The present observational study was conducted at Nucleobase Life Sciences Research Laboratory, Bengaluru, India, from March 2013 to March 2016. This study was approved by the Ethical Committee of the PMNM Dental College, Bagalkot, affiliated to Rajiv Gandhi University of Health Sciences, Karnataka, India. The plaque samples from 65 caries active subjects aged between 35 to 44 years were collected using sterile wooden toothpicks and then transferred to vials containing 1 ml sterile phosphate buffer saline (HiMedia, India). The vials were vortexed for one minute and diluted by 100-fold in 1X phosphate buffer saline. The processed plaque samples were then plated on Mitis Salivarius Bacitracin (MSB) agar (HiMedia, India) and anaerobically incubated at 37°C for 48 hours [10,11]. Obtained cutures were subjected to sequencing.

Genomic DNA isolation: The bacterial DNA was isolated according to the methodology described by Moreira M et al., with a few modifications [12]. Briefly, 1 ml of 24-hours-old culture broth was centrifuged at 49,000 g for two minutes and the supernatant was discarded. The pellet was washed with sterile distilled water and then centrifuged at 49,000 g for two minutes. Approximately 675 μl of TE buffer (100 mM Tris-Cl and 100 mM Ethylenediaminetetraacetic acid), 1.4 M NaCl, 1% Cetyl Trimethyl Ammonium Bromide (CTAB) and 0.03 μg/μl proteinase K (Sigma, India) was added and incubated at 37°C for 30 minutes. Almost 75 μl of sodium dodecyl sulfate (20%) was added and incubated at 65°C for two hours. After incubation, the solution was centrifuged at 49,000 g for two minutes and the supernatant was collected in a sterile Eppendorf tube. Chloroform: isoamyl alcohol (24:1 v/v) was added and centrifuged at 49,000 g for seven minutes. The aqueous phase was transferred to another vial and the solution was homogenized by adding isopropyl alcohol (0.6 v/v) and incubated at room temperature for one hour. After centrifuging at 49,000 g for seven minutes, 70% of cold ethanol was added. The solution was centrifuged at 49,000 g for seven minutes and the supernatant was discarded. The vial was left open in air chamber to dry the pellet and later suspended in 25 μl of ultra-pure water and stored at -20°C until further use. The concentration of eluted bacterial DNA was determined by measuring at A260 using UV spectrophotometer (Sartorius stedim biotech, Germany) and later the purity of DNA was assessed from A260/A280 ratio.

16S rDNA sequencing: The clinical isolate was identified by 16S rDNA sequencing, the PCR was performed using universal primer 16S FP5’-AGAGTTTGATCCTGGCTCAG-3’, 16S RP5’-AAGGAGGTGATCCAGCCGCA-3’ [11]. The following conditions were maintained: initial denaturation 94°C for two minutes, denaturation at 94°C for 50 seconds, annealing at 48°C for 30 seconds, extension at 72°C for 90 seconds and final extension at 72°C for six minutes. The GenBank accession number was obtained from the National Centre for Biotechnology Information (NCBI) database after the submission of the 16S rDNA sequences.

Phylogenetic analysis: The phylogenetic analysis of the S. dentapri H14 was performed along with 14 members of MS species, whose determined 16S rDNA sequences were retrieved from GenBank nucleotide sequence database. The phylogenetic tree was performed based on the Kimura 2-parameter model [13]. The tree was constructed by maximum likelihood method. The tree with the highest log based likelihood (-5486.8001) was considered for the analysis. The percentage of the tree in which the related taxa grouped together was depended on 1000 resampling. Primary tree in the heuristic search was achieved by applying the neighbour-joining method to a matrix of pairwise distances estimated using the maximum composite likelihood approach and the analyses were carried out in MEGA 6 [14]. Based on the measurement of branch lengths in the number of replacements per site, the tree was drawn to scale. The acquired positions with less than 85% site coverage were removed, e.g. fewer than 15% absent data, alignment gaps and vague bases. There were a total of 1423 positions in the final data set.

Biochemical characterization: Biochemical characterization was performed as described by Takada K et al., with a few modifications [9]. The biotype of S. dentapri was determined as described by Yoo SY et al., [10]. A phenol red broth base (HiMedia, India) was used as the basal medium for the fermentation of mannitol, melibiose, salicin, trehalose, sorbitol, inulin, raffinose and lactose discs (HiMedia, India). Arginine dihydrolase and esculin fermentation broth (HiMedia, India) were also employed in biochemical characterization. After incubation at 37°C for 48 hours the tubes were observed for colour change to distinguish the positive and negative reaction. Reproducibility and reliability of the results were affirmed by repeating the experiments in triplicate.

Among the 65 clinical isolates, one isolate designated as H14 was detected as S. dentapri by 16S rDNA sequencing. The GenBank accession number provided by NCBI for the strain H14 was KP975177. The 16S rDNA sequences of the H14 strain demonstrated highest similarity with S. dentapri NUM 1529 (99 %). From the phylogenetic analysis, it was observed that S. dentapri is closely related to S. macacae than other members of MS [Table/Fig-1]. The colony characteristics of S. dentapri H14 was found to be small, raised, irregular, rough, dark-blue in colour and glistening on the MSB agar [Table/Fig-2]. The S. dentapri H14 expressed biotype II biochemical characterization of MS. The biochemical characteristics of S. dentapri are outlined in [Table/Fig-3] and the biochemical test results are represented in [Table/Fig-4].

[Table/Fig-1]:

Phylogenetic analysis depicting the relationship of S. dentapri with other MS species based on the 16S rDNA sequences. The tree constructed by maximum likelihood method generated using Kimura-2-parameter model in MEGA 6.0 software. The accession numbers are shown in parenthesis. The numbers above the branches referred to bootstrap values based on 1000 resampling.

[Table/Fig-2]:

Colonies of S. dentapri H14 on MSB agar.

[Table/Fig-4]:

Biochemical tests of S. dentapri H14.

Mn: Mannitol, Mb: Melibiose, Sa: Salicin, Tr: Trehalose, Sb: Sorbitol, In: Inulin, Rf: Raffinose, La: Lactose, Ar: Arginine, Es: Esculin Fermentation Broth.

To the best of our knowledge, this is the first reported study of isolation of S. dentapri from human active caries. In context to the morphological characterization, S. dentapri could not be differentiated from other MS species. In contrast, molecular identification based on 16S rDNA sequencing showed a high level of sensitivity for detection of MS species [15]. Phylogenetically, S. dentapri is closely related to S. macacae when compared with other MS [Table/Fig-1] which is in agreement with the previous study [9].

In the present study, the clinical strain of S. dentapri displayed different biochemical characteristics when compared with the type strain S. dentapri NUM 1529 [Table/Fig-3]. The reason for the strain metabolic variability might be due to heterogeneity of S. dentapri or dietary habits of the host. S. dentapri H14 demonstrated biotype II characteristics which was earlier detected in few strains of MS species namely S. ratti, S. downei and S. mutans [10,16,17]. However, within a species, strains can be differing with respect to biochemical characteristics and the same strains can exhibit unreliable metabolic activity [18]. The biochemical characteristics of S. dentapri were also compared with the results of an earlier study with reference to S. macacae and S. mutans [Table/Fig-3] [9,19]. S. dentapri and S. macacae displayed contrary biochemical results for melibiose fermentation and arginine hydrolysis, while S. dentapri and S. mutans demonstrated variation for inulin fermentation and arginine hydrolysis.

As this is the first finding of S. dentapri in caries active subject, this study recommends further investigations to be carried out for determining the virulence factors of S. dentapri.