Molecular characterization of cariogenic and non-cariogenic Streptoccoi species in dental plaque biofilm using 16S-23S rRNA gene spacer region amplification

Abstract: Interspecies competitive exclusion of microbial diversity explained using 16S-23S rRNA gene spacer region amplification .Virulent and non-virulent strains of streptococci strain were isolated from dental plaque biofilm for analysis. DNA isolation done from plaque strains from dental biofilms of caries-active and caries-free subjects, respectively .Two pairs of primers were designed from the 16S–23S rRNA gene spacer region to amplify the variable and species-specific non-coding regions. The results revealed that the ITS lengths (246 to 391 bp) and sequences were highly conserved among strains within a species. The precision of utilising intergenic spacer sequencing for recognisable proof of viridian group streptococci was checked by 16S rDNA sequencing for all strains apart from strains of Streptococcus oralis and Streptococcus mitis, which were hard to separate by their 16S rDNA arrangements. Comparative sequence analysis of the 16S-23S rRNA spacer region sequences revealed that not only Streptococcus mutans, but also Streptococcus salivarius, Streptococcus oralis and Streptococcus mitis play equal role in oral disesase progression as compare to Streptococcus mutans which is reported in previous studies.

KEY WORDS : 16S-23S rRNA, Streptococcus oralis , Streptococcus mitis, Dental plaque,Biofilm.

Introduction
Genetics and molecular basis for virulence in oral disease analysed using the 16S r RNA gene  sequencing technique. Application of 16S r RNA gene  sequencing technique to study     the   oral   microbiome   will   continue   to contribute to our knowledge of the oral microbiome, and with new technologies, the depth and breadth of the information collected should be remarkable [1].  In a study it is  revaled that the  species that were previously thought to be associated primarily with health were also elevated in caries-active individuals, leading the authors to   conclude   that   “the   relationship   of   acid-base metabolism   to   16S   r   RNA   gene-based   species assignments appears to be ‘complex’G’’ 30 [2].  Another reason   why   the   16S   sequence   alone   appears inadequate to predict association of particular bacetria with   oral   diseases   is   related   to   the   concept   of phenotypic   plasticity. To explore  the relationship  of  Genotype  and Cariogenic Potential  in  Streptococcus  mutans  numerous  studies have demonstrated  [3,4].  Biofilm in the form of supragingival and subgingival plaque is the etiologic agent in dental caries and periodontal diseases. The ability of bacteria to survive and persist in a given environment will depend, in part, on their inherent genetic plasticity, which determines their ability to respond to fluctuating local environmental condition or stresses [5]. 
Dental caries is a transmissible infectious disease in which Streptococcus mutans plays the major role. As in many infectious diseases, colonization by pathogens is required before the disease can occur. Streptococcus mutans are generally considered to be the principal etiological agent of dental caries [6].There is a range of virulence factors important for the establishment of Streptococcus mutans in the complex microbial community of dental biofilm. Studies of the virulence factors of Streptococcus mutans and their correlation with species biodiversity are fundamental to understanding the role played by colonization by different genotypes in the same individual, and the expression of characteristics that may or may not influence their virulence capacity and survival ability under different environmental conditions. Studies using phenotyping and/or genotyping methods strongly suggest that the mother is the major primary source of infection for children who carry S.mutans and/or S.sobrinus strains (2-10) and the saliva is the principal vehicle by which transfer of Streptococcusmutans may occur [7]. examination of the genetic structure of  Streptococus mitis biovar 1 in its natural habitat in the human oral cavity and pharynx, and  investigated  the role of  selected microbial properties (IgA1 protease production and the ability to bind salivary α-amylase) and host, spatial, and temporal factors play in determining the structure of the bacterial population, they reported the  Genetic diversity in nine structural gene loci by MLEE analysis of 19 and 13 isolates representing different REA types of Streptococcus mitis biovar 1 isolates from two individuals, compared to the diversity in the two collections combined [8]  .In previous finding it is demonstrated tht Streptococcus mutans, the microbial species most strongly associated with dental caries [3,9,10,12,13].
Material and methods 
DNA isolation from plaque strain from dental biofilm of caries active and caries free subject respectively.following the who norms for dental plaque sample collection and isolation. DNA isolation was carried out by using Cetyltrimethylammonium bromide (CTAB) method of DNA extraction [14,15]. The cultured single colony was centrifuged at 12500 rpm for 2 min, and the supernatant was discarded. The bacterial cells pellets were washed twice using Tris EDTA (TE) buffer. A 600 µl of CTAB buffer was added to the bacterial pellet, and the entire solution was kept in a heat bath at 65 °C for 30 min to ensure lysis of cell walls. Afterwards, 600 µl of a Chlorophorm: Isoamyl Alcohol solution (24:1, v/v) was added and mixed thoroughly by inverting 20-30 times. The mix was centrifuged at 12500 rpm for 7 min. Supernatent above the interphase was transferred to fresh tube to which 2.5 parts of cold 100% Ethanol was added. All the components were mixed thoroughly by inverting tubes and incubated at -20 °C for 30 min. Nucleicacids were allowed to pellet down as precipitate by centrifuging the mixture at 12500 rpm for 20 min at 4°C. Finally, the isolated nuclec cids were washed with 70% Ethanol. The pellet was air dries at 37°C until all the alcohol content evaporates and dried pellet was resuspended and dissolved in 50 μl of nuclease free water and stored at – 4°C for downstream PCR analysis.

