Original Article Braz J Oral Sci. January/March 2009 - Volume 8, Number 1 Genetic polymorphism of Streptococcus mutans strains associated with incomplete caries removal Cristiane Duque1, Thais de Cássia Negrini2, Nancy Tomoko Sacono3, Marcelo Fabiano Gomes Boriollo4, José Francisco Hofling5, Josimeri Hebling6, Denise Madalena Palomari Spolidorio7 1 DDS, PhD, Post-doctorate student, Department of Microbiology and Immunology, Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas (Unicamp), Piracicaba (SP), Brazil; Department of Orthodontics and Pediatric Dentistry, Faculdade de Odontologia de Araraquara, Universidade Estadual Paulista “Júlio de Mesquita Filho” (Unesp), Araraquara (SP), Brazil 2 DDS, MS, Doctorate Student; Department of Clinical Analysis, Faculdade de Ciências Farmacêuticas, Unesp, Araraquara (SP), Brazil 3 DDS, MS, Doctorate Student, Department of Orthodontics and Pediatric Dentistry, Faculdade de Odontologia de Araraquara, Unesp, Araraquara (SP), Brazil 4 DDS, MS, PhD, Professor, Department of Microbiology, Universidade José do Rosário Vellano, Alfenas (MG), Brazil 5 BSc, PhD, Professor, Department of Microbiology and Immunology, Faculdade de Odontologia de Piracicaba, Unicamp, Piracicaba (SP), Brazil 6 DDS, PhD, Professor, Department of Orthodontics and Pediatric Dentistry, Faculdade de Odontologia de Araraquara, Unesp, Araraquara (SP), Brazil 7 BSc, PhD, Professor, Department of Physiology and Pathology, Faculdade de Odontologia de Araraquara, Unesp, Araraquara (SP), Brazil Received for publication: March 15, 2009 Accepted: April 22, 2009 Correspondence to: Cristiane Duque Department of Microbiology and Immunology Faculdade de Odontologia de Piracicaba Universidade Estadual de Campinas Av. Limeira, 901 CEP 13414-903 – Piracicaba (SP), Brazil e-mail: cristianeduque@yahoo.com.br Abstract Aim: Despite the antibacterial properties of dental materials, the survival of residual bacteria under restorations has been demonstrated after incomplete caries removal. The aim of this study was to evaluate the genetic polymor- phism of Streptococcus mutans strains isolated from deep dentinal lesions before and three months after incomplete caries removal. Methods: Samples of carious dentin were collected from 33 primary and/or permanent molars be- fore and after indirect pulp treatment and processed for microbiological isolation of mutans streptococci (MS). After three months of the dental treatment, positive cultures for MS were detected in only ten of these teeth. DNA of MS isolates were obtained and subjected to polymerase chain reaction (PCR) for identification of S mutans. The arbitrary primed-PCR method (primer OPA-13) was used to detect the genetic polymorphism of S. mutans strains. Results: Identical or highly related S. mutans genotypes were observed in each tooth, regardless of the collect. Considering each tooth separately, a maximum of nine genotypic patterns were found in each tooth from all the collects. In addition, at least one genotypic pattern was repeated in the three collects. Genetic diversity was observed among the S. mutans isolates, obtained from different teeth after three months of the dental treatment. Conclusions: The persistence of identical genotypic patterns and the genetic similarity among the isolates, from the same tooth in distinct collects, showed the resistance of some S. mutans strains after incomplete caries removal treatment. Keywords: Streptococcus mutans, polymerase chain reaction, polymorphism, genetic, dental caries. Introduction The wide distribution and variety of oral bacteria demonstrate their ability to survive among their human hosts, as a result of the efficient transmission of strains and their persistence in the oral cavity1. There are several microenvironments within the mouth that harbor communities of pathogenic or non-pathogenic bacteria, called biofilms, which are mainly found in the hard sur- faces of the teeth2. The biochemical instability between tooth substance and the overlying