Open Access

Genotypic and phenotypic heterogeneity in Streptococcus mutans isolated from diabetic patients in Rome, Italy

SpringerPlus20165:1794

https://doi.org/10.1186/s40064-016-3470-0

Received: 7 December 2015

Accepted: 4 October 2016

Published: 13 October 2016

Abstract

Our study focuses on the antimicrobial susceptibility, genotypic and phenotypic heterogeneity, and serotype classification of the Streptococcus mutans isolated from type II diabetic patients (n = 25; age 42–68). Eighty-two percent of isolates were classified as serotype c. No serotype k was present. Macrorestriction analysis of genomic DNA of the isolates exhibited a clonal diversity that paralleled the phenotypic heterogeneity, which was also assessed in terms of biofilm forming ability. Isolates were susceptible to all the classes of antibiotics. In conclusion a great heterogeneity and no antimicrobial resistance were apparent in the considered S. mutans strains from diabetic patients.

Keywords

Caries Plaque PFGE Antimicrobial susceptibility

Background

Globally, dental caries is a major public health concern imposing an economic impact among developed countries. Italy was estimated with the highest percentage of Gross National Product spent on oral health in 2010 (Patel Reena 2012). Predisposition of diabetic patients to dental caries may result as sequelae of poor glycemic control, due to high levels of glucose in saliva in conjunction with xerostomia, although a strong association has not been found hitherto (King et al. 1998; Ship 2003; Taylor 2004). Streptococcus mutans, one of the main causative agents of caries (Loesche 1986), is a commensal inhabitant of the tooth surface, participating in the formation of a complex multispecies microbial community (i.e. a biofilm) called dental plaque (Kuramitsu et al. 2007). It adheres to the pellicle of tooth enamel surface through adhesins (AgI/II) or extracellular polysaccharide (EPS) formed by glucosyltransferases (GtfB, GtfC, and GtfD) in presence of sucrose (Krzyściak et al. 2014). Increased intake of dietary carbohydrates acts as source of sugar increasing the production of metabolic acid end-products, and thus demineralization of teeth enamel and dentine at low pH (Loesche 1986). The EPS also serves as a reservoir of sugar and maintains acidic pH in the depths of plaque (Dibdin and Shellis 1988), and a continuous flush of salivary sugar in the EPS may pose a greater caries threat in diabetics. In a biofilm, S. mutans expresses competence and bacteriocin genes that can facilitate integration of various dispensable genes (cnm, gbpA), endowing a competitive advantage and a wide array of genome heterogeneity (Waterhouse and Russell 2006; Waterhouse et al. 2007). In general, Horizontal Gene Transfer (HGT) is known to be strongly favored in biofilm increasing the acquisition rate of exogenous genetic material. Among this material are the determinants of antibiotic resistance (Davies and Davies 2010), posing a continuous threat to the antimicrobial susceptibility of S. mutans (Baddour et al. 2005). In this context, diabetic individuals are subjected to various long term medications escalating the chances of acquiring resistance to these medicines by the indigenous flora of diabetics. Frequent use of antibiotics against infections in such immune-compromised patients has shown increased risk of antibiotic resistance among pathogens (Boyanova and Mitov 2013).

Based on the rhamnose glucose polysaccharide surface antigen (RGP) S. mutans is classified into four serotypes (c, e, f, k), among which k exhibits reduced cariogenecity and immunogenicity (Hamada and Slade 1980; Nakano et al. 2010). In addition, infective endocarditis (IE) is reportedly caused at higher frequency by non-c serotype S. mutans (Nakano et al. 2010). Also DNA fingerprinting methods such as RFLP, AP-PCR, PFGE have demonstrated significant genotypic diversity among S. mutans isolates (Caufield and Walker 1989; Brady et al. 1991; Napimoga 2004; Lembo et al. 2007; Moser et al. 2010).

In the present study, we have focused on the antimicrobial susceptibility as well as genotypic and phenotypic heterogeneity of S. mutans isolates from type II diabetic patients aged >40 and bring the importance of such studies from patients with metabolic disorders to the forefront.

