteriocins) produced bybacteria can suppress thegrowth of other non-compatible species, e.g.bacteriocins fromStreptococcus salivarius andStreptococcus sanguis cansuppress Streptococcusmutans and theperiodontopathic organismActinobacillus actinomycetem-comitans, respectively.Inorganic nutrients - e.g.magnesium, nitrogen,sulphur, potassium,phosphate, as well asselenium and other traceelementsEnvironmental factors –Temperature, pH, and Redoxpotential (eH). The fallinglevels of pH (with acidproduction) and eH (withthickening of the plaque) helpprovide ecological niches deepwithin the plaque for highlyfermentative facultativespecies such as the mutansstreptococci (MS). Acidsproduced deep within theplaque not only demineralizeenamel, but suppress thegrowth of bacterial specieswhich are not aciduric. Theshift toward aciduricorganisms also promotes anincrease in the proportion oflactobacilli, which are highlyacidogenic in their own right,producing lactic acid fromglucose fermentation.

Although lactobacilli are notregarded as important in theinitiation of caries, theirpresence in large numbersindicates that the necessaryenvironmental conditions forproducing dental caries exist2-

3.Salivary factors - resting andstimulated flow, pH, fluoride,calcium and bicarbonatelevels; salivary anti-bacterialproperties (antibodies,lysozyme, lactoferrin,peroxidase)1,4 .Tooth factors - anatomicalfeatures such as hypoplasiaand other surface defects(which are influenced bymaternal and peri-natalhealth and nutrition), andfixed orthodontic appliances,which provide increasedsurface area for retention ofthe biofilm5.

Manipulating the physical, ionic,and metabolic factors whichmodulate the properties of thebiofilm provides a powerful approachto caries prevention and control6,7.However, the question must beasked, are such strategies intendedto target just one or two particularspecies of bacteria, or the entirebiofilm?

Is dental caries a specificinfection caused by mutansstreptococci?

Since the mid-1970’s when the“specific plaque hypothesis” was firstproposed, the key pathogens indental caries have traditionally beenconsidered to be the mutansstreptococci (MS) family, with S.mutans and S. sobrinus thought tobe the major initiators of thedisease.

Problems with the specific plaquehypothesis for dental caries aremany, and in summary form includethe following:



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MS levels correlate with cariesincidence at the populationlevel, but not necessarily atthe individual level. MS countsin saliva and plaque are notlinearly associated with cariesincidence in an individualpatient, despite evidence for alinear caries progression overtime.8

MS negative individuals withcoronal or root surface cariescan be found, albeit at lowrates (typically 2 percent)9,10.When used as a survey toolfor large population cohorts,culture-based tests for MStend to give greater negativethan positive predictivevalues, i.e., they are moreeffective in identifying healthyindividuals (low bacterialcounts) than patients withdisease who requiretreatment11.MS counts alone may varyaccording to the site sampledas well as the caries riskstatus. A study in Dundeewhich compared thefrequency of isolation of S.mutans and S. sobrinus fromthe saliva of 12 month oldinfants with and withoutdental caries collected salivasamples using the tongue-loop method, for subsequentmicrobiological culture12. Ofthe cohort of 1393 infants,some 39 were diagnosed withcaries. S. mutans was foundat low frequency even ininfants with caries, but wasisolated more often fromthose infants with cariescompared to those who werecaries-free (29.7 vs 9.8%),however differences in theisolation frequencies of S.sobrinus (2.7 vs 1.3%) werenot significant.MS are not unique in the oralflora in being able to secreteglucosyl- transferase enzymesor in producing extracellularpoly-saccharides.

Many organisms (bothbacteria and fungi) in thenormal oral microflora areboth acidogenic and aciduric,and produce organic acids invitro following exposure tofermentable substrates.MS evokes a host immuneresponse involving both IgAand IgG antibodies, but this isnot always protective13.Immunization against MS insome animal models reducesthe incidence of new cariouslesions but does notnecessarily eliminate thedisease.While cariogenic plaque maycontain more than 200 millionbacteria per mg wet weight,MS counts as a proportion oftotal plaque bacteria aretypically very low (1%)10,14.Patients who have high levelsof both major species of MS(i.e. S. mutans and S.sobrinus) tend to have ahigher caries rate than thosewith only one species, but it iscommon to find adult andelderly patients with S.mutans only and not S.sobrinus15.There are 300-plus species ofbacteria in the non-cultivablenormal oral flora in humans(i.e. those which at presentcannot be grown in thelaboratory, but can beidentified by moleculargenetic methods), and manyof these are found insignificant numbers incariogenic biofilms16.There is no obligaterequirement for MS in termsof generating a pH drop inresponse to sucrose and otherfermentable substrates.Moreover, many bacterialspecies other than MS cangrow and survive at pH valueslow enough to demineralizeenamel, e.g. Veillonella disparand Enterococcus faecalis, aswell as Lactobacillus spp17.