biofilm, determined by intense production of acids derived from bacterial metabolism, and may lead to dissolution of the dental hard tissues and consequently to the development of a carious lesion3. Among the oral pathogens, mutans streptococci (MS) comprise a group of seven bacterial species of which only Streptococcus mutans, S. sobrinus, S. rattus and S. cricetus can be found on the human oral cavity4. The highest prevalence of S. mutans (74 to 94%) among oral streptococci, 3Genetic polymorphism of Streptococcus mutans strains associated with incomplete caries removal Braz J Oral Sci. 8(1): 2-8 isolated from carious lesions demonstrates their association with car- ies development5. S. mutans has the ability of tolerating continuous cycles of acid shock, produced in the oral environment by mecha- nisms of proton extrusion from the cell via membrane-associated pro- cesses and by acid end product efflux, protecting important cellular components, especially DNA, from aggressive effects of acidification6. In addition, in adverse environments with nutrient restriction, some bacteria, such as S. mutans, are able to obtain carbohydrates from host-derived saliva or serum glycoproteins7-8. Genetic diversity among strains of the same species may reflect their ability to survive extreme environmental changes. Therefore, selection of strains best fitted to a given environment can be related to the generation of genetic variants, representing clonal popula- tions, which conserve DNA into single cell lines, or non-clonal pop- ulations, which incorporate DNA from other cells9. Several studies have shown genetic heterogeneity, among S. mutans strains, using modern molecular typing techniques, such as arbitrary primed poly- merase chain reaction (AP-PCR), multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphisms (RFLP) and re- petitive extragenic palindromic PCR (REP-PCR)9-15. AP-PCR is a valuable DNA analysis tool that have been used mainly in streptococcal epidemiology and transmission studies, by virtue of its rapidity, efficiency and reproducibility in generating genetic fingerprints of bacterial isolates10,16. Several studies using AP-PCR have demonstrated that caries-active children have greater genotypic diversity of S. mutans, compared to caries-free children14,17. In addition, a site-specific colonization pattern of S. mutans geno- types in coronal and root caries lesions has been reported12,18. The survival of oral streptococci has been observed even months or years after incomplete caries removal and cavity sealing19-21, sug- gesting the generation of treatment-resistant strains. Paddick et al.15 have detected different genotypes of Streptococcus oralis and Actinomyces naeslundii, five months after incomplete carious dentin removal, with reduced phenotypic and genotypic diversity compared to strains isolated from the initial samples before treatment. There- fore, the aim of this study was to evaluate the genetic polymorphism of S. mutans strains isolated from deep dentinal lesions before and three months after incomplete caries removal. Materials and methods Thirty-three molars (27 primary and 6 young permanent) with deep occlusal caries lesions were obtained from 20 children of both gen- ders, aged four to ten years and subjected to indirect pulp treatment. The absence of irreversible pulpal or periapical diseases was deter- mined through clinical and radiographic examinations of all se- lected teeth. This study was conducted under a protocol approved by the Research Ethics Committee of Faculdade de Odontologia de Ara- raquara of Universidade Estadual Paulista, in Brazil, “Júlio de Mes- quita Filho” (Unesp) and a signed informed consent was obtained from the legal tutors for the the children’s participation. Clinical procedures and microbiological sampling Indirect pulp treatment was divided in two sessions. At the first ses- sion, anesthesia was delivered and a rubber dam applied to isolate the tooth and prevent contamination by saliva. Rubber cup pumice