Identification of a novel bacterium Scardovia wiggsiae in high caries risk adolescence: A metagenomic and melt curve analysis.

Background: Mutans streptococci which comprise only 2% have long been presumed to be the specific pathogen responsible for caries. A novel caries associated bacterium namely Scardovia wiggsiae is recognized to be ecologically competitive in active caries lesions. The actual pathogen needs to be identified, so as to specifically target and reduce the prevalence of caries in a given community. Aim: The aim of the study is to evaluate the presence of S. wiggsiae in combination with other bacteria in caries risk adolescence.
Methods: Sixty adolescent subjects were screened. Phase I-to determine the prevalence of S. wiggsiae in saliva, plaque and dentinal caries samples of low and high caries risk individuals (n = 30 each) using polymerase chain reaction (PCR); Phase-II-to identify its presence by 16SrRNA metagenomic analysis and quantitatively evaluate the cariogenic pathogen using high-resolution melt curve analysis and real-time PCR.
Results: Highest prevalence of S. wiggsiae was observed in dentinal caries followed by plaque and saliva samples of high caries risk individuals under PCR analysis. Metagenomic analysis showed two-fold statistically increased presence of Lactobacilli and Bifidobacteriaceae (S. wiggsiae) in dentinal samples compared to plaque samples (P = 0.05). Mutans streptococcus recorded the minimum.
Conclusion: Scardovia wiggsiae is identified as one of the predominant microorganism. Copyright:

INTRODUCTION

Oral diseases unlike other infectious diseases, are generally associated with complex microorganisms.[1] An unprecedented increase in prevalence and severity of dental caries (DC) is being experienced in most of the developing countries for the past two decades.[2] An extensive survey by National Health, conducted in 2004 throughout India observed the prevalence of DC to be 51.5% in 5-year-old children, 53.8% in 12-year-old children and 63.1% in 15-year-old teenagers. (National Oral Health Survey and Fluoride Mapping, 2004).[3] Further, it was also observed that DC was always higher in children living in urban and cosmopolitan places due to increased demand for caloric requirements and simultaneous decrease in professional and maternal care in oral hygiene practices.[456]

There has been a controversial research reports regarding actual major pathogens causing DC. Much research has identified the major pathogen of DC to be Mutans streptococci.[7] However, Van Houte et al.,; Sansone et al., found that M. streptococci may persist on tooth surfaces without development of lesion and is not necessarily high in caries associated biofilms.[89] Unexpectedly, Mutans streptococcus comprise only 2% or less of the initial population, irrespective of the caries activity. Rather nonmutans aciduric and acidogenic bacteria namely Lactobacillus and Bifidobacterium have been proposed to co-prevail within the niche area such as in plaque and dentinal caries.[10111213] This concept is being strengthened by recent molecular analyses showing that the microflora associated with white spot lesions (WSL) and early childhood caries (ECC) is more diverse and the novel phylotypes and species including Streptococcus sobrinus, Streptococcus vestibularis, Streptococcus salivarius; Lactobacillus; Bifidobacterium dentium; and Scardovia wiggsiae may also play a role.[141516]

Given these circumstances the specific, nonspecific and ecological plaque hypothesis by Loesche, Theilade, and Marsh, have been reconsidered; with Takahashi and Nyvad, proposing the “extended caries ecological hypothesis” which explains the relationship between acidogenic and aciduric shifts in the composition of dental biofilm and the change in the mineral balance of the dental hard tissues.[17181920] Acidification of biofilm can be a driving force for Lactobacilli and Bifidobacterium than M. streptococci, to colonize and proliferate in caries lesions. Further, extensive research resulted in isolation of 197 Bifidobacteria strains constituting 73.3% and 68.4% in plaque and dentinal caries; confirming plaque and dentinal caries to possibly constitute a habitat for Bifidobacteria.[21] This microbial acid-induced adaptation (phenotypic) and selection (genotypic) disturbs the demineralization/remineralization balance from “net mineral gain” to “net mineral loss” and initiate lesion development.

