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We have previously isolated a gram-negative microaerophilic strain, PAGU2000 T from a patient presenting with a fever in Kumamoto Prefecture, Japan. The present study aimed to comprehensively analyze the taxonomy of the isolated strain using a polyphasic approach. The 16S rRNA gene sequence analysis indicated that the strain was a member of enterohepatic Helicobacter. The strain PAGU2000 T shared a 97.5% 16S rRNA gene nucleotide identity with Helicobacter valdiviensis, and this taxonomic position was confirmed by phylogenetic analysis of the GyrA amino acid sequences. The proposed strain PAGU2000 T has a 1.482 Mbp chromosome with a DNA G + C content of 31.3 mol% and encodes 1520 predicted coding sequences. The average nucleotide identity between the strain PAGU2000 T and type strain of H. valdiviensis was 70.3%, which was lower than the recommended threshold of 95% for species delineation. The strain PAGU2000 T was a motile, non-spore-forming, and spiral-shaped bacterium, exhibiting catalase and oxidase activities but not urease and nitrate reduction. This study demonstrates that the isolate represents a novel species within enterohepatic Helicobacter, for which the name Helicobacter higonensis is proposed (type strain: PAGU2000 T  = GTC 16811 T  = LMG 33095 T ). In this study, we describe the phenotypic and morphological features of this strain and propose an emended description of some biochemical traits of H. valdiviensis., (© 2024 The Societies and John Wiley & Sons Australia, Ltd.)

Introduction: In 2010, oral fluoroquinolone tosufloxacin (TFX) granules were released as the first oral respiratory quinolone for children in Japan., Methods: To investigate the recent trend of H. influenzae strains with low susceptibility to quinolones in children, we analyzed the gene sequences of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE of 23 clinical isolates from 15 patients aged <15 years with an MIC of ≥0.5 μg/mL for TFX from 2010 to 2018., Results: Amino acid substitutions were observed in both GyrA and ParC in 13 strains (81%, 13/16), except two strains with a TFX MIC of 0.5 μg/mL with amino acid substitution in only GyrA and one strain with a TFX MIC of 1 μg/mL with no amino acid substitution. Four ST422 strains were observed in 2018, the detection age range was wide (0-7 years), and the residential city was varied. A total of 3/15 patients had a clear history of TFX treatment., Conclusions: Even for the strain with an MIC of 0.5 μg/mL for TFX, it is highly possible that it harbors a mutation in gyrA, which is the first step toward quinolone resistance, and it may also harbor mutations in both gyrA and parC. Furthermore, several specific sequence type quinolone-resistant H. influenzae strains, particularly ST422, may be widespread among children in Japan. It is necessary to investigate changes in resistance both at the MIC and gene levels. The continuous monitoring of strains and the use of antimicrobial drugs in treatment should be carefully observed., (Copyright © 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Although fluoroquinolones are considered as alternative therapies of pulmonary Mycobacterium avium complex (MAC) disease, the association between fluoroquinolone resistance and MAC genotypes in clinical isolates from individuals not previously treated for MAC infection is not fully clear., Methods: Totals of 154 M. avium isolates and 35 Mycobacterium intracellulare isolates were obtained from treatment-naïve patients with pulmonary MAC disease at the diagnosis of MAC infection at 8 hospitals in Japan. Their susceptibilities of moxifloxacin were determined by broth microdilution methods. Moxifloxacin-resistant isolates were examined for mutations of gyrA and gyrB. Variable numbers of tandem repeats (VNTR) assay was performed using 15 M. avium VNTR loci and 16 M. intracellulare VNTR loci., Results: Moxifloxacin susceptibility was categorized as resistant and intermediate for 6.5% and 16.9%, respectively, of M. avium isolates and 8.6% and 17.1% of M. intracellulare isolates. Although the isolates of both species had amino acid substitutions of Thr 96 and Thr 522 at the sites corresponding to Ser 95 in the M. tuberculosis GyrA and Gly 520 in the M. tuberculosis GyrB, respectively, these substitutions were observed irrespective of susceptibility and did not confer resistance. The VNTR assays showed revealed three clusters among M. avium isolates and two clusters among M. intracellulare isolates. No significant differences in moxifloxacin resistance were observed among these clusters., Conclusions: Although resistance or intermediate resistance to moxifloxacin was observed in approximately one-fourth of M. avium and M. intracellulare isolates, this resistance was not associated with mutations in gyrA and gyrB or with VNTR genotypes., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: The use of non-β-lactam agents has increased in Japan due to the prevalence of β-lactam-resistant pathogens. This study aimed to clarify the recent trend of antimicrobial susceptibility and molecular epidemiological features in Haemophilus influenzae., Methods: Fifty-seven Haemophilus influenzae isolated from a Japanese teaching hospital in 2017 were characterised, and the data were compared with those of a previous study. The MICs were determined using the broth dilution method. Genetic backgrounds were compared by multilocus sequence typing. The bactericidal activity of tosufloxacin at, or near, the therapeutic Cmax was determined in vitro, with susceptible isolates and quinolone low-susceptible isolates by time-kill assay., Results: The results of the susceptibility tests showed that >90% of isolates were susceptible to cephalosporins and carbapenems, whereas ampicillin-susceptible and clarithromycin-susceptible isolates decreased. Regarding quinolones, low-susceptible isolates were noted in 2017, although all isolates were judged as susceptible. All low-susceptible isolates had an amino acid substitution in GyrA, and two isolates had an additional substitution in ParC. These isolates had different genetic backgrounds. Furthermore, the time-kill kinetic assay using the Cmax of tosufloxacin indicated that the low-susceptible isolates could persist for at least 8hours., Conclusions: This study revealed that Haemophilus influenzae has demonstrated multidrug low-susceptibility in recent years. The low-susceptible isolates had genetic diversity, meaning that resistance occurred independently., (Copyright © 2019 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Objectives: Campylobacter jejuni (C. jejuni) is one of the most common pathogens that causes gastroenteritis. Because there is currently insufficient epidemiological information about the antimicrobial susceptibility and molecular characterisation of clinical isolates of C. jejuni in Japan, this study carried out antimicrobial susceptibility testing and multilocus sequence typing (MLST) of clinical C. jejuni isolates in Tokyo between 2000-2017., Methods: Antimicrobial susceptibility to erythromycin and ciprofloxacin was tested using the broth microdilution method in 430 C. jejuni clinical isolates collected over 18 years, between 2000-2017, at a Tokyo general hospital. To observe the sequence type (ST) evolution, 82 isolates were chosen from three non-consecutive years (16 isolates from 2000, 25 isolates from 2008, and 41 isolates from 2017) and analysed by MLST as a molecular characterisation test. Mutations in the quinolone resistance-determining region of the gyrA and gyrB genes were identified., Results: The rate of resistance to erythromycin was low, but that of ciprofloxacin resistance was 34.9% in 2000-2008 and 41.9% in 2009-2017. The most common clonal complex (CC) identified during the entire period was CC21; ST4526 with ciprofloxacin resistance was highly prevalent in 2017 (6 of 11; 54.5%)., Conclusion: The results indicate that the rate of resistance to quinolone has gradually increased. Since ST4526 was not isolated in 2000 and 2008, it is likely that ST4526 is rapidly increasing in Japan., (Copyright © 2019 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Purpose: Haemophilus influenzae strains with low susceptibility to quinolones have recently emerged in the paediatric field in Japan. These strains are judged as 'susceptible' in routine susceptibility tests, although they may survive after quinolone treatment. Therefore, we aimed to construct a simple and cost-effective identification method for low-susceptibility strains using disc diffusion assays., Methodology: A total of 33 H. influenzae clinical isolates and a control strain were used. For the disc diffusion assay, levofloxacin, norfloxacin, nalidixic acid and pipemidic acid were employed. Correlations between the inhibition zone diameter and amino acid substitutions were evaluated., Results: All of the tested strains formed clear inhibition zones on both levofloxacin and norfloxacin discs. By contrast, none of the low-susceptibility strains showed inhibition zones against nalidixic acid, while the low-susceptibility strains with amino acid substitutions in both GyrA and ParC did not show inhibition zones against pipemidic acid discs, indicating that low-susceptibility strains can be detected with high sensitivity and specificity by the presence or absence of inhibition zones for earlier quinolones., Conclusion: A disc diffusion test combining results from nalidixic acid and pipemidic acid can detect low-susceptibility strains harbouring amino acid substitutions without the need for genetic analysis. This test can help reduce inappropriate and unnecessary fluoroquinolone use.