prophylaxis and antisepsis of operative area, using 0.2% chlorhexi- dine digluconate, were performed. If necessary, access to infected dentin was gained using a sterile # 245 carbide bur (KG Sorensen, Barueri, SP, Brazil) at high speed. After removing the superficial necrotic dentin, an initial collect (A) of carious dentin was sampled with a sterile excavator, imme- diately immersed in 1 mL of saline and maintained in refrigerated boxes (4 ºC). A sterile round steel bur at low speed was used to clean all carious tissue from the dentinoenamel junction, leaving a layer of soft dentin on the cavity floor to avoid pulp exposure. After washing and air-drying the cavities to remove debris, a second collect (B) of carious dentin was obtained from the mesial portion of the cavity floor for microbiological analysis, as previously described. In order to standardize the amount of collected dentin, a small cavity was created at the flat end of an amalgam plugger, which was completely filled with dentin. The remaining carious dentin was covered with resin-modified glass ionomer cements (Vitrebond; 3M/ESPE, St. Paul, MN, USA or Fuji Lining LC; GC Corp., Tokyo, Japan) or a cal- cium hydroxide-based cement (Dycal; Dentsply, Milford, DE, USA). The cavities were restored with temporary zinc oxide-eugenol ce- ment (IRM; Dentsply). After three months, the second session of the indirect pulp treatment was undertaken. Under the same initial conditions of anesthesia and isolation, teeth were reopened and the liner materials, carefully and completely removed. A third collect (C) of carious dentin was sampled from the distal portion of the cav- ity floor, as already described. Later, the teeth were lined with glass ionomer cement and restored definitively with silver amalgam (Dis- persalloy, Dentsply International, USA). Microbiological procedures The tubes containing the carious dentin samples were vortexed for two minutes and the suspension was serially diluted with saline. For each dilution, 25 μL of the samples were placed duplicated in agar sacarose bacitracin (SB) – SB-2022 and incubated at 37 oC for 24 hours. After incubation, six to eight representative colonies of strep- tococci were collected and inoculated individually in 5 mL of brain heart infusion (BHI) (Brain Heart Infusion, Difco Laboratories, BD, Sparks, MD, USA) broth for 24 hours. The purity of the cultures were confirmed using Gram technique and aliquots of the subculture fro- zen at -20 oC, in 10% glycerol BHI for posterior molecular analysis of bacterial isolates. Polymerase chain reaction The extraction of chromosomal DNA was described by Nociti et al.23 and modified by Nascimento et al.18. Briefly, overnight cultures in 4 Duque C, Negrini TC, Sacono NT, Boriollo MFG, Hofling JF, Hebling J, Spolidorio DMP Braz J Oral Sci. 8(1): 2-8 BHI broth were centrifuged, followed by washing twice with TRIS- EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH = 8.0) and boiled for ten minutes. After centrifugation, 60 μL of supernatant was collect- ed and used as templates for the PCR. In order to confirm S. mutans molecular identity, DNA from MS isolates obtained from carious dentin collects was submitted to PCR method, using specific prim- ers for portions of the glucosyltransferase B gene (gtf B) following the bases sequences’: 5’ – ACT ACA CTT TCG GGT GGC TTG G – 3’ e 5’ – CAG TAT A AG CGC CAG TTT CAT C – 3’, to amplify a517 bp DNA fragment24. Each PCR mixture contained 5 μL of the DNA template, 5 µL of 10x PCR amplification buffer (100 mM Tris-HCl, 500 mM KCl, pH = 8.3), 0.2 mM of dNTPs (DNA polymerization mix), 3.0 mM MgCl 2 , 1 μM of each primer, 2.5 U of Taq DNA polymerase and sterile dis- tilled water, in order to make a final volume of 25 μL. All PCR re- agents were obtained from Invitrogen, Life Technologies, São Paulo, Brazil. Positive and negative controls of PCR were purified in genom- ic DNA of S. mutans (ATCC 25175) and sterile water, respectively. The amplification of DNA was performed in a