Enumeration of Salivary Streptococci and Lactobacilli in Children with Differing Caries Experiences in a Rural Indian Population

Objectives. Compare the total salivary Streptococci and Lactobacilli counts in cle and noncle children with differing caries experiences, correlate the bacterial counts with dm/DMFT status and identify the different biotypes ofMutans Streptococci (MS). Patients. Group I included thirty subjects with dental caries (DC) and cle lip and palate (CL/P); Group II had thirty subjects with DC but without CL/P. Group III comprised a control of thirty subjects with neither DC nor CL/P. Methodology. Enumeration of total salivary Streptococci and Lactobacilli was done by the plate count method and correlation of counts with dm/ DMFT status examined. Differences in biochemical reactions were used to identify the biotypes. Results. Streptococci colonies in CL/P children with caries (64.30± 24.52)was signi�cantly higher than in childrenwith noCL/P or caries (45.57± 16.73). No signi�cant differences in the Lactobacilli count were observed. dm/DMFT status and Streptococci counts showed a strong positive correlation whereas Lactobacilli counts showed a moderate correlation. S. mutans was the predominant biotype. Conclusions. Higher total salivary Streptococci and Lactobacilli counts exist in cle subjects with caries than in the non-cle subjects. Positive correlation between dm/DMFT scores and salivary Streptococci reinforces its role in DC. S. mutans and S. sobrinus are the biotypes more frequently associated with dental caries in children.


Introduction
Dental caries is a complex process shrouded by many indirect factors which obscure the direct causes.Bacteria are thought to play an integral role in this process.Cariogenic microorganisms colonize on tooth surfaces, cause a marked reduction of pH in the presence of a sugar substrate, and consequently induce dental caries.Studies have shown that Streptococcus mutans is an efficient cariogenic microorganism [1,2].Most populations with a high caries experience have also reported positive associations between caries experience and salivary levels of Lactobacilli [3,4].
CL/P individuals, in addition to the presence of anatomic and functional de�ciencies, also have a higher prevalence of dental disease than normal children.e literature on CL/P subjects and dental caries is con�icting with reports suggesting that individuals born with oral cles have a higher risk of caries [5][6][7][8][9][10][11] and there also exist studies that did not �nd any difference in caries experience between individuals born with cles and unaffected controls [12][13][14][15][16].A recent meta-analysis on the frequency of caries in individuals born with cles was inconclusive [17].
Among the cariogenic microbes, the roles of Streptococci and Lactobacilli have been investigated in CL/P subjects as well [18,19].In addition to a sugar rich diet, poor oral hygiene maintenance, lack of motivation to perform regular preventive dental home care, low �uoride exposure, and malaligned teeth have been put forward as possible explanations [20].Since it is not possible to encompass all these factors in a limited time frame study, we focused on the vital microbial aspect of the caries process.Most studies on the microbial �ora of CL/P patients have been carried out in developed countries, with little microbial data available from an Indian population [21][22][23].erefore, we compared T 1: Scheme of biochemical tests (adapted from references [25,37]).Mutans Streptococci (MS) have been grouped into six biotypes of which four, namely, S. mutans, S. sobrinus, S. cricetus, and S. rattus have been identi�ed as the primary hosts in humans [24,25].Some bacterial strains are more likely to be associated with the caries process than others.Hence, we also investigated the different MS biotypes present in the cle and noncle children in order to gain a better insight into the caries process.

Materials and Methods
e study was set in Department of Oral Maxillofacial Pathology and Microbiology in a rural part of central India.Examination for dental caries in all the subjects was carried out by a single examiner under natural light using a mouth mirror and explorer.Sum of decayed, missing due to caries and �lled teeth in the deciduous dentition (dm) and sum of decayed, missing due to caries and �lled teeth in the permanent dentition (DMFT) were used to assess caries experience as per the WHO criteria [26].Caries prevalence (dm/DMFT > 0) and severity (mean dm/DMFT) were calculated.
A total of 90 subjects aged 6-12 years were divided into 3 groups: Group I included 30 subjects with DC and CL/P (4 BCLP, 24 UCLP and 2 CP subjects were enrolled.Presence of labial and/or palatal �stulae was noted in 18 of the 30 subjects), Group II included 30 subjects with DC without CL/P, and Group III included 30 subjects with no DC or CL/P.Group I children were consecutively drawn from subjects reporting for secondary surgical repairs, speech therapy or seeking de�nitive orthodontic treatment as a part of their CL/P follow-up visits.Non cle subjects of Groups II and III were consecutively selected from a regular pool of dental patients.Subjects with history of drug therapy three months prior to the saliva sampling, any systemic disease, or undergoing active orthodontic treatment were excluded from the study.Informed consent was obtained from the study subjects or their parents prior to inclusion in the study and clearance obtained from the institutional ethical committee.

