Distribution of amoxicillin-resistant oral streptococci in dental plaque specimens obtained from Japanese children and adolescents at risk for infective endocarditis.

BACKGROUND: Infective endocarditis (IE) is known to be a life-threatening disease and prevention of its onset is important. Oral amoxicillin (AMPC) is generally prescribed to patients at risk for IE prior to undergoing risky procedures, such as invasive dental treatments. We previously found that approximately 5% of systemically healthy Japanese subjects harbor strains highly resistant to AMPC. In the present study, the prevalence of strains in patients at risk for IE was investigated. METHODS AND RESULTS: Thirty-four Japanese children and adolescents designated at risk for IE by their cardiovascular surgeons participated. Dental plaque specimens were obtained at recall examinations for dental checkups and placed in sterile phosphate-buffered saline, then diluted and streaked onto selective media for oral streptococci and also media containing AMPC. Nine strains with a minimum inhibitory concentration of AMPC of 16µg/mL or more were isolated from 7 of the subjects (20.6%), each of which was also resistant to other antibiotics analyzed except for new quinolone drugs. The 16S rRNA sequence of each strain demonstrated that all were oral streptococcal species. In addition, dental plaque specimens collected from 5 subjects after an additional interval of 3-4 months showed that 2 harbored the same clones at different time points. CONCLUSIONS: These findings suggest a higher prevalence of AMPC-resistant strains in children and adolescents at risk for IE as compared to systemically healthy subjects. Thus, alternative antibiotics should be considered for such subjects when performing prophylaxis procedures.

Multilocus sequence typing of Streptococcus mutans strains with the cbm gene encoding a novel collagen-binding protein.

OBJECTIVE: Streptococcus mutans, an oral pathogen associated with infective endocarditis (IE), possesses two genes encoding collagen-binding proteins, namely cnm and cbm. In this study, we used multilocus sequence typing (MLST) of S. mutans with the cbm gene. DESIGN: Forty-five S. mutans strains including 15 strains with the cnm gene, 15 strains with the cbm gene, and 15 strains without these two genes were analysed by MLST. In addition, the collagen-binding properties as well as the abilities to adhere to and invade human umbilical vein endothelial cells (HUVEC) were also evaluated for all strains. RESULTS: In the groups of cnm-positive and cbm-positive strains, all properties, including collagen binding, adhesion, and invasion were significantly greater than those of the cnm-cbm-negative group. Moreover, MLST revealed three clonal complexes of S. mutans possessing the cbm gene. These three clones showed no close relatedness with clones of strains containing the cnm gene. Among three clones harbouring the cbm gene, two clones belong to serotype k, and appeared to be associated with the pathogenesis of IE due to their strong collagen binding and relatively enhanced abilities to adhere to and invade endothelial cells. However, such properties were relatively weak in the other non-serotype k clone possessing the cbm gene. CONCLUSIONS: MLST indicated a difference in evolution between S. mutans strains with the cbm gene and those with the cnm gene. In addition, this technique also suggested the importance of cbm-positive S. mutans clones relative to the pathogenesis of IE.

Amoxicillin-resistant oral streptococci identified in dental plaque specimens from healthy Japanese adults.

BACKGROUND: Infective endocarditis (IE) is known to be a life-threatening disease and invasive dental procedures are considered to be important factors. Oral amoxicillin (AMPC) is widely used for prophylaxis in patients with heart disorders who are at risk for IE. However, there is only limited information regarding the inhibition of oral bacteria by AMPC. METHODS AND RESULTS: Dental plaque specimens were obtained from 120 healthy Japanese adult subjects, then diluted and streaked onto selective medium for oral streptococci. The minimum inhibitory concentration (MIC) of AMPC was evaluated using a macro-dilution method by Clinical Laboratory Standard Institute (2006). Seven strains with an MIC of AMPC of 16µg/mL or more were isolated from 5 subjects. The bacterial species were confirmed by sequence analysis of 16S rRNA from each strain, which demonstrated that most were Streptococcus sanguinis, followed by Streptococcus oralis. Dental plaque specimens collected from these 5 subjects again after an interval of 2-3 months possessed no strains with an MIC of AMPC of 16µg/mL or more. CONCLUSIONS: These findings suggest that strains with a high MIC of AMPC are present in the oral cavities of Japanese adults, though they may be transient rather than inhabitants.