thermocycler (GeneAmp PCR System 2400, Perkin-Elmer’s Applied Biosystems Division, Fos- ter City, CA, USA) with initial denaturation at 95 oC for five minutes, followed by 30 cycles of denaturation at 95o C for 30 seconds, anneal- ing at 59 oC for 30 seconds, extension at 72 oC for one minute, ending with final extension at 72 oC for seven minutes24. The PCR amplifica- tion products were separated by electrophoresis in 1% agarose gels in Tris-borato-EDTA (TBE), running buffer (pH = 8.0) at 75 V for two hours. Gels were stained with 0.5 μg of ethidium bromide/mL and visualized under ultraviolet light illumination (UltraLum; Labtrade do Brasil, São Paulo, Brazil). A 100 bp DNA ladder was included as a molecular-size marker in each gel. AP-PCR Strains identified as S. mutans by PCR method were used for geno- typing. AP-PCR amplification was performed with primer OPA-13 (5’- CAGCACCCAC – 3’)10,14,18. All reactions were processed in a vol- ume of 25 μL, containing 2.5 μL of 10x PCR amplification buffer (100 mM Tris-HCl, 500 mM KCl, pH = 8.3), 7 mM MgCl 2 , 0.2 μM of dNTP, 1 μM of primer, 2.5 U of Taq DNA polymerase, 50 ηg of DNA and dis- tilled water18. The amplification was performed in the same thermo- cycler with an initial denaturation at 94 oC for five minutes and 45 cycles of denaturation at 94 oC for 30 seconds, annealing at 36 oC for 30 seconds, extension at 72 oC for one minute, ending with final ex- tension at 72 oC for three minutes. Amplicons generated by AP-PCR were analyzed electropho- retically in 1.4% agarose gel, in TBE running buffer and stained in 0.5 μg/mL ethidium bromide. A 1 Kb DNA ladder was used as mo- lecular-size marker. The gels were photographed and their images, captured with a digital imaging system (Kodak Digital Science 1D; Eastman Kodak Company, Rochester, N Y, USA). The molecular weights for each band or amplicon were computed and analyzed using the Sigma Gel software ( Jandel Scientific, San Rafael, CA, USA). The amplicons were converted in binary data and submit- ted to NTSYS-pc software (Applied Biostatistics, Inc., Setauket, N Y, USA), using the simple matching coefficient (S SM ), and the un- weighted pair group method with mathematic average (UPGMA) cluster analysis to generate similarity dendrograms. Results Only ten deciduous teeth were included in the study that showed positive culture in all collects (A, B and C). From the 429 select- ed MS colonies, 377 (87.9%) were identified as S. mutans. Table 1 shows the number of MS isolates and S. mutans strains, accord- ing to the collected period and material group. One hundred and seventy-three S. mutans isolates were chosen for AP-PCR analysis considering the three collects (A, B and C). Because the distribu- tion of the teeth was not similar among the material groups, data analysis was performed for all teeth, independent of the lining ma- terial. All of them were subjected to AP-PCR. Each isolate received a specific code according to the following sequence: number of the tooth (from 1 to 10), collect (A, B and C) and number of the bacterial isolate (from one to eight), for example: 5A2. The amplification of genomic DNA of this species resulted in fragments (amplicons or eletrophoretic bands), ranging from 0.3 to 2.2 Kb in size. Some of them were species-specific, whereas others were found in only one or few S. mutans strains. The AP-PCR finger- printing profile analysis with primer OPA-13 showed distinct geno- types patterns of S. mutans obtained from caries samples. In this study, the same genotypic pattern was considered for iden- tical or highly related samples with genetic similarity (S SM ) ≥ 0.869 (threshold). Considering each tooth separately, a maximum of nine genotypic patterns were found in each tooth from all the collects (A, B and C). In addition, at least one genotypic pattern was repeated in the three collects. Table 2 shows the total number of genotypic pat- terns detected in each tooth, without repetitions among the collects (genetic similarity S SM ≥ 