Collection of Saliva.
Paraffin-stimulated whole saliva of subjects in the different groups was collected following the protocol previously described [27].Participants were requested to void saliva into a wide-mouthed disposable cup two hours aer their last meal in a mid morning session.
2.2.Culture Method.Saliva (0.5 mL) was diluted with 9.5 mL of sterile water and tenfold serial dilutions of saliva were made up to 10 −6 , before plating.Mitis Salivarius Agar (MSA) supplemented with potassium tellurite and Rogosa SL were used as culture media for isolation of total salivary Streptococci and Lactobacilli, respectively.Appropriate amounts of semisolid media was poured onto petri-plates in a laminar air �ow unit and allowed to solidify at room temperature.To each plate 100 L of the diluted sample was spread evenly and the plates were sealed, placed in an anaerobic jar with AnaeroGas Pack system (Himedia), and incubated at 37 ∘ C for 48 hours.Bacterial colonies were observed under a dissecting microscope and deduced on the basis of colony morphology, shape and color� and in case of doubt con�rmation for Streptococci and Lactobacilli was done by gram staining or catalase tests, respectively.Colony counting was done under a colony counter and the number of CFU were multiplied by the number of times the original mL of sample was diluted (the dilution factor of the plate counted) and expressed as the number of colony forming units per milliliter (CFU/mL) of saliva.

2.�. Mor�holo�ical and �iochemical �denti�cation of �solates.
e colonies showing resemblance to the Mutans Streptococci on the basis of morphologic appearance, size, and color characteristics were subjected to a biochemical scheme of tests (Table 1) for identi�cation and differentiation of the biotypes.Each of the different colony morphologies obtained were four-way streaked on MSA media and incubated in anaerobic gas jar with AnaeroGas pack for 24 hours.e colonies obtained were subcultured on a 7.5 mL of slant containing MSA media for pure isolation and again incubated for 48 hours anaerobically.e pure isolates obtained on the slants were then subjected to the biochemical tests.Strains were distinguished based on their ability to ferment different carbohydrates (Mannitol, Sorbitol, Inulin, Mellibiose, Raf-�nose), the ability to hydrolyze arginine, aerobic growth, and susceptibility to bacitracin.e biochemical tests were repeated with cultures in order to con�rm reproducibility and reliability.
All the data were recorded and subjected to statistical analysis in SPSS 17. Descriptive statistical tests including the mean Streptococci and Lactobacilli counts in the different groups were determined.One-way ANOVA compared the mean salivary Streptococcal counts and Lactobacilli counts in the different groups.Tukey post hoc test was applied to determine the groups in which the signi�cant differences were.Pearson product-moment correlation was run to determine the relationship between dm/DMFT status of the children

Results
dm/DMFT scores of greater than 3 were noted in nine subjects with cle lip and palate.e twenty-one remaining cle subjects had dm/DMFT scores between 1-3.Eight of the noncle subjects had dm/DMFT scores more than 3. e remaining twenty-two noncle subjects had dm/DMFT scores between 1 and 3. Mean dm/DMFT score (2.17) in the cle and noncle groups was similar (Table 2).At sample dilutions beyond the concentration factor 10 3 the number of CFU that could be counted tended to be less than 30, hence dilution factor 10 3 was chosen for calculation of the mean Streptococci and Lactobacilli to avoid false positive results.In Table 3, the mean and standard deviations of the salivary Streptococci and Lactobacilli levels in the different groups are detailed; in the last two columns, the results of the statistical analysis are reported.ere was a statistically signi�cant difference between the Streptococci count in the different groups as determined by one-way ANOVA.A Tukey post hoc test revealed that the Streptococci count in the group of cle children with caries (643 ± 24 CFUs) was signi�cantly higher than in the control subjects without cle or caries (4 ± 167 CFUs) (  4).No statistically signi�cant differences were noted in the Streptococci counts in subjects with no cles but having caries and the control group (  4).Mean Lactobacilli counts of the cle children with caries (238 ± 18 CFUs), the subjects with no cles but having caries (271 ± 18 CFUs), and control subjects without cle or caries (184 ± 18 CFUs) showed no statistical signi�cant difference.
Pearson correlation was used to determine the relationship between dm/DMFT status of the children and their salivary Streptococci and Lactobacilli numbers.Streptococci count and the dm/DMFT scores in the subjects with cle and caries showed a strong, positive correlation (  81).Similarly a strong positive correlation was observed for the Streptococci count and the dm/DMFT scores for subjects without cles but having caries (  84).Lactobacilli counts and dm/DMFT scores in Groups I and II showed a moderate positive correlation (  42 and   1, resp.)(Figures 1  and 2).
In children with both caries and cles S. mutans was identi�ed in nearly two-thirds (63%) of the subjects followed by S. sobrinus from 20% of the subjects.In children with no cles but having decayed teeth S. mutans and S. sobrinus were the biotypes identi�ed from �3% and 10% of the subjects, respectively.In less than half (40%) of the children from the control group biotype S. mutans was recovered, and in only a small fraction (3.33%) was S. sobrinus identi�ed.Interestingly, in none of the subjects S. sobrinus was identi�ed in isolation and it was always identi�ed along with S. mutans.e biotypes S. cricetus and S. rattus were not identi�ed in any of the subjects.In certain subjects (60%, 76.6%, and 90% in Groups I, II, and III resp.)biochemical categorization of the strains, although resembling the Mutans group on the basis of morphologic characteristics, was inconclusive (Figure 3).