Results and discussion

Dental health analysis of diabetes

The DMF index is a key measure of the caries experience in dental epidemiology. The mean DMFT values among the Italian middle aged diabetic patients was 16.3 (SD = 9.0) while the median plaque index was 0.544. Previous oral health studies among Type II diabetic population in Sudan also found a significant higher plaque index (Mohamed et al. 2013). Moreover, Taylor et al. (2004) found six large cohort studies reporting an association of poor glycemic control with high caries experience. Diabetic subjects are usually restricted to refined carbohydrates and proteinaceous diet. Nevertheless, poor glycemic control, xerostomia and repeated food intake may disrupt the microbial homeostasis of plaque and increase the incidence of caries due to acidogenic bacteria (Taylor 2004; Marsh 2009).

Screening of S. mutans isolates and serotyping

500 bp amplicons obtained using gtfB primers confirmed the putative S. mutans clinical isolates from diabetics. Only 68 % (n = 17) isolates from the serially diluted plaque or saliva samples showed positive amplification. Selection of adult patients in a particular age group with no other medical complications except diabetes might have restricted to end up with low number of patients and consequently isolating few clinical strains. Even though possessing a higher plaque index, the viable count of S. mutans was low presumably due to proper maintenance of their oral health by 2 times brushing and usage of mouth washes among 48 % respondents (data not shown). Screening of the serotype by a molecular approach classified 14 isolates as c type, while 2 and 1 as type e and f, respectively. We did not record any k serotype among our isolates. This result is in accordance with the global prevalence of serotype c S. mutans in the oral cavity (Nakano et al. 2006). Serotype e, f, k are commonly found in atheromatous plaque (Abranches et al. 2011). People with diabetes are usually more prone to heart disease and atherosclerosis (Chait and Bornfeldt 2009), which may follow dental surgical procedures favoring the access of S. mutans in sufficient numbers in the blood-stream. Hence, a thorough stomatological study of the diabetic subjects can serve as a prophylaxis to such health disorder.

Antimicrobial susceptibility testing

Diabetic foot infection, urinary tract and lower respiratory tract infections are a constant threat to poor glycemic control, which has demonstrated an increase in antibiotic prescription rates in Netherlands (Venmans et al. 2009). Notwithstanding this fact prompts a warning situation, pointing towards increased antimicrobial resistance among indigenous flora, all the bacterial strains isolated from diabetics were susceptible to all antibiotics tested (Table 1). According to EUCAST clinical breakpoint, the average zone diameter against penicillin, clindamycin and vancomycin were far from the breakpoints (12, <19 and 15 mm, respectively). Concomitantly, the susceptibility against other antibiotics (erythromycin, oxacillin, rifampicin, tetracycline, gentamicin, cefoxitin, linezolid, norfloxacin and levofloxacin) was compared with the performance standard values published by CLSI for streptococci other than S. pneumoniae (CLSI 2011). Clindamycin, erythromycin, rifampicin and cefoxitin exhibited higher inhibition compared to others with a zone diameter ranging between 35–41, 30–38, 29–36 and 28–34 mm respectively. Penicillin resistance was not observed, which countertrends the previously recorded 14 % penicillin-resistant S. mutans isolates from dental patients in Rome (Pasquantonio et al. 2012). Hence, with scarce available information on antimicrobial disc susceptibility tests based on EUCAST and CLSI guidelines, our work may aid as a reference scale for determining reduced susceptibility to various class of antibiotics for future studies.
Table 1

Distribution of S. mutans isolates from diabetic patients according to the inhibition zone diameters (mm) exhibited by various antibiotics (EUCAST format)

Antibiotic

Disk Content (μg)

Inhibition zone diameter (mm)