Purpose: To prevent severe invasive pneumococcal infection, pneumococcal conjugate vaccines (PCVs) were introduced in Japan in 2010, and in 2013 a pneumococcal 13-valent conjugate vaccine (PCV13) was included in the routine vaccination schedule for infants. In this study, we analysed the antimicrobial susceptibilities and capsular types of pneumococci isolated from non-invasive patient sites from 2007 to 2016 to assess the impact of the introduction of PCV13., Methodology: A total of 618 pneumococcal isolates collected at a teaching hospital from 2007 to 2016 were used. These isolates were characterized by capsular typing, multilocus sequence typing and antimicrobial susceptibility testing., Results: Capsular typing indicated that, after the introduction of the PCV, the proportion of PCV13 serotypes decreased (P<0.01), while non-PCV13 serotypes became diverse. In particular, increases in 22 F, 15A and 23A were noted among non-PCV13 serotypes. Regarding antimicrobial susceptibility, the non-susceptibility rate to penicillin of pneumococci that showed higher minimum inhibitory concentrations (MICs) than the susceptibility breakpoint decreased, and pneumococci tended to become susceptible. However, all type 23A pneumococci and 77.8  % of type 15A pneumococci showed the reverse trend, with low susceptibility to penicillin. Furthermore, all 15A and 23A isolates had macrolide resistance genes., Conclusion: These data suggest that PCVs can prevent infections caused by PCV serotypes. However, since non-PCV13-type pneumococci, in particular 15A and 23A, which have acquired multidrug resistance, have already emerged over time, the development of a novel vaccine targeting a broader spectrum of pneumococci is warranted.

Pneumococcal isolates from adult patients in northern Japan in 2016 were subjected to molecular investigation related to pneumococcal surface protein A (PspA) and drug resistance determinants. Of the 51 isolates, serotype 3/ST180 was the most prevalent (17.6%), followed by 35B (ST2755/ST558) (11.8%) and 15A (ST63/ST7874/ST13068/ST13785) (9.8%). Coverage of serotypes by 13-valent conjugate vaccine and 23-valent polysaccharide vaccine was 27.5% and 49%, respectively. All the isolates expressed PspA family 1 or 2 (51% and 49%, respectively). Each serotype was associated with either of the PspA families ( e.g. , serotype 3, PspA family 1; serotypes 35B and 15A, PspA family 2). Multidrug resistance (MDR) was found in 84.3% of the isolates. Minimum of one altered penicillin-binding protein gene was detected in 82.4% of isolates, indicating 25.5% non-susceptibility to penicillin. Serotypes 15A and 35B were predominant and demonstrated MDR. An isolate of serotype 15A/ST13785 (single-locus variant of ST242) was resistant to fluoroquinolones associated with double mutation in the quinolone resistance-determining regions of gyrA and parC . The present study indicates the spread of MDR pneumococci represented by isolates of serotypes 3, 15A, and 35B, and prevalence of both PspA family 1 and 2 in isolates obtained from adult patients.

Clostridium difficile is a major nosocomial pathogen in humans with an increasing incidence in the community. The "one-health" approach of research is needed to investigate possible reservoirs of C. difficile and route of its transmission. The objective of this study is to investigate the occurrence of C. difficile in pigs in the Czech Republic with characterisation of the isolates to determine their genetic relatedness to C. difficile isolates from European and Asian pigs. A total of 198 pig faeces samples from 23 farms were investigated and of those 57 samples (55 piglets, 2 sows) from 11 farms were confirmed as C. difficile positive. The majority of C. difficile isolates belonged to the sequence type 11 and clade 5. The predominant ribotypes were 078 (n = 23), 078-variant (n = 5), 033 (n = 10) followed by RTs 150 (n = 7), 011 (n = 5), 045 (n = 4), 126, 014, 002 (n = 1, each). All isolates were susceptible to metronidazole, vancomycin and tetracycline. Isolates of RTs 150 and 078-variant were moxifloxacin resistant (MIC≥32 mg/L) and carried the amino acid substitution Thr82Ile in the GyrA. A multi-locus variable number tandem-repeats analysis (MLVA) revealed a clonal relatedness of isolates within individual farms and in C. difficile RT078 isolates between two Czech farms. Czech C. difficile RT078 isolates clustered with German C. difficile RT078 isolates and Czech C. difficile 078-variant isolates clustered with C. difficile RT078 isolates from Japan and Taiwan. This study found an emergence of C. difficile RT078 in Czech piglets that was related genetically to C. difficile RT078 isolates from Germany, Japan and Taiwan., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Emergence of antimicrobial resistance in Neisseria gonorrhoeae is a major public health concern globally, and new antimicrobials for treatment of gonorrhea are imperative. In this study, the in vitro activity of sitafloxacin, a fluoroquinolone mainly used for respiratory tract or urogenital infections in Japan, and additional newer generation fluoroquinolones were determined against ciprofloxacin-resistant N. gonorrhoeae isolates. Minimum inhibitory concentrations (MICs) of ciprofloxacin, levofloxacin, moxifloxacin, sitafloxacin, pazufloxacin, and tosufloxacin against 47 N. gonorrhoeae isolates cultured in 2009 in Japan were determined by agar dilution method. The quinolone resistance-determining region (QRDR) of gyrA and parC was sequenced. The in vitro potency of sitafloxacin was substantially higher compared with all other tested fluoroquinolones. The MICs of sitafloxacin ranged from 0.03 to 0.5 mg/L for 35 ciprofloxacin-resistant N. gonorrhoeae isolates (ciprofloxacin MICs from 2 to 32 mg/L). No identified mutations in GyrA and ParC QRDR resulted in higher sitafloxacin MIC than 0.5 mg/L. Sitafloxacin had a high activity against N. gonorrhoeae isolates, including strains with mutations in DNA gyrase and topoisomerase IV, resulting in high-level resistance to ciprofloxacin and all other newer generation fluoroquinolones examined. However, it was still to a lower extent affected by GyrA and ParC QRDR mutations resulting in sitafloxacin MICs of up to 0.5 mg/L. This indicates that sitafloxacin should not be considered for empirical first-line monotherapy of gonorrhea. However, sitafloxacin could be valuable in a dual antimicrobial therapy and for cases with ceftriaxone resistance or allergy.

Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans., (© 2016 The Societies and John Wiley & Sons Australia, Ltd.)

A total of 29 Campylobacter jejuni and C. coli strains were isolated from Thai and Japanese children with diarrhea using the Loop-mediated Isothermal Amplification method. The samples were evaluated for mutations in gyrA and 23S rRNA in order to assess resistance against fluoroquinolones and macrolides, respectively. Among the isolated strains, 9 (8 C. jejuni and 1 C. coli) were from Thai children, and the other 20 (C. jejuni) were isolated from Japanese children. High fluoroquinolone resistance rates were observed in Thai (66.7%) and Japanese (90%) children. Macrolide resistance was not observed in Japanese children but was observed at a considerable rate of 12.5% of C. jejuni isolated in the Thai cohort. The results indicate that continuous monitoring of resistance of Campylobacter strains to fluoroquinolones and macrolides is definitely necessary.

A total of 349 Salmonella enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) strains, which were isolated between 2008 and 2012 from 349 pigs at two slaughterhouses in Okinawa Prefecture, Japan, were investigated for antimicrobial susceptibility and the presence of antimicrobial resistance genes. All isolates were resistant to at least four antimicrobial agents. The antimicrobial agents for which isolates showed a high incidence of resistance were as follows: ampicillin (100%) and streptomycin (100%), followed by gentamicin (99.7%), oxytetracycline (99.7%), sulfamethoxazole/trimethoprim (99.4%), nalidixic acid (40.1%) and oxolinic acid (40.1%). All isolates were sensitive to cefuroxime, ceftiofur, colistin, fosfomycin, enrofloxacin, orbifloxacin and danofloxacin. The predominant resistance phenotypes and genotypes were: resistance to ampicillin, streptomycin, gentamicin, oxytetracycline and sulfamethoxazole/trimethoprim (58.5%, 204/349) and blaTEM-strA-strB-aadA1-aadA2-aacC2-tet (B)-sul1-sul2-dhfrXII-dhfrXIII (36.1%, 126/349). The quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE of the quinolone-resistant isolates (n=12) showed amino acid substitutions of Ser-83→Phe or Asp-87→Tyr in GyrA and Ser-107→Ala in ParC. To our knowledge, this is the first report on the molecular characterization of antimicrobial resistance among S. Choleraesuis strains in Japan.

Leptospirosis is a worldwide zoonosis. The importance of urban leptospirosis is recognized in Japan: urban rats carry pathogenic leptospires and people acquire these pathogens through contact with surface water or soil contaminated by the urine of the infected animals. To determine the current Leptospira carriage rate in urban rats, 29 wild rats were trapped in the central area of Fukuoka and strains isolated from their kidneys and urine analyzed. When semi-solid Korthof's medium containing 0.1% agar was used for isolation, 72.2% and 30.8% of the kidney and urine cultures, respectively, were found to be Leptospira-positive. The isolates belonged to Leptospira interrogans, and were classified into two groups (serogroups Pomona and Icterohaemorrhagiae) based on the results of gyrB sequence analysis and microscopic agglutination testing (MAT). Strains belonging to serogroup Icterohemorrhagiae grew well in liquid medium. On the other hand, serogroup Pomona isolates multiplied very little in liquid medium, but did grow in a semi-solid medium. Although strains belonging to serogroup Pomona have not been recognized as native to Japan, this strain may be widely distributed in urban rats. Representative strains from each group were found to be highly pathogenic to hamsters. Our findings should serve as a warning that it is still possible to become infected with leptospires from wild rats living in inner cities of Japan. Furthermore, the use of semi-solid medium for culture will improve the isolation rate of leptospires from the kidneys of wild rats., (© 2015 The Societies and Wiley Publishing Asia Pty Ltd.)

Ureaplasma spp. cause several disorders, such as nongonococcal urethritis, miscarriage, and preterm delivery with lung infections in neonates, characterized by pathological chorioamnionitis in the placenta. Although reports on antibiotic resistance in Ureaplasma are on the rise, reports on quinolone-resistant Ureaplasma infections in Japan are limited. The purpose of this study was to determine susceptibilities to five quinolones of Ureaplasma urealyticum and Ureaplasma parvum isolated from perinatal samples in Japan and to characterize the quinolone resistance-determining regions in the gyrA, gyrB, parC, and parE genes. Out of 28 clinical Ureaplasma strains, we isolated 9 with high MICs of quinolones and found a single parC gene mutation, resulting in the change S83L. Among 158 samples, the ParC S83L mutation was found in 37 samples (23.4%), including 1 sample harboring a ParC S83L-GyrB P462S double mutant. Novel mutations of ureaplasmal ParC (S83W and S84P) were independently found in one of the samples. Homology modeling of the ParC S83W mutant suggested steric hindrance of the quinolone-binding pocket (QBP), and de novo prediction of peptide structures revealed that the ParC S84P may break/kink the formation of the α4 helix in the QBP. Further investigations are required to unravel the extent and mechanism of antibiotic resistance of Ureaplasma spp. in Japan., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Objectives: We determined the prevalence of macrolide and fluoroquinolone resistance-associated mutations in Mycoplasma genitalium DNA specimens from men with non-gonococcal urethritis (NGU) and analysed their effects on antibiotic treatments of M. genitalium infections., Methods: In this retrospective study, we examined antibiotic resistance-associated mutations in the 23S rRNA, gyrA and parC genes of M. genitalium and the association of the mutations with microbiological outcomes of antibiotic treatments in men with M. genitalium-positive NGU., Results: No macrolide resistance-associated mutations in the 23S rRNA gene were observed in 27 M. genitalium DNA specimens in 2011 and in 24 in 2012. However, 5 of 17 in 2013 had 23S rRNA mutations. Three of 15 in 2011, 6 of 19 in 2012 and 8 of 17 in 2013 had fluoroquinolone resistance-associated alterations in ParC. Three in 2013 had both the antibiotic resistance-associated alterations coincidentally. In two men with M. genitalium harbouring 23S rRNA mutations, the mycoplasma persisted after treatment with a regimen of 2 g of extended-release azithromycin (AZM-SR) once daily for 1 day. All nine men with mycoplasma harbouring ParC alterations were microbiologically cured with a regimen of 100 mg of sitafloxacin twice daily for 7 days., Conclusions: Macrolide- or fluoroquinolone-resistant M. genitalium appears to be increasing, and the increase in fluoroquinolone-resistant mycoplasmas is especially remarkable in Japan. Mycoplasmas harbouring 23S rRNA mutations would be resistant to the AZM-SR regimen, but those harbouring ParC alterations would still be susceptible to the sitafloxacin regimen., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Acinetobacter baumannii is generally the most frequently isolated Acinetobacter species. Sequence analysis techniques allow reliable identification of Acinetobacter isolates at the species level. Forty-eight clinical isolates of Acinetobacter spp. were obtained from blood cultures at Tohoku University Hospital. These isolates were identified at the species level by partial sequencing of the RNA polymerase β-subunit (rpoB), 16S rRNA, and gyrB genes. Then further characterization was done by using the PCR for detection of OXA-type β-lactamase gene clusters, metallo-β-lactamases, and carO genes. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were also performed. The most frequent isolate was Acinetobacter soli (27.1%). Six of the 13 A. soli isolates were carbapenem nonsusceptible, and all of these isolates produced IMP-1. PFGE revealed that the 13 A. soli isolates were divided into 8 clusters. This study demonstrated that A. soli accounted for a high proportion of Acinetobacter isolates causing bacteremia at a Japanese tertiary hospital. Non-A. baumannii species were identified more frequently than A. baumannii and carbapenem-nonsusceptible isolates were found among the non-A. baumannii strains. These results emphasize the importance of performing epidemiological investigations of Acinetobacter species.