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There is substantial variationin the microflora at differentsub-sites within approximaldental plaque. As plaquedevelops in the interproximalregion, MS preferentiallycolonize a specific sub-sitebelow the contact point, atwhich S. mutans is morecommon than S. sobrinus18.S. sobrinus is rarely found inisolation, and is more oftenfound in association with S.mutans (if present at all).However, S. sobrinus is themore potent of the twospecies in terms of acidproduction and synthesis ofboth intra- and extra-cellularpolymers14.Non-MS aciduric bacteria suchas Actinomyces naeslundii areinvolved in the pathogenesisof root surface caries19,20. Inroot surface caries, neitherMS nor Actinomyces speciessecrete collagenases, howeverproteolytic enzymes originatefrom other biofilm speciessuch as Porphyromonasgingivalis21.Strains of MS differ in theirvirulence traits such as thepotential to synthesize water-insoluble glucan polymersfrom sucrose. This is onereason why colonization withMS does not always lead todental caries activity. Strainswhich synthesize smallamounts of insoluble glucansshow reduced adherencewithin the biofilm and sloweraccumulation of plaque, withless expression of disease22.

A further and major philosophicalproblem with the specific plaquehypothesis is that MS are normal(commensal) members of the oralmicroflora, rather than externalpathogens. While transmission of MSfrom mother to child via a spoon wasdemonstrated conclusively in thelandmark studies of Kohler and

Bratthall in the late 1970’s23, theconcept of a “window of infectivity”at 26 months of age 24,25 has nowbeen disproven in a series of studies26-28 which demonstrated thatinoculation with mutans streptococciand other oral bacteria occurs withinthe first three months of life, withrepeated exposure from carers(particularly the mother) and siblingsthrough saliva micro droplets.Moreover, the traditional conceptthat MS lack a suitable ecologicalniche in the pre-dentate oral cavity,and cannot establish until theprimary teeth erupt, has also beenformally disproven by studies whichdemonstrate pre-dentate acquisition,i.e. before hard tissue surfaces arepresent28,29.

Once acquired, the oral flora withrespect to dental caries appears tobe relatively stable, adding furtherweight to the premise that so-calledcariogens are normal parts of theoral flora. Genotypes (strains) ofmutans streptococci are relativelystable within the one individual, andtend to persist for several years30.

Biofilm ecology and its implications

The “ecological concept” of dentalcaries is based on the view that acatastrophic change in the normalplaque biofilm is responsible fordisease31,32. The main advantages ofthis approach 17,33 are that it:

Deals with the problems inthe specific plaquehypothesis, as summarizedabove.Recognizes the possibility thatbacteria yet to be culturedcan participate in the cariesprocess.Aligns with a comprehensiveapproach to caries as adisease, which results fromthe interplay of host,microbial, lifestyle andbehavioral factors.Supports a concept of “cariesrisk management throughout



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life”, rather than focusing onlyon “windows of infection” orassumed high risk period. Inso doing, it recognizes theclinical reality that caries candevelop at any time aftertooth eruption.Leads to novel and improvedstrategies for riskassessment, risk reductionand clinical management.

Applying this “ecological catastrophe”concept to the issue of pH within thebiofilm, it is clear that the low pHenvironment generated fromcarbohydrate metabolism is themajor factor responsible for theshifts observed in the oral micro florawith high carbohydrate diets.Laboratory studies using chemostatsystems in which pH conditions canbe manipulated 34 provide usefulinsights into how the microbiallygenerated pH changes can causesuch shifts. In this study, threechemostats were used. In the first,following glucose metabolism, the pHfall was restricted to a minimumvalue of pH 5.5, while the pH fall wasarrested in the other two chemostatsat either pH 5. 0 or 4.5. When thepH was allowed to fall, the numbersand proportions of Streptococcusmutans and Lactobacillus rhamnosusincreased. Importantly, this increasewas directly related to the magnitudeof the pH fall. The increase inproportions of acidogenic bacteriawas accompanied by a fall in theproportions of acid-sensitive species(such as Fusobacterium nucleatum,Streptococcus gordonii andStreptococcus oralis). Thus, a fall inpH to between 5.5 and 4.5 will allowthe enrichment of potentiallycariogenic species whilst permittingspecies associated with health toremain relatively unaffected.However, a further reduction in pH(<pH 4.5) will not only enhance thecompetitiveness of cariogenicorganisms, but will inhibit the growthand metabolism of non-caries-associated species17.

In patients with low salivary flowrates (regardless of the cause ofthis), the concurrent reduction insalivary flow and pH can be expectedto provide a more favorable growthenvironment in which aciduricorganisms can flourish1,4. While thiseffect is well recognized for salivarydysfunction induced by medications,irradiation of salivary glands, andsalivary gland diseases such asSjogren's syndrome1,35,36, it shouldalso be remembered that the sameacidic conditions can be created bothby a diet with high intakes of acidicdrinks and foods (exogenous acids),and by eating disorders orgastrointestinal conditions (such aschronic gastric reflux) in which thesource of the acid is endogenous(hydrochloric acid from thestomach). The latter patients willtypically present with dental erosionand occasionally with frank caries6,37.The more dramatic and unusualnature of dental erosion may welldistract the clinician from realizingthe likelihood of greatly increasedcounts of aciduric bacteria becauseof their preferential growth underacidic conditions. Several studies 38,39

have reported that patients witheating disorders are more susceptibleto both dental caries and erosion,and that such patients with lowresting salivary flow rates have veryhigh counts of MS and lactobacilli.Clearly, the preventive program forsuch patients must be targeted toaddress the global risk factor of lowpH that underpins both dental cariesand dental erosion.