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

Vancomycin

5

0

0

0

0

0

1

6

5

1

4

1*

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Erythromycin

30

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

2

2

1

5

2

2

1

2*

0

0

0

0

Oxacillin

1

0

0

0

0

0

0

0

0

0

0

0

0

3

3

2

5*

3

2

0

0

0

0

0

0

0

0

0

0

0

0

0

Penicillin

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

2

3

4

4*

2

1

1

0

0

0

0

0

0

0

0

Rifampicin

5

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3

2

3

3

0

3

3*

1

0

0

0

0

0

0

Tetracycline

30

0

0

0

0

0

0

0

0

0

0

0

0

1

1

0

1

4

4

1

2

2*

1

1

0

0

0

0

0

0

0

0

Gentamicin

10

0

0

0

2

1

2

3

1

3

4

0

0

2*

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Cefoxitin

30

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

2

2

0

6

4

3*

0

0

0

0

0

0

0

0

Linezolid

10

0

0

0

0

0

0

0

0

0

2

0

1

1

0

4

5*

2

1

2

0

0

0

0

0

0

0

0

0

0

0

0

Norfloxacin

10

1

1

3

1

1

3

2

3

2

1*

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Clindamycin

10

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2

1

6

5

0

0

3*

1

Levofloxacin

5

0

0

0

0

0

0

0

1

1

0

4

4

2

3

2*

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

* Zone diameter exhibited by the control strain UA159

Macrorestriction analysis

Macrorestriction analyses using SmaI and BssHII of S. mutans isolates were studied previously to identify maternal transmission as well as genotypic uniformity in an individual (Mineyama et al. 2007; Mitchell et al. 2011). This work illustrates genotypic heterogeneity of S. mutans from caries of diabetic patients of a specific region. The two clustered dendogram along with the schematic representation of the digested bands in Fig. 1 illustrates the clonal diversity of the S. mutans clinical strains. One major cluster accounts for 88 % of the clinical strains along with reference strain UA159 in it. PD27 and PDC7 formed the other cluster. PFGE of PD11, PD17 and PD20 projected similarity, but PD17 and PD20 showed one less band in the lower part of the gel. Conversely, SDC12 and PD1 exhibited complete overlapping of the digested bands. SDC21A–SDC21B and SDC4–SDC4B were isolated from the oral cavity of the same patient and were included in the study due to their differences in growth behavior in broth. As SDC21A and SDC21B belonged to the same serotype and showed the same macrorestriction pattern, they may be considered as variants of the same strain. SDC4 and SDC4B were instead dissimilar in both the characteristics and may be, therefore, considered as different strains. Genotypic variability is well known in S. mutans and records suggests 23 % gene content divergence through whole genome sequence comparison, which validates the diversity found in our study (Cornejo et al. 2013). Although PFGE is considered as the gold standard for discerning clonal relatedness (Birren and Lai 2012), a robust conclusion about the phylogenetic relationship between the strains can be reached only through Whole Genome Sequencing (WGS) studies. WGS of our set of clinical strains are underway, among which the genome sequence of SDC21A has been deposited in GenBank as S. mutans AD01 (Accession number: LGAC00000000).
Fig. 1

Dendogram and schematic representation of PFGE pattern. Scale represents Pearson distance

Phenotypic features of isolates

Biofilm formation, aciduricity and acidogenesis are the key virulence factors which have been extensively compared among large number of clinical isolates with genotypic differences, demonstrating phenotypic variability at the same time (Palmer et al. 2013). Wide genetic variability as observed by PFGE urged us to compare few virulence characteristics among isolates. The strains showed high variability in the capacity to develop mature biofilm. While SDC21A, SDC21B, PD20 and PD5 exhibited high amount of biomass, PD27 and PD11 were poor biofilm formers in presence of glucose Fig. 2. A sucrose independent biofilm formation is governed by various surface antigens among which WapA, P1 and competence factors are known to aid in initial adherence of S. mutans to the tooth surface, and a potential variability in these genes among the strains may be associated with such phenotypic heterogeneity (Bowen et al. 1991; Zhu et al. 2006; Senadheera and Cvitkovitch 2008). Gene sequence comparative studies of these gene loci upon WGS may reveal the underlying cause for such phenotypic variation.
Fig. 2

Percentage of biofilm formation (bars) and cell aggregation (squares and dotted lines) among isolates. Error bar represents %Relative Standard deviation (RSD)

Co-aggregation is an important event in the initial stages of biofilm development (Kolenbrander 2000). Apparently there was merely any correlation between amount of biofilm biomass and aggregation (Pearson Correlation r = −0.1), as exemplified by SDC21A and SDC21B, which were high biofilm formers but had a contrasting low aggregation capability Fig. 2. PD27 and PD11 showed an opposite behavior. Hence, co-aggregation is not a major factor determining biofilm formation in S. mutans.