Cellulolytic bacteria A191(T), A192 and A193 isolated from the soil of Sakhalin fir forest in Hokkaido, Japan were studied phenotypically, genotypically and phylogenetically. Analysis of their 16S rRNA gene and gyrB sequences and DNA base composition suggested that these isolates were conspecific and members of the genus Streptomyces. However, levels of 16S rRNA gene and gyrB sequence similarity between the isolates and the type strains of their closest relatives in the genus Streptomyces were no higher than 97.9 and 95.0 %, respectively, implying that these isolates were distinctive. Moreover, the results of DNA-DNA hybridization experiments and physiological characterization clearly differentiated these isolates from their closest neighbours. It is therefore concluded that these isolates represent a novel species of the genus Streptomyces, for which the name Streptomyces abietis is proposed. The type strain is A191(T) ( = NBRC 109094(T) = DSM 42080(T)).

Infection from fluoroquinolone-resistant Enterobacteriaceae is an increasing health problem worldwide. In the present study, we developed a pyrosequencing-based high-throughput method for analyzing the nucleotide sequence of the quinolone resistance-determining regions (QRDRs) of gyrA and parC. By using this method, we successfully determined the QRDR sequences of 139 out of 140 clinical Escherichia coli isolates, 28% of which were nonsusceptible to ciprofloxacin. Sequence results obtained by the pyrosequencing method were in complete agreement with those obtained by the Sanger method. All fluoroquinolone-resistant isolates (n = 35; 25%) contained mutations leading to three or four amino acid substitutions in the QRDRs. In contrast, all isolates lacking a mutation in the QRDR (n = 81; 57%) were susceptible to ciprofloxacin, levofloxacin, and nalidixic acid. The qnr determinants, namely, the qnrA, qnrB, and qnrS genes, were not detected in the isolates, and the aac(6')-Ib-cr gene was detected in 2 (1.4%) of the isolates. Multilocus sequence typing of 34 randomly selected isolates revealed that sequence type 131 (ST131) (n = 7; 20%) is the most prevalent lineage and is significantly resistant to quinolones (P < 0.01). The genetic background of quinolone-susceptible isolates seemed more diverse, and interestingly, neighboring STs of ST131 in the phylogenetic tree were all susceptible to ciprofloxacin. In conclusion, our investigation reveals the relationship between fluoroquinolone resistance caused by mutations of QRDRs and the population structure of clinical extraintestinal E. coli isolates. This high-throughput method for analyzing QRDR mutations by pyrosequencing is a powerful tool for epidemiological studies of fluoroquinolone resistance in bacteria.

Little information is available on the distribution of antimicrobial resistance genes in anaerobes in Japan. To understand the background of antimicrobial resistance in anaerobes involved in intra-abdominal infections, we investigated the distribution of eight antimicrobial resistance genes (cepA, cfiA, cfxA, ermF, ermB, mefA, tetQ, and nim) and a mutation in the gyrA gene in a total of 152 organisms (Bacteroides spp., Prevotella spp., Fusobacterium spp., Porphyromonas spp., Bilophila wadsworthia, Desulfovibrio desulfuricans, Veillonella spp., gram-positive cocci, and non-spore-forming gram-positive bacilli) isolated between 2003 and 2004 in Japan. The cepA gene was distributed primarily in Bacteroides fragilis. Gene cfxA was detected in about 9 % of the Bacteroides isolates and 75 % of the Prevotella spp. isolates and did not appear to contribute to cephamycin resistance. Two strains of B. fragilis contained the metallo-β-lactamase gene cfiA, but they did not produce the protein product. Gene tetQ was detected in about 81, 44, and 63 % of B. fragilis isolates, other Bacteroides spp., and Prevotella spp. isolates, respectively. The ermF gene was detected in 25, 13, 56, 64, and 16 % of Bacteroides spp., Prevotella spp., Fusobacterium spp., B. wadsworthia, and anaerobic cocci, respectively. Gene mefA was found in only 10 % of the B. fragilis strains and 3 % of the non-B. fragilis strains. Genes nim and ermB were not detected in any isolate. Substitution at position 82 (Ser to Phe) in gyrA was detected in B. fragilis isolates that were less susceptible or resistant to moxifloxacin. This study is the first report on the distribution of resistance genes in anaerobes isolated from intra-abdominal infections in Japan. We expect that the results might help in understanding the resistance mechanisms of specific anaerobes.

Fluoroquinolones (FQs) have been detected in aquatic environments in several countries. Long-term exposure to low levels of antimicrobial agents provides selective pressure, which might alter the sensitivity of bacteria to antimicrobial agents in the environment. Here, we examined FQ levels and the resistance of Escherichia coli (E. coli) to FQs by phenotyping and genotyping. In the aquatic environment in Osaka, Japan, ciprofloxacin, enoxacin, enfloxacin, lomefloxacin, norfloxacin, and ofloxacin were detected in concentrations ranging from 0.1 to 570 ng L(-1). FQ-resistant E. coli were also found. Although no obvious correlation was detected between the concentration of FQs and the presence of FQ-resistant E. coli, FQ-resistant E. coli were detected in samples along with FQs, particularly ciprofloxacin and ofloxacin. Most FQ-resistant E. coli carried mutations in gyrA, parC, and parE in quinolone resistance-determining regions. No mutations in gyrB were detected in any isolates. Amino acid changes in these isolates were quite similar to those in clinical isolates. Six strains carried the plasmid-mediated quinolone resistance determinant qnrS1 and expressed low susceptibility to ciprofloxacin and nalidixic acid: the minimum inhibitory concentrations ranged from 0.25 μg mL(-1) for ciprofloxacin, and from 8 to 16 μg mL(-1) for nalidixic acid. This finding confirmed that plasmids containing qnr genes themselves did not confer full resistance to quinolones. Because plasmids are responsible for much of the horizontal gene transfer, these genes may transfer and spread in the environment. To our knowledge, this is the first report of plasmid-mediated quinolone resistance determinant qnrS1 in the aquatic environment, and this investigation provides baseline data on antimicrobial resistance profiles in the Osaka area., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Clinical efficacies of mutant prevention concentration (MPC) and mutant selection window (MSW) hypotheses have been evaluated for human clinical isolates. We tested the MSW hypothesis by evaluating the relationships between MPCs and mutation frequencies against enrofloxacin for avian pathogenic Escherichia coli (APEC) isolates. Mutation frequencies of strains with MPC:MIC ratios of 8 to 16 were significantly higher than those of strains with an MPC:MIC ratio of 4. Mutation frequencies and MPCs of serogroup O2 strains were lower than those of the other strains; these results may correlate with the absence of fluoroquinolone-resistant O2 strains. Our results support the MSW hypothesis that the range of the MSW is involved in selection of resistant mutants.