MS levels as a surrogate measurefor a cariogenic dental plaquebiofilm

By acknowledging that MS are nolonger regarded as sole ornecessarily dominant pathogens indental caries, it follows thatassessment of the dental plaquebiofilm should be based onparameters such as acid productionby fermentation under conditions ofsubstrate challenge6,17 and perhaps



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by bacterial growth and survivalunder conditions of low pH. Theseglobal assessments will, bydefinition, be inclusive of all bacteriainvolved in the caries process, andnot only MS. Taking a broadapproach to the biofilm ecologyrecognizes the so-called “insurancehypothesis”40, i.e. that thebiodiversity of the biofilm insures itagainst declines in function overtime, and makes it more resistant toexternal stressors.

Because of the properties ofthe cariogenic plaque biofilm, thepresence of MS is still useful as asurrogate measure for:

Adverse changes in thebiofilm ecology driven by lowsalivary pH 9.Behavioral factors such assnacking with sucrose, whichlower biofilm pH6,17.Lifestyle factors which maytransfer significant loads ofpathogens to infants bysalivary micro droplets (suchas kissing and on-demandfeeding with sweetenedbovine milk)26-28.Development of a plaquebiofilm with tolerance of lowoxygen environments.

MS are facultativemicroorganisms, which can tolerateoxygen levels in normal mouth airand in the general atmosphere, butprefer oxygen-poor growthconditions. This feature is essentialnot only to their survival deep withinthe plaque biofilm but is also criticalin terms of favoured growth sitessuch as fissures and interproximalspaces.

The ability to thrive in ananaerobic environment is also linkedto the ability of bacteria involved indentine caries to survive in thedentine in lesions that do not haveobvious entry points on the enamelsurface. For these so-called "occult"or hidden lesions, it is likely that thecariogens gain access to the dentine-

enamel junction via lamellae orcracks in the enamel. These lamellaeare distributed throughout enamel inboth deciduous and permanentteeth. By entering these toothsurface defects, the cariogenicbacteria are at a strategic advantageover the host since they areprotected from saliva and itscomponents as well as oral hygieneand other interventions41.

The facultative nature of MSexplains in part the interactionbetween smoking and caries risk.Smoking creates anaerobiosis withinthe oral cavity, and the low oxygenenvironment favors the growth ofmutans streptococci. There are twoadditional components involved inthe impact of smoking on dentalcaries. The first is salivarydysfunction, which is the result ofthe pharmacological effects ofnicotine on salivary gland flow.Reduced flow is linked with reducedpH, which is also a favorableparameter for growth. The secondfactor is the direct effect of nicotinein the salivary milieu on mutansstreptococci. There is now someevidence that nicotine itself candirectly affect the growth ofStreptococcus mutans, withconcentrations of nicotine in theorder of 0.1-1.0 mmol/L able tostimulate growth, although higherconcentrations are inhibitory42. Avalue of 1.0 mmol/L approximatesthe salivary levels achieved withsome tobacco products, includingsmokeless tobacco.

The tendency for MS to occupyan anaerobic ecological niche has aninteresting consequence in terms ofperiodontal therapy. It is well knownthat both attachment loss per se androot surface exposure from surgicaland non-surgical periodontaltreatment are associated positivelywith root surface caries. Thesusceptibility for root surfaces forcaries after periodontal therapy hasbeen attributed to a loss of thefluoride-rich outer layers of



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cementum and dentine43, howeverthe microbial effects of periodontaltherapy should also be considered.Through soft tissue changes such asrecession, the available supragingivalarea for dental plaque biofilm isincreased. There is clear evidencethat following periodontal therapy,the severity of root surface caries isassociated strongly with high salivarycounts of MS44,45. This effect iscompounded in very elderly patients,in whom polypharmacy causesreductions in the resting salivary flowrate and salivary pH, andcorresponding increases in bothsugar clearance time 15 and levels ofMS 15,46,47.

A longitudinal study which examinedthe oral flora in patients with severeperiodontitis after thorough scalingand root planing in combination withoptimal plaque control raises someinteresting observations48. Atbaseline and after 4 and 8 months,samples were taken from the saliva,the tongue dorsum and thesupragingival interdental spaces.These samples were cultured bothaerobically and anaerobically, inorder to determine the total numberof colony forming units (CFU) persample as well as the numbers ofStreptococcus mutans andlactobacilli. Although the totalnumber of aerobic and anaerobicCFU in samples from the tongue andthe saliva remained nearly constantover the 8 month period, there weremarked increases in the number ofS. mutans detected in samples fromthe teeth, despite a decrease in thetotal number of anaerobic CFU insamples from the teeth, meaningthat the relative proportion of theStreptococcus mutans had increaseddramatically. Thus, although theperiodontal conditions improved forall patients, the development of acariogenic plaque biofilm continued.