Furthermore, the strains were equally acidogenic with a mean final pH of 4.4 ± 0.08 after 72 h (pH range of 4.3–4.6). This may reflect the acidogenic potential of the caries causing bacteria in various patients as well as significant criteria in the multifactorial nature of dental caries. With a mean pH far below the pH of saliva, these bacteria in co-operation with other acidogenic bacteria may pose a serious threat.

Conclusion

Numerous studies have been conducted on etiology of dental caries in children and young adults (Lembo et al. 2007; Sgolastra et al. 2010). But with an increasing average age of individuals and a globally ascending level of diabetes, it is very essential to make cohort studies specifically focused on oral epidemiology of adult diabetics. Future studies will aim at WGS and comparative sequence analysis of the virulence genes of the clinical strains. Our study also encourages and acts as a reference for future epidemiological studies of S. mutans isolates from Italian diabetic patients.

Methods

Sampling and growth conditions

Patients were enrolled during the period October 2012–March 2013. Ethical approval was granted by Comitato Etico Indipendente Fondazione PTV Policlinico Tor Vergata (prot. n. 101/12). All the diabetic patients were ≥40 years of age, had ≥10 natural teeth and had no history of long-term antibiotic use (≥14 days) in the 6 months preceding the study. The considered diabetic hosts (n = 25) where diagnosed with a Type II diabetes and had an average age of 58 (SD = 7.2 years). All patients older than 70 (n = 6) were excluded from the study due to various associated combordities. The tooth examination was performed by the same clinician and the dichotomous plaque index (PI) was assessed at four sites per tooth (Löe 1967). For the evaluation of caries experience, the DMFT index (number of decayed, missing and filled teeth) was used. Supragingival plaque on buccal and lingual surface was collected in 10 mL of distilled water. About 5 mL of unstimulated saliva was obtained from all subjects 2 h after the breakfast and 10 min after mouthwash with 10 mL of deionized water. The plaque and saliva samples were homogenized by ultrasonic dispersion for 20 s at 0 °C. 100 µL of these dispersed samples were spread plate on selective agar medium TSY20B (Trypticase Soy agar with 1 % yeast extract, 20 % sucrose and 200 IE of bacitracin) or MSB (Mitis-Salivarius agar containing 15 % sucrose, 1 % tellurite and 200 IE bacitracin) at appropriate dilutions (Gold et al. 1973; Schaeken et al. 1986). All plates were incubated at 37 °C, in an atmosphere of 91 % N2, 5 % CO2, and 4 % H2 for 5 days.

PCR screening and serotyping

Colonies obtained on selective media were transferred on BHI agar and subsequently screened by colony PCR using S. mutans specific primers for gtfB (Oho et al. 2000). Similarly, PCR based serotypic classification of the clinical isolates were determined using rhamnose–glucose polysaccharide specific primers (Shibata et al. 2003; Nakano et al. 2004). S. mutans UA159 was always used as a positive or negative control as appropriate.

Susceptibility tests

The antimicrobial susceptibility of the clinical isolates was determined by disc diffusion method based on the protocol prescribed by European Committee on Antimicrobial Susceptibility Testing (EUCAST) on viridans group streptococci (Matuschek et al. 2014). Filter discs (Oxoid Ltd., Hants, United Kingdom and Liofilchem, Italy) containing the antimicrobial agents penicillin (1 U), rifampicin (5 µg), oxacillin (1 µg), clindamycin (10 µg), cefoxitin (30 µg), erythromycin (30 µg), levofloxacin (5 µg), linezolid (10 µg), gentamicin (10 µg), norfloxacin (10 µg), tetracycline (30 µg) and vancomycin (5 µg) were used for the study. The zone diameters were measured at three positions for each disc using a caliper. The antimicrobial susceptibility of S. mutans UA159 was evaluated as a reference strain.

