Background: Fluoroquinolone-resistant and extended-spectrum β-lactamase (ESBL)-carrying multidrug-resistant Escherichia coli have become severely problematic. In particular, a lineage of multilocus sequence-type ST131 which belongs to O25:H4 and carries ESBL CTX-M-15 has spread worldwide. Methods: Fluoroquinolone-resistant E. coli strains were isolated from various clinical specimens in a commercial clinical laboratory in 2008 and 2009 in Hokkaido Prefecture, Japan. Results: Among 478 clinical isolates, 112 strains (23.4%) showed levofloxacin (LVX) resistance. About 80% of the fluoroquinolone-resistant strains (88 strains) showed common features, namely O25:H4-ST131, phylogenetic group B and the same mutation pattern in quinolone resistance-determining regions. Pulsed field gel electrophoresis patterns suggested numerous lineages of O25:H4-ST131. The fluoroquinolone-resistant strains, including strains of O25:H4-ST131 and other types, more frequently shared CTX-type ESBL genes than did fluoroquinolone-susceptible strains. The ESBL genes fell into the CTX-M-9 and CTX-M-2 groups. CTX-M-15 (CTX-M-1 group) was not found among any of the strains isolated in this study. Sitafloxacin showed markedly potent activity against E. coli isolates compared with LVX, ciprofloxacin and ulifloxacin. Conclusion: The most prevalent fluoroquinolone-resistant strains of E. coli isolated in Hokkaido Prefecture, Japan, are O25:H4-ST131. However, similar to other areas of Japan, the ST131 clones represent distinct lineages from the general worldwide dispersal of multidrug-resistant clones which carry CTX-M-15.

We identified and genetically characterized seven fluoroquinolone-resistant Streptococcus pneumoniae strains among 293 clinical strains isolated from 1999 to 2001 in Japan. The resistant strains were isolated only from adults, and 7 of 31 isolates (22.6%) were from patients more than 20 years old. Resistant strains were not found in 262 isolates from children under age 10.

Prevalence and molecular characteristics of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) were studied in Hokkaido, the main northern island of Japan. Among the 1,015 S. aureus isolates derived from clinical specimens of outpatients collected in 2009, methicillin resistance gene mecA was detected in 189 isolates (18.6%). The most frequent staphylococcal cassette chromosome mec (SCCmec) type in MRSA was II (83.1%), followed by IV (6.9%) and V (3.2%). MRSA with type II-SCCmec showed multiple drug resistance and harbored various toxin and virulence factor genes except for Panton-Valentine leucocidin (PVL) gene. These isolates were mostly classified into sequence type 5 (ST5) (or other STs in CC5) and coagulase genotype II and were thus genetically similar to hospital-acquired MRSA, which have been predominating in Japan (New York/Japan clone). PVL gene was detected in three MRSA strains belonging to ST6 (two strains) and ST59 (one strain), having type IVa- and Vt-SCCmec, respectively, and also in two methicillin-susceptible S. aureus ST121 and ST188. The arcA gene within the arginine catabolic mobile element (ACME) was detected in the two PVL-negative ST5 MRSA strains, which had type IIa- or V-SCCmec. The PVL gene-positive ST6 and ST59 CA-MRSA strains were susceptible to more antimicrobials and had less virulence factor genes than the PVL-negative ST5 MRSA, including the ACME-arcA-positive strains. In the present study, ST6 was identified as a lineage of PVL-positive CA-MRSA, the ACME-arcA was first detected in ST5 MRSA with type V-SCCmec, and ST59 Taiwanese CA-MRSA strain was isolated in Hokkaido for the first time. These findings suggest a potential spread of these emerging CA-MRSA clones in Japan.