Later Tanner et al., (2011a, 2011b, 2012) during their research on microbiota of ECC, uncovered an extraordinary diverse ecosystem and reported a new predominant caries pathogen namely S. wiggsiae (S. wiggsiae) which is evident in higher level, even in the absence of S. mutans.[101415] Scardovia (Scar. do’vi. a. N. L. fem. n. named after Vittorio Scardovi, an Italian microbiologist who has made many contributions to our knowledge of Bifidobacteria) (Jian and Dong, 2002); wiggsiae (wiggs’i. ae. N. L. fem. gen. n. named after Lois Wiggs, American microbiologist, for her contributions to anaerobic microbiology) is a gram-positive, anaerobic, nonspore forming and nonmotile bacilli.[22] S. wiggsiae is one of the 7 genera of Bifidobacteriaceae family. It is a new bacterial genus and was removed from the genus Bifidobacteriaceae in 2002 due to difference in its genome sequence.[2324] It is existing as 195th human oral taxon in Human Oral Microbiome Database (HOMD).[25] Concurrently with these changes in the interpretation of the microbial etiology of caries, novel concepts have evolved around the caries itself suggesting, it is necessary to establish its prevalence in a given community.

We believe that its paramount to understand and recognize the diverse microbiota and identify the actual predominant genera in the microbial communities causing caries, such that, preventive strategies can be targeted specifically to eradicate the same before there is an irredeemable damage to the tooth. This can bring about decreased level of prevalence, thereby lowering the caries experience in a given population. Though the presence S. wiggsiae in ECC of European population has been noted, there is no evidence in literature on the prevalence of S. wiggsiae in Indian urban adolescence population so far.

With many limitations in the culture studies, molecular methods has been a major leap in identifying diverse taxa's in varied population. A metagenomic approach namely 16S rRNA in which the total DNA segments can be obtained from a microbial community seems to be promising. The microflora of a given sample can be reliably identified in a quantitative manner based on the platform provided by 16S rRNA metagenomic assay which provides a rapid and extensive analysis.[25] Hence, the study was done in two folds; to determine the prevalence of S. wiggsiae in saliva, plaque, and dentinal caries using polymerase chain reaction (PCR) in low and high caries risk individuals; to identify the presence of microbiota in the samples of caries risk individuals by 16S rRNA metagenomic analysis and quantitatively evaluate the cariogenic pathogens in the samples of caries risk individuals of adolescent urban population in southern India using real-time PCR.

METHODS

Study design and participants

Ethical clearance was obtained from Institutional Review Board. The study was conducted among 60 systemically healthy adolescents (10–19 years of age) school children of Chennai city, South India. All the experiments were performed in accordance with relevant guidelines and regulations. Permission to conduct the study was obtained from school authorities and informed consent was obtained from the parents of children willing to participate. Participants who used chemical mouthwash, antibiotic therapy up to 6 months prior to the study, undergoing orthodontic treatment, mentally challenged patients and patients with xerostomia, were excluded from the study. The study was subjected to two Phases I and II.

Phase I

The participants were categorized based on ADA risk assessment criteria into two groups, low caries risk (Group I; n = 30) and high caries risk (Group II; n = 30).

Salivary samples (IS = 30; IIS = 30): The participants were asked to chew paraffin wax (0.5 cm × 0.5 cm) for 3 min to obtain stimulated saliva. Saliva was collected using a sterile disposable syringe

Plaque samples (IP = 30; IIP = 30): Supragingival plaque samples (1.5 mg) were taken using sterile Gracey curettes (Acharya, Manipal, India) from the buccal and interproximal surfaces of all molars

Dentinal caries samples (IID = 30): Samples were taken from the teeth with caries involving not more than one-third of dentin (selection done using ICDAS criteria [5 and 6] and verified radio graphically) using sterile spoon excavator (larger diameter in accordance with size of the cavity-#17, #18, #19 GDC, Mumbai, India). Local anesthesia (LIGNOX 2%A, 1: 80,000 adrenaline, DJ Lab, India) was administered. Before excavation, the involved teeth were isolated with rubber dam (Hygiene dental dam, Coltene, Switzerland) or in case the application of rubber dam is not possible the teeth were isolated using cotton rolls and excavation was done under high vacuum evacuation to minimize salivary contamination. The carious lesion was removed using sterile water-cooled round bur (larger diameter in accordance with size of the caries lesion-#3, #7, #9 Mani Inc., Japan) in a high speed air-turbine hand piece till the dentino-enamel junction to refine the walls of the cavitated lesion. The superficial layers of the dentinal sample were then sequentially excavated using separate sterile spoon excavators and care was taken to minimize contamination of the superficial bacteria with the deeper layers. Then the samples (10 mg wet weight of dentin) were taken at the end of excavation with the criterion of hardness to probe. Once the sample was collected, the involved teeth were restored with temporary restoration. This deeper caries sample represents the advancing front of the caries lesion.

