Molecular characterization of Streptococcus mutans gtfB gene isolated from families (original) (raw)
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Brazilian Journal of Oral Sciences, 2014
Aim: To characterize the genetic variability of Streptococcus mutans isolates and to correlate this variability with different colonization profiles observed during dental caries in a sample of children. Methods: S. mutans samples were isolated from the saliva of 30 children with varying histories of dental caries, and they were characterized according to morphological and biochemical markers and the sequences of their 16S-23S intergenic spacer region. The genetic variability of the isolates was first assessed using Random Amplified Polymorphic DNA (RAPD) markers. Next, the isolates were differentiated by sequencing a specific region of the gene encoding the enzyme glucosyltransferase B (gtfB). Results: Characterization using RAPD markers uncovered significant genetic variability among the samples and indicated the existence of clusters, which allowed us to reconstruct both the origin and clinical history of the disease. By sequencing the 16S-23S intergenic region, it was found that all of the isolates belonged to the species S. mutans. Based on the genetic similarity of the isolates and pattern of amino acid variations identified by partial sequencing of the gtfB gene, base-pair changes were identified and correlated with different virulence patterns among the isolates. Conclusions: The partial sequencing of the gtfB gene can be a useful tool for elucidating the colonization patterns of S. mutans. As amino acid variations are likely to be correlated with differences in biological risk, molecular characterization, such as that described in this paper, could be the key for assessing the development of dental caries in children.
Journal of Medical Microbiology, 2004
The present study evaluated the relationship between clonal diversity and some virulence traits of Streptococcus mutans isolated from eight caries-free and eight caries-active subjects. A total of 155 S. mutans isolates from caries-free subjects and 144 isolates from caries-active subjects were obtained from samples of saliva, dental plaque and tongue surface and identified by PCR. The isolates were submitted to arbitrarily primed (AP)-PCR (OPA-2 and OPA-13) and multilocus enzyme electrophoresis (MLEE) to establish the genotypic diversity. Production of water-insoluble glucan (WIG) (monitored by SDS-PAGE), final pH of cultures and the ability of bacterial cells to adhere to smooth glass in the presence of sucrose were measured. High and comparable abilities of MLEE and AP-PCR were found to distinguish S. mutans genotypes, using Simpson's index of discrimination (0 . 971 and 0 . 968, respectively). The results showed a significant difference (P , 0 . 01) in the number of genotypes when caries-free and caries-active groups were compared by both fingerprinting methods used. Final pH (P ¼ 0 . 32) and the percentage of adherence to a glass surface (P ¼ 0 . 62) did not show differences between the two groups; however, the intensities of WIG bands from the caries-active group were greater than those from the caries-free group (P , 0 . 01). In addition, WIG was positively correlated with the ability of S. mutans to adhere to a glass surface (r ¼ 0 . 34, P ¼ 0 . 02) from caries-active subjects. These data showed that AP-PCR analysis and MLEE are both effective methods for assessing the genetic relatedness of S. mutans. Using these techniques, it was found that there is a larger number of genotypes of S. mutans with increased ability to synthesize WIG in caries-active individuals.
Brazilian Archives of Biology and Technology, 2013
The objective of this study was to analyze the genotypic diversity, frequency of serotypes and the detection of mutacins from Streptococcus mutans isolates in caries-free and caries-active individuals. A total of 260 S. mutans isolated from 28 individuals with and without dental caries were subjected to AP-PCR and PCR screening of glucosyltransferase B, mutacin and serotype genes, which showed the presence of. 70 different genotypes. There was no statistically significant association between the presence of genes for serotypes and mutacins with dental caries. However, there was a statistically significant and a strong association between the higher genotypic diversity in the subjects with caries (r = 0.72, p = 0.001). There was an increase in the number of genotypes with increasing age (p <0.01).
