Antibiotic Resistance Patterns and Related Mobile Genetic Elements of Pneumococci and ß-Hemolytic Streptococci in Thai Healthy Children.

Transmission of antibiotic resistance genes among Streptococcus pneumoniae and beta-hemolytic streptococcus (BHS) was generally associated with transmissible genetic elements. The objectives of this study were to investigate carriage rate, antibiotic resistance and related mobile genetic elements of pneumococci and BHS from school-children. The pneumococci and BHS were recovered from 220 Thai school-children, and then tested for antibiotic susceptibility pattern by disc diffusion. Antibiotic resistance genes and related genetic elements were detected by PCR with specific primers. A total of 77 pneumococcal isolates were resistant to erythromycin (42 %), tetracycline (44 %), clindamycin (8 %), or penicillin (3 %). Fifty-four BHS isolates were resistant to erythromycin (28 %), tetracycline (52 %), or clindamycin (13 %). All isolates tested were 100 % sensitive to penicillin and levofloxacin. Among erythromycin-resistant streptococcal isolates showed different phenotypes of clindamycin resistance. It was found that isolated pneumococci showed constitutive clindamycin resistance (19 %), and inducible clindamycin resistance (12 %). The BHS isolates exhibited constitutive clindamycin resistance (40 %), and inducible resistance (20 %) phenotypes. The predominant erythromycin resistance genes in pneumococci and BHS were mefE and ermB, while the most common tetracycline resistance gene in this population was tetM. Furthermore, almost all erythromycin- and tetracycline-resistant streptococci (97 %) mainly contained various genetic elements, including mega elements and six different transposon types (Tn2009, Tn2017, Tn917, Tn3872, Tn6002 and Tn916). Therefore, carriages of pneumococci and BHS with multidrug resistance in children might be important reservoirs of antibiotic-resistance genes carried by transposons. Tn916-like elements could lead to dissemination of the antibiotic resistance genes among genus streptococcus in human oral cavity and nasopharynx.

Identification and characterization of a collagen-binding protein, Cbm, in Streptococcus mutans.

Streptococcus mutans, a major pathogen of dental caries, is occasionally isolated from the blood of patients with infective endocarditis. Bacterial attachment of exposed collagen tissue in the impaired endothelium is an important step in the onset of infective endocarditis. In our previous studies, some S. mutans strains were shown to possess collagen-binding activities and most of them had an approximately 120-kDa cell-surface collagen-binding protein called Cnm. However, several strains without Cnm proteins show collagen-binding properties. In the present study, another collagen-binding protein, Cbm, was characterized and its coding gene cbm was sequenced in these strains. The amino acid alignment in the putative collagen-binding domain of Cbm was shown to have approximately 80% identity and 90% similarity to the comparable region of Cnm. Cbm-deficient isogenic mutant strains constructed by insertional inactivation of the cbm gene, lacked collagen-binding properties, which were recovered in the complemented mutant. Analyses of a large number of clinical isolates from Japan, Thailand and Finland revealed that cbm-positive strains were present in all of these countries and that cnm-positive and cbm-positive strains were detected in the oral cavity of approximately 10 and 2% of systemically healthy subjects, respectively. In addition, cnm-positive strains were predominantly identified in the serotype f group, whereas cbm-positive strains were frequently detected in serotype k. These results suggest that Cbm as well as Cnm are major cell surface proteins of S. mutans associated with binding to type I collagen and predominantly identified in serotype k strains.

Molecular characterization of Streptococcus mutans strains containing the cnm gene encoding a collagen-binding adhesin.

OBJECTIVE: Streptococcus mutans, known to be a major pathogen of dental caries, is also considered to cause infective endocarditis. Its 120-kDa Cnm protein binds to type I collagen, which may be a potential virulence factor. In this study, we characterized S. mutans clinical strains focusing on the cnm gene encoding Cnm. DESIGN: A total of 528 S. mutans strains isolated from Japanese, Finnish, and Thai subjects were investigated. Using molecular techniques, the distribution frequency of cnm-positive strains and location of the inserted cnm were analyzed. Furthermore, isogenic mutant strains were constructed by inactivation of the cnm gene, then their biological properties of collagen-binding and glucan-binding were evaluated. Southern hybridization of the genes encoding glucan-binding proteins was also performed. RESULTS: The distribution frequency of cnm-positive strains from Thai subjects was 12%, similar to that previously reported for Japanese and Finnish subjects. Furthermore, the location of insertion of cnm was the same in all cnm-positive clinical isolates. As for the cnm-inactivated mutant strains constructed from 28 clinical isolates, their collagen-binding activity was negligible. In addition, glucan-binding activity in the cnm-positive clinical isolates was significantly reduced and corresponded to a lack of gbpA encoding glucan-binding protein A. CONCLUSIONS: Our results indicate that strains with cnm genes, the most crucial factor for the collagen-binding property of S. mutans, are detectable at similar frequencies over several different geographic locations. In addition, the common properties of these strains are a high level of collagen-binding activity and tendency for a low level of glucan-binding activity.

