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Table of Contents   
ORIGINAL RESEARCH  
Year : 2019  |  Volume : 30  |  Issue : 4  |  Page : 579-582
Detection and characterization of Streptococcus downei, a rare bacterial species of mutans streptococci from caries-active patients


1 Department of Microbiology, J.J. College of Arts and Science, Pudukkottai, Affiliated to Bharathidasan University, Tamil Nadu, India
2 Biotechnology Division, Department of Applied Science, University of Technology, Baghdad, Iraq
3 Department of Biotechnology, Krupanidhi Degree College; Nucleobase Life Sciences Research Laboratory, Bengaluru, Karnataka, India

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Date of Submission19-Jul-2017
Date of Decision14-May-2018
Date of Acceptance14-Jun-2018
Date of Web Publication18-Nov-2019
 

   Abstract 


Background: The oral bacteria, mutans streptococci (MS), are an etiological agent of dental caries. Of MS, Streptococcus downei are rarely isolated bacteria. Aim: The aim of this study was to isolate and characterize S. downei from caries-active subjects. Materials and Methods: In all, 65 dental plaque samples were collected from dental caries-active subjects. All the isolates were further identified and characterized using 16S rDNA sequencing, biochemical tests, antibiogram, and minimum inhibitory concentration (MIC). Results: Five isolates have been identified as S. downei using 16S rDNA sequencing. Phylogenetic analysis showed that S. downei was closely related to S. sobrinus. The biotype traits of these five isolates were IV (n = 3), V (n = 1), and variants (n = 2). The study proposed one new biotype, classified as biotype VIII for the variant strain. The antibiogram tests revealed that all the strains of S. downei were susceptible to all the antibiotics used in the study with higher sensitivity to penicillin and ampicillin. The MIC of ampicillin and erythromycin against S. downei was 0.047 and 0.39 μg/mL, respectively. Conclusion: The study reports the prevalence of S. downei in caries-active subjects and recommends further investigations to determine its role in the disease.

Keywords: Antibiogram, biotyping, cariogenic bacteria, oral streptococci, phylogenetic

How to cite this article:
Salman HA, Senthilkumar R, Mahmood BS, Imran K. Detection and characterization of Streptococcus downei, a rare bacterial species of mutans streptococci from caries-active patients. Indian J Dent Res 2019;30:579-82

How to cite this URL:
Salman HA, Senthilkumar R, Mahmood BS, Imran K. Detection and characterization of Streptococcus downei, a rare bacterial species of mutans streptococci from caries-active patients. Indian J Dent Res [serial online] 2019 [cited 2019 Dec 9];30:579-82. Available from: http://www.ijdr.in/text.asp?2019/30/4/579/271058



   Introduction Top


Dental caries is a polymicrobial transmissable disease that is popular with all the age groups of mankind.[1] Among the oral bacteria, mutans streptococci (MS) have been associated as a major cariogenic bacteria.[2],[3]

MS consists of 17 species, namely, Streptococcus mutans, S. ratti, S. sobrinus, S. dentapri, S. macacae, S. downei, S. dentirousetti, S. devriesei, S. orisratti, S. ferus, S. orisuis, S. criceti, S. ursoris, S. troglodytae, S. dentasini, S. orisasini, and S. oriloxodontae.[4],[5],[6] Of these, S. mutans and S. sobrinus are strongly implicated with human dental caries.[3] However, recently S. dentapri was isolated from caries-active subjects.[7]

The first incident of isolation of S. downei was from the dental plaque of monkeys, primarily categorized as S. mutans serotype h,[8] and later reclassified as a novel species of MS.[9] S. downei was believed to be one of the main causative agents of dental caries.[9],[10] The understanding of the genetic diversity and the characterization of MS may assist in developing new preventative measurements of caries so as to promote health and to avoid the disease.

S. mutans and S. sobrinus are usually detected from human dental plaque, particularly in the carious lesion. The occurrence of S. downei in the human dentine cavity is still not well investigated since rare studies have been dedicated to isolate and characterize S. downei. To date, only four strains of S. downei have been isolated from human dental plaque.[11] Therefore, the objective of this study was to isolate and characterize S. downei, based on phylogenetic analysis, biotype traits, antibiogram tests, and minimum inhibitory concentration (MIC), isolated from caries-active subjects.