All the salivary, plaque and dentinal caries samples were collected in SDS/Triton bacterial lysis buffer containing 2% SDS (SIGMA-ALDRICH, Cat# 71736) and 10% Triton-X100 (SRL Fine Chemicals, Cat#64518), which were then taken to the laboratory for further microbial assessment (PCR and Metagenomic analysis). The collected samples were used for both Phase I (salivary, plaque and dentinal caries) and II (plaque and dentinal caries)

DNA extraction

The collected plaque and dentinal caries samples were collected in SDS/Triton bacterial lysis buffer containing 2% SDS (SIGMA-ALDRICH, Cat# 71736) and 10% Triton-X100 (SRL Fine Chemicals, Cat#64518). Bacterial cells were lysed by heating the samples at 95°C for 10 min followed by centrifugation at 12,000 rpm at room temperature for 5 min to pellet undigested cells and debris. The supernatant containing bacterial genomic was transferred to a fresh 0.5 ml tube.

Quantification of DNA

The DNA extracted from samples was quantified by QUBIT Fluorometer to determine the total DNA concentration.

Bacterial specific polymerase chain reaction analysis

The prevalence of S. wiggsiae was determined by species specific PCR. Amplification of 16S rRNA gene specific region by PCR indicates the presence of S. wiggsiae in a given sample. PCR was done in 50 μg of total genomic DNA with the forward and reverse primer pair. The following PCR programs (Takara, Shiga, Japan, #TP600) were used: After an initial denaturation at 94°C for 2 min, the samples were subjected to 40 cycles of denaturation at 94°C for 20 s, annealing at 51°C for 20 s and elongation at 72°C for 20 s, with a final extension at 72°C for 5 min.

Post polymerase chain reaction analysis

The amplified region of 16S rRNA of S. wiggsiae were verified by electrophoresis on 1.5% agarose gels stained by ethidium bromide and visualized by UV light.(Gel documentation unit, Medicare scientific industry, Chennai, India)

Following the findings of PCR analysis observed in Phase I, Phase II were carried out in plaque and dentinal caries samples of high caries risk individuals only.

Phase II

The plaque and dentinal caries samples (n = 30) collected from the high caries risk individuals were further subjected to the following microbial assessment

To identify the presence of microbiotas in * Dentinal caries samples of high caries risk patients, by metagenomic analysis.

To quantify the most prevalent microbiotas identified by metagenomic analysis, in * Plaque samples of high caries risk patients.

*Dentinal caries samples of high caries risk patients of urban adolescent population by Real Time PCR.