Polymerase Chain Reaction Setup
Species of bacterial isolates were identified by the combined use of universal primers for 16S-23S rRNA gene spacer amplification (RSA), and sequencing of the amplicons. We used Phusion high fidelity DNA polymerase (Thermo Scientific) to ensure the high fidelity of the amplicons. PCR reactions were conducted in a final volume of 20 µl containing: 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs each, 0.5 µM primer each, 0.02U/µl reaction of Phusion DNA polymerase supplemented with 1 µl of bacterial genomic DNA.

Thermal program for PCR reaction was kept as below; 
98°C -3 min
98°C -15 sec
58°C -20 sec
72°C -30 sec, from step 2, 35 cycles
72°C - 5 min 
4°C - hold”  (Gene Amp 9600, Perkin-Elmer, Norwalk, USA).

  Primer Design
 Two pairs of primers were designed from the 16S-23S rRNA Gene spacer region to amplify the variable and species specific non-coding regions. First pair contains two primers i.e. G1 (GAAGTCGTAACAAGG) and L1 (CAAGGCATCCACCGT) was prepared according to Jensen et al. (1993), which is to investigate the diversity of bacterial species. Second primer pair ITSF (GTCGTAACAAGGTAGCCGTA) and ITSR (GCCAAGGCATCCACC) are complimentary to 1443 position of 16S rRNA and 23 position of 23S rRNA of E. coli and used to analyze microbial diversity of soil samples [16].

RESULT AND DISCUSSION
Molecular Analysis of strain isolated from caries active and caries free subject

To study the microbial diversity of virulent strain isolated from dental plaque biofilm of caries active and caries free subject, the following molecular test conducted. Bacterial strains used in this study were- Strains C1 - C2 isolated from dental plaque biofilm from caries active subject and strain CF1 - CF2 isolated from dental plaque biofilm from caries free subject . Colony selection done by focusing on maximum percentage of strain recovered on mitis salivarius agar by conventional culture technique.
Isolated strain from caries active and caries free dental plaque biofilm sample were identified by morphological characteristics and biochemical identification method, further strain recovered maximum in number were selected for microbial characterization. In the present examination, we used a PCR strategy to recognize bacterial strain of known morphology and biochemical charecterisctics from dental plaque biofilm sample of caries active and caries free subject.

Figure 1 PCR assay by Phusion high fidelity DNA polymerase (Thermo Scientific). Gel profile of PCR products using the Caries avtive (CR1 and CR2) and Caries free (CF1 and CF2) bacterial sample from dental plaque biofim and template and two pairs of primers used to amplify 16s rRNA and 23 position of 23S rRNA of species, M , Molecular size maker (100 bp DNA ladder, Gene direx company). bands correspond to ~350 bp for sequencing.

PCR techniques with preliminaries built dependent on the 16S rRNA arrangement are broadly used for their fast and delicate recognition of bacteremia [17]. The PCR of present investigation as shown in Fig 1 , illustrates clear full reaction by using the two pairs of primers which were designed from the 16S-23S rRNA Gene spacer region to amplify the variable and species specific non- coding regions. The result yielded amplicons of expected sizes , reaction stored with proofreading enzyme and sended for sequencing.