   Materials and Methods Top


Isolation of bacteria

The institutional ethics committee of P.M.N.M Dental College, Bagalkot, Karnataka, India, approved this study in the meeting held on 07 February 2013 (Ref. No. PMNMDCH/2576/2013-14). In all, 65 caries-active subjects participated in the study, from which plaque samples were collected from each individual. The subjects of this study were between 35 and 44 years of age as per the guidelines of the World Health Organization.[12] The nature of the study was initially explained to all the subjects who participated in this study. A written informed consent was collected from each subject, before sample collection. It was ensured that the subjects participating in the study did not have chronic disease other than caries and also had not received antibiotics for at least 6 weeks before sample collection. The samples were processed in sterile phosphate-buffered saline, and then streaked on mitis salivarius bacitracin (MSB) agar (HiMedia, India).[13] The bacterial colonies showing typical colony morphologies of MS were pure cultured and subjected to further analysis.

Extraction of genomic DNA and 16S rDNA sequencing

The genomic DNA of the bacterial cells was extracted using Cetyltrimethylammonium bromide (CTAB) method as described by Salman et al.[7] Species identification of clinical isolates was determined by 16S rDNA sequencing. Polymerase chain reaction (PCR) amplification of 16S rDNA region was done in 20 μL of reaction mixture containing 10.75 μL of nuclease-free water, 2 μL of 10 × reaction buffer with 1.5 mM MgCl2, 2 μL of 2.5 mM dNTP mix, 2 μL of 10 picomoles 16S forward primer (5′-AGAGTTTGATCCTGGCTCAG-3′), 2 μL of 10 picomoles 16S reverse primer (5′-AAGGAGGTGATCCAGCCGCA-3′), 0.25 μL of 5 U Taq DNA polymerase, and 1 μL of 50 ng/μL DNA template. PCR temperature condition for 30 cycles was as follows: initial denaturation at 94°C for 2 min, denaturation at 94°C for 50 s, annealing at 48°C for 30 s, extension at 72°C for 90 s, and final extension at 72°C for 6 min. The 16S rDNA sequences were submitted to National Centre for Biotechnology Information (NCBI) database to obtain GenBank accession numbers.

Phylogenetic analysis

Phylogenetic analysis of 16S rDNA sequences of S. downei strains was performed along with reference strains of S. downei ATCC 33748, S. mutans ATCC 25175, and S. sobrinus ATCC 33478. The 16S rDNA sequences of the reference strains were retrieved from NCBI GenBank database. Phylogenetic analysis was constructed by Phylogeny.fr, a web-based program, using “one click” mode.[14] Sequence alignments were prepared by MUSCLE program, and the poorly aligned position and divergent regions of the aligned DNA were eliminated by Gblocks program. The branch support value having less than 50% or a number of bootstrap less than 85% were collapsed. Phylogenetic tree was constructed by maximum likelihood method using PhyML3.0 software.[15] The tree was drawn and rendered using TreeDyn.

Biochemical characterization

Biochemical tests were performed to determine the biotypes of isolates as described by Shklair and Keene [16] and Yoo et al.[13] A phenol red broth base (HiMedia) was used as the basal medium for fermentation of mannitol, melibiose, raffinose, and sorbitol. Arginine dihydrolase (HiMedia) was also used. Biochemical tests were performed in triplicate and repeated to confirm the reproducibility and reliability.

Antibiogram

The antibiotics selected in this study were as follows: penicillin-G 10 units/disc, ampicillin 10 μg/disc, cefotaxime 30 μg/disc, cephalothin 30 μg/disc, erythromycin 15 μg/disc, cefazolin 30 μg/disc, chloramphenicol 30 μg/disc, and methicillin 5 μg/disc (HiMedia). A single colony was picked from 24-h culture BHI agar plate and inoculated into BHI broth.[17] The test tubes were incubated at 37°C for 24 h. The inoculum was then adjusted to match the turbidity of 0.5 McFarland standards (HiMedia). The test organism was aseptically swabbed on BHI agar using a sterile cotton swab.[18] The plates were kept for 10 min to dry, and then antibiotic discs were aseptically placed on the inoculated BHI agar. The plates were anaerobically incubated at 37°C for 24 h. The inhibition zones were measured in millimeter (mm). The experiment was performed in triplicate and repeated to confirm the reliability and reproducibility.

Determination of MIC

The MIC of ampicillin and erythromycin (HiMedia) was determined by microdilution method as previously described by Salman et al.[3] The experiment was repeated to confirm the reliability and reproducibility of the results.

Statistical analysis

One-way analysis of variance and post hoc test were used using SPSS version 17 to determine the P value, means, and standard deviation and to study the multiple comparison of antibiotics within S. downei strains. Statistical analysis result with P value ≤0.05 was considered as significant.