16S metagenomic analysis

From a pool of DNA which possess six decadent forward and reverse primers, a total of 10 ng of DNA was extracted where the PCR was performed using 16S rRNA gene sequencing method of hypervariable region V6. Due to selective sequence vagueness, the V6 hypervariable region of all culturable, nonculturable, aerobic and anaerobic bacteria of the given samples were amplified using these primers. Under the following circumstances, the PCR amplification of V6 was executed. Denaturation started initially at 94°C for 4 min, succeeded by 94°C of which 30 cycles were completed for 30 s, 58°C where it also done for 30 s, 72°C also done for 30 s, and a final extension at 72°C for 5 min. As the 5’ ends of the above degenerate primers along with an adaptor sequence as per the protocol proposed by Ion Torrent-sequencing library preparation, the PCR amplicons can be used directly to generate amplicon libraries in the subsequent emulsion PCR step. The primer dimers and products of nonspecific amplification using purified gel elution kit and Track It 1 kb Plus DNA Ladder (PN 10488-085;Life Technologies) was utilized among 1.5% agarose gel from which the estimated size of amplicon from the first PCR was confirmed. Subsequently after purification using Qubit ds DNA HS assay kit (Qubit V2, Vienna) the amplicon libraries of the eluted PCR were evaluated. In order to obtain a diluted amount of 2.8 × 108of fragments of DNA, the total quantity of DNA molecules which has been eluted by PCR amplicon library was evaluated from overall quantity and concentration of the amplicon. Based on the manufacturers information, the emulsion PCR (i.e., second PCR) was taken in order to provoke the bar coded libraries with the help of Ion Xpress Plus fragment library kit (Catalog #4471269; Life Technologies). Based on the following conditions, the mixed emulsion was augmented: 94°C for 6 min, succeeded by 40 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 90 s and then 5 cycles at 94°C for 30 s and 68°C for 6 min. The percent prevalence was exposed after sequencing of the augmented first PCR product which was obtained from the previous amplification step. Based on the manufacturer's information, with the help of Ion Torrent Personal Genome Machine (PGM) along with Ion Sequencing 200 kit (Cat #4482006, Life Technologies) the clonal library was squelched on a 314 chip which was previously generated. With the aid of the PGM software, the sequences that exhibit low partiality, cryptic, and polyclonal characteristics were removed by processing and filtering the DNA sequence from each of the clones.

Sequence selection parameter

To find the highly positive target species, the raw sequences were refined under rigid conditions. The filtration prototype used to remove the low quality reads were as follows: (1) <150 bp was the diameter of the target sequence (appropriate length of 150–200 bp was chosen). (2) Reads with adapter sequences that exceeds three errors. (3) Reads that exceeds two errors or those that are not identical with the decanted PCR primers. (4) Sequences of cryptic bases (repeats which are long and >6 bp) and (5) Reads which exhibits <95% confidence interval. Using the QIIME and Ribosomal Database Project which was obtained from a reference dataset, the Operational Taxonomic Units (OTUs) of 16S rRNA were gathered independently at 97% to calculate their distinctiveness. Using UPGMA gathering (Unweighted Pair Group Method with Arithmetic mean, known as average linkage) the hierarchical gathering was done for the taxa which was copious and most accepted where the OTUs were also assigned. Those bacterial genera that passed above the filtering process were tabulated along with their respective OTU identifier and read count. Read count represents true bacterial sequence. The reference sequences were taken based on the reference sequences preserved at (ncbi.nlm.nih.gov/nucleotide) a common database. The arrangement of bacteria should be at least 95% homologous to the respective reference sequences. The genus of bacteria should show its read count greater or equal to 500. Higher read count, higher is the population of particular genus. The fluorescent signal was monitored which provides the information on the absence and presence of a specific target sequences by quantifying the target. When the fluorescent signal goes through the melting phase, it establishes genotype and verifies the specific target bacteria.

16S rRNA amplification and real-time polymerase chain reaction analysis

Equal concentration of genomic DNA samples were subjected to PCR amplification of the 16S rRNA gene hypervariable regions V1 to V9 with the following set of primers: Forward: AGTTTGATCCTGGCTCAG, and Reverse: TACCTTGTTACGACTT under the following conditions. After an initial denaturation at 94°C for 4 min, the samples were subjected to 40 cycles of 94°C for 45 s, 48°C for 45 s, 72°C for 2 m, with a final extension at 72°C for 5 min. The PCR amplified products were cleaned with QI quick PCR purification kit (Cat# 28104) to remove primer dimers, which otherwise may interfere with real-time PCR analysis. The purified PCR amplicons were then subjected to real-time PCR analysis with S. wiggsiae specific primers to determine its presence in the samples in a quantitative manner in rotor gene Q real time PCR unit. The following set of primers that are present within the 16S rRNA gene were used:

SCARW Forward: GTGGACTTTATGAATAAGC

SCARW Reverse: CTACCGTTAAGCAGTAAG

10 μM of each of the above primers were added to HRM RT-Master Mix (Cat# 206542, Qiagen, Germany) in 20 μl reaction, and samples were analyzed in Rotor Gene Q real time PCR equipment (Qiagen, Germany). The following amplification condition was used: after an initial denaturation at 95°C for 10 min, samples were amplified for 25 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, followed by high resolution melt curve analysis between 60°C to 95°C to determine the specificity of the amplifications. The following six standards were used to identify the copy number of amplicons in each of the samples-106, 105, 104, 103, 102, and 101. Upon completion of the real time run, the samples were quantified with in-system software in reference to standards, and presence of bacteria was expressed as copy numbers.