Comparative sequence analysis of the 16S-23S rRNA spacer region sequences

Purified PCR amplicons were sent for sequencing with ITSF and 1443F primers in a single pass reaction from Eurofins. The obtained sequences were trimmed for bad-quality mixed reads from both the ends in SeqMan tool of DNA STAR (Fig. 2). 

Fig. 2: Chromatogram showing good and bad quality reads in raw sequences
The high-quality sequences were aligned in a local CLUSTAL-W alignment tool of BioEdit programme with selecting “full multiple alignment” and “bootstrap NJ tree” options (Fig. 2). Fully aligned sequences were checked for variations among two replicates like C1/C2 and CF1/CF2 with both primers, separately.
 In these, presence of SNP’s/indels were examined manually, which was further confirmed with base calling values in chromatogram (.abi) files. It is obvious, that single pass sequences would show ambiguities in base calling. Thus, we modified only those SNP’s/Indels which are present among replicates and showed bad quality in the chromatogram. Indels which are present among two samples i.e. Caries (C) and Caries Free (CF) were considered as true differences and further used in downstream analysis.   

The conserveness of blocks of 13, 20, 24 and 27 bases from 1-13, 52-77, 107-131 (Red underlined) and 159-186 nt position, indicates that all the samples are originated from same bacterial genera (Fig. 3). Presence of variations of >1 base was considered as indels that indicate that sequences are originated from a minimum of two different species (Fig. 3). Like, sequences of C1 and C2 samples are originated from one species and sequences of CF1/CF2 samples are originated from another species. 
From NCBI BLAST-N analysis, we identified that the species representing two samples are Streptococus salivarius, Streptococcus oralis and Streptococcus mitis (Fig. 3and 4). BLAST-N alignment for high similarity search indicates that the samples collected from caries Free (CF) mouth cavity belongs to S. salivarius with high identity (97%) and E-value (9e-152) (Fig. 3A). Few Gaps and SNPs are visible in alignment due to strain specific variations (Fig. 3A and B). 

A

B

Fig. 3  BLAST-N analysis of the sequence obtained from Caries Free (CF) sample (A) and number of hits to the top hit species/strains (B)

On the contrary, sample collected from the caries containing moth cavity have high similarity to S. oralis and S. mitis with high identity (98%) and E-value (2e-138) (Fig. 4 A and B). Similarly, gaps and SNPs are visible indicating variations among strains (Fig. 4A-C). 

A

     B

C

Fig. 4  BLAST-N analysis of the sequence obtained from Caries (CF) containing sample (A and B) and number 
of hits to the top hit species/strains (C)

The finding of present studies were in full agreement with the study in which it is reported that the application of 16S r RNA gene  sequencing  to analysis   of   the   oral   microbiome   will   continue   to contribute to our knowledge of the oral microbiome, and with new technologies, the depth and breadth of the information collected should be remarkable [18]. Earlier finding demonstrated the natural   transformation   of   Streptococus strains that    have   allowed investigators to analyze the functions of many genes in particular concern strain that are isolated from specific sources . Induction of genetic competence in these streptococci is mediated by quorum sensing, which depends on a competence stimulating peptide (CSP) signaling system [19]. 

 In previous finding it is demonstrated that Streptococcus mutans, the microbial species most strongly associated with dental caries [3,9,10]. Similarly , Streptococcus mutans, the microbial species most strongly associated with carious lesion, is naturally present in the human oral microbial flora [2]. The result of present finding is in full agreement with [21] who assayed and demonstrated the cariogenic property of Mutans streptococci that they were not among the aciduric isolates. Subjects without root caries harbored less microscopic organisms, and Streptococcus anginosus (pH 4.8) and Streptococcus oralis (pH 5.2) were the prevalent aciduric microscopic organisms as compare to s.mutans. The result of the present studies was also in the agreement with Chen et al., (2004) who evaluated the attainability of sequence analysis of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (ITS) for the identification and demonstration of clinically applicable viridans group streptococci
is solid and could be utilized as an option exact strategy for analyzing of streptococci viridans group.
 Comparative sequence analysis of the 16S-23S rRNA spacer region sequences revealed that not only Streptococcus mutans but also Streptococcus salivarius and Streptococcus mitis play equal role in oral disesase progression as compare to  Streptococcus mutans which is reported in various studies. 
Conclusion: Comparative sequence analysis of the 16S-23S rRNA spacer region sequences revealed that not only Streptococcus mutans but also Streptococcus salivarius and Streptococcus mitis play equal role in oral disease progression as compare to Streptococcus mutans which is reported in various studies. Current findings align strongly with previous studies, confirming that 16S rRNA gene sequencing remains a robust and expanding tool for characterizing the oral microbiome. Studies also substantiate the complex role of Streptococcus mutans in dental caries. Study demonstrate—that S. mutans, S. salivarius, and S. mitis are all implicated in oral disease progression, and that viridans group streptococci can be effectively differentiated using 16S–23S ITS sequencing—supports and advances this existing body of work. In particular, it verifies the utility of high-resolution molecular techniques for identifying functional contributors to oral disease, beyond the classic focus on S. mutans alone.
Disclaimer (Artificial intelligence)
Option 1: 
Author(s) hereby declare that NO generative AI technologies such as Large Language Models (ChatGPT, COPILOT, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. 
Option 2: 
Author(s) hereby declare that generative AI technologies such as Large Language Models, etc. have been used during the writing or editing of manuscripts. This explanation will include the name, version, model, and source of the generative AI technology and as well as all input prompts provided to the generative AI technology
Details of the AI usage are given below:
1.
2.
3.