Macrorestriction and PFGE

Genomic DNA extraction from clinical isolates was performed by modification of a protocol described previously (Ripa et al. 2001). A high resolution low background gel picture was used to analyze the DNA fingerprint of clinical isolates. PFGE patterns were analyzed and compared using Gel Analyzer. The molecular weight and retention (Rf) values of the digested bands were calculated based on the molecular weight of λ phage ladder. The band size values of the isolates were compared to create a similarity matrix using Pearson correlation coefficient and subsequently cluster analysis using UPGMA in Infostat Professional version 2014 (Di Rienzo et al. 2014).

Cell aggregation assay

Clinical isolates were grown in BHI broth till the mid exponential phase (OD600 nm = 0.7) from an overnight culture, harvested, washed twice in PBS and re-suspended in the same buffer to obtain an OD600 = 0.6 U. One mL of suspension was added with 5 µL of 0.1 M CaCl2, vortexed and transferred to a cuvette. After equilibrating at room temperature for 1 min, the decrease in OD600 of the samples was recorded for 120 min in a spectrophotometer (Cary 100) at 37° C. The percent of aggregation was calculated as [(OD600 at 0 min–OD600 at 120 min)/(OD600 at 0 min)] × 100 (Ahn et al. 2008).

Biofilm Assay

Biofilm formation was assessed in polystyrene 96-well (flat bottom) cell culture clusters (Costar 3595; Corning Inc., NY). An overnight culture of each isolate was transferred in pre-warmed BHI and grown at 37 °C in microaerophilic condition till the mid-exponential phase and then diluted 100 fold in Semi Defined Minimal medium containing 0.8 % glucose (Ahn et al. 2008). An aliquot of culture was dispensed in a microtiter plate and incubated at 37 °C, in 5 % CO2. After 20 h incubation, the culture medium was decanted and the wells were washed thrice with saline. The adhered cells were stained for 15 min using 200 µL of 0.1 % crystal violet at room temperature. Wells were then rinsed twice with saline (0.9 % NaCl). The bound dye was extracted from the adherent cells using 200 µL of 99 % ethanol and quantified at 495 nm. The assay was performed in triplicates.

Final pH analysis

Final pH analysis measures pH at which the growth of each isolate is completely inhibited (van Houte et al. 1996). 50 µL of an overnight culture was inoculated in 5 mL of Phenol Red Dextrose broth (Difco) supplemented with 1 % glucose and incubated at 37 °C, 10 % CO2 for 3 days. The final pH was determined using a pH meter. Non-inoculated medium was used as a control. The tests were done in triplicates.

Statistical analysis

Statistical data analysis was done in Statgraphics Centurion ver. XIV and Infostat Professional version 2014 (Di Rienzo et al. 2014). All the data set were analyzed by non parametric Kruskal–Wallis one way ANOVA at 95 % confidence interval.

Declarations

Authors’ contributions

AD performed the microbiology experiments and wrote the manuscript, LC and ML performed isolation and first identification of bacteria and contributed to the editing of the manuscript, GP supervised the study and contributed to manuscript editing, DP supervised the molecular microbiology section and contributed to the editing of the manuscript, DL designed the study and enrolled patients, LAV designed and supervised the study, wrote and edited the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We acknowledge sincere thanks to Daniela Bencardino for her assistance in PFGE. This work was supported by a Grant from Ministry of University and Research MIUR-ITALY-PRIN Project 2009 No. 2009P5EKH4_004 (to G.P.) and by the University of Camerino Grant No. FPA00057 (to V.L.A.).

Competing interests

The authors declare that they have no competing interests.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee (Ethical approval was granted by Comitato Etico Indipendente Fondazione PTV Policlinico Tor Vergata—prot. n. 101/12) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

(1)
Microbiology Unit, School of Pharmacy, University of Camerino
(2)
Department of Clinical Science and Translational Medicine, University of Rome Tor Vergata
(3)
School of Biosciences and Veterinary Medicine, University of Camerino
(4)
Department of Systems Medicine, University of Rome Tor Vergata

