Genotypic Variations of Mutans Streptococci Isolated from Dental Caries by REP-PCR

Mutans streptococci (MS) are a group of oral bacteria considered as the main cariogenic organisms. MS consists of several species of genus Streptococcus which are sharing similar phenotypes and genotypes. The aim of this study is to determine the genetic diversity of the core species of clinical strains of Streptococcus mutans, Streptococcus sobrinus and Streptococcus downei by using repitative extragenic palindromic (REP) primer. The DNA of the clinical strains of S. mutans (n=10), S. sobrinus (n=05) and S. downei (n=04) have been employed in the present study, which have been previously isolated from caries active subjects. The DNA of the clinical and reference strains was subjected to PCR amplification using REP primer. The phylogenetic dendrogram is constructed from the REP PCR banding profile by neighbourjoining method using PyElph 1.4 software. The size of the DNA amplicons generated by using REP primer were S. mutans (1500 bp to 250 bp), S. sobrinus (6000 bp to 250 bp) and S. downei (5000 bp to 400 bp). The results present common band at 480 bp in all the clinical strains of S. sobrinus. The current study is the first to demonstrate the genetic variety of S. sobrinus and S. downei by using REP primer. REP-PCR have been found to be a powerful method to study the molecular diversity of S. mutans, S. sobrinus and S. downei. Additionally, further studies are suggested to analyze the species specific bands and also to find the possibility to produce a new specific primer for S. sobrinus.


Introduction:
Dental caries is ubiquitous and pandemic disease affecting all the age groups of humans. However, due to the fact that dental caries is cumulative process, infected individuals are increased with ageing (1-3). Among adult population, dental caries demonstrating a higher level of severity affecting 5 to 10 teeth per individual (4,5). An extensive and comprehensive National Health Survey performed throughout India revealed that 80 % of the population in the age group 35-44 years old affected by dental caries (6). Mutans streptococci (MS), Streptococcus mutans and Streptococcus sobrinus, are the principal causative agents of the formation of dental caries (7,8). S. mutans is considered as the solitary pioneer cariogenic determinant (7,9). Recent studies validate that the conjoined action of S. mutans and S. sobrinus have intensified the process of oral caries (10). Even though the incident of isolation of S. downei is infrequent, latest report confirmed the isolation of S. downei from caries active subjects (11).
The majority of people harbor MS in their oral cavities; nevertheless, not all acquire dental caries. This leads to the theory that these bacteria are genetically diverse and possess variable virulence prospective. Genetic diversity referred as the number of genotypes present within an organism. The genotyping can illuminate the phenotypic diversity in microorganisms, such as antibiotic resistance, geographic dissemination, host specificity, pathogenicity, transmission and virulence factors (8,12). The genetic diversity can also study the heterogeneity of the MS (13,14) and the possibility to design a vaccine. Many genotypic tools were used in favor of studying the genetic diversity of MS, e.g. pulse field gel electrophoresis (15), arbitrarily-primed PCR (16), multi-locus sequence typing (17) and repetitive elements based PCR (rep-PCR) (18). Rep-PCR amplifying repetitive elements in genomic bacteria to generate genetic markers (19). Repetitive DNA elements are non-coding genes generally located in eubacteria. One of the main types of rep-PCR is repetitive extragenic palindromic (REP) (20). REP consists of 33-40 bp of conserved palindromic sequences (21). REP-PCR produces a highly sensitive, specific and steady gene profile out of nanogram DNA to amplify many unique bands (22,23).
In the previous published studies, we have detected strains of S. mutans, S. sobrinus and S. downei isolated from dental caries active subjects aged between 35 to 44 years (8,11). Insight to this context, the objective of the existing study was focused to investigate the genetic diversity of those species using REP-PCR.

Molecular Identification
All the clinical strains were identified previously (8,11)  The extraction and purification of the DNA have been performed by cetyl trimethyl ammonium bromide method as previously described (24). The DNA amplification was conducted according to the methodology explained by Versalovic et al (25). Amplification was carried out in 25 µl of reaction mixture containing: 5 µl 5x Gitschier buffer [83 mM (NH4)2SO4, 335 mM Tris-HCL (pH 8.8), 33.5 mM MgCl2, 32.5 µl EDTA and 150 mM βmercapto-ethanol], 10 % DMSO, 160 µg/ml BSA, 1.25 mM of each dNTPs, 0.3 µg/ml of each REP primer (REP1R: 5′-IIIICGICGICATCIGGC-3′, REP2I: 5′-ICGICTTATCIGGCCTAC-3′) (Sigma-Genosys, UK), 2 U of DNA polymerase and 50 ng of the each DNA of reference and clinical samples. DNA amplifications were performed in the thermal PCR cycler (G-Storm, UK) using 30 cycles PCR with the following conditions: initial denaturation at 95 ºC for 7 min, initiation 94 ºC for 1 min, annealing 52 ºC for 1 min, extension 65 ºC for 8 min and final extension 65 ºC for 16 min. The final PCR products were resolved in 1.5 % agarose in 1X TAE buffer at 4 ºC for 16 h at 55 V. The PCR genomic fragments were visualized under a UV transilluminator (BioBee, India) followed by digital capturing of the picture using gel documentation system.