This stresses the need for abroad approach to assessing patientrisk, and highlights where the overallassessment of plaque biofilm

properties can be useful. A range ofproducts are now available for thispurpose (Table 1). The Snyder test isbased upon the fermentation ofglucose by bacteria from a sample ofstimulated saliva. The resulting acidproduction over 72 hours lowers thepH of the medium and changes thecolour of a pH indicator. This testdemonstrates total acid productionby cariogenic microorganisms butdoes not delineate between mutansstreptococci and lactobacilli61.

Table 1. Techniques for assessing biofilmbehaviour

Biomass (plaque thickness andmaturity)

Disclosing with erythrosin dye2-Tone disclosing (GC Plaque-Check)Fluorescence (KaVoDIAGNOdentTM; DurrVistaProof)

Acid productionFermentation test (GCPlaque-Check+pH)

Level of MSCultural assays (IvoclarVivadent Dentocult-SM)Immunoassays (GC Saliva-Check SM)

Modifications of this same principle,in which the pH-lowering potential ofpooled plaque samples wasmeasured in an adapted glucosebroth showed some association withcaries prevalence49, however site-specific tests for fermentation suchas the GC Plaque-Check+pH™ test17

overcome the disadvantages ofpooled plaque samples or usingsaliva samples.

Measuring MS levels at the chairside

Numerous studies over the past twodecades have established a stronglink between the presence of



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pathogenic bacteria at a young ageand greater caries experience. Bothcross-sectional and longitudinalstudies of dental caries haveestablished the value of salivary MSas a surrogate marker in caries riskassessment. Thus, while not a solepathogen, undertaking periodic (e.g.annual) assessments of MS levelsmay be useful for identifying patientswith long-term caries risk in both theprimary, mixed and permanentdentitions1,50. Today, the range ofmethods for measuring MS levelsincludes culture-based tests,metabolic tests, antigenic tests suchas direct immunoassays, andmolecular genetic methods.

Culture-based tests

Culture-based methods for MS andalso for lactobacilli have beenavailable commercially for manyyears, with a wide number of samplecollection strategies and culturemedia being suggested as optimal(Table 2)51. For culture of oralstreptococci, the most common basemedium is Mitis-Salivarius agar, orMitis Salivarius agar with bacitracin(MSB). This is inoculated withstimulated saliva or dental plaque.

In most contemporary culture-basedtests, this same MSB medium isinoculated with stimulated saliva,which is elicited by chewing smallblocks of paraffin to dislodge plaqueso that it is dispersed into the saliva(Figure 1).

All culture-based tests used incaries risk assessment have severallimitations:

Live, viable bacteria arenecessary.An incubator is required toprovide the optimum temperaturefor growth (37 degrees Celsius).It takes a substantial time(typically 48 hours) to obtain aresult.The shelf life of the kit is limitedbecause of temperature-relateddecline in some components ofthe growth medium (particularlythe antibiotic bacitracin).MSB has been shown to underestimate the actual levels of S.mutans in samples.The growth media are semi-selective and there is little or nodiscrimination between S. mutansand S. sobrinus.

Figure 1. Various cultural testsused for mutans streptococci. A,Tongue blade imprint onto MSBagar. This was a common methodused in the 1980’s. B, “Stripmutans” test strips showing (fromleft to right) high, medium, andlow levels of MS. This was apopular test in the 1990’s. C.Contemporary (2008) version ofthe Dentocult tests with separatemedia for MS and lactobacilli,known as the CRT. D, DentocultSM test strip from the CRT after72 hours, showing multiplecolonies in a saliva sample from ahigh caries risk patient. E,Biochemical test panel (RapidID32 Strep) results forStreptococcus mutans. F,Identical panel with Streptococcussobrinus. Differences betweenbiochemical results allow cleardifferentiation between these twospecies.



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A recent comprehensiveassessment of the most suitableculture media for theenumeration of S. mutans fromclinical samples determined thespecificity and sensitivity of fiveso-called selective media82.Tryptone-yeast-cysteine-sucrose-bacitracin (TYCSB) was found tobe the medium of choice for theisolation of S. mutans from salivasamples, giving the bestdelineation between S. mutansand both S. sobrinus and non-mutans streptococci (Figure 2),however this medium is notavailable currently for chairsideuse. Confirmation of identitytypically requires follow-upbiochemical tests conducted in apanel using a set of fermentationand enzymatic tests, such asproduction of acid from N-acetylglucosamine, arbutin andmelibiose, and the presence ofalpha-galactosidase and alpha-glucosidase activities83. (Figure 1,panels E and F).Atmospheric conditions forculture stipulate a low oxygentension. For clinical samples, thisoften entails using expired airfrom the mouth, sodiumbicarbonate pellets, or depletingoxygen using combustion or acatalyst. In contrast, in thelaboratory setting, incubation ofculture plates for mutansstreptococci can be undertaken inan anaerobic environment (e.g.95% nitrogen and either 5%carbon monoxide or 5% carbondioxide).