References

  1. Abranches J, Miller JH, Martinez AR et al (2011) The collagen-binding protein Cnm is required for Streptococcus mutans adherence to and intracellular invasion of human coronary artery endothelial cells. Infect Immun 79:2277–2284. doi:https://doi.org/10.1128/IAI.00767-10 View ArticlePubMedPubMed CentralGoogle Scholar
  2. Ahn SJ, Ahn SJ, Wen ZT et al (2008) Characteristics of biofilm formation by Streptococcus mutans in the presence of saliva. Infect Immun 76:4259–4268. doi:https://doi.org/10.1128/IAI.00422-08 View ArticlePubMedPubMed CentralGoogle Scholar
  3. Baddour LM, Wilson WR, Bayer AS et al (2005) Infective endocarditis: diagnosis, antimicrobial therapy, and management of complications: a statement for healthcare professionals from the Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Cardiovascular Disease in the Young. Circulation 111:e394–e434. doi:https://doi.org/10.1161/CIRCULATIONAHA.105.165564 View ArticlePubMedGoogle Scholar
  4. Birren B, Lai E (2012) Pulsed field gel electrophoresis: a practical guide. Academic Press, CambridgeGoogle Scholar
  5. Bowen WH, Schilling K, Giertsen E et al (1991) Role of a cell surface-associated protein in adherence and dental caries. Infect Immun 59:4606–4609PubMedPubMed CentralGoogle Scholar
  6. Boyanova L, Mitov I (2013) Antibiotic resistance rates in causative agents of infections in diabetic patients: rising concerns. Expert Rev Anti Infect Ther 11:411–420. doi:https://doi.org/10.1586/eri.13.19 View ArticlePubMedGoogle Scholar
  7. Brady LJ, Crowley PJ, Ma JK et al (1991) Restriction fragment length polymorphisms and sequence variation within the spaP gene of Streptococcus mutans serotype c isolates. Infect Immun 59:1803–1810PubMedPubMed CentralGoogle Scholar
  8. Caufield PW, Walker TM (1989) Genetic diversity within Streptococcus mutans evident from chromosomal DNA restriction fragment polymorphisms. J Clin Microbiol 27:274–278PubMedPubMed CentralGoogle Scholar
  9. Chait A, Bornfeldt KE (2009) Diabetes and atherosclerosis: is there a role for hyperglycemia? J Lipid Res 50(Suppl):S335–S339. doi:https://doi.org/10.1194/jlr.R800059-JLR200 PubMedPubMed CentralGoogle Scholar
  10. CLSI (2011) Performance standards for antimicrobial susceptibility testing; Twenty First Informational Supplement. Clinical and Laboratory Standards Institute, WayneGoogle Scholar
  11. Cornejo OE, Lefébure T, Bitar PDP et al (2013) Evolutionary and population genomics of the cavity causing bacteria Streptococcus mutans. Mol Biol Evol 30:881–893. doi:https://doi.org/10.1093/molbev/mss278 View ArticlePubMedGoogle Scholar
  12. Davies J, Davies D (2010) Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 74:417–433. doi:https://doi.org/10.1128/MMBR.00016-10 View ArticlePubMedPubMed CentralGoogle Scholar
  13. Di Rienzo JA, Casanoves F, Balzarini MG et al (2014) InfoStat versión 2014. InfoStat Group, Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Argentina. http://www.infostat.com.ar
  14. Dibdin GH, Shellis RP (1988) Physical and biochemical studies of Streptococcus mutans sediments suggest new factors linking the cariogenecity of plaque with its extracellular polysaccharide content. J Dent Res 67:890–895View ArticlePubMedGoogle Scholar
  15. Gold OG, Jordan HV, van Houte J (1973) A selective medium for Streptococcus mutans. Archs oral Biol 18:1357–1364View ArticleGoogle Scholar
  16. Hamada S, Slade HD (1980) Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Rev 44:331–384PubMedPubMed CentralGoogle Scholar
  17. King H, Aubert R, Herman W (1998) Global burden of diabetes 1995–2025. Diabetes Care 21:1414–1431View ArticlePubMedGoogle Scholar