REFERENCES:
1.	Ranjan, R., Rani, A., Metwally, A., McGee, H. S., & Perkins, D. L. (2016). Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing. Biochemical and Biophysical Research Communications, 469(4), 967–977. https://doi.org/10.1016/j.bbrc.2015.12.083
2.	 Bowman, J. S., & Ducklow, H. W. (2015). Microbial communities can be described by metabolic structure: A general framework and application to a seasonally variable, depth-stratified microbial community from the coastal West Antarctic Peninsula. PLoS ONE, 10(8), e0135868. https://doi.org/10.1371/journal.pone.0135868
3.	Agnello, M., Cen, L., Shokeen, B., He, X., Shi, W., Wong, D. T. W., Lux, R., & Tran, N. C. (2022). Oral microbiome: Streptococcus mutans/caries concordant-discordant children. Frontiers in Microbiology, 13, 782825. https://doi.org/10.3389/fmicb.2022.782825
4.	Butler, R. R., Soomer-James, J. T., Frenette, M., & Pombert, J. F. (2017). Complete genome sequences of two human oral microbiome commensals: Streptococcus salivarius ATCC 25975 and S. salivarius ATCC 27945. Genome Announcements, 5(24), e00536-17. https://doi.org/10.1128/genomeA.00536-17
5.	 Benítez-Páez, A., Belda-Ferre, P., Cabrera-Rubio, R., & Mira, A. (2014). Streptococcus dentisani sp. nov., a novel member of the mitis group. International Journal of Systematic and Evolutionary Microbiology, 64(Pt_1), 60–65. https://doi.org/10.1099/ijs.0.052548-0
6.	 Russell, S. L., Ickovics, J. R., & Yaffee, R. A. (2010). Parity and untreated dental caries in U.S. women. Journal of Dental Research, 89(10), 1091–1096. https://doi.org/10.1177/0022034510375292
7.	Napimoga, M. H., Kamiya, R. U., Rosa, E. A., Rosa, R. T., Hirata, R. D. C., & Hirata, M. H. (2005). Transmission, diversity and virulence factors of Streptococcus mutans genotypes. Journal of Oral Science, 47(2), 59–64. https://doi.org/10.2334/josnusd.47.59
8.	  Hohwy, J., Reinholdt, J., & Kilian, M. (2001). Population dynamics of Streptococcus mitis in its natural habitat. Infection and Immunity, 69(10), 6055–6063. https://doi.org/10.1128/IAI.69.10.6055-6063.2001
9.	Baron, E. J. (1996). Microbiology of dental decay and periodontal disease. In Medical Microbiology (pp. 99–110). University of Texas Medical Branch at Galveston.
10.	Cornejo, O. E., Lefébure, T., Bitar, P. D., Pavinski Bitar, P. D., Lang, P., & Richards, V. P. (2013). Evolutionary and population genomics of the cavity-causing bacteria Streptococcus mutans. Molecular Biology and Evolution, 30(4), 881–893. https://doi.org/10.1093/molbev/mst003
11.	 Alsaimary, I. E. (2008). Efficiency of some antibacterial agents against Streptococcus mutans associated with tooth decay. African Journal of Biotechnology, 8(2), 219–222
12.	 Agnello, M., Cen, L., Shokeen, B., He, X., Shi, W., & Tran, N. C. (2022). Oral microbiome: Streptococcus mutans/caries concordant-discordant children. Frontiers in Microbiology, 13, 143. https://doi.org/10.3389/fmicb.2022.782825
13.	  Wood, D. N., Weinstein, K. E., Podbielski, A., Kreikemeyer, B., Gaughan, J. P., Valentine, S., & Buttaro, B. A. (2009). Generation of metabolically diverse strains of Streptococcus pyogenes during survival in stationary phase. Journal of Bacteriology, 191(20), 6242–6252. https://doi.org/10.1128/JB.00440-09