We investigated the contribution of quinolone resistance-determining region (QRDR) mutations to fluoroquinolone (enrofloxacin, orbifloxacin and danofloxacin) susceptibility in 58 Mycoplasma bovis isolates from dairy cattle in Japan. Fluoroquinolone non-resistant isolates (fluoroquinolone-MICs≤2 μg/ml) possessed no QRDR mutations (19 isolates) or Ser83Leu in GyrA (32 isolates). Fluoroquinolone-resistant isolates (fluoroquinolone-MICs≥4 μg/ml) possessed Ser83Leu in GyrA and Ser81Pro in ParC (3 isolates) or Ser83Phe in GyrA and Ser80Ile in ParC (4 isolates). Laboratory-derived fluoroquinolone-resistant mutants selected from 2 isolates (possessing Ser83Leu in GyrA) had an amino acid substitution in ParC at the same position (Ser80Ile or Ser81Tyr) as fluoroquinolone-resistant isolates, suggesting a concurrent amino acid substitution in ParC (Ser80 or Ser81) is important in fluoroquinolone resistance in M. bovis isolates.

A rubber-degrading bacterium, the strain NS21, that was isolated from a soil sample in a botanical garden in Japan (Imai et al., 2011) was examined by phenotypic, phylogenetic and chemotaxonomic approaches to determine its taxonomic position. The strain NS21 was motile, possessing a single polar flagellum and a facultatively anaerobic straight rod. Analysis of the 16S rRNA and gyrB gene sequences of NS21 revealed a close relationship to the genus Rhizobacter. The predominant quinone type was Q-8. The G+C content of the NS21 genomic DNA was 70.8 mol%. The major fatty acids were C16:0, C17:0 cyclo, C18:1ω7c and C16:1ω7c and/or iso-C15:0 2-OH. C12:0 2-OH was present. The DNA-DNA hybridization experiments indicated that the DNA relatedness values of the strain NS21 to R. dauci H6(T) and R. fulvus Gsoil 322(T) were lower than 24%. The phenotypic characteristics showed obvious dissimilarities when compared to closely related species. On the basis of these taxonomic properties, a novel species is proposed as Rhizobacter gummiphilus sp. nov., with the strain NS21(T) (NBRC 109400(T), BCC 58006(T)) as the type strain. The emended description of the genus Rhizobacter was also presented.

Helicobacter cinaedi colonizes the colons of human and animals and can cause colitis, cellulitis, and sepsis in humans, with infections in immunocompromised patients being increasingly recognized. However, methods for analyzing the molecular epidemiology of H. cinaedi are not yet established. A genotyping method involving multilocus sequence typing (MLST) was developed and used to analyze 50 H. cinaedi isolates from Japanese hospitals in addition to 6 reference strains. Pulsed-field gel electrophoresis (PFGE) results were also compared with the MLST results. Based on the genomic information from strain CCUG18818, 21 housekeeping genes were selected as candidates for MLST and were observed to have high homology (96.5 to 100%) between isolates. Following a comparison of the 21 housekeeping genes from 8 H. cinaedi isolates, 7 genes were chosen for MLST, revealing 14 sequence types (STs). The isolates from 3 hospitals belonged to the same STs, but the isolates from the other 4 hospitals belonged to different STs. Isolates belonging to ST6 were analyzed by PFGE and showed similar, but not identical, patterns between isolates. Isolates belonging to ST9, ST10, and ST11, which belonged to the same clonal complex, had the same pattern. All isolates were found to contain mutations in GyrA and the 23S rRNA gene that confer ciprofloxacin and clarithromycin resistance, respectively, in H. cinaedi. These results raise concerns about the increase in H. cinaedi isolates resistant to clarithromycin and ciprofloxacin in Japan.

In 2010, a total of 1327 clinical Escherichia coli isolates from five hospitals in the Kyoto and Shiga regions of Japan were analysed by PCR. The prevalences of plasmid-mediated AmpC β-lactamase (pAmpC)-producers, extended-spectrum β-lactamase (ESBL)-producers and co-producers of pAmpC and ESBL were 1.7%, 9.7% and 0.3%, respectively. Less than one-half of the pAmpC-producers were reported to be resistant to third-generation cephalosporins, cephamycins and β-lactam/β-lactam inhibitors using the old 2009 Clinical and Laboratory Standards Institute (CLSI) breakpoints. CMY-2 was the most prevalent pAmpC type (95%), and CTX-M-14 (38%), CTX-M-15 (26%) and CTX-M-27 (19%) were the most prevalent ESBL types. The worldwide O25b-ST131-B2 clone accounted for 11% of pAmpC-producers and 41% of ESBL-producers. The O25b-ST131-B2 clone was characterised by a CTX-M-27- or CTX-M-15-type ESBL and ciprofloxacin-non-susceptibility with quadruple mutations in the quinolone resistance-determining regions (S83L and D87N in GyrA and S80I and E84V in ParC). A significant proportion of pAmpC-producers and the O25b-ST131-B2 clone were found in Japan by a recent regional surveillance programme., (Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

We developed a simple genotyping method for Flavobacterium psychrophilum for analysing two single nucleotide polymorphisms (SNPs) in the gyrA gene and to distinguish between isolates that are virulent and avirulent to ayu, Plecoglossus altivelis altivelis (Temminck & Schlegel). The genotyping method is an on/off switch assay and is based on the polymerase chain reaction technique with phosphorothioated primers. We classified 232 isolates from four families of fish (i.e. Plecoglossidae, Osmeridae, Cyprinidae and Salmonidae) into four genotypes (G-C, A-T, A-C and G-T). The G-C type isolates exhibited strong pathogenicity to ayu, whereas the A-T and G-T types did not show any pathogenicity to this species. The A-C type exhibited no or weak pathogenicity to ayu. These results indicate that genotyping F. psychrophilum isolates with two SNPs from gyrA can clearly distinguish between isolates potentially harmful to ayu (G-C type) and those that are potentially not harmful or less harmful (A-C, A-T and G-T type). The on/off switch assay provides a quick, simple, and very powerful DNA genotyping technique for F. psychrophilum isolates., (© 2012 Blackwell Publishing Ltd.)