Distribution of oral streptococci highly resistant to amoxicillin in dental plaque specimens from Japanese children and adolescents.

Oral streptococci are major pathogens of infective endocarditis. Prophylactic antibiotics are commonly given to subjects with certain kinds of heart disorders when invasive dental treatments are performed, with amoxicillin (AMPC) being widely used for this purpose. However, there is little information regarding AMPC-resistant oral streptococci. Here, a total of 344 dental plaque specimens collected from 253 healthy Japanese children, adolescents and young adults (aged 2-22 years) were diluted and streaked onto culture medium containing high-dose AMPC. The MICs for the isolated strains were evaluated using a macrodilution broth method described by the Clinical and Laboratory Standards Institute. Bacterial DNA was extracted from each strain and the entire sequences of the 16S rRNA gene were compared with those in GenBank to identify the species. The results showed that strains with AMPC MICs >16 µg ml(-1) were isolated from 18 specimens from 14 patients. Analyses of the 16S rRNA gene sequences of these strains identified them as major oral streptococcal species, including Streptococcus oralis and Streptococcus mitis. These findings indicate that oral streptococci with elevated MICs for AMPC exist in certain small populations of healthy children, and highlight the need for further studies to determine risk factors that lead to the appearance of such strains.

Distribution of periodontopathic bacterial species in Japanese children with developmental disabilities.

BACKGROUND: Recent developments in molecular biological techniques have enabled rapid detection of periodontopathic bacterial species in clinical specimens. Accumulated evidence suggests that detection of specific bacterial species enables identification of subjects at high risk for the onset of periodontitis. We investigated the distribution of 10 selected periodontopathic bacterial species in dental plaque specimens obtained from children with disabilities who were attending daycare centers. METHODS: A total of 187 children (136 boys, 51 girls) aged 1-6 years old and diagnosed with such disabilities as mental retardation, cerebral palsy, and autism, participated in the study. Subgingival dental plaque specimens were collected from the buccal side of the maxillary left second primary molar after a clinical examination. Bacterial DNA was extracted from the specimens and PCR analyses were carried out to detect 10 selected periodontopathic species using specific primers for each. In addition, statistical analyses were performed to analyze the correlations among clinical parameters and the detected species. RESULTS: The most frequently detected species was Capnocytophaga sputigena (28.3%), followed by Aggregatibacter actinomycetemcomitans (20.9%) and Campylobacter rectus (18.2%). Eikenella corrodens, Capnocytophaga ochracea, and Prevotella nigrescence were detected in approximately 10% of the specimens, whereas Treponema denticola, Tannerella forsythia, and Prevotella intermedia were rarely found, and Porphyromonas gingivalis was not detected in any of the subjects. The total numbers of detected species were positively correlated with the age of the subjects. There were 10 subjects with positive reactions for T. denticola and/or T. forsythia, in whom the total number of bacterial species was significantly higher as compared to the other subjects. Furthermore, subjects possessing C. rectus showed significantly greater values for periodontal pocket depth, gingival index, and total number of species. CONCLUSION: We found that approximately one-fourth of the present subjects with disabilities who possessed at least one of T. denticola, T. forsythia, and C. rectus were at possible risk for periodontitis. Follow-up examinations as well as preventive approaches should be utilized for such individuals.

Molecular and clinical analyses of the gene encoding the collagen-binding adhesin of Streptococcus mutans.

Streptococcus mutans is a known pathogen of dental caries and its major cell surface antigens have been widely investigated. Recently, an approximately 120 kDa Cnm protein with binding properties to type I collagen was identified, and its encoding gene (cnm) cloned and sequenced. In the present study, we sequenced cnm from 47 different clinical S. mutans strains and found that the nucleotide alignment of the collagen-binding domain was well conserved. We devised a PCR method for identifying the cnm gene, examined the prevalence of cnm-positive S. mutans strains in various mother-child groups, and assessed the significance of such strains for transmission and dental caries. The detection rate of cnm-positive strains was significantly lower in strains isolated from Japanese children in the 2000s (8.0 %) as compared to those isolated in the 1980s (15.8 %) (P<0.05). Furthermore, the presence of S. mutans possessing cnm in salivary specimens collected from 55 S. mutans-positive mother-child pairs was 40 and 32.7 % in the mothers and children, respectively. The frequency of cnm-positive children whose mothers were also positive was 72 %, which was significantly higher than that of cnm-positive children with negative mothers (P<0.0001, odds ratio 17.5). In addition, clinical parameters indicating dental caries were significantly increased in children with cnm-positive S. mutans in saliva (n=13), as compared to those with cnm-negative S. mutans (n=15) and S. mutans-negative children (n=20) (P<0.01). These results indicate that cnm-positive S. mutans strains are closely correlated with dental caries, while vertical transmission in cnm-positive mother-child pairs was also demonstrated.