Statistics

A simple percentage prevalence of the above bacterium in the samples were determined and statistically analyzed using Chi-square test (inter group comparison) and Fisher's exact test (intra group comparison) for Phase I. Values were considered statistically significant with P < 0.05 and the power of the study was calculated to be 85%. The data were analyzed by using IBM SPSS Software version 22 (Statistical package for social sciences IBM Corp. USA). Nonparametric Mann–Whitney U-test was used in Phase II. Statistical significance was observed among the groups and the P value was set at 0.05.

RESULTS

Intra-group comparative percentage prevalence of S. wiggsiae is given in Table 1. Mean percentage of Scardovia wiggsiae in dentinal samples are depicted in Table 2. Total genomic analysis of the tested dentine samples are given in Table 3. Graphs 1-3 represents High resolution Melt Curve Analysis.

Table 1

Intragroup comparative percentage prevalence of Scardovia wiggsiae in saliva, plaque and dentinal caries samples of high caries risk subjects

Specimen	Organism		P
	Absent	Present
Saliva
n	8	6
%	57.10%	42.90%
Plaque
n	17	13	=0.039
%	56.7%	43.3%
Dental caries
n	7	23
%	23.3%	76.7%
Total
n	34	43
%	44.2%	55.8%

Table 2

Mean percentage of bacteria in tested (dentine) samples

Bacterial genus	Samples (In counts)						Mean (%)
	1	2	3	4	5	6
Lactobacillus	7362	3722	1176	8390	2908	4920	47.46%
Bifidobacteriaceae Parascardovia	_	2198	4472	3619	_	8274	30.93%
Bifidobacteriaceae Scardovia		1735	3908		2700	5839	23.63%
Bifidobacteriales	22				1920		3.23%
Mutans Streptococcus				88		29	0.19%

Table 3

Total genomic analysis of the tested dentine samples

Samples	Bacterial Genus	Bacterial counts	Percentage	???	???
Sample 1	Lactobacillus; otu_1845	7362	95.57315332
	Leuconostoc; otu_1850	310	4.024406076
	Bifidobacteriales; otu_598	22	0.285603012
	Bacillus; otu_1353	9	0.116837596	Sum=	7703
Sample 2	Lactobacillus; otu_1845	3722	45.39577997
	Bifidobacteriaceae; Parascardovia; otu_606	2198	26.80814734
	Bifidobacteriaceae; Scardovia; otu_607	1735	21.16111721
	artificial sequences; Unclassified; otu_4025	514	6.269057202
	Facklamia: out_1802	14	0.170752531
	Selenomonas; Unclassified; otu_1708	13	0.158555921
	Bacillus; otu_1353	3	0.036589828	Sum=	8199
Sample 3	Bifidobacteriaceae; Parascardovia; otu_606	4472	44.38709677
	Bifidobacteriaceae; Scardovia; otu_607	3908	38.78908189
	Lactobacillus; otu_1845	1176	11.67245658
	artificial sequences; Unclassified; otu_4025	407	4.039702233
	Weissella; Unclassified; otu_1863	91	0.903225806
	Bacillaceae; Oceanobacillus; otu_1376	21	0.208436725	Sum=	10075
Sample 4	Lactobacillus; otu_1845	8390	54.34993846
	Bifidobacteriaceae; Parascardovia; otu_606	3619	23.44367429
	Bifidobacteriaceae; Scardovia; otu_607	2700	17.49044503
	artificial sequences; Unclassified; otu_4025	538	3.485133122
	Selenomonas; Unclassified; otu_1708	102	0.660750146
	Streptococcus; Unclassified; otu_1858	88	0.570058949	Sum=	15437
Sample 5	Lactobacillus; otu_1845	2908	44.61491255
	Bifidobacteriales; otu_598	1920	29.45688862
	artificial sequences; Unclassified; otu_4025	1473	22.59895674
	Weissella; Unclassified; otu_1863	114	1.749002762
	Bacillaceae; Oceanobacillus; otu_1376	71	1.089291194
	Bacillus; otu_1353	32	0.490948144	Sum=	6518
Sample 6	Bifidobacteriaceae; Parascardovia; otu_606	8274	42.55735007
	Bifidobacteriaceae; Scardovia; otu_607	5839	30.03291842
	Lactobacillus; otu_1845	4920	25.30603847
	artificial sequences; Unclassified; otu_4025	339	1.743647773
	Leuconostoc; otu_1850	41	0.210883654
	Streptococcus; Unclassified; otu_1858	29	0.149161609	Sum=	19442