14.	  Medina, E., Goldmann, O., Toppel, A. W., & Chhatwal, G. S. (2003). Survival of Streptococcus pyogenes within host phagocytic cells: A pathogenic mechanism for persistence and systemic invasion. The Journal of Infectious Diseases, 187(4), 597–603. https://doi.org/10.1086/373998
15.	Tel-Zur, N., Abbo, S., Mizrahi, Y., & Propis, E. (1999). Modified CTAB procedure for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus (Cactaceae). Plant Molecular Biology Reporter, 17, 249–254. https://doi.org/10.1007/BF0277213
16.	 Petersen, F. C., Tao, L., & Scheie, A. A. (2005). DNA binding-uptake system: A link between cell-to-cell communication and biofilm formation. Journal of Bacteriology, 187(13), 4392–4400. https://doi.org/10.1128/JB.187.13.4392-4400.2005
17.	 Cardinale, M., Brusetti, L., Quatrini, P., Borin, S., Puglia, A. M., Rizzi, A., Zanardini, E., Sorlini, C., Corselli, C., & Daffonchio, D. (2004). Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities. Applied and Environmental Microbiology, 70(10), 6147–6156. https://doi.org/10.1128/AEM.70.10.6147-6156.2004
18.	Clarridge, J. E., III. (2004). Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clinical Microbiology Reviews, 17(4), 840–862. https://doi.org/10.1128/CMR.17.4.840-862.2004
19.	 Sousa, V., Diniz-Freitas, M., Gallas-Torreira, M., Limeres, J., & Diz, P. (2017). Peri‐implant and periodontal microbiome diversity in aggressive periodontitis patients: A pilot study. Clinical Oral Implants Research, 28(5), 558–570. https://doi.org/10.1111/clr.12854
20.	  Marchant, S., Brailsford, S. R., Twomey, A. C., Roberts, G. J., & Beighton, D. (2001). The predominant microflora of nursing caries lesions. Caries Research, 35(6), 397–406. https://doi.org/10.1159/000047483
21.	Paddick, J. S., Brailsford, S. R., Kidd, E. A. M., Beighton, D., & Tickle, M. (2003). Effect of the environment on genotypic diversity of Actinomyces naeslundii and Streptococcus oralis in the oral biofilm. Applied and Environmental Microbiology, 69(11), 6475–6480. https://doi.org/10.1128/AEM.69.11.6475-6480.2003
22.	Lal, D., Verma, M., & Lal, R. (2011). Exploring internal features of 16S rRNA gene for identification of clinically relevant species of the genus Streptococcus. Annals of Clinical Microbiology and Antimicrobials, 10, 28. https://doi.org/10.1186/1476-0711-10-28
23.	Ucuncu, M. Y., Ucuncu, M. K., Karacan, I., & Topcuoglu, N. (2024). Genome resequencing and comparative analysis of Streptococcus mutans isolates obtained from patients with high and low caries risk. Figshare. https://doi.org/10.6084/m9.figshare.25866511.v1
24.	Alanjary, M., Steinke, K., & Ziemert, N. (2019). AutoMLST: An automated web server for generating multi-locus species trees highlighting natural product potential. Nucleic Acids Research, 47(W1), W276–W282. https://doi.org/10.1093/nar/gkz276
25.	Bedoya-Correa, C. M., Rodríguez, R. J. R., & Parada-Sánchez, M. T. (2019). Genomic and phenotypic diversity of Streptococcus mutans. Journal of Oral Biosciences, 61(1), 22–31. https://doi.org/10.1016/j.job.2018.11.001
26.	Baker, S. P., Nulton, T. J., & Kitten, T. (2019). Genomic, phenotypic, and virulence analysis of Streptococcus sanguinis oral and infective-endocarditis isolates. Infection and Immunity, 87(1), e00703-18. https://doi.org/10.1128/IAI.00703-18