Discussion
Dental caries is a simple process in concept, but complicated in detail and microbes are a vital cog in the wheel of this process.Detection and quantitation of microbes is necessary to understand the disease process.Several molecular methods have been developed for the detection and quantitation of microorganisms on the basis of differences in DNA and RNA; that are simple, rapid in detection, and technically sensitive.Conventional culture has been employed since long for detection and quantitation of microbes and if the strict adherence to procedures is followed such as sterile lab conditions, strict anaerobic/aerobic protocols maintained then conventional plating and enumeration also produces predictive results.However, there are certain drawbacks to the conventional  methods including long time span, labor intensiveness, and reduced level of sensitivity when compared to the molecular methods [28].Saliva samples were used in the study to assess the microbial aspect of dental caries.Questions concerning reliability of saliva over plaque or oral swab samples can be raised.e reports of Mundroff et al. (1990) and Dasanayake et al. (1995) nullify this doubt as they found that detection of Streptococci and Lactobacilli in stimulated saliva was in excellent agreement with either pooled plaque or oral swab samples [29,30].Studies have shown that differences in culture may result when different media are used [31,32].Mitis-Salivarius (MS) agar was developed as a selective and differential medium for oral Streptococci [33].e addition of Bacitracin and sucrose to MS agar (MSB agar) led to better selective recovery and isolation of S. mutans [34].However, Schaeken et al. (1986) have shown that addition of bacitracin is inhibitory to certain MS biotypes, especially S. sobrinus and S. cricetus.Chair-side methods exist that are rapid and simple in detection [35,36].However they do not permit biotyping of MS.We chose a biochemical scheme based on the papers of Hamada and Slade (1980) and Coykendall (1989) to permit the identi�cation and differentiation of biotypes [25,37].
Caries experience was assessed using the popular dm/DMFT index.Both measures were used as the age group of subjects in the study was 6-10 years, and in this age group children having both primary and permanent teeth are present.Interestingly, the caries severity (mean dm/DMFT) was identical in the cle and noncle groups.
It needs to be kept in mind that the use of dm/DMFT scores is not without limitations.e "M/m" and "F/f " component of the score may be affected not only due to new caries lesions, but also due to dental units missing due to reasons like trauma.Appropriate steps were taken in the study and reasons why teeth may have been extracted were investigated to avoid counting teeth missing or �lled for reasons other than caries.Strong positive numerical association was noted between caries experience and Streptococci levels in children having caries, both cle and noncle.Studies by Hegde et al. (2005) and Ali et al. (1998) have reported similar results of caries experience and S. mutans levels in Indian populations [23,38].In contrast studies by Dasanayake and Cau�eld (2002) in SriLankan children and Gudkina and Brinkmane. (2008) in Riga failed to �nd positive correlation between caries and salivary S. mutans levels [39,40].
e Streptococcal count in CL/P subjects with DC was higher than the counts in non CL/P subjects with DC.Studies have proposed that the presence of oral cles leads to poor oral hygiene [5][6][7][8][9][10][11]. Presence of �stulae was noted in more than half of the CL/P subjects in the study.Cle subjects with unrepaired �stula are noted to have a poorer oral hygiene status and increased prevalence of caries than fully repaired subjects [7,41].Ahluwalia et al. (2004) proposed that the longer clearance time of food from the oral cavity of subjects with palatal cles was responsible for the high caries prevalence.ough the caries experiences in subjects with and without cles in our study were similar, the Streptococci counts were higher in the cle children.Food substrates for the cariogenic bacteria tend to remain for a longer duration in cle subjects with residual �stulae, due to reasons like nasal regurgitation and food impaction in the �stula.is in turn may probably lead to an increase in colonisation of cariogenic bacteria.Additionally, Turner et al. (1998) suggested that the presence of nasal �uid draining into the oral cavity leads to increased adherence of plaque bacteria onto tooth surfaces.ese factors could possibly explain the higher Streptococcal counts in CL/P subjects in the current study.