Graph 1

Amplification curves of Scardovia wiggsiae in plaque and dentinal caries samples

Graph 3

High resolution melt curve analysis of Scardovia wiggsiae in plaque and dentinal caries samples of high risk participants

Melt curve analysis of Scardovia wiggsiae in plaque and dentinal caries samples (df/dt)

Phase I

None of the saliva and plaque samples of group-IS, IP showed amplification around the 171 base pair region indicating total absence of S. wiggsiae in low caries risk participants. Groups IS, IP were significant with Groups IIS (P = 0.006) and IIP (P < 0.001). Group IIS was not significant with Group IIP (P = 0.99) and Group IID (P = 0.08). Group IIP was found to be significant with Group IID (P = 0.04). Group IID indicated higher percentage prevalence of S. wiggsiae in dentinal caries samples than Group IIS and IIP with significant P value of 0.039. Thus, the percentage prevalence of S. wiggsiae were found to be highest in dentinal caries samples followed by plaque and salivary samples of high caries risk participants, which were confirmed by the presence of amplification around the 171 base pair region.

Phase II

To determine the relative copies of S. wiggsiae in the plaque and dentinal caries samples, 1 nanogram of total genomic DNA was first amplified with 16S rRNA gene universal primers. This step was included to ensure enhanced detection of S. wiggsiae as the amount of plaque and caries samples obtained yielded only a few nanograms of total genomic DNA.

The presence of S. wiggsiae in both plaque and dentinal carious samples were quantified. It was expressed as OTU and was identified as read counts in a given sample.

Quantitative PCR analysis showed amplification threshold (computed tomography [CT]) between 20°C to 25°C. A few of the samples showed delayed cycle threshold after 30°C. As the CT value difference was >5°C, these were considered as false positives.

The analysis of the melt curve showed a sharp peak for S. wiggsiae between 77.5°C to 78.5°C in both plaque and dentine samples which was further confirmed by high resolution melt curve analysis with similar melt curve behavior [Graphs 1-3]. Melt curves indicates the rate of change of fluorescence with respect to temperature (df/df). When comparing the plaque and the dentine samples, data analysis revealed 2 fold increased presence of S. wiggsiae in the dentinal caries samples relative to plaque samples which was statistically significant with the P value of 0.05.

16S rRNA gene sequencing on the PGM-Ion Torrent sequencing stage analysis showed presence of S. wiggsiae in 4 out of 6 dentinal caries samples tested. The read counts were in the range of 1500–8500. Almost all the dentinal caries samples carried Lactobacillus. Variation in the read count values was observed in all samples, which indicated that the overall quantity of bacteria varied in and among the tested fragments [Tables 2 and 3].

DISCUSSION

Many adolescents suffer from early caries, which remains a public health issue worldwide. Such lesions progress rapidly increasing the risk of caries, sometimes causing irreparable damage to the permanent dentition. Successful eradication depends on identifying and targeting the specific bacteria. Tanner et al. in 2011 evaluated the plaque samples of both caries free and ECC children by culturing in blood agar and acid agar.[14] Tanner et al. observed that though S. mutans, S. sorbinus, Parascardovia denticolens and S. wiggsiae were found to be predominant, S. mutans and S. wiggsiae were the principle organisms associated with severe ECC. Again in 2012, he evaluated the microbiota associated with WSL using HOMM microarray, and found that Veillonellaceae, Granulicatella, Bifidobacteriaceae, Prevotella and S. wiggsiae were predominant.[16] In contrast to the previous findings, it was concluded that S. wiggsiae were observed in higher level with the absence of M. streptococcus and S. sorbinus in WSL.[16]