Genetic polymorphism of Streptococcus mutans strains associated with incomplete caries removal
2009
The aim of this study was to determine whether random amplified polymorphic DNA (AP-PCR) analysis is able to differentiate genetically different clones of mutans streptococci, in 22 Brazilian family members. Stimulated saliva samples were collected from fathers, mothers and infants. For 5-18 months babies with erupting primary dentition, plaque samples were collected using sterile tooth pick tips. From these samples, mutans streptococci were isolated on SB-20 agar plates. After growth, representative colonies were identified by biochemical methods on the basis of carbohydrate fermentation. Streptococcus mutans isolates were obtained from all family members and AP-PCR typed separately with a random primer (OPA-13). Bacterial cell lysates were used as template in PCR reactions and the amplified DNA fragments obtained were compared by agarose gel electrophoresis. Results demonstrated that the father shared the baby's genotype in three families and the mother shared the baby's genotype in 12 families seven babies harbored Streptococcus mutans strains similar to those of their siblings. The technique was able to demonstrate the genetic Streptococcus mutans in Brazilian family members.
Oral Microbiology and Immunology, 2009
Streptococcus mutans is a bacterial resident of the oral cavity and is considered to be the principal etiological agent of dental caries in humans (13, 23). As S. mutans can be isolated from individuals either with or without a history of caries (4, 25), differences in the colonizing S. mutans may contribute to the eventual development and progression of caries among different individuals. One difference may be in the bacterial load. High levels of S. mutans have been associated with caries (7, 12, 18, 33); however, many people with a high S. mutans load because of highlevel sucrose consumption do not develop caries (25, 29). Another important factor may be the characteristics of S. mutans. Certain S. mutans attributes that are believed to be important in caries development, including acidogenicity, aciduricity, biofilm-formation potential and production of glucans, are known to vary among isolates of S. mutans and may determine the differences in caries
Journal of Clinical Microbiology, 2008
Streptococcus mutans is one of several members of the oral indigenous biota linked with severe early childhood caries (S-ECC). Because most humans harbor S. mutans, but not all manifest disease, it has been proposed that the strains of S. mutans associated with S-ECC are genetically distinct from those found in caries-free (CF) children. The objective of this study was to identify common DNA fragments from S. mutans present in S-ECC but not in CF children. Using suppressive subtractive hybridization, we found a number of DNA fragments (biomarkers) present in 88 to 95% of the S-ECC S. mutans strains but not in CF S. mutans strains. We then applied machine learning techniques including support vector machines and neural networks to identify the biomarkers with the most predictive power for disease status, achieving a 92% accurate classification of the strains as either S-ECC or CF associated. The presence of these gene fragments in 90 to 100% of the 26 S-ECC isolates tested suggested their possible functional role in the pathogenesis of S. mutans associated with dental caries. The mutans streptococci (MS) are strongly associated with dental caries by virtue of their metabolic, ecological, and epidemiological attributes (23, 46). Among the MS, Streptococcus mutans appears to be a predominant bacterial species in the microbiota of preschool children with severe early childhood caries (S-ECC) (4-6, 49). Although the association between S. mutans and S-ECC seems convincing, most children colonized by S. mutans do not manifest the disease (8), suggesting that among other possibilities, S. mutans vary in their ability to initiate caries. In our previous study, we demonstrated that strains of S. mutans strains associated with S-ECC differ in their genomic composition compared to caries-free (CF) controls (42). Using the power of suppressive subtractive DNA hybridization (SSH), several unique gene segments were identified from a strain of S. mutans (AF199) that was isolated from a child with S-ECC. The presence of unique genetic loci among S. mutans strains is consistent with the recent work by Waterhouse and Russell (51), as they described the presence of "dispensable genes" distributed among strains of S. mutans. These segments include mobile genetic elements that are widely distributed in S. mutans (2) and have been shown to modulate sucrose (31) and melibiose metabolism (40). S. mutans strains also vary in content in terms of the presence of plasmids (10, 32), mutacin I, II, III, and IV operons (3, 19, 35-37), serotypic antigens (43); competence (34), the comBCD genes (28), and gtfBC (14, 48, 52), among other genetic loci. Based on the wide diversity of genotypes and genetic loci within S. mutans, different strains of S. mutans apparently comprise both common and unique ge