Phylogenetic analysis
Phylogenetic analysis based on REP-PCR results was constructed by the neighbour-joining method using PyElph 1.4 software as described by Pavel and Vasile (26). The banding patterns of the clinical strains were also evaluated.

Results:
The genetic banding pattern of S. mutans and S. sobrinus by REP primer is shown in Fig. 1, while the genetic banding pattern of S. downei is shown in Fig. 2. The banding pattern of REP primer in both the reference and clinical strains of S. mutans demonstrated bands ranged from molecular size 1500 bp to 250 bp, while for S. sobrinus from 6000 bp to 250 bp. The banding pattern of REP-PCR in the clinical strains of S. downei demonstrated bands between 5000 bp to 400 bp. REP primer demonstrated its ability to generate bands for all the tested species which can refer as a strong tool for genetic diversity. Figure 1 revealed the presence of common bands in the strains of S. mutans and S. sobrinus at the molecular weights 1500 bp, 1300 bp, 1100 bp, 880 bp, 750 bp and 250 bp. All the clinical strains of S. sobrinus have characteristic band at molecular weights 6000 bp, 5000 bp, 4000 bp and 480 bp. While, clinical and reference strains of S. mutans lacked bands at the same molecular weights. All the strains of S. downei showed the presence of monomorphic bands at molecular weights 1500, 1400 and 1100 bp (Fig. 2). The data suggest that these monomorphic bands can be further analyzed and used as a species specific primers for S. sobrinus and S. downei. The results also revealed the absence of bands at different The results of the phylogenetic dendrogram inferred from REP-PCR banding pattern using the neighbour-joining method is presented in Fig. 3 for S. mutans and S. sobrinus, and in Fig. 4 for S. downei. Two groups of S. mutans were clustered with the strains of S. sobrinus. Strains number H5 and H17 of S. mutans were genetically close to S. mutans ATCC 25175 and S. mutans MTCC 497.
While, reference strain of S. sobrinus is clustered with other strains of the same species. In S. downei, the genetic distance of strain number H62 was far than the rest of the other strains of the same species.   The dendrogram constructed by neighbourjoining method using PyElph 1.4 software. The genetic distances are demonstrated above the branches.

Discussion
Due to the fact that S. mutans and S. sobrinus are the prime agents of dental caries (7,8), the genetic diversity of the representative clinical strains of S. mutans and S. sobrinus was studied using rep-PCR with REP primer. However, clinical strains of S. downei were recently isolated from caries active subjects (11). As per literatures survey, this is the first study to determine the genetic diversity of S. sobrinus and S. downei by using REP primer.
REP-primer employed in the present study generated bands for all the strains of S. mutans, S. sobrinus and S. downei (Figs. 1 and 2). This is in contrary to the previous report stating that REP primer did not produce any amplicons with S. mutans and S. sobrinus (27). The reason for this might be due to the variation of the primers employed or the heterogeneity of the strains. Moreover, no indication of banding patterns among alpha hemolytic streptococci was observed by using REP primer (28). However, our results are in agreement with the previous reports stating that rep-PCR with different primers i.e. REP, ERIC, SERE, and BOX are reproducible, diverse among strains and appropriate for strain-typing (22,29).
Among the bands generated by REP primer, bands at 1500 bp, 1300 bp, 1100 bp and 250 bp were common in both S. mutans and S. sobrinus. Moreover, similar bands were observed between S. mutans, S. sobrinus and S. downei at molecular weights 1500 bp, 1400 bp and 1100 bp (Figs. 1 and  2). All the clinical strains of S. sobrinus had a specific bands at 6000 bp, 5000 bp, 4000 bp and 480 bp. Thus, these bands could be further studied to generate species-specific primer for identification of S. sobrinus. Recent studies reported the genotypic, diversity, commonality, and stability of the S. mutans using REP-PCR (30,12). In few strains, bands were missing; the reason for this may be attributed to different serotypes. The present results indicate that the clinical strains of both S. mutans and S. sobrinus were more diverse compared with the reference strains (Fig. 1).
The correlation among species was well presumed from the banding pattern dendrogram. Phylogenetic trees reconstructed by the neighbourjoining method, established the genetic settlement of representative strains of MS. The reference strains of S. mutans and S. sobrinus are assembled with its own strains (Fig. 3). Clinical strains of S. downei H45 and H50 are genetically related and clustered together with strains no. H47 and H62 (Fig. 4). The genetic distance of strain number H62 was attributed to the variation in gene composition among the members of MS. Genetically, MS species are closely related to each other, in particular, S. sobrinus, S. downei and S. mutans (8,31).

Conclusion:
The findings presented herein show the usefulness of REP-PCR to study the diversity and genotypic of S. mutans, S. sobrinus and S. downei. However, the present study recommend more research to find the possibility to produce genetic markers for MS species.