  18. Kolenbrander PE (2000) Oral microbial communities: biofilms, interactions, and genetic systems. Annu Rev Microbiol 54:413–437. doi:https://doi.org/10.1146/annurev.micro.54.1.413 View ArticlePubMedGoogle Scholar
  19. Krzyściak W, Jurczak A, Kościelniak D et al (2014) The virulence of Streptococcus mutans and the ability to form biofilms. Eur J Clin Microbiol Infect Dis 33:499–515. doi:https://doi.org/10.1007/s10096-013-1993-7 View ArticlePubMedGoogle Scholar
  20. Kuramitsu HK, He X, Lux R et al (2007) Interspecies interactions within oral microbial communities. Microbiol Mol Biol Rev 71:653–670. doi:https://doi.org/10.1128/MMBR.00024-07 View ArticlePubMedPubMed CentralGoogle Scholar
  21. Lembo FL, Longo PL, Ota-Tsuzuki C et al (2007) Genotypic and phenotypic analysis of Streptococcus mutans from different oral cavity sites of caries-free and caries-active children. Oral Microbiol Immunol 22:313–319. doi:https://doi.org/10.1111/j.1399-302X.2007.00361.x View ArticlePubMedGoogle Scholar
  22. Löe H (1967) The gingival index, the plaque index and the retention index systems. J Periodontol 38:610–616. doi:https://doi.org/10.1902/jop.1967.38.6.610 View ArticlePubMedGoogle Scholar
  23. Loesche WJ (1986) Role of Streptococcus mutans in human dental decay. Microbiol Rev 50:353–380PubMedPubMed CentralGoogle Scholar
  24. Marsh PD (2009) Dental plaque as a biofilm: the significance of pH in health and caries. Compend Contin Educ Dent 30:76–78, 80, 83–87; quiz 88, 90Google Scholar
  25. Matuschek E, Brown DFJ, Kahlmeter G (2014) Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories. Clin Microbiol Infect 20:O255–O266. doi:https://doi.org/10.1111/1469-0691.12373 View ArticlePubMedGoogle Scholar
  26. Mineyama R, Yoshino S, Maeda N (2007) DNA fingerprinting of isolates of Streptococcus mutans by pulsed-field gel electrophoresis. Microbiol Res 162:244–249. doi:https://doi.org/10.1016/j.micres.2006.06.014 View ArticlePubMedGoogle Scholar
  27. Mitchell SC, Ruby JD, Moser S et al (2011) Maternal transmission of mutans Streptococci in severe-early childhood caries. Pediatr Dent 31:193–201Google Scholar
  28. Mohamed HG, Idris SB, Ahmed MF et al (2013) Association between oral health status and type 2 diabetes mellitus among sudanese adults: a matched case-control study. PLoS One 8:e82158. doi:https://doi.org/10.1371/journal.pone.0082158 ADSView ArticlePubMedPubMed CentralGoogle Scholar
  29. Moser S, Mitchell SC, Ruby JD et al (2010) Repetitive extragenic palindromic PCR for study of Streptococcus mutans diversity and transmission in human populations. J Clin Microbiol 48:599–602. doi:https://doi.org/10.1128/JCM.01828-09 View ArticlePubMedGoogle Scholar
  30. Nakano K, Nomura R, Shimizu N et al (2004) Development of a PCR method for rapid identification of new Streptococcus mutans serotype k strains. J Clin Microbiol 42:4925–4930. doi:https://doi.org/10.1128/JCM.42.11.4925-4930.2004 View ArticlePubMedPubMed CentralGoogle Scholar
  31. Nakano K, Inaba H, Nomura R et al (2006) Detection of cariogenic Streptococcus mutans in extirpated heart valve and atheromatous plaque specimens. J Clin Microbiol 44:3313–3317. doi:https://doi.org/10.1128/JCM.00377-06 View ArticlePubMedPubMed CentralGoogle Scholar
  32. Nakano K, Nomura R, Matsumoto M, Ooshima T (2010) Roles of oral bacteria in cardiovascular diseases—from molecular mechanisms to clinical cases: cell-surface structures of novel serotype k Streptococcus mutans strains and their correlation to virulence. J Pharmacol Sci 113:120–125. doi:https://doi.org/10.1254/jphs.09R24FM View ArticlePubMedGoogle Scholar
  33. Napimoga MH (2004) Genotypic diversity and virulence traits of Streptococcus mutans in caries-free and caries-active individuals. J Med Microbiol 53:697–703. doi:https://doi.org/10.1099/jmm.0.05512-0 View ArticlePubMedGoogle Scholar