Sampling methods for culture-based tests

An important variable in culture-based methods is the requirementfor viable organisms, which replicateto form colonies. For precisemeasurement of colony forming units(CFU), standardized conditions ofculture are required. This makesboth clinical and field studiesproblematic in that storage and

transport of growth media mayinfluence viability and thus colonycounts.

Figure 2. Bacterial recovery after inoculationof five growth media with 100 million CFU ofStreptococcus mutans type strain NCTC10449, expressed as a percentage. Recoveryon TCYSB more than twice that of othermedia, including MSB, the basis of manycommercial tests. Data derived from Ref. 82.

There are several possible methodsof sampling, which can be used whenestimating levels of MS in the oralcavity:

Direct sampling of plaque (e.g.overlying carious lesions) usingan absorbent cotton tip orsponge.Sampling of stimulated wholesaliva using a plastic stick orwooden spatula which is thenwithdrawn through the lips, as inthe "strip mutans " method.Collection of stimulated wholesaliva in a cup, the contents ofwhich are then poured over asolid agar growth medium orused to inoculate a liquid culture.Sampling of unstimulated salivausing an absorbent cotton tip orsponge (e.g. in neonates).Sampling of both unstimulatedsaliva and plaque using anabsorbent tip, which is gentlyrubbed over the dorsum of thetongue, alveolar ridges, and labialand lingual surfaces of all eruptedteeth (e.g. in young children).Sampling from the dorsum of thetongue using a loop, which isdrawn across the surface, basedon work, which shows that levelsof MS in whole unstimulated



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saliva correlate significantly withlevels in debris collected with aloop from the dorsum of thetongue84.Sampling from the dorsum of thetongue using a wooden tonguedepressor, which is thenimpressed directly onto cultureplates.An oral rinse using a fixedvolume of transport medium,which is then used as theinoculum.

Sampling of the stimulated saliva ispreferred because of its simplicity,however it must be stated once morethat salivary levels are a surrogatemeasure for plaque levels ofcariogenic bacterial species. Whileorganisms from plaque will bedispersed into the saliva by chewing,deeper portions of the plaquebiomass, particularly the less aerobicenvironments in fissures andinterproximal spaces, will contributerelatively less to salivary levels bysimple mechanical dispersion duringchewing. It is for this reason thatsalivary counts and levels in plaquedo not correlate absolutely.

There is also the effect ofsurface area, in that an increase inthe available hard surfaces will tendto result in elevated counts simplybecause hard surfaces are thepreferred ecological niche for mutansstreptococci. This is amplydemonstrated by reports of dramaticincreases in salivary levels of mutansstreptococci following the placementof orthodontic brackets85. Furtherevidence is seen in a study of cariesrisk factors in elderly patients, whichreported the salivary levels of MS asa function of the number of teeth86.When the raw data for salivary levelswere adjusted for the number ofteeth in the mouth, levels of MS(reported as CFUs/ml saliva pertooth) were significantly associatedwith coronal surface caries. It wassuggested that reporting salivarybacteriological data as a function oftooth number and per mL of saliva

could improve the reliability ofbacteriological data in that it wouldaccount at least in part for surfacearea dispersion effects involved insalivary sampling.

Antigen-specific assays

These tests utilize highly specificmonoclonal antibodies, givingabsolute specificity for the bacteria ofchoice, e.g. S. mutans or S.sobrinus. These antibodies can beused in number of diagnosticmethods, includingimmunofluorescence, flow cytometry,latex agglutination, immunoblots andsolid phase immunoassays. Theformer methods are laboratory-based, while immunoassays havebeen brought to clinical application indental practice.

The GC Saliva-Check SM ™uses a combination of three highlyspecific anti-S. mutans monoclonalantibodies (SWLA-1, 2, and 3)87,88, toincrease binding and reduce thedetection limit to 100,000 bacteriaper mL of saliva, which is therecognized level for increased cariesrisk (Figure 3). The tests areundertaken at chairside within ashort time frame, and no specialapparatus or techniques arerequired. Unlike culture-based tests,viable bacteria are not needed.Samples of saliva are collected andreacted with buffers to establish aconstant pH (and thus charge forproteins), and detergents, for properdispersal of the sample. The sampleis then placed on a strip ofnitrocellulose or other suitablematerial, which is impregnated withmonoclonal antibodies, which trap S.mutans bacteria, triggering adetection reaction. Because this iscomplete within 5 minutes, theresults can be discussed with thepatient at the same appointment9.Control reactants ensure that thedetection chemistry is workingproperly, allowing the clinician tointerpret a negative result withconfidence (Figure 4).