Minimum inhibitory concentrations of sitafloxacin, gatifloxacin, moxifloxacin, sparfloxacin, levofloxacin and ciprofloxacin against 59 ciprofloxacin-resistant clinical isolates of Mycobacterium tuberculosis from Japan were determined. The isolates were most susceptible to sitafloxacin and gatifloxacin. To understand better the basis for drug resistance, nucleotide sequences encoding the gyrA and gyrB quinolone resistance-determining region were determined. Predicted amino acid sequences revealed distinct mutational patterns likely to be responsible for fluoroquinolone resistance. Double gyrA mutations as well as mutations in both gyrA and gyrB correlated with increased resistance to all fluoroquinolones., (Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

The third-line treatment regimen for Helicobacter pylori after failure of clarithromycin- and metronidazole-based therapies is not yet established. Sitafloxacin (STX) is a quinolone that possesses potent in vitro activity against H. pylori. In this study, the susceptibility of H. pylori isolates to STX was examined and the efficacy of STX-based triple therapy as a third-line regimen was evaluated. STX showed minimum inhibitory concentrations (MICs) of ≤1 μg/mL against all 100 H. pylori isolates, and the MIC(90) (MIC for 90% of the organisms) of STX was 5 log(2) dilutions lower than that of levofloxacin (LVX). The MIC(50) (MIC for 50% of the organisms) of STX against gyrA mutants was 0.12 μg/mL and was significantly lower than that of LVX (8 μg/mL). The activity of STX at pH 5.5 was significantly less than that at pH 7.0. In the clinical trial, 28 patients with two eradication failures were treated with STX-based triple therapy [rabeprazole 10 mg twice daily (b.i.d.), amoxicillin 750 mg b.i.d. and STX 100mg b.i.d. for 7 days]. The eradication rate was 75% using intention-to-treat analysis and 80% using per-protocol analysis. Two gyrA mutant strains were eradicated. Amongst participants, a low pepsinogen I/II ratio was associated with successful eradication. These results suggest that STX could be active against most clinical H. pylori isolates and that STX-based triple therapy is a promising and safe third-line therapy., (Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

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., (Copyright © 2012 S. Karger AG, Basel.)

Background and Aim: As bacterial resistance to clarithromycin limits the efficacy of clarithromycin-based regimens for Helicobacter pylori infection, attention has turned to quinolone-based rescue therapies. Resistance of H. pylori to both clarithromycin and quinolone can be predicted by genetic testing. Here, we used this approach to evaluate the prevalence of clarithromycin- and quinolone-resistant strains of H. pylori in Japan., Methods: DNA was extracted from gastric tissue samples obtained from 153 patients infected with H. pylori (103 naive for eradication therapy and 50 with previous eradication failure following triple proton pump inhibitor/amoxicillin/clarithromycin therapy). Mutations in H. pylori 23S rRNA and gyrA genes associated with resistance to clarithromycin and quinolones, respectively, were determined., Results: Of 153 patients, 85 (55.6%) were infected with clarithromycin-resistant strains. The prevalence of clarithromycin-resistant strains in patients with previous eradication failure (90.0%, 45/50) was significantly higher than that (38.8%, 40/103) of those naive for eradication therapy (P < 0.001). Fifty-nine patients (38.6%) were infected with strains resistant to quinolones. The incidence of quinolone-resistant strains also appeared higher in patients with eradication failure (48.0%, 24/50) than in those who had not undergone therapy (34.0%, 35/103); however, the difference was not statistically significant (P = 0.112). The incidence of quinolone-resistance in clarithromycin-resistant strains (44/85, 51.8%) was significantly higher than that in clarithromycin-sensitive strains (15/68, 22.1%) (P < 0.001)., Conclusions: A high incidence of quinolone-resistance was found in clarithromycin-resistant strains of H. pylori, particularly in patients with previous eradication failure. Our results suggest that testing for susceptibility of H. pylori to quinolones is useful for determining the optimal rescue eradication regimen., (© 2011 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.)

Objectives: Streptococcus pyogenes causes various diseases in humans. While the prevalence of fluoroquinolone-resistant S. pyogenes isolates has been increasing since 2000 in the USA and Europe, it has remained very low in Japan. We isolated a fluoroquinolone-resistant S. pyogenes strain and analysed its genetics., Methods: TU-296, a strain of S. pyogenes resistant to levofloxacin (MIC 16 mg/L), was isolated from the throat of a patient in their thirties with pharyngitis in autumn 2007. We carried out susceptibility tests for various antimicrobial agents and PCR analysis of the genes gyrA, gyrB, parC and parE in the quinolone resistance-determining region, followed by sequencing of the PCR products to find mutation(s) and the resulting amino acid substitution(s). We then sequenced the PCR product of the emm gene and determined the emm genotype., Results: S. pyogenes TU-296 was found to have the following mutations and amino acid substitutions: adenine 476 to cytosine in gyrA and cytosine 367 to thymine in parC, resulting in Glu-85→Ala in GyrA and Ser-79→Phe in ParC. The genotype of the isolate was emm11., Conclusions: Amino acid substitutions in fluoroquinolone-resistant S. pyogenes have already been reported from Europe and the USA, including Ser-81→Phe or Tyr and Met-99→Leu in GyrA, as well as Ser-79→Phe, Tyr or Ala and others in ParC. Numerous point mutations were found in parC and parE of S. pyogenes TU-296. In addition, a new amino acid substitution was detected (Glu-85→Ala in GyrA). To our knowledge, there have been no previous reports of this substitution in a clinical isolate of S. pyogenes.

The aim of this study was to establish the importance of detecting fluoroquinolone (FQ) resistance in multidrug resistant (MDR) Mycobacterium tuberculosis, and to show the usefulness of a hybridization-based line probe assay (LiPA) for detecting gyrA mutations. Thirty-three MDR M. tuberculosis isolates were collected from a total of sixty MDR isolates identified in Japan over 6 months during a national surveillance study in 2002. Seventeen MDR isolates were collected by the National Center for Global Health and Medicine in Japan over 6 years from 2003 to 2008. These 50 isolates were examined for FQ susceptibility, and analysed by LiPA and gyrA sequencing. Among them, 22 (44 %) showed FQ resistance. All FQ-resistant isolates had at least one mutation in gyrA. The results of the LiPA were fully consistent with the DNA sequencing results. Given that on the basis of our results almost half of the MDR M. tuberculosis isolates in Japan might have resistance to FQ, it is important to monitor FQ resistance in patients with MDR tuberculosis (TB), as well as with drug-susceptible TB, prior to commencing treatment. For the detection of FQ resistance, LiPA is useful and can rapidly and efficiently assess FQ resistance.

Surveillance for antimicrobial resistance in Mycoplasma genitalium clinical strains is extremely limited as culturing of strains from clinical specimens is still difficult. We therefore conducted a non-cultural assessment of fluoroquinolone resistance of M. genitalium clinical strains by analysing the quinolone-resistance determining regions (QRDRs) of the gyrA and parC genes. The QRDRs amplified from M. genitalium DNA taken from urine specimens of 28 men with non-gonococcal urethritis positive for M. genitalium by polymerase chain reaction were sequenced. An amino acid change (Phe-108-->Iso) in GyrA was found in one specimen, and the same change was accompanied by an amino acid change (Lys-97-->Arg) in ParC in another specimen. A single amino acid change (Ser-83-->Asn, Asp-87-->Tyr or Asp-87-->Val) in ParC was also found in three other respective specimens without alterations in GyrA. No alterations in GyrA and ParC were found in the remaining 23 specimens. The alterations of Ser-83-->Asn, Asp-87-->Tyr and Asp-87-->Val in ParC found in 3 (10.7%) of 28 specimens were analogous to those commonly observed in fluoroquinolone-resistant mutants of other Mycoplasma and Ureaplasma spp. M. genitalium harbouring mutations associated with fluoroquinolone resistance in the parC gene may have emerged clinically and the prevalence may be ca. 10% in Japan., (Copyright (c) 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

We established the distribution of amino acid alterations in quinolone resistance-determining regions (QRDRs) of Streptococcus pneumoniae isolates in Japan and described the correlation of these alterations with serotypes determined by multilocus sequencing typing. Among 141 S. pneumoniae isolates, five levofloxacin-resistant isolates harbored mutations in both gyrA and parC and/or parE and were clonally unrelated. Among 136 levofloxacin-susceptible isolates, one isolate (MIC = 2 mg/l) had a first-step parC mutation at Asp78. Twenty isolates had Lys137Asp in parC and Ile460Val in parE and contained nine serotypes and eight clonal complexes (CCs), including all eight Colombia(23F)-26 (CC138) isolates. Eighty-one isolates had Ile460Val in parE alone and contained 14 serotypes and 16 CCs, including 36 of 37 Netherlands(3)-31 (CC180) isolates and all 22 Taiwan(19F)-14 (CC271) isolates. In contrast, seven of ten Taiwan(23F)-15 (CC242) isolates were wild-type. Although each QRDR genotype contained various serotypes and CCs, prevalent clones were mostly associated with a single QRDR genotype.