Distribution of serotypes, prevalence of resistance to penicillin and/or erythromycin (EM), and its genetic traits were analyzed for a total of 1,061 noninvasive or colonization isolates of Streptococcus pneumoniae (998 and 61 isolates from children and adults, respectively) in Hokkaido, northern main island of Japan, in the year 2011, the pre-PCV7 routine immunization period. Serotype deduction was performed by sequential multiplex polymerase chain reaction (PCR), employing mutagenic PCR-restriction fragment length polymorphism for discrimination of 6A/C and 6B/D. Unaltered three PBP genes and macrolide resistance genes erm(B) and mef(A/E) were detected by multiplex PCR. Among isolates from children, 25 serotypes, including the prevalent types 6B (17.5%), 19F (15.6%), 23F (12.2%), and 6C (11.6%), were identified, revealing the PCV7 and PCV13 coverage rates as 48.2% and 60.3%, respectively, while serotype 3 was the most frequent (19.0%) among isolates from adults. Most of the pediatric isolates (96.8%) exhibited resistance to EM (minimum inhibitory concentration [MIC], ≥1 μg/ml), with a higher prevalence of erm(B) (67.2%) than mef(A/E) (39.7%). erm(B) was associated with high-level EM resistance (MIC, ≥128 μg/ml) and distributed at high detection rates to major serotypes 23F (85.2%) and 6B (85.1%), as well as minor serotypes 3, 10A, 14, 15B, 15C, 19A, and 23A (>90%). While penicillin-resistant S. pneumoniae (PRSP) (penicillin G-MIC, 2-3 μg/ml) was detected in 7.8% of isolates from children, the most common PBP gene genotype was gPRSP (three altered genes pbp1a, 2x, and 2b; 38.3%), which was detected at higher rates (>60%) in the dominant serotypes 23F, 6B, and 19F, and minor serotypes 6D and 15A. Dominant serotypes in the S. pneumoniae isolates were generally similar to those reported for invasive strains, despite lower coverage rates by PCV7/13. The importance of further surveillance on incidence and drug resistance in the post-PCV7 period was suggested for non-PCV7/13 serotypes 6C, 6D, 10A, 15A, 15B, 15C, 23A, and 35B.

Panton-Valentine leukocidine (PVL) is a distinctive virulence factor of community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA), and arginine catabolic mobile element (ACME) is a staphylococcal genomic island that enhances fitness and the ability of bacterial cells to colonize on skin and mucous membranes. ACME is characteristically found in USA300, which is a predominant CA-MRSA clone [sequence type (ST) 8] in the USA and is spreading globally, and has also been detected in non-ST8 MRSA at low frequency. In Japan, spread of MRSA with PVL and/or ACME and their genetic traits have not yet been well characterized. In the present study, the prevalence and genetic diversity of PVL + /ACME + MRSA were investigated for 422 MRSA clinical isolates collected from outpatients in northern Japan over a period of 1 year. All the isolates were genotyped for the staphylococcal cassette chromosome mec (SCCmec) and coagulase genes (coa), and screened for PVL and ACME genes. The PVL + /ACME + isolates were studied further by genetic analysis, including single-nucleotide polymorphism (SNP) analysis based on PVL genes (lukS-PV-lukF-PV), ACME (arc and opp3 clusters) and the sarU promoter region. Among all the isolates examined, PVL genes and ACME were detected in eight (SCCmec-II, n51; SCCmec-IV, n56; SCCmec-V, n51) and 20 (SCCmec-II, n514; SCCmec-IV, n55; SCCmec-V, n51) isolates, respectively. Five isolates were found to have both PVL genes and ACME (type I), and were classified into ST8/spa-t008/agr-I/coa-IIIa, which is the same genetic traits as USA300. Fifteen PVL " /ACME + isolates had type DII-ACME, belonging to either ST5 or ST764 [clonal complex (CC) 5], and spa-t001, -t002 or -t3557. All the ST8 PVL + /ACME-I + MRSA had identical sequences of PVL genes (haplotype R) and ACME arc/opp3 clusters as those of USA300. In contrast, in the CC5 PVL " /ACME-DII + MRSA, SNPs in the arc cluster were detected in 11 sites (four haplotypes), with some different profiles of virulence/resistance factors. These results indicated single clonality of ST8 PVL + /ACME-I + MRSA and heterogeneity of CC5 PVL " /ACME-DII + MRSA, and suggest their potential spread in northern Japan.