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Such tests are particularly useful fortracking transfer of MS from mothersto infants, as part of assessingstrategies designed to reduce therisk of early childhood caries89. Usingsuch tests provides data, whichagree with conventional culturaltests, but within the sameappointment rather than afterseveral days9. Typical results withimmunoassays reveal that one thirdof an adult population would havelevels above 100,000 per mL, andthat a small proportion of thepopulation would have more thanone million per mL. In a large studyconducted by Shi et al. in whichnearly 2,000 human saliva sampleswere examined using the threespecies-specific monoclonalantibodies to detect and quantify S.mutans levels in human saliva,values ranged from less than 10,000to a high of 36 million cells/mL. Over15% of the saliva samples examinedhad counts over 500,000 cells/mL88.Contemporary laboratory studies foranalyzing cariogenic plaque typicallyemploy molecular methods withspecific primers for 16S rRNA genesand amplification in the polymerase

chain reaction (PCR), for example,competitive PCR, nested PCR, andreal time PCR, with the latter givingthe most rapid results90-94. Whilegiving accurate bacterial countsdown to low detection limits of 100organisms or less, because thesemethods require DNA extraction andcomplex thermo-cycling equipment,they cannot be used in the dentaloffice setting.

Novel strategies for manipulatingcariogenic biofilms

The final section of this paperfocuses on novel means forinterfering with changes in biofilmsthat increase their cariogenicity,highlighting recent developments indental research which may holdpotential for application in clinicalpractice for high risk patients, wherecombinations of various agentswould be used with appropriatelifestyle changes (Table 2). Theunderlying concept is that, from thecaries perspective, dental plaquebiofilms range from benign (thin,immature) through to more harmful(thick, fermenting).

Figure 3. GC Saliva-CheckSM chair side diagnostic kitbased on a solid phaseimmunoassay, with threemonoclonal antibodiesspecific for Streptococcusmutans. A, Components ofthe kit, showing the testdevice and the tworeagents. B, Addition of thefirst pH adjusting reagent.C, Addition of the secondpH adjusting reagent. D,Agitation of the sample. E,Loading onto the test strip.The liquid moves down thestrip by capillary action. F,A positive result withcolour reactions seen inboth the test (T) andcontrol (C) wells.



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Figure. 4. Typical immunoassay resultsobtained in clinical practice and verified byculture on specific media. A, Control sample ofbacteria-free filtered saliva, showing lack ofreaction in the test well, but a positive controlresult. B, Saliva sample from a patient with1000 CFU/mL is below the caries risk level anddoes not cause a positive test result. C, Salivasample from a patient with 100,000 CFU/mLgives a clear positive test result.

A range of commercial products mayassist in determining surrogateendpoints for agents, which couldinfluence biofilms (Table 2). Withregard to infants, caries preventionshould target specific behaviors,which will influence the biofilm (Table4), taking into account the ease withwhich cariogenic biofilms develop onthe teeth in the early years of life(Figure 5).

One important implication ofthe belief that caries is a multi-pathogen biofilm disease is that“single pathogen” approaches suchas vaccines to S. mutans or itscomponents (such as glucosyltransferase), and the use ofgenetically modified S. mutans (withdeletions of glucosyltransferase

enzymes (B/C/D), thephosphoenolpyruvate-dependentphosphotransferase system (PTS), orof lactate dehydrogenase) would nolonger be appropriate.

Figure. 5. Time course of colonization withStreptococcus mutans after tooth eruption,using tryptone-yeast-cysteine-sucrose-bacitracin (TYCSB) agar to assess samples ofsaliva. Bacterial identification was verifiedusing the Rapid ID32 biochemical test. Datashow the frequency of colonized individuals ina cohort of 111 infants (35 pre-term and 76full-term). By the age of 24 months, 84% ofinfants harbored S. mutans. Based on datafrom Ref. 28.

Table 3. Caries prevention agents, whichinfluence biofilm behaviour

Traditional agents

Mechanical oral hygiene (biofilm disruption)Surfactants (in dentifrices and mouthrinses)High concentration and low pH topicalfluoridesMetallic ions (Cu, Fe, Zn, Sn, Ag)Oxygen radicals (ozone, hydrogen peroxide)Bisguanides - chlorhexidineNaturally occurring phenolics (includingessential oils)Synthetic phenolics (Triclosan)

Novel agents

XylitolCasein PhosphopeptidesOther natural plant-derived products(ingestible antimicrobials)Inhibitory bacterial species (probiotics)Alkalinizing approaches (arginine, urea)Synthetic peptides with antibacterialpropertiesAgents, which modulate biofilm behaviour(quorum sensing)Photosensitization



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A. Xylitol

Xylitol is a naturally occurring polyol,which is taken up by MS but is notfermentable. Numerous studies havedocumented the non-cariogenicity ofxylitol95, however more recentlyattention has been directed to theeffects of prolonged consumption ofxylitol on MS. Chewing a xylitol gumthree times daily for a minimum offive minutes each time for threemonths may give a 10 fold reductionin salivary levels of MS96,97, astrategy which would be useful inhigh caries risk patients, orthodonticpatients and mothers of infants, asan adjunct to other suppressiveapproaches such as chlorhexidinetherapy. Habitual consumption ofxylitol in the diet appears to selectfor MS with impaired adhesionproperties, i.e., they bind poorly toteeth and shed easily from plaque tosaliva.

