Genotypic and phenotypic characterization of Streptococcus mutans isolated from dental caries

Streptococcus mutans, considered as principal causative agent of dental caries, maintains a biofilm lifestyle in the dental plaque. The oral cavity harbors numerous S. mutans strains, which displayed remarkable genotypic and phenotypic diversity. This study evaluated the genotypic and phenotypic diversity of 209 S. mutans strains isolated from 336 patients with dental caries and compared with the universal reference strain UA159. Our study has revealed a high degree of genotypic and phenotypic variability among the clinical strains. We observed significant differences in colony morphology, generation time, biofilm formation, bacteriocin and acid production while growing in culture medium. All the clinical isolates were able to lower pH while growing in THY broth. In consistent with phenotypic variations, we also observed tremendous level of genotypic variation by AP-PCR and gene specific PCR. AP-PCR analysis suggested that most of the patients with dental caries have distinct type of S. mutans strains. Genes related to various two component systems were highly conserved among the strains, however, bacteriocin encoding genes such as nlmAB, nlmC were absent in half of the clinical isolates. In sum, our study highlights the genotypic and phenotypic diversity of S. mutans clinical isolates and indicates the presence of diverse mechanism to initiate and establish the biofilm lifestyle which leads to tooth decay.

Introduction important for S. mutans, which is naturally competent bacterium and therefore has the potential 80 for rapid genome diversification through horizontal gene transfer (48). In a previous study, Palmer The aim of the present study was to investigate the genotypic and phenotypic heterogeneity 86 of S. mutans isolates from 336 patients with dental caries from Bangladesh. We found that S. 87 mutans strains isolated from dental caries have high level of genotypic and phenotypic 88 heterogeneity.   Typing of clinical isolates by AP-PCR. 119 The genetic diversity of S. mutans isolates was analyzed by arbitrarily primed PCR (AP-120 PCR) by using the primers set OPA 02 (5'-TGCCGAGCTG-3') and OPA 13 (5'-121 CAGCACCCAC-3') as described previously (21 (Table-4). In addition to the strains being tested, purified genomic 132 DNA from S. mutans UA159 was used as a positive control and distilled water was used as a 133 negative control in each PCR. The PCR products were analyzed by electrophoresis in a 1.5% 134 agarose gel. gyrA gene was used as an internal control.

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Biofilm assay 136 For biofilm assay, overnight grown bacteria in THY broth were diluted 1:20 and inoculated 137 into fresh THY medium supplemented with 1% sucrose into wells of polystyrene flat-bottom 24-138 well microtiter plate and incubated for 48-hr at 37°C in microaerophilic condition. After 139 incubation, the culture medium was decanted, and the wells were washed thrice with distilled water 140 and stained with 1% crystal violet for ten minutes. The plates were further washed with distilled 141 water twice to remove the unabsorbed dye. The cells were then resuspended into 1-ml 95% ethyl 142 alcohol and absorbance was taken at 550 nm. Each experiment was performed in triplicates.

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While growing on sugar, S. mutans produces acid which, in turn, reduces the pH that causes 150 tooth decay (17). In order to investigate the acid production capacity of the clinical isolates, 151 overnight grown S. mutans culture was inoculated in 10 ml of THY broth (pH 8.32) and the pH 152 was determined at different time intervals (0-hr, 24-hr, 48-hr and 72-hr) using a pH meter. Each 153 experiment was performed in duplicate.

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Deferred antagonism bacteriocin assay 155 To investigate the bacteriocin production by the clinical isolates, isolated colonies were 156 stabbed into THY agar plates with a toothpick and grown overnight (~18-hr) at 37°C under 157 microaerophilic conditions. Indicator strains were grown to mid exponential growth phase in THY around the mutacin-producing strains was measured. The isolates were recorded as bacteriocin 162 producer against the indicator bacteria if the zone of diameter was 5-mm or greater.

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Dental health analysis of diabetes 165 The DMFT (decay-missing-filled-Teeth) index is widely used to assess the epidemiology 166 of dental caries status.  To investigate the growth kinetics of various clinical isolates, we performed the growth 207 curve analysis for 12 hours in compare to the reference strain UA159. We observed noticeable 208 variation in growth rate in some strains, however, most of the strains grew at similar rate as like 209 UA159 (Fig 2a). Some of the isolates demonstrated very slow growth rate with long dividing time 210 (>150 minutes) and took three to four days to have distinct colony on the agar plate at 211 microaerophilic condition. Similarly, final growth at OD630 after 24-hour incubation was also less 212 for the slow grower (Fig 2b). Growth pattern of the UA159 was in the middle among the isolated 213 strains (dividing time 57 minutes and final growth at OD630 was 1.768).    (Fig 3). Some strains grew faster than UA159.  (Table 2   247 and Fig 4) and turned the initial pH of the media from 8.34 to more acidic pH (up to pH 5.0). Most 248 of the clinical isolates displayed better acid production ability than the reference strain, UA159 249 which has turned the THY broth from pH 8.34 to pH 6.25.  (Table 2 and Fig 5).  where serotype c was shown to be most prevalent S. mutans in the oral cavity (7, 56). 304 We also performed AP-PCR of 40 selected strains to investigate the genotypic diversity of  (63, 64). In another study, Nakano et al. also found that 328 gbpA gene is absent in some S. mutans strains (65). We also investigated the growth kinetics of 329 the isolated strains (Fig 2) and found a wide variation in growth kinetics pattern among the strains.  (Fig 3). In addition to acidurity, we also investigated the acidogenesis character of the isolated 337 strains and found noticeable variation among acid production while growing on THY media.

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However, all the strains could turn the initial medium pH of 8.32 to more acidic pH from 5.02 to incubation. When we assessed the correlation of acid production with status of dental caries, we 346 did not find any correlation between acidogenecity and tooth decay status (data not shown).

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Production of bacteriocins to inhibit closely related bacteria are assumed to be important virulence In this study, we tested the clinical isolates against S. pyogenes and L. lactis by 352 deferred antagonism bacteriocin assay and observed a wide variation in bacteriocin production.

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Although a minor fraction (17%, and 12% respectively) was able to display antagonistic activity, 354 both nlmAB and nlmC genes were present in majority of the isolates (63% and 48% respectively).  respectively in our (Table 3). This is in agreement with previous studies, which found the mutacin 397 IV encoding nlmA and nlmB genes in 50% of a population of 70 clinical isolates (31) and nlmC 398 was found in 60% of the isolates (19,29). We did not find any cnm genes, encodes the collagen-399 binding protein, among the isolated strains, which is inconsistent with a previous report where cnm 400 gene was not detected in a collection of 33 clinical isolates (29). However, two recent studies