Infection, Genetics and Evolution, 2014
Comparative genomics is a popular method for the identification of microbial virulence determinants, especially since the sequencing of a large number of whole bacterial genomes from pathogenic and non-pathogenic strains has become relatively inexpensive. The bioinformatics pipelines for comparative genomics usually include gene prediction and annotation and can require significant computer power. To circumvent this, we developed a rapid method for genome-scale in silico subtractive hybridization, based on blastn and independent of feature identification and annotation. Whole genome comparisons by in silico genome subtraction were performed to identify genetic loci specific to Streptococcus mutans strains associated with severe early childhood caries (S-ECC), compared to strains isolated from caries-free (CF) children. The genome similarity of the 20 S. mutans strains included in this study, calculated by Simrank k-mer sharing, ranged from 79.5% to 90.9%, confirming this is a genetically heterogeneous group of strains. We identified strain-specific genetic elements in 19 strains, with sizes ranging from 200 to 39 kb. These elements contained protein-coding regions with functions mostly associated with mobile DNA. We did not, however, identify any genetic loci consistently associated with dental caries, i.e., shared by all the S-ECC strains and absent in the CF strains. Conversely, we did not identify any genetic loci specific with the healthy group. Comparison of previously published genomes from pathogenic and carriage strains of Neisseria meningitidis with our in silico genome subtraction yielded the same set of genes specific to the pathogenic strains, thus validating our method. Our results suggest that S. mutans strains derived from caries active or caries free dentitions cannot be differentiated based on the presence or absence of specific genetic elements. Our in silico genome subtraction method is available as the Microbial Genome Comparison (MGC) tool, with a user-friendly JAVA graphical interface.
BMC oral health, 2017
Early childhood caries (ECC) is an aggressive condition that can affect teeth of young children. This study aimed to evaluate genotypic diversity and phenotypic traits of S. mutans isolated from dental biofilms of children with different caries status in comparison with caries free (CF) children. Streptococcus mutans strains were isolated from supragingival biofilm samples of CF, ECC and severe-ECC (S-ECC) children and genotyped by arbitrary-primer polymerase chain reaction - AP-PCR. S. mutans genotypes were tested for their ability to reduce the suspension pH through glycolysis, to tolerate extreme acid challenge and by their ability to form biofilm. Response variables were analyzed by ANOVA/Tukey or Kruskal-Wallis/Mann-Whitney tests at a 5% of significance. There was an increase in the prevalence of Streptococcus mutans in biofilms with the severity of dental caries. No differences in genotypic diversity and in acidogenicity of genotypes were found among CF, ECC and S-ECC children...
Genotypic profiles by AP-PCR of streptococcus mutans in caries-active and caries-free preschoolers
Acta odontologica latinoamericana : AOL, 2010
Streptococcus mutans, an acidogenic and aciduric microorganism that colonizes the oral cavity is recognized as the main causal agent of dental caries. Epidemiological studies have shown a strong correlation between the number of S. mutans in the oral cavity and prevalence and incidence of caries. At present, different genotypic and phenotypic methods are known to determine the profiles of settling and epidemiological distribution of S. mutans. The aim of this study was to investigate the profiles of S. mutans isolated from children with and without dental caries by using the AP-PCR (arbitrarily primed polymerase chain reaction) and api-Zym methods. In the AP-PCR method, random DNA segments of the target bacterium are amplified with single primers of arbitrary sequence. The api-Zym system (bioMirieux, Marcy-létoile, France) is a phenotypic micro-method that allows simultaneous detection of 19 enzymatic activities from bacterial inoculum. A transversal observational study was conducte...