Table 4. Risk factors for early colonizationwith cariogenic bacteria

Infant factors

Premature birthEnamel hypoplasiaSweetened fluids taken at bedtimeDrinking only from bottlesOn-demand formula feedingDelayed start for solid foodsSucrose exposure more than 3X perdaySharing food and utensils with adultsRegularly kissed on the lipsIrregular tooth brushing by carers

Maternal factors

High plaque levelsSucrose exposure more than 3X perdayHigh salivary levels of S. mutans

Based on Ref. 28.

One important advantage of thisapproach is that it is suitable formaintaining long-term suppression ofpathogens without concerns of safetywith prolonged use. Use of highxylitol containing gums such asXylimax™ (0.6 grams per pellet ofgum) is a simple measure toincorporate into a daily routine, andbrings the important advantages ofincreased salivary clearance,buffering and remineralization. Thisneeds to be balanced against theissue of adaptation to xylitol in thediet.

B. Natural peptides

Casein phosphopeptides (CPP) arenaturally occurring molecules frombovine milk, which are able to bindcalcium and phosphate ions andstabilize amorphous calciumphosphate (ACP). Under acidicconditions, CPP are able to releasecalcium and phosphate ions andthereby maintain a state of supersaturation with respect to toothenamel, reducing demineralizationand enhancing remineralization98.The delivery of CPP or complexes ofCPP and ACP (Recaldent ™) to theplaque fluid can be achieved by arange of vehicles, including chewinggums, dentifrices and topical gels,such as GC Tooth Mousse and ToothMousse Plus (designated MI Pasteand MI Paste Plus in somecountries). CPP bind strongly todental plaque, and are able to slowor prevent the diffusion of calciumions from enamel during episodes ofacid challenge, and serve as a sourceof calcium for subsequentremineralization99-101. CPP may alsoaffect adhesion of MS and modulatefermentation by dental plaquebacteria101-102. An important aspectof CPP is their long half-life in saliva,which is due to their unique aminoacid composition and to the releaseof phosphate ions, which may inhibitproteolytic breakdown of thesepeptides. Their metabolism in plaque(with a half life of 2.8 hours) alsoresults in a pH elevating effect



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because of their substantial argininecontent.

C. Synthetic peptides

Peptides with antimicrobialproperties can also be synthesized,with inherent antimicrobial propertiesor as delivery systems forestablished biocides or toxins. Issueswith these synthetic agents include:low binding affinity to plaque, poorpenetration, limited stability in thebiofilm because of proteolysis, lowspecific activity, and high cost. Forthese reasons, antibacterial peptides(bacteriocins) from oral bacteriawould seem a more fruitful avenue toexplore.

D. Natural plant products

A number of plant-derived extracts,which has been shown to possesssignificant anti-cariogenic properties.Propolis, essential oils and flavonoidshave been of interest for manyyears, with recent attention beingdirected towards cranberry extractsand the combination of the agentsapigenin and farnesol, which blockexpression and secretion of theglucosyl transferase enzyme fromMS, preventing their synthesis ofinsoluble glucans. Combinations ofthese agents with fluoride may allowa change in biofilm behaviourwithout necessarily reducing thelevels of MS or other bacteria103,104.

Cacao bean husk extract(CBH) is one such material whichunder laboratory conditions canreduce the growth rate of MS andother oral streptococci, reduce acidproduction by MS, inhibit thesynthesis of insoluble glucans by theglucosyltransferases from S. mutansand S. sobrinus, and thereby impairsucrose-dependent cell adherence. Inthe rat animal model of dental caries,administration of CBH in drinkingwater at concentrations exceeding1.0 mg/mL has been shown to causesignificant reductions in both plaqueaccumulation and in dental caries105.

Chewing sticks from the treeJuglandaceae regia have been usedin the Indian subcontinent tomaintain oral hygiene. Laboratoryand clinical studies of the aqueousextracts from these sticks show avariety of anti-cariogenic properties,including reduced growth andadherence of S. mutans for up to 3hours; reduced acid production by S.mutans for 90 minutes; reducedglucan-induced aggregation of S.mutans, and direct bactericidaleffects on S. mutans106. Anassessment of the aqueous extractfrom Terminalia chebula by the sameauthors yielded similar properties.The extract strongly inhibited thegrowth, acid production, sucrose-induced adherence and glucaninduced aggregation of S. mutans107.

E. Alkalinization strategies

Increasing alkali production in dentalplaque is a simple means of applyingecological pressure to the biofilm. S.sanguis and S. gordonii use argininedeiminase to elevate pH throughmetabolism of amino acids, whileurease breaks down urea releasingammonia which again elevates pH.Supplying substrate for these twoenzyme pathways is the basis forpoly-arginine (argininebicarbonate/calcium carbonate,CaviStat ™) and V-6™ chewing gum,respectively. While the urease genefrom S. salivarius has been insertedinto a genetically modified strain ofS. mutans to make it ureolytic108,109,acceptance of genetically modifiedbacteria by the community as adental intervention would likely posemajor problems.