Molecular characterization of Streptococcus mutans strains isolated from the heart valve of an infective endocarditis patient.

Streptococcus mutans, known to be an aetiological agent of dental caries, is occasionally isolated from patients with infective endocarditis (IE). S. mutans strains with a defect in all three types of glucosyltransferase (GTF) obtained from an infected heart valve extirpated from an IE patient have been reported previously. In this study, molecular analyses of strains detected in heart valve (strain V1) and dental plaque (strain P1) samples taken from the same patient were performed. Complete nucleotide alignments of the gtfB, gtfC and gtfD regions in strains V1 and P1, as well as in the reference strain MT8148, were determined, which revealed the existence of alignments with a high similarity to erythromycin- and spectinomycin-resistance genes in the middle of the gtfB-gtfC and gtfD genes, respectively, of V1. Strain V1 also showed a higher MIC for these two antibiotics compared with strain P1. Next, primers to detect the specific sequences of the antibiotic-resistance genes in strain V1 were constructed and PCR amplification was performed with template DNA from dental plaque and infected valve tissue samples taken from the patient. Attenuated expression of GTFs in V1 caused a significantly lower susceptibility to phagocytosis by human polymorphonuclear leukocytes compared with the reference strain. These results suggest that the blood isolate V1 found in the oral cavity invaded and survived in the bloodstream for a long duration and that this was related to its virulence in IE in our patient.

Repeated bacteraemia caused by Streptococcus mutans in a patient with Sjogren's syndrome.

Streptococcus mutans, considered to be a pathogen for dental caries, is known to cause bacteraemia and infective endocarditis. Herein, an unusual case of repeated bacteraemia caused by S. mutans identified in a 71-year-old male is described. The patient visited Itami City Hospital with the major complaint of a fever, and a subsequent clinical examination led to a diagnosis of possible infective endocarditis without specific vegetation formation around the heart valve. A bacteriological examination of blood taken at the first visit showed the presence of S. mutans. Antimicrobial treatment was provided, which successfully eliminated the pathogenic bacteria from the blood. However, the patient returned and was hospitalized twice more with a recurrent fever, and S. mutans was again detected. Analyses of the biological properties of the S. mutans isolates showed that they possessed cariogenic properties and had a low susceptibility to phagocytosis by human polymorphonuclear leukocytes. Since the patient had Sjögren's syndrome, in which a reduction of saliva secretion is a characteristic feature, a great number of dental caries lesions were identified. The findings indicated that S. mutans present in those dental caries lesions caused repeated bacteraemia in this case.

Streptococcus mutans clonal variation revealed by multilocus sequence typing.

Streptococcus mutans is the major pathogen of dental caries, a biofilm-dependent infectious disease, and occasionally causes infective endocarditis. S. mutans strains have been classified into four serotypes (c, e, f, and k). However, little is known about the S. mutans population, including the clonal relationships among strains of S. mutans, in relation to the particular clones that cause systemic diseases. To address this issue, we have developed a multilocus sequence typing (MLST) scheme for S. mutans. Eight housekeeping gene fragments were sequenced from each of 102 S. mutans isolates collected from the four serotypes in Japan and Finland. Between 14 and 23 alleles per locus were identified, allowing us theoretically to distinguish more than 1.2 x 10(10) sequence types. We identified 92 sequence types in these 102 isolates, indicating that S. mutans contains a diverse population. Whereas serotype c strains were widely distributed in the dendrogram, serotype e, f, and k strains were differentiated into clonal complexes. Therefore, we conclude that the ancestral strain of S. mutans was serotype c. No geographic specificity was identified. However, the distribution of the collagen-binding protein gene (cnm) and direct evidence of mother-to-child transmission were clearly evident. In conclusion, the superior discriminatory capacity of this MLST scheme for S. mutans may have important practical implications.