Lactobacilli are notable organisms in dental caries and clinical studies have shown positive correlation of Lactobacilli number in the plaque and saliva of caries active individuals [3,4].Available data suggest that in plaque from the smooth surface of the teeth and as well as in plaque beneath which caries is initiated, Lactobacilli constitute generally low or negligible proportion of plaque microbiota.In contrast, Lactobacilli are found in relatively high proportions in cavitated lesions suggesting that their role in dental caries probably lies in progression and in advanced carious lesion rather than in initiation of the disease [42].ough a differentiation of carious lesions was not made in the study on the basis of duration, it is possible that a majority of caries active subjects in the cle and non cle groups may have presented with carious lesions that were in advanced stages.is is because many subjects included in this study were from a rural population and the likelihood that they were exposed to timely treatment strategies is minimal.Additionally, visits to the dentist to address concerns related to dental pain were more oen resolved through extractions.Lack of timely preventive dental strategies and access to basic dental care for the rural population in this study may possibly be responsible factors.
e human mouth is usually sterile at birth but soon acquires a predominantly streptococcal microbiota and with age the microbiota of streptococcal species differentiates .Rogers (1969), isolated 82 streptococcal strains from the mouth of individuals with active caries [43].Mutans streptococci are harbored by virtually all dentate humans, albeit in relatively low proportions compared with other oral streptococci.e biotypes identi�ed were S. mutans and S. sobrinus of which S. mutans were recovered in greater numbers.Additionally, S. sobrinus was not identi�ed in isolation and was always identi�ed together with S. mutans similar to reports of Loesche [44].ough we failed to recover S. cricetus and S. rattus, reports of �nding these strains have also been published [45].
Some of the colonies when subjected to the biochemical tests failed to show categorical results.Hamada and Slade [37] reported the characteristic colony morphologies S. mutans and S. sobrinus form when grown on MSA.Presumptive iden-ti�cation of the colonies on the basis of color size and shape characteristics was done initially prior to biochemical testing.However, lack of conclusive results from these colonies in the biochemical scheme is probably because they did not �t into the MS group, although they were initially labeled as MS on the basis of appearance.In an interesting report [46], So et al. recovered non-MS from one-third of the plaque samples they plated on MSB agar, though it is popularly used as a selective medium.No previous literature exists regarding the predominant biotypes of MS in CL/P subjects and so comparison with other studies is not possible.
Caution should be exercised in extrapolation of the results as differences in microbial growth and enumeration tend to result when different microbial methods are chosen.Also differences in bacterial isolation could result due to differences in dilution of salivary samples, tooth brushing prior to visiting the clinic or repeated swallowing leading to clearing of bacteria from saliva [47,48].

Conclusions
Higher streptococcal and lactobacilli counts were observed in subjects with CL/P and DC than in subjects with DC but no CL/P and the least counts were in the caries free subjects.Streptococcal counts showed strong positive correlation with dm/DMFT whereas Lactobacillus counts were only weakly correlated with dm/DMFT scores in the groups with dental caries.Positive correlation between dm/DMFT scores and streptococci probably reinforces its strong association with DC development.ough there was lack of a clear relationship between levels of particular biotypes and DC, the biotypes of S. mutans and S. sobrinus were found to be overwhelmingly associated with the subjects with DC.

42 F 1 :F 2 :
Correlation of dm/DMFT with Streptococci and Lactobacilli in Group I. Correlation of dm/DMFT with Streptococci and Lactobacilli counts in Group II.

F 3 :
S. mutans S. sobrinus S. rattus S. cricetusBiotypes of MS II I III Others * * * * Inconclusive results from the biochemical tests Biotypes in the different groups.
T 2: Caries and age data of the subjects.
T 3: Mean salivary Streptococci and Lactobacilli counts in the different groups.Bacterial count (CFU/mL saliva * ) Group I mean ± SD Group II mean ± SD Group III mean ± SD