0.869). Lining material MS colonies Total S. mutans strains Total (%)Baseline Reentry Baseline Reentry A B C A (%) B (%) C (%) Vitrebond 70 51 14 136 57 (81.5) 44 (86.3) 14 (100) 115 (84.6) Fuji lining LC 57 55 47 154 55 (96.5) 55 (100) 37 (78.7) 142 (92.2) Dycal 59 51 25 135 49 (83.1) 47 (92.3) 19 (76) 115 (85.2) Total 186 157 86 429 161 (86.6) 146 (93) 70 (81.4) 377 (87.9) Table 1. MS colonies isolated from SB-20 medium and S. mutans strains identified by PCR 5Genetic polymorphism of Streptococcus mutans strains associated with incomplete caries removal Braz J Oral Sci. 8(1): 2-8 Based in the matrices generated by the UPGMA analysis us- ing coefficient S SM , the genetic similarity levels obtained among the S. mutans strains are illustrated in Figure 1, which shows represen- tative dendrograms of S. mutans isolates obtained for each tooth (1 to 10). The genetic similarity values ranged from 0.555 ≤ S SM ≤ 1. Two to four S. mutans groups (clusters) containing identical or highly related isolates (S SM ≥ 0.869) were found in each tooth. These groups can be identified by tonalities of gray in the dendrograms. Identical or highly related isolates were found in all collects in the same tooth. Comparing the genot y pic patterns in Col lect C, which were obta ined three months af ter denta l treatment , a dendrog ram w ith the SM isolates is show n in Fig ure 2 and the genetic simi- larit y index obser ved was 0.621 ≤ S SM ≤ 1. U PGM A ana lysis re- vea led 11 g roups conta ining identica l or hig h ly related isolates (0.869 ≤ S SM ≤ 1). Seven of these g roups had isolates correspond- ing to a specif ic tooth (Groups 1, 3, 7, 8, 9, 10 and 11). Other three g roups had isolates f rom t wo d istinct teeth (Groups 4, 5 and 6) and one g roup had isolates f rom four teeth (Group 2), demon- strating genetic similarit y among S . mutans stra ins, obta ined f rom the same tooth and g reater poly mor phism among isolates f rom d if ferent teeth. Discussion S. mutans is the major pathogen associated with dental caries in humans. Several studies have demonstrated its capacity to toler- ate extreme pH changes, varying from alkalinity 25 to high levels of acidity6. Furthermore, in situations of environmental nutrient stress, such as after cavity sealing, S. mutans is capable of produc- ing glycosidic enzymes that release carbohydrates from serum glycoproteins7-8 present in the dentinal tubules15. Therefore, it is plausible to suggest that specific phenotypic characteristics could be expressed by genotypes best fitted to survive and/or grow in ad- verse environments1. In the present study, three months after incomplete caries re- moval, genetic diversity of S. mutans strains was observed in samples of deep caries lesions even after the contact with antibacterial mate- rials, such as glass-ionomer cements26. Analyzing each tooth individ- ually, a maximum of nine different genotypic patterns was observed from all the collects. Several studies have demonstrated genetic het- erogeneity among S. mutans strains, obtained from saliva or dental biofilm samples10,13, especially in caries-active individuals14,17. Some investigators have suggested that the genotype frequency can even vary among the oral sites, but a site-specific colonization of geno- types seems to exist in dental caries lesions12,18. Some identical or highly genotypic patterns were isolated in more than one collect, in a same tooth. Although few clones (S SM = 1) have been detected, the great genetic similarity could indicate resistance of S. mutans strains. Genetic groups containing highly related S. mu- tans isolates are thought to have derived from a single ancestral cell9, which could have originated new strains with genetic variations. It is commonly accepted that genetic polymorphism between close spe- cies is determined by modifications in base pairing, by deletion or insertion of new genetic sequences27, in addition to clone transmis- sion to external sources28. However, the frequency of these events in vivo