Serotype distribution of Streptococcus mutans a pathogen of dental caries in cardiovascular specimens from Japanese patients.

The involvement of oral bacteria in the pathogenesis of cardiovascular disease has been studied, with Streptococcus mutans, a pathogen of dental caries, detected in cardiovascular lesions at a high frequency. However, no information is available regarding the properties of S. mutans detected in those lesions. Heart valve specimens were collected from 52 patients and atheromatous plaque specimens from 50 patients, all of whom underwent cardiovascular operations, and dental plaque specimens were taken from 41 of those subjects prior to surgery. Furthermore, saliva samples were taken from 73 sets of healthy mothers (n=73) and their healthy children (n=78). Bacterial DNA was extracted from all specimens, then analysed by PCR with S. mutans-specific and serotype-specific primer sets. The detection rates of S. mutans in the heart valve and atheromatous plaque specimens were 63 and 64 %, respectively. Non-c serotypes were identified with a significantly higher frequency in both cardiovascular and dental plaque samples from the subjects who underwent surgery as compared to serotype c, which was detected in 70-75 % of the samples from the healthy subjects. The serotype distribution in cardiovascular patients was significantly different from that in healthy subjects, suggesting that S. mutans serotype may be related to cardiovascular disease.

Detection of cariogenic Streptococcus mutans in extirpated heart valve and atheromatous plaque specimens.

The involvement of oral bacteria in the pathogenesis of cardiovascular diseases has been the focus of attention in many studies, and several periodontal pathogens have been detected in diseased cardiovascular lesions, suggesting relationships between oral microorganisms and cardiovascular diseases. However, no information is available regarding the involvement of cariogenic pathogens such as Streptococcus mutans. The presence of oral streptococcal species and periodontitis-related bacteria in 35 heart valve and 27 atheromatous plaque clinical specimens, as well as 32 dental plaque specimens from the same subjects, was analyzed using a PCR method. Furthermore, broad-range PCR with DNA sequencing analysis was employed to identify the bacterial species in those samples. Streptococcus mutans was frequently detected in the heart valve (69%) and atheromatous plaque (74%) specimens, while other bacterial species, including those related to periodontitis, were detected with much lower frequencies. The bacterial composition in cardiovascular tissues was found to be markedly distinct from that in dental plaque, with only a limited number of species, including S. mutans, in the cardiovascular regions shown to have possibly originated from the oral cavity. Semiquantitative assay results revealed that S. mutans was detected in significant quantities in the heart valve (40%) and atheromatous plaque (48%) specimens, whereas the quantities of all other tested bacterial species, including several related to periodontitis, were negligible in the cardiovascular samples. These results indicate that S. mutans is a possible causative agent of cardiovascular disease.

Isolation and characterization of Streptococcus mutans in heart valve and dental plaque specimens from a patient with infective endocarditis.

Streptococcus mutans, known to be an aetiologic agent of dental caries, also causes infective endocarditis (IE), although a comparison of isolates from the oral cavity and infected heart valve of the same patient has not been reported. In the present study, infected heart valve and dental plaque samples from a patient with IE were analysed. Broad-range PCR with DNA sequencing revealed that 50 clones from the dental plaque isolates were composed of oral streptococci and periodontopathic bacteria, whereas only Streptococcus mutans was detected in 50 clones from the heart valve. Eighteen strains of Streptococcus mutans were isolated from dental plaque and seven from the heart valve, and the biochemical properties of each were in accordance with those of Streptococcus mutans. DNA fingerprinting analysis revealed that all the oral isolates of Streptococcus mutans had similar patterns, which were different from those of the isolates from the infected heart valve. Western blotting using glucosyltransferase (GTF)-specific antiserum showed that the seven strains from the heart valve lacked the three types of intact GTF. In addition, the sucrose-dependent adhesion rates of these isolates were significantly lower than those of the oral isolates (P<0.001). Furthermore, the isolates from the heart valve were less susceptible to erythromycin and kanamycin. These results indicate that the properties of the Streptococcus mutans strains isolated from the infected valve were different from those of typical oral strains, which may be related to the effects of IE.