Oral health is generally considered as a mirror of one's general health and sometimes associated with several systemic diseases . Dental caries, commonly known as tooth decay, is the most common oral health problem worldwide and S. mutans is considered to be the primary causative agents of dental caries . S. mutans resides in the dental plaque, a multispecies biofilm community that harbors more than 700 different types of microorganisms . As the biofilm matures, the pioneer colonizers, which are comprised mostly of mitis streptococci, are replaced with early colonizers, such as S. mutans . The ability to establish biofilm lifestyle, production of organic acid and ability to survive at low pH, outstanding ability to outcompete other bacteria by the production of bacteriocin and generation of genetic diversity by natural transformation are attributed as the prime driving force for its ability to adapt and survive in the rapidly changing environment of the oral cavity [7–11, 14]. In this study, we show that the phenotypic and genotypic properties that are associated with the virulence of S. mutans are diverse and vary significantly among 209 newly isolated clinical strains.
We found that types and colony morphology of the isolated strains on mitis salivarius agar vary considerably from patient to patient. We observed multiple strains in the same sample with various colony morphology, which further confirmed that dental plaques indeed contain multispecies biofilm structures and intimate association of all the species are required for causing dental caries.
We also performed AP-PCR of first 40 strains to investigate the genotypic diversity of the isolated strains and found that 26 different genotypes are present among the strains. High levels of genotypic variations were also found previously by several groups [16, 52, 53]. Zhou et al. classified 730 S. mutans isolates into 337 distinct genotypes by AP-PCR fingerprint analysis . In a study with young adults, Emanuelsson et al.  noticed only seven genotypes in subjects who had previously experienced dental caries. Napimoga et al. found eight genotypes in caries-active subjects using AP-PCR . However, it has been reported that children harbor only one to five distinct genotypes of S. mutans . The high prevalence of genotypic variations can be attributed to diversified horizontal gene transfer, various nutritional behavior, and chemical environment in the oral cavity.
As like the genotypic diversity, our phenotypic studies revealed that isolated S. mutans strains have wide variation in phenotypic diversity. Ability to form biofilm, to sustain the growth at low pH and production of acids are considered as key virulence factors in S. mutans and were studied extensively . We observed high variability of these virulence factors among the isolated strains. Some strains displayed better sucrose-dependent biofilm forming capacity than the universal reference strain, UA159 (Fig. 5) and some were crippled in biofilm forming capacity. Biofilm formation capacity of S. mutans is aided by various genes, which encode several surface antigens to attach the teeth surface [56, 57]. The variation in biofilm forming capacity can be due to the presence or absence of various biofilm associated genes, prevalence of polymorphism among these genes and differential epigenetic regulation. We also investigated the presence of biofilm associated gene, gbpA in the clinical isolates, however, this gene was present in all strains.
Acid resistance of S. mutans strains is conferred by the F1FO-H+-translocating ATPase and the activity and optimum pH of this ATPase enzymes are correlated with acid tolerance of oral bacteria . For instance, lactobacilli which are strong aciduric organism exhibit better activity and lower pH optima for the ATPase than the acid-sensitive species, S. sanguinis . Our results suggest that isolated strains have differential response to acid stress. Most of the clinical strains suffered from growth constraints and individual strain exhibited distinct growth kinetics at pH 5.5 (Fig. 3). In addition to acidurity, we also investigated the acidogenesis character of the isolated strains and found noticeable variation among acid production while growing on THY media. However, all the strains could turn the initial medium pH of 8.32 to more acidic pH from 5.02 to 6.54. A large proportion of the clinical isolates displayed better acid production than UA159 although some of the strains were either equal or poor acidogenic as like UA159. Our results showed little variation from a previous study where equal acidogenicity was observed among the S. mutans isolates . The apparent variation in acid production may be due to different methods and growth medium used in the studies. When we assessed the correlation of acid production with status of dental caries, we did not find any correlation between acidogenicity and tooth decay status (data not shown).
Production of bacteriocins to inhibit closely related bacteria are assumed to be important virulence attributes in S. mutans, which encodes several bacteriocin encoding genes to inhibit the growth of various bacteria in vitro . Mutacin IV and V are two important non-lantibiotic bacteria produced by S. mutans to inhibit S. pyogenes, S. gordonii, S. oralis, L. lactis and other streptococci . In this study, we tested the clinical isolates against S. pyogenes and L. lactis by deferred antagonism bacteriocin assay and observed a wide variation in bacteriocin production. Although a minor fraction (11%, and 9.56% respectively) was able to display antagonistic activity, both nlmAB and nlmC genes were present in 63% and 48% of the isolates. This is in agreement with previous studies, which found the mutacin IV encoding nlmA and nlmB genes in 50% of a population of 70 clinical isolates (31) and nlmC was found in 60% of the isolates [14, 25].
Previous genetics and biochemical study indicated that the buildup of a processed form of comC gene product (CSP) results in activation of a two-component system (ComD and ComE), which induces the expression of bacteriocins . In this study, we investigated the presence or absence of comC, comD, and comE genes among the clinical isolates and found that comDE two component systems is present in all the isolates. However, comC was absent in 28% of the strains, which might be due to either absence of this gene in the isolates or presence of different version of this gene which was not amplified by the primer sequences. Previously, it has been found that significant numbers of the sequenced strains either lack the comCDE genes or contained various mutations that could lead to failure to produce functional ComCDE proteins [61, 62].
In this study, we also investigated the distribution of several putative virulence genes with an aim to identify the genetic elements associated with observed phenotypes. Although the presence or absence of the genetic elements tested did not correlate with caries status, their distribution was strongly associated with the virulent phenotypes. For example, strains lacking nlmAB, nlmC or comC genes were unable to display antagonistic activity against the indicator bacteria. In addition, some strains with nlmAB, nlmC or comC genes were also unable to display antagonistic activity against the indicator bacteria. This might be due to the presence of bacteriocin immunity protein, lack of specific target protein in the indicator bacteria or the mutation in bacteriocin transporter or signaling molecules involved in quorum sensing pathway. IN our study we found that most of the strains are genetically homogenous for genes associated with two-component systems which is in accordance with a previous report which reported a significant level of genetic homogeneity among S. mutans strains . However, our results are in contrast with Palmer et al. who reported that wide variations exist among strains of S. mutans in the pathways involved in quorum sensing, genetic competence and non-lantibiotic bacteriocins . Variation in the same species is prevalent in several bacterial species, either by sharing genes by some but not all isolates or by strain-specific genes that are unique to each isolates [25, 63]. In a genome wide comparison, it was revealed that S. mutans strains, UA159 and NN2025, differ in 10% of the genes and 20% of the open reading frames (ORFs) .
The phenotypic and genotypic properties of S. mutans clinical isolates presented here imply that clinical strains have undergone intense evolutionary changes to cope up with the rapidly changing environment in the oral cavity. Further genomics and transcriptomics studies are warranted to get insight into the tremendous variation exists among the S. mutans clinical isolates and their possible interactions with symbiotic and antagonistic neighbors prevalent in the oral cavity. It would also be worthwhile to employ metagenomic approaches to understand the complex architecture of dental caries associated microbiome and their ability to cause tooth decay.