is not known yet8. As a part of the evolution process, bacteria have the ability to gain genetic material from other cells using the mechanism of trans- formation. For recombination to occur, the foreign DNA must share between 70 and 100% identity with the sequence in the recipient strain’s chromosome29. Some S. mutans strains may acquire several cariogenic properties30, fluoride31 and antibiotic resistance32 by trans- formation. It is possible that S. mutans could act as a donor of DNA to another species, such as S. sanguis and S. milleri33. Cvitkovitch2 suggested that bacterial transformations can occur in environments which experience extreme changes and fluctuations in population dynamics, such as the oral cavity. Li et al.34 have demonstrated that S. mutans cells are hypertransformable when grown in biofilms in vitro. Bacteria in these environments are frequently exposed to vari- ous stress conditions, such as nutrient excess or shortage, low pH, high osmolarity and the consumption of antimicrobial agents by the host9,34. Therefore, natural genetic transformation could be consid- ered an important mechanism of cell’s adaptation to environmental changes, providing microbial resistance, genetic variation and rapid evolution of the virulence factors2,29,34. Comparing S. mutans genotypic patterns isolated from carious samples after three months of the dental treatment, genetic diver- sity was observed among the teeth and high intra-tooth similarity was detected. These results may suggest that different resistant S. mutans strains have tooth-specific colonization, because it was not possible to verify a common genetic pattern within the sample sub- jected to the treatment proposed in this study. Some investigators have shown genetic similarity among S. mutans genotypes obtained from members of the same family11,13,35-36 or individuals that cohab- it in environments, such as nursery schools, denoting horizontal transmission37. Generally, non-related subjects rarely share identical S. mutans genotypes10. Nascimento et al.18 have compared 40 genetic types determined by AP-PCR, isolated from nine different patients and it was observed a great diversity among them, demonstrating that the maximum value of the similarity indices (S SM = 0.960) was observed only in isolates from the same individual. Despite some deficiencies, such as the difficulty to visualize low-intensity bands and the need for more than one primer to in- crease the technique accuracy14, studies have shown the efficacy of AP-PCR in the detection of genetic polymorphism of various bacte- Teeth 1 2 3 4 5 6 7 8 9 10 Isolates 15 12 15 16 21 15 18 20 21 20 Genotypic patternsa 2 3 4 3 7 5 2 9 6 3 Table 2 - Total of S. mutans strains and genotypic patterns found in each tooth from all collects (A, B and C) a genotypic patterns (genetic similarity S SM ≥ 0.869) among the collects were considered only once. 6 Duque C, Negrini TC, Sacono NT, Boriollo MFG, Hofling JF, Hebling J, Spolidorio DMP Braz J Oral Sci. 8(1): 2-8 0.00 0.25 0.50 0.75 1.0 Tooth 1 Tooth 2 Tooth 3 Tooth 4 Tooth 5 0. 968 PC 1B2 1B3 1B5 1B6* 1C2 1C1 1C3 1C7 1C5 1A2 1B1 1A3 1C9 1A5 1C8 PC 2C3 2C2 2C8 2C5 2C6* 2C7 2C1* 2A1 2A2 2A8 2A7 2A5 PC 3A2 3A5 3C5 3C4* 3B7 3B1* 3A6 3B2 3B5 3C3 3A1 3A4 3A3 3B6 3C2 PC 4C3 4C4 4C5* 4B4 4B2 4B3 4A4 4A6 4C1 4A3 4C2 4C6* 4B1 4A1 4B5 4A2 PC 5B6 5B7 5A2 5A3 5A4 5A5 5B1 5B3 5C6* 5C7 5C3 5C4 5C5 5C2 5C1 5A1 5C8* 5B4* 5A6* 5B5* 5A7* 0.584 S StrainsSM 1.000 0.941 1.000 0.837 0.941 1.000 1.000 1.000 0.918 1.000 0.941 1.000 1.000 1.000 -------- 0.662 0.941 1.000 0.922 1.000 0.812 0.941 0.790 1.000 1.000 1.000 1.000 ------- 0.576 0.941 0.912 0.824 0.814 0.941 0.840 0.941 1.000 1.000 1.000 1.000 1.000 0.912 0.941 -------- 0.804 1.000 0.941 0.760 0.941 1.000 0.892 1.000 1.000 0.941 1.000 1.000 0.856 0.882 1.000 1.000 -------- 0.429 1.000 0.941 1.000 1.000 1.000 1.000 1.000 1.000 0.800 1.000 0.941 1.000 1.000 0.878 0.912 0.941 0.846 0.768 0.709 0.682 ------- 0.00 0.25 0.50 0.75 1.0 Tooth 6 Tooth 7 Tooth 8 Tooth 9 Tooth 10 0. 