Detection of serotype k Streptococcus mutans in Thai subjects.

INTRODUCTION: Streptococcus mutans, known to be a pathogen of dental caries as well as bacteremia and infective endocarditis, is classified into four serotypes, c, e, f and k, based on the structures of serotype-specific polysaccharides. Serotype k was recently designated using blood isolates from Japanese subjects and such strains are considered to be virulent in the bloodstream. The purpose of the present study was to analyse the serotype distribution of strains isolated from Thai subjects and determine whether serotype k strains were present. METHODS: A total of 250 S. mutans strains were isolated from 50 Thai subjects, and serotypes of all strains were determined. Then, molecular and biological analyses were carried out for serotype k strains. RESULTS: Immunodiffusion and polymerase chain reaction analyses showed that serotype c was the most prevalent (70%), followed by serotypes e (22.8%), f (4.4%) and k (2.8%), which indicated that serotype k S. mutans strains occurred in Thai individuals at a similar rate to that previously reported for Japanese and Finnish populations. Molecular analyses of the seven serotype k strains showed extremely low expression of rgpE, which is related to glucose side-chain formation in serotype-specific rhamnose-glucose polymers, similar to previous reports for those other populations. In addition, analysis of the biological properties of the seven serotype k strains demonstrated low levels of sucrose-dependent adhesion, cellular hydrophobicity, dextran-binding activity and phagocytosis susceptibility by human polymorphonuclear leukocytes, which are characteristics similar to those of serotype k strains previously isolated in Japan. CONCLUSION: Our results indicate the possibility of a worldwide prevalence of serotype k strains with properties in common with those of previously reported strains.

Demonstration of mother-to-child transmission of Streptococcus mutans using multilocus sequence typing.

A new reliable genotyping method, multilocus sequence typing (MLST), was used to evaluate vertical transmission of the cariogenic pathogen Streptococcus mutans. A total of 136 S. mutans strains were isolated from saliva samples of 20 Japanese mother-child pairs, including 5 girls and 5 boys with primary dentition, and 5 girls and 5 boys with mixed dentition. The nucleotide sequences of 8 partial housekeeping genes, aroE, murI, gltA, glnA, glk, tkt, lepC, and gyrA, were analyzed and a similarity for all of those sequences between strains from a mother-child pair was regarded as indicating transmission, which was shown in 70% of the pairs. Interestingly, the rate of transmitted strains from mothers was significantly higher in the girls (90%) than in the boys (p = 0.001). Furthermore, the S. mutans sequence type (ST) with the highest distribution percentage in each maternal saliva sample was found to be transferred to their children. In addition, variations in two large conjugative-transfer associated regions, TnSmu1 and TnSmu2, were determined and compared with the STs defined by MLST. No variations in those two regions shown by PCR patterns were present in any of the strains isolated from the same families with the same STs, though isolates of some STs from different families showed distinct patterns for TnSmu2. Our results indicate that mothers are the main source for transmission of S. mutans to their children, while the present MLST method was also shown to be useful for investigating bacterial transmission.

Protein antigen in serotype k Streptococcus mutans clinical isolates.

Streptococcus mutans, a major pathogen of dental caries and infective endocarditis, is classified into serotypes c, e, f, and k, with serotype k strains recently reported to be frequently detected in persons with infective endocarditis. Thus, we hypothesized that common properties associated with infective endocarditis are present in those strains. Fifty-six oral S. mutans strains, including 11 serotype k strains, were analyzed. Western blotting analysis revealed expression of the 3 types of glucosyltransferases in all strains, while expression of the approximately 190-kDa cell-surface protein (PA) was absent in 12 strains, among which the prevalence of serotype k (7/12) was significantly high. Furthermore, cellular hydrophobicity and phagocytosis susceptibility were lower in the group of serotype k strains. These results indicate that the absence of PA expression, low cellular hydrophobicity, and phagocytosis susceptibility are common bacterial properties associated with serotype k strains, which may be associated with virulence for infective endocarditis.