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A typing system for Clostridium difficile using sequencing of the surface-layer protein A encoding gene (slpA) was evaluated and used to analyse clinical isolates in Japan. A total of 160 stool specimens from symptomatic patients in Japan was examined and 87 C. difficile isolates were recovered. slpA sequence typing was found to have reliable typability and discriminatory power in comparison with PCR ribotyping, and the typing results were highly reproducible and comparable. slpA sequence typing was used to type C. difficile in DNA extracted directly from stool specimens. Among the 90 stool specimens in which direct typing results were obtained, 77 specimens were positive for C. difficile culture, and typing results from isolated strains agreed with those from direct typing in all 77 specimens. The slpA sequence type smz was dominant at all four hospitals examined, and this endemic type was detected by culture and/or direct typing in 61 (62 %) of 99 stool specimens positive for toxic culture and/or direct slpA sequence typing. Comparison of epidemic strains reported throughout the world revealed one isolate identified as slpA sequence type gc8, which was found to correspond to PCR ribotype 027 (BI/NAP1/027), whereas no isolates were found with the slpA gene identical to that of PCR ribotype 078 strain. slpA sequence typing is valuable for comparison of C. difficile strains epidemic in diverse areas because the typing results are reproducible and can easily be shared. In addition, slpA sequence typing could be applied to direct typing without culture.

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Biofi lms can be defi ned as communities of microorganisms attached to a surface. Those bacterial biofi lms cause serious problems, such as antibiotic resistance and medical devicerelated infections. Nontypeable Haemophilus infl uenzae (NTHi) is an important pathogen in respiratory infections, as it forms biofi lms both in vitro and in vivo such as human middle ear. Recent reports indicate that otitis media, paranasal sinusitis and lower respiratory tract infections caused by Haemophilus infl uenzae have become more diffi cult to treat with oral antibiotic therapy. However, there has been no attention given to antibiotic eradication of NTHi biofi lm. To investigate the antimicrobial effect of various antibiotics against NTHi biofi lm formation, we conducted the following comparative study using both β -lactamase-negative ampicillin (AMP)-susceptible (BLNAS) and AMP-resistant (BLNAR) NTHi strains. In a microtiter biofi lm assay, both levofl oxacin and gatifl oxacin, of the fl uoroquinolone antibiotic group, signifi cantly inhibited biofi lm formation by BLNAS and BLNAR NTHi in a dose-dependent fashion compared to ampicillin of the penicillin antibiotic group, cefotaxime of the cephalosporin antibiotic group, and both erythromycin and clarithromycin of the macrolide antibiotic group. Furthermore, in fl ow cell chamber studies, confocal laser scanning microscopy counted survival bacteria in mature biofi lm had been treated with gatifl oxacin, ampicillin, cefotaxime and erythromycin. Only gatifl oxacin completely killed the BLNAR NTHi isolates within biofi lms without regard to the thickness of biofi lm formation. The results of this study suggest that fl uoroquinolones potentially have a role in therapy against diseases caused by both BLNAS and BLNAR NTHi isolates within biofi lms.

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