F. Inhibitory organisms andprobiotic approaches

A number of novel strategies exploitcompetition between bacterialspecies. This can be direct (throughsecretion of bacteriocins, toxins,enzymes or waste products) orindirect (through environmentalchanges in pH). Organisms of



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interest have included clinicalisolates of S. sanguis, S. salivarius(strain K12) and S. oligofermentans.The probiotic approach makes soundbiological sense, provided theorganisms can be delivered oftenenough to establish their presenceover their commensal counterparts.

In fact, there is increasingevidence of an important interactionbetween oral bacteria within thesanguinis (sanguis) group and MS110.These bacteria have the advantageof being naturally derived, normalcommensals, and thus come underthe “generally recognized as safe”(GRAS) regulatory classification. Arecent study demonstrated thatthere might be competition andantagonism between S. sanguinisand MS111. Children who did notharbour detectable levels of MS hadhigher levels of S. sanguinis in theirsaliva than children colonized withMS. If S. sanguinis (or otherbacterial species) were to be used asa probiotic therapy in a topicalproduct, issues of shelf life (for aproduct containing live bacteria)would provide an important technicalchallenge to overcome.

Screening libraries andculture collections of food-derivedbacteria for inhibitory effects ondental caries has also become amajor area of investigation in recentyears, aiming to find agents, whichare GRAS and thus free of safetyconcerns for repeated ingestion.Several species have been identifiedwhich can cause lectin-glycoproteinadhesion to S. mutans, raising thepossibility of reducing levels of thesein saliva and the superficial layers ofdental plaque after using rinses ordentifrices to aggregate S. mutansand the probiotic bacteria intoclumps which are then ingested orexpectorated112. These adhesiveinteractions should be effective evenif the probiotic bacteria have beeninactivated (for long term storage)by pasteurization, as the lectinswould be heat resistant. The same

approach could also be taken withdelivery using sugar-freeconfectioneries. Current species ofinterest include certain isolates ofLactobacillus paracasei andLactobacillus rhamnosus.

G. Altering biofilmcommunication pathways

Biofilm properties may also bemanipulated by affecting thepathways of biofilm manipulation, orof cell to cell signaling within thebiofilm. Blocking this “quorumsensing” would reduce the ability ofthe biofilm to tolerate stresses suchas reductions in nutrients or assaultby external chemical agents (such asbiocides). Slowing the biofilmaccumulation rate may be possibleusing agents such as furanone, whichaffect quorum sensing113.

H. Targeted therapies

“Magic bullet” and “smart bomb”therapies are of ongoing interest.Antibodies to particular bacterialspecies in the biofilm could beconjugated to a toxin or biocide,however the challenges remain ofgaining penetration and resistingdegradation by proteases within theplaque biofilm, which may prove tobe insurmountable. Usingendogenous photosensitization ofprotoporphyrins in biofilm bacteria114-117 with light as the vector mayovercome the penetration problem,and remains an important area forfurther investigation.

Conclusions

There are now multiple lines ofevidence, which indicate that dentalcaries is a multi-pathogen diseaseand that MS may participate but arenot critically essential for disease tooccur. Taking a “whole of biofilm”ecology approach to dental cariesintroduces a number of newstrategies for controlling caries risk.Clinical diagnostic kits can bevaluable in patient management



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provided their interpretation assurrogate measures of the diseaseprocess is borne in mind. Moderntechnology for plaque fermentationtesting and for chairsideimmunoassays fits well withincontemporary caries managementstrategies such as STEM (System forTotal Environmental Management)6

and CAMBRA (Caries management byrisk assessment)118-119. Dentalhygienists, dental therapists anddental chairside assistants can assistthe dentist or dental specialist in thecaries management process throughtheir delegated work using chairsidetests for patient workup andmonitoring.

Disclosure

LJ Walsh was responsible fordeveloping the GC Plaque-Check+pHtest for dental plaque fermentation,and has a commercial interest in thisdiagnostic kit. Both authors havebeen involved in clinical trials usingculture-based and immunoassaytests from several manufacturers,but neither have any commercialinterest in these products.

STEM

Int Dent S Afric 2008; 10: 50-65

Resumen

Los enfoques modernos de lacaries dental, tales como elSistema para Administración

Ambiental Total - STEM (Systemfor Total EnvironmentalManagement), se basan en unenfoque ‘integral del paciente’ e‘integral del biofilm’ en relacióncon la enfermedad. Existen hoyen día múltiples líneas deevidencia que indican que lacaries dental es una enfermedadmultipatógena y que losStreptococcus mutans puedenser parte del desarrollo de laenfermedad, aunque no sonnecesariamente esenciales. Seestán explorando nuevasestrategias para controlar elriesgo de adquisición de caries,las mismas que se basan en elconcepto moderno del biofilm.Las herramientas de diagnósticoclínico que examinan placa osaliva en busca de característicasdel biofilm que son portadoras(marcadoras) del proceso de laenfermedad, pueden ser útiles enel manejo clínico del pacientepuesto que pueden seracometidas in situ. Publicadoprimero en Int Dent S Afric 2008; 10 50-65.

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Chairside testing for cariogenic bacteria: current ... · The biofilm which causes dental caries arises from the normally thin dental plaque biofilm which is present continuously