Six Bordetella pertussis strains isolated from children in Japan from 2004 to 2006 showed high-level resistance to nalidixic acid (NAL; MIC, >256 microg/ml) and decreased susceptibilities to fluoroquinolones. All of the NAL-resistant strains had the same D87G mutation in gyrA.

Two novel Psychrobacter-like bacteria, strains KC 40(T) and KC 65, were isolated from a marine crustacean specimen collected from the Sea of Japan, and were characterized by using a polyphasic approach. Strains were selected on the basis of their ability to produce black-brown diffusible pigments on commonly used organic media, which appears to be a unique characteristic of recognized members of the genus Psychrobacter. Phylogenetic analyses based on both 16S rRNA and gyrB gene sequences showed that the novel isolates formed a separate cluster within the genus Psychrobacter. Strains KC 40(T) and KC 65 shared highest levels of 16S rRNA gene sequence similarity with Psychrobacter urativorans DSM 14009(T) (98.0 %), Psychrobacter pulmonis CCUG 46240(T) (97.9 %), Psychrobacter cibarius JG-219(T) (97.9 %), Psychrobacter faecalis Iso-46(T) (97.8 %), Psychrobacter aquimaris SW-210(T) (97.6 %), Psychrobacter namhaensis SW-242(T) (97.6 %) and Psychrobacter nivimaris 88/2-7(T) (97.6 %). DNA-DNA hybridization experiments revealed 84 % DNA-DNA relatedness between strains KC 40(T) and KC 65 but much lower levels of relatedness (7-35 %) between the novel strains and the type strains of recognized Psychrobacter species, confirming their assignment to a single novel species of the genus Psychrobacter. The two novel strains could be distinguished from recognized species of the genus Psychrobacter based on a combination of physiological and biochemical characteristics. On the basis of phenotypic and molecular properties, strains KC 40(T) and KC 65 are considered to represent a novel species of the genus Psychrobacter, for which the name Psychrobacter fulvigenes sp. nov. is proposed. The type strain is KC 40(T) (=KMM 3954(T)=NRIC 0746(T)=JCM 15525(T)).

In total, 83 avian pathogenic Escherichia coli (APEC) isolates from avian colibacillosis during a period from 2001 to 2006 in Japan were investigated for serogroups, typical virulence factors, antimicrobial susceptibility, and genetic relatedness. The most common serogroup was O78 (30.1%); 80.7% of isolates harbored the iss gene and 55.4% of isolates harbored the tsh gene. Antimicrobial resistance of the isolates was found for ampicillin (77.1%), oxytetracycline (75.9%), kanamycin (36.1%), fradiomycin (33.7%), trimethoprim (25.3%), enrofloxacin (21.7%), and florfenicol (6.0%). Although multiple antimicrobial-resistant phenotypes (three or more antimicrobials) accounted for 54.2% of isolates, no isolate exhibited resistance to all agents tested. The fluoroquinolone-resistant isolates had point mutations in GyrA (Ser83 --> Leu, Asp87 --> Asn) and ParC (Ser80 --> Ile, Glu84 --> Gly). Of 18 enrofloxacin-resistant E. coli isolates, nine isolates belonged to serotype O78. In PFGE analysis, eight of the nine enrofloxacin-resistant O78 isolates were classified into an identical cluster. This suggests that a specific genotype of fluoroquinolone-resistant O78 APEC may be widely distributed in Japan.

Gram-negative bacteria, Photorhabdus luminescens and P. temperata, form a mutualistic association with entomopathogenic heterorhabditid nematodes while P. asymbiotica is known as an opportunistic human pathogen that causes disseminated bacteremic spread on two continents, the United States and Australia. In the course of our phylogenetic study of Photorhabdus bacteria associated with Japanese Heterorhabditis nematodes, we found two Photorhabdus isolates (Photorhabdus sp. Cbkj163 and OnIr40) whose partial 16S rRNA gene sequence showed high similarities to clinical isolates of this pathogen from Heterorhabditis indica. The phylogenetic study, based upon the gyrase subunit B gene sequences of the two isolates, revealed clustering with these clinical isolates of P. asymbiotica from both the United States and Australia but not with other Photorhabdus bacteria associated with nematodes. The two bacterial isolates were also found to share microbiological and biochemical characteristics with clinical and entomopathogenic Photorhabdus strains. Moreover, not only the two novel Photorhabdus isolates but also an Australian clinical isolate of P. asymbiotica formed mutualistic association with H. indica isolates. These data suggest that the bacteria isolated from H. indica CbKj163 and OnIr40 are a novel subspecies of P. asymbiotica, and that some clinical isolates of P. asymbiotica could have originated from bacteria associated with entomopathogenic nematodes.

Eight strains, 002-165T, 002-079T, B0021T, Hojyo2, RB603B, RB677T, 002-074T and RB678, isolated from the environment of food-processing factories in Japan, were characterized using a polyphasic approach. The isolates were Gram-negative, strictly aerobic, pink-pigmented, facultatively methylotrophic, non-spore-forming rods. The chemotaxonomic characteristics of these isolates included the presence of C18 : 1omega7c as the major cellular fatty acid and ubiquinone Q-10 as the predominant ubiquinone. The DNA G+C content was 67.1-71.1 mol%. Phylogenetic analyses of 16S rRNA and DNA gyrase B subunit (gyrB) nucleotide sequence confirmed that the eight strains belonged to the Methylobacterium clade. Moreover, a DNA-DNA hybridization analysis showed that the eight isolates represented five novel species. On the basis of their phenotypic and phylogenetic distinctiveness, the isolates represent five novel species within the genus Methylobacterium, for which the names Methylobacterium persicinum sp. nov. (type strain 002-165T =DSM 19562T =NBRC 103628T =NCIMB 14378T), Methylobacterium komagatae sp. nov. (type strain 002-079T =DSM 19563T =NBRC 103627T =NCIMB 14377T), Methylobacterium brachiatum sp. nov. (type strain B0021T =DSM 19569T =NBRC 103629T =NCIMB 14379T), Methylobacterium tardum sp. nov. (type strain RB677T =DSM 19566T =NBRC 103632T =NCIMB 14380T) and Methylobacterium gregans sp. nov. (type strain 002-074T =DSM 19564T =NBRC 103626T =NCIMB 14376T) are proposed.