With this background of available data and with the observation of diverse microbiota, the actual microorganisms responsible for the initiation and progression of caries seem varied and controversial. This study was undertaken to examine the prevalence of cariogenic bacteria from plaque and dentinal caries of adolescent patients with high caries risk index using 16SrRNA genomic analysis and real time quantitative PCR. Since oral hygiene behavior and diet related choices adopted at this stage usually lasts into adulthood, and as there is still a lack of epidemiological studies about the prevalence of DC in adolescents, this age group was targeted.[2627]

Metabolic output of the microbial community dictates the risk of the oral disease based on the organisms involved in the process, thus providing us an understanding in risk predictions involved for an individual. It becomes very relevant to know the list of cariogenic players, because the microbial composition of caries lesions is so variable and the combinations of possible consortia are so numerous. Thus, disruption in the development of early colonizers and other key players during biofilm formation would affect the entire process and eliminate the need for the presence of cariogenic or periodontal pathogens. Hence, the change in paradigm in the etiology of tooth decay must be identified to translate to a focused appropriate therapy.

The presence or absence of S. wiggsiae was analyzed in all the 60 adolescents in this study. The quantitative PCR analysis showed complete absence of S. wiggsiae in low caries risk individuals. However, a bacterial complex including Lactobacilli and S. wiggsiae without M. streptococci characterized high caries risk adolescents with active caries.

In high caries risk individuals, among 60 salivary samples, 26 samples were contaminated and only15 of 34 samples were positive for S. wiggsiae. Although not statistically significant with plaque samples, it was significant with dentinal caries samples.

Of the 60 plaque sample, only 26 were positive for S. wiggsiae. The cascade of ecological events in the biofilm can be reversible or irreversible based on the acidification of plaque due to frequent intake of sugared beverages/snacks. In general, bacterial composition of plaque is similar to that found in caries lesion, but as the caries progresses, the proportion of specific genera either decreases or increases as a result of more specialized niche in the deeper layers of the dentinal caries.[28] Although potentially cariogenic bacteria may exist naturally in dental plaque, these organisms may be weakly competitive at neutral pH and may be found to be in smaller proportion in the total plaque community.[29] This conforms with our findings of lesser percentage of S. wiggsiae in plaque samples than that of dentinal caries, thus validating the “Extended caries ecological hypothesis.” This further supports the findings of Marsh, who stated that the immature plaque undergoes sequence of events lacking total specificity in the microbial etiology, and the pattern of bacterial succession goes on for a longer period of time.[29] The collected samples for this study could have been from either an immature or mature plaque. Furthermore, compliance of the patience would be another variable factor which counts for the limitations of the present study.

77.05% of dentinal caries samples were identified with S. wiggsiae, indicative of higher percentage prevalence of S. wiggsiae [Table 1]. Similar observations were found in dentine samples of children and in adults in a study done by Vacharaka et al., emphasizing the role played by S. wiggsiae in the progression of caries.[30] Moreover it has been reported that “open” dentin cavities were estimated to have the highest bacterial diversity than enamel carious lesions, averaging to about 251 species and 177 species-level phylotypes per samples respectively. Even “hidden” dentin cavities, with minimal or no contact with the oral cavity have been observed to have 201species-level phylotypes.[28]

Since saliva does not represent the real bacterial diversity of a diseased state and also not a real indicator for microbial assessment, only plaque and dentinal caries samples were subjected for real-time PCR and melt curve analysis for verifying the target bacteria.[3132]

Different genomic approaches have been formulated to study the holistic functional output. Metagenomic analysis is basically a DNA sequencing technique, which gives us an insight about taxonomic diversity. Descriptive level of taxonomic resolution or a previously determined bacteria of similar description are sequenced using microarray method. 16S rRNA gene sequencing used in multiple taxonomic stints, as in HOMD, the common database for oral bacteria, has been established as the “gold standard” for identification of bacterial species. It serves as a rapid and reliable alternative method for bacterial identification quantitatively.[3334]