968 PC 6C4* 6B4 6B1 6B2 6A4 6A6* 6B6* 6B3* 6C3 6A1 6A3 6B5 6A2 6A5 PC 7C1 7A2 7B6 7A4 7B1 7B2 7C5 7A3 7B5 7A1 7C7 7C2 7C3 7C6 7B3 7B4* 7A6 7A5 PC 8A6 8A5 8C4 8C2 8B5 8B7 8B8* 8B1 8B2 8A4 8B3* 8A8 8B6* 8A7 8C3 8A3* 8C5* 8A1* 8C1* 8B4* PC 9B1 9B6 9B7* 9A3 9C2 9C3 9A4 9A6 9A5 9C4 9C7 9A1 9C5 9C8 9C6 9C1* 9B2* 9B3 9B4 9B5* 9A2* PC 10A1 10A3 10C4 10A2 10A5 10A6 10B1 10B6 10B5 10B4 10B3 10B2 10C5 10C1 10C2 10C3* 10C8* 10C6 10A4 10C7 0.941 0.865 0.941 1.000 0.922 1.000 0.778 0.790 0.863 0.912 0.941 0.882 0.941 1.000 --- 0.702 0.941 0.912 0.941 1.000 1.000 1.000 0.888 1.000 0.941 0.880 0.941 1.000 1.000 0.926 0.918 0.555 0.941 ------ 0.693 0.941 0.876 1.000 0.941 1.000 1.000 0.839 1.000 0.941 0.922 0.868 0.941 0.827 0.882 0.882 0.812 0.737 0.670 0.706 ------ 0.774 1.000 0.941 0.787 0.941 1.000 0.922 1.000 0.882 0.941 0.912 0.874 0.941 1.000 1.000 0.926 0.817 0.843 1.000 0.941 0.739 ------ 0.747 0.941 1.000 0.876 0.941 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.935 0.929 1.000 1.000 0.809 0.843 0.941 1.000 ------- Strains SSM Figure 1. Genetic similarity indices (AP-PCR method, primer OPA-13) verified among S. mutans strains sampled from caries lesions of each teeth, submitted to indirect pulp treatment. A: dendrograms obtained to isolates of teeth 1 to 5. B: dendrograms obtained to isolates of teeth 6 to 10. Individual bands were analyzed by matrices generated by UPGMA analysis using coefficient SSM (simple matching). Tonalities of gray in the dendrograms illustrate identical or highly related isolates (S SM ≥ 0,869) found in each tooth. * Different genotypic patterns obtained in each tooth (S SM < 0,869). A B 7Genetic polymorphism of Streptococcus mutans strains associated with incomplete caries removal Braz J Oral Sci. 8(1): 2-8 Figure 2. Genetic similarity indices (AP-PCR method, primer OPA-13) verified among S. mutans strains sampled from caries lesions three months after dental treatment – Collect C. Dendrogram generated from UPGMA analysis, using coefficient S SM (simple matching). Different tonalities of gray in the dendrograms illustrate identical or highly related isolates (S SM ≥ 0,869). PC 6C4 4C3 4C4 4C5 7C1 3C4 9C1 7C5 9C5 9C8 9C6 8C5 9C7 9C4 9C2 9C3 3C5 2C7 2C1 4C1 4C2 4C6 6C3 10C4 10C1 10C3 10C2 10C5 3C3 3C2 7C2 7C3 7C6 7C7 10C6 10C7 10C8 8C3 8C4 8C2 1C2 1C1 1C3 1C5 1C7 1C8 1C9 2C3 2C2 2C8 2C5 2C6 8C1 5C6 5C7 5C3 5C4 5C5 5C1 5C2 5C8 5B4 0.00 0.25 0.50 0.75 1.0 0. 968 0.941 0.838 1.000 0.941 0.863 0.809 1.000 0.926 0.941 1.000 1.000 0.873 1.000 0.941 0.856 1.000 0.789 0.762 0.941 0.892 0.941 1.000 0.833 0.941 0.912 1.000 1.000 0.871 0.802 0.882 0.941 1.000 1.000 0.926 0.853 0.941 0.846 0.733 0.882 1.000 0.710 0.941 1.000 1.000 1.000 0.929 1.000 0.621 0.941 1.000 0.922 1.000 0.776 0.731 1.000 0.926 1.000 1.000 0.941 0.873 0.849 0.750 --- SSM Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Group 10 Group 11 0.869 SSM 1 Strains rial species10,16, obtaining similar results to more sophisticated tech- niques like MLEE14. In this study, genetic heterogeneity was verified among S. mutans strains isolated form caries lesions, even three months after incom- plete caries removal. The persistence of some identical genotypes and high genetic similarity among isolates of the same tooth, in dis- tinct collects, denoted resistance of some S. mutans strains to dental treatment. The polymorphism observed in different teeth may sug- gest that resistant strains are specific to each tooth, since a common genetic pattern among individuals was not found. However, further studies are necessary to evaluate the phenotypic characteristics of different S. mutans genotypes resistant to indirect pulp treatment or another incomplete caries removal technique, observing possible similarities in the production of enzymes that participate of several virulence mechanisms of this species. Acknowledgements The authors thank Andréia Cristina Celi and Carina Bento Luis Macera for laboratorial assistance. 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