  34. Oho T, Yamashita Y, Shimazaki Y et al (2000) Simple and rapid detection of Streptococcus mutans and Streptococcus sobrinus in human saliva by polymerase chain reaction. Oral Microbiol Immunol 15:258–262View ArticlePubMedGoogle Scholar
  35. Palmer SR, Miller JH, Abranches J et al (2013) Phenotypic heterogeneity of genomically-diverse isolates of Streptococcus mutans. PLoS One 8:e61358. doi:https://doi.org/10.1371/journal.pone.0061358 ADSView ArticlePubMedPubMed CentralGoogle Scholar
  36. Pasquantonio G, Condò S, Cerroni L et al (2012) Antibacterial activity of various antibiotics against oral streptococci isolated in the oral cavity. Int J Immunopathol Pharmacol 25:805–809PubMedGoogle Scholar
  37. Patel Reena (2012) The State of Oral Health in EuropeGoogle Scholar
  38. Ripa S, Zampaloni C, Vitali LA et al (2001) SmaI macrorestriction analysis of Italian isolates of erythromycin-resistant Streptococcus pyogenes and correlations with macrolide-resistance phenotypes. Microb Drug Resist 7:65–71. doi:https://doi.org/10.1089/107662901750152828 View ArticlePubMedGoogle Scholar
  39. Schaeken MJ, van der Hoeven JS, Franken HC (1986) Comparative recovery of Streptococcus mutans on five isolation media, including a new simple selective medium. J Dent Res 65:906–908View ArticlePubMedGoogle Scholar
  40. Senadheera D, Cvitkovitch DG (2008) Quorum sensing and biofilm formation by Streptococcus mutans. In: Bacterial signal transduction: networks and drug targets. Springer, New York, pp 178–188Google Scholar
  41. Sgolastra F, Fidanza F, Carosi D et al (2010) An interdisciplinary approach to a survey on dental caries in a group of 3-year-olds in Ascoli Piceno (Italy). Eur J Paediatr Dent 11:137–140PubMedGoogle Scholar
  42. Shibata Y, Ozaki K, Seki M et al (2003) Analysis of loci required for determination of serotype antigenicity in Streptococcus mutans and its clinical utilization. J Clin Microbiol 41:4107–4112. doi:https://doi.org/10.1128/JCM.41.9.4107 View ArticlePubMedPubMed CentralGoogle Scholar
  43. Ship JA (2003) Diabetes and oral Health. J Am Diet Assoc 134:54–55. doi:https://doi.org/10.1024/0301-1526.32.1.54 Google Scholar
  44. Taylor GW (2004) Diabetes, periodontal diseases, dental caries and tooth loss: a review of the literature. Compend Contin Educ Dent 25:179–192PubMedGoogle Scholar
  45. Van Houte J, Lopman J, Kent R (1996) The final pH of bacteria comprising the predominant flora on sound and carious human root and enamel surfaces. J Dent Res 75:1008–1014View ArticlePubMedGoogle Scholar
  46. Venmans LMAJ, Hak E, Gorter KJ, Rutten GEHM (2009) Incidence and antibiotic prescription rates for common infections in patients with diabetes in primary care over the years 1995 to 2003. Int J Infect Dis 13:e344–e351. doi:https://doi.org/10.1016/j.ijid.2008.12.003 View ArticlePubMedGoogle Scholar
  47. Waterhouse JC, Russell RB (2006) Dispensable genes and foreign DNA in Streptococcus mutans. Microbiology 152:1777–1788. doi:https://doi.org/10.1099/mic.0.28647-0 View ArticlePubMedGoogle Scholar
  48. Waterhouse JC, Swan DC, Russell RRB (2007) Comparative genome hybridization of Streptococcus mutans strains. Oral Microbiol Immunol 22:103–110. doi:https://doi.org/10.1111/j.1399-302X.2007.00330.x View ArticlePubMedGoogle Scholar
  49. Zhu L, Kreth J, Cross SE et al (2006) Functional characterization of cell-wall-associated protein WapA in Streptococcus mutans. Microbiology 152:2395–2404. doi:https://doi.org/10.1099/mic.0.28883-0 View ArticlePubMedGoogle Scholar

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