   Results Top


Among the study population, 5 (7.69%) isolates were identified as S. downei based on 16S rDNA sequencing, and NCBI GenBank accession numbers assigned are listed in [Table 1]. The colony morphology of these five strains of S. downei was glistening bubble colony with moderate size. Phylogenetic analysis displayed that S. downei is closely related to S. sobrinus than to S. mutans [Figure 1]. The biotype traits of five S. downei strains displayed that two strains belong to biotype IV, one expressed biotype V, while the other two strains were variant biotypes [Table 1]. Among the two variant strains, one new biotype VIII was proposed to one strain H64 (KP975212). All the strains of S. downei were susceptible to the selected antibiotics [Table 2]. Among the antibiotics, the highest zone of inhibition was shown by penicillin and the least was methicillin. It is statistically significant (P < 0.05) with penicillin compared with other antibiotics except with ampicillin, cefotaxime, and cefazolin which nearly have similar values. The MIC of ampicillin and erythromycin against S. downei was 0.047 and 0.39 μg/mL, respectively.
Table 1: GenBank accession numbers and biotyping of S. downei strains

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Figure 1: Phylogenetic analysis of the S. downei strains along with reference strains based on 16S rDNA sequences. The tree was constructed by maximum likelihood method. GenBank accession numbers are demonstrated next to strain numbers

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Table 2: Antibiogram test results of S. downei

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   Discussion Top


In this study, five strains of S. downei were detected from five subjects of dental caries. To the best of our knowledge, this is the second reported study of isolation of S. downei from human dental plaque and probably the first isolation from caries-active subjects. The 16S rDNA sequencing identification showed a high level of accuracy for detection and differentiation of S. downei from other closely related members of MS.

In this study, S. downei was isolated on MSB agar. The colony morphology of S. downei was unable to be differentiated from other species of MS due to its high similarity.[3] However, S. mutans, S. sobrinus, and S. dentapri were previously isolated on MSB agar.[3],[7],[13]

Phylogenetically, S. downei and S. sobrinus were noted to be more closely related to each other than to S. mutans [Figure 1], and this finding is in agreement with a previous report.[9]

Four S. downei strains isolated from human dental plaques in an earlier study demonstrated biotypes I and II, whereas the reference strain of S. downei displayed biotype VI.[11] In this study, strains of S. downei demonstrated biotypes IV and V and variant types. These biotype results of S. downei strains clearly display heterogeneity among strains. The reason for this variability might be due to the host's oral environments, ethnic diversity, and the dietary habits of the studied subjects or the heterogeneity of S. downei. Of these variants, S. downei H64 (KP975212) was similar to biotype V,[16] except that it was negative to arginine dihydrolase broth, and hence, a new biotype, VIII, was proposed. The other variant biotype, H47 (KP975212), was negative to all the biochemical tests, and therefore no biotype was proposed and this is in agreement with that of the results of an earlier study.[13] However, the first five biotypes (I–V) of MS were classified by Shklair and Keene,[16] followed by biotype VI, which was proposed by Beighton et al.[9] and biotype VII proposed by Yoo et al.[13]

Resistance to antibiotics by the viridans streptococci group has increasingly been reported over the past decades.[19],[20],[21] However, reported studies on antibiotic susceptibility testing of MS species are limited. In this study, the susceptibility of the antibiotics was tested against S. downei. All the clinical strains of S. downei were susceptible to the selected antibiotics [Table 2]. To the best of the literature survey, this is the first report of determining the susceptibility of S. downei by antibiotics. S. downei is more susceptible to the antibiotics when compared with S. sobrinus and S. mutans, respectively, as shown in an earlier report.[22] Among the antibiotics used, penicillin and ampicillin showed the highest zone of inhibition against S. downei strains [Table 2].

Among the tested antibiotics, six were β-lactam (penicillin, ampicillin, cefazolin, methicillin, cefotaxime, and cephalothin) and two were non-β-lactam (erythromycin and chloramphenicol). Ampicillin and penicillin were the highest in β-lactam, while erythromycin was the highest in non-β-lactam [Table 2]. Therefore, MIC of ampicillin and erythromycin was determined. In this study, the MIC of ampicillin and erythromycin against S. downei strains was 0.047 and 0.39 μg/mL, respectively. These values are similar for the MIC of ampicillin and erythromycin against S. mutans and S. sobrinus.[3]


   Conclusion Top


This investigation reveals the possibility of S. downei to inhabit the human oral niche and may somehow take part in the initiation or development of caries. Thus, further investigations are necessary to understand the role of S. downei in dental caries.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Correspondence Address:
Dr. Khalid Imran
Department of Biotechnology, Krupanidhi Degree College, Bengaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_400_17

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