The PGM-Ion Torrent sequencing platform used for this process is ideal for small genome sequencing and more specific in targeted DNA and RNA sequencing. Basically 16S rRNA gene contains 9 hypervariable regions (V1-V-9), each region constituting species-specific sequences. These regions are considered useful targets for species identification for various diagnostic assays. Among these regions, V6 region identifies and distinguishes most bacterial species. It is one of the shortest regions with the most nucleotide diversity in the sequences among the bacterial analyses. Added to the above, fluorescent signal was monitored to quantify the target bacteria. Specific peaks of S. wiggsiae was observed in the melt curve analysis was observed [Graphs 1-3].

Furthermore, in order to determine the bacterial population in the dentinal carious lesions, total genomic analysis of 6 dentinal samples was analyzed. Data analysis showed presence of varying percentage of Lactobacilli, Bifidobacteriaceae-Parasacardovia, Bifidobacteriaceae-Scardovia, Leuconostoc, Bacillus, Selenomonas, Facklamia and M. streptococcus, with higher percentage of Lactobacilli and Bifidobacteriaceae [Table 3]. M. streptococcus recorded the minimum, with its presence only in 2 out of 6 samples [Table 3]. Presence of S. wiggsiae was observed in 4 out of 6 samples tested. Almost all the samples carried Lactobacillus (11%–95%) [Table 3]. The screening of Lactobacilli in this study is consistent with the previous reports of Obata and Byun et al.[3536] In metagenomic analysis of 16S rRNA gene, the taxonomic resolution below descriptive level cannot be analyzed, hence the species determination of the bacteria was not possible in this study.

Bacteria colonizing carious dentine survive in an ecosystem that is complex and continually changing. Lactobacilli being the late colonizer are associated more with advanced caries, as the pH shifts with the mature microbial communities.[1236] The low pH in the biofilm due to bacterial metabolism is considered an important factor in the development of caries lesion, activating the aciduric and acidogenic bacteria.[3738] Apart from their characteristics, these bacteria is said to contribute to further drop in pH, contributing to their dominance as observed in this study.[11]

Despite limited information regarding the metabolic pathways of S. wiggsiae, it is being suggested that it metabolizes sugars through a unique metabolic pathway called fructose-6-phosphate pathway (F6PPK shunt) and produce acetic acid as an acidic end product in contrast to lactic acid by the glycolytic pathway of M. streptococci. Interestingly, it has been observed that acetic acid is produced in large proportions in a nonionized form. Non ionized form of acetic acid is more likely to penetrate into enamel decalcifying from within, facilitating carious progression than lactic acid. It explains that S. wiggsiae exhibits high acid productivity reducing the environmental pH to 3.5 and indicates tolerance to acidic conditions.[39] The findings clearly justifies, why S. wiggsiae was found in high number than Lactobacilli, M. streptococci and other bacteria in the deeper layers of dentin caries, though percentage variation among plaque and dentinal carious samples varied from individual to individual depending on the oral environment. Lower counts of M. streptococci in such lesions could be due to the absence of salivary glycoproteins, which is essential for its survival. In addition, S. wiggsiae is primarily an anaerobic bacterium whereas M. streptococci is a facultative anaerobic bacterium. However, the proteolytic, slow growing anaerobes namely Lactobacilli will continue to sustain and grow due to the diffusion of serum like nutrients in tubular spaces from the pulp chamber.[40] Hence, due to shift in local environmental condition that alters the balance of resident microflora, bacterial profiles differ and change with the progression of caries.[41]

CONCLUSION

Bacterium S. wiggsiae was one among the predominant bacteria in plaque and dentinal caries samples of high caries risk urban adolescence population

S. wiggsiae was found in high number than Lactobacilli, M. streptococci in the deeper layers of dentin caries. M. streptococcus recorded the minimum

This study showed the plausibility of applying S. wiggsiae as a microbial marker, for caries risk assessment. Hence, alternative targets to intervene biologically and therapeutically may reflect the success in preventive care in a society.

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Conflicts of interest

There are no conflicts of interest.