Objectives: We investigated the antimicrobial susceptibilities and resistance mechanisms of cattle-adapted Salmonella enterica serovar Dublin isolated in Japan in the past 30 years. This study is an example of evaluation of the impact of introduction of antimicrobials in veterinary medical practice on the selection of resistance in S. enterica., Methods: The antimicrobial susceptibilities and prevalence of R-plasmids in Salmonella Dublin isolated in Japan from 1976 to 2005 were investigated. To evaluate the importance of gyrA mutation and active efflux, we derived the gyrA revertants and acrAB deletion mutants, and then compared with their parental strains the MICs of quinolone antimicrobials such as nalidixic acid, enrofloxacin, ofloxacin and ciprofloxacin., Results: Salmonella Dublin isolates with R-plasmids and resistance to more than three antimicrobials were predominant between 1981 and 1995. From the latter half of the 1990s to the present, Salmonella Dublin isolates without R-plasmids became dominant. The introduction of nalidixic acid into the veterinary field in the mid-1980s was followed by the emergence of nalidixic acid-resistant isolates, which are now predominant. We found only a single gyrA mutation (Asp-87-->Tyr) among the nalidixic acid-resistant isolates. Although the reduced susceptibilities to the fluoroquinolones were observed among the nalidixic acid-resistant isolates, none of the isolates was resistant to the fluoroquinolones used in this study. The MIC data for the fluoroquinolones differed up to 4-fold. Results of the susceptibility test using gyrA revertants and acrAB mutants suggest that the isolates with the gyrA mutation were selected by the use of nalidixic acid, and the AcrAB-TolC system accounts for the decreased fluoroquinolone susceptibilities., Conclusions: These data suggest that the introduction of nalidixic acid in veterinary medicine seemed to affect the susceptibilities of Salmonella Dublin among the cattle population in Japan, whereas the introduction of enrofloxacin has not caused any additional effect. The prudent use of antimicrobials in the veterinary field should be continuously stressed.

A polyphasic taxonomic approach was applied to determine the taxonomic position of a hydrocarbon-degrading actinomycete, strain Hou&#95;blueT, which was isolated from soil samples collected from an oil spring in Niigata, Japan. The results of 16S rRNA and gyrB gene sequence comparisons indicated that strain Hou&#95;blueT represented a novel lineage in the suborder Corynebacterineae. Colonies were malachite green-like in colour on 1/10 trypticase soy agar and the cell morphology was coccoid in all growth phases. The cell-wall diamino acid and sugar indicated chemotype IV and variation A1gamma. The sugars of the peptidoglycan were glycolated. The polar lipids were composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and some unspecified glycolipids. The organism contained two novel cyclic forms of menaquinone, smaragdiquinone A-8(H4, omega-cycl) and smaragdiquinone B-8(H4, dicycl). The major fatty acids were cis-9-18 : 1 (34.46 %) and 16 : 0 (25.1 %). Small amounts of 10-methyl-branched fatty acids were also present (10-methyl-17 : 0, 0.17 %), but not tuberculostearic acid (10-methyl-18 : 0), which has been shown to be present in all nocardiae. Gas-chromatographic analysis of the mycolic acid revealed a carbon-chain length of C43-C49. The DNA G+C content was 63.7 mol%. On the basis of phenotypic and phylogenetic distinctness, the organism is proposed to represent a novel genus and species, Smaragdicoccus niigatensis gen. nov., sp. nov., with the type strain Hou&#95;blueT (=MBIC 06267T=DSM 44881T).

A study was carried out to clarify the taxonomy of four Gram-positive, heterotrophic mesophiles isolated from marine lakes in the Republic of Palau. The strains, designated YM3-251(T), YM3-653, YM3-671 and YM11-542(T), formed aerial and substrate mycelia. The cell-wall peptidoglycan contained meso-diaminopimelic acid, glutamic acid and alanine. The G+C content of their genomic DNA was approximately 45 mol%. The major fatty acid was iso-C(15 : 0) and the major isoprenoid quinone was MK-9. The strains formed a distinct group in the 16S rRNA gene tree and shared a range of phenotypic properties that distinguished them from members of related genera in Thermoactinomycetaceae fam. nov. The name proposed to accommodate the new isolates is Mechercharimyces gen. nov., comprising two species based on genotypic and phenotypic criteria, including comparative gyrB and DNA-DNA relatedness data. The names proposed for these taxa are Mechercharimyces mesophilus sp. nov., the type species, and Mechercharimyces asporophorigenens sp. nov., with the type strains YM3-251(T) (=MBIC06230(T)=DSM 44894(T)) and YM11-542(T) (=MBIC06487(T)=DSM 44955(T)), respectively.

Background: Recent years have witnessed a decrease in the rate of Helicobacter pylori eradication due to antimicrobial resistance, clarithromycin or metronidazole resistance in particular. As one of the alternatives to the standard regimens, levofloxacin-containing therapy has been considered a promising regimen. Nevertheless, there is a little information concerning the prevalence of levofloxacin resistance and this resistance mechanism., Materials and Methods: Levofloxacin susceptibility was examined using E-test in 507 H. pylori strains clinically isolated in Japan from 2001 to 2004. Mutation patterns in the quinolone resistance-determining regions of the gyrA and gyrB genes were evaluated, performing direct sequencing of 68 levofloxacin-resistant and 50 susceptible strains., Results: Primary levofloxacin resistance was found in 76 (15.0%) strains. Fifty-seven (83.8%) of 68 levofloxacin-resistant strains analyzed had point mutations in gyrA at Asn-87 or Asp-91, while seven (14.0%) of 50 susceptible strains had gyrA mutations. There was a significant difference in the occurrence of gyrA mutations between levofloxacin-resistant and -susceptible strains (p < .001). In levofloxacin-resistant strains, the occurrence of gyrA mutations at Asn-87 was most common regardless of minimal inhibitory concentration levels, and that of gyrA mutations at Asp-91 tended to be associated with low-level resistance. A double gyrA mutation at Asn-87 and Asp-91 might have an additional impact. As for gyrB, three (4.4%) of 68 levofloxacin-resistant strains with no susceptible strains had mutations., Conclusions: Primary levofloxacin resistance was common in Japan and primarily related to gyrA mutations at Asn-87 and Asp-91.

Strain DY(T), which was isolated from garden soil in Japan, was subjected to a polyphasic taxonomic study. Sequence analysis of the 16S rRNA gene and the GyrB protein revealed that the closest relative of strain DYT was [Flavobacterium] ferrugineum Sickles and Shaw 1934, with 94.8 and 90.1 % similarity, respectively. The two strains had similar chemotaxonomic characteristics, with menaquinone 7 as the major quinone system, 47.2-48.9 mol% DNA G+C content and 15 : 0 iso, 15 : 1 iso, 17 : 0 iso 3-OH and summed feature 3 as the major fatty acids. Based on genotypic and phenotypic characteristics, [Flavobacterium] ferrugineum IAM 15098T could be clearly differentiated from other members of the genus Flavobacterium. Strain DYT and [Flavobacterium] ferrugineum IAM 15098T could be easily distinguished from neighbouring taxa by morphological features (non-motile, non-gliding and non-filamentous single cells). Therefore, it is proposed that [Flavobacterium] ferrugineum IAM 15098T and strain DYT represent two separate species of a new genus, Terrimonas gen. nov., with the names Terrimonas ferruginea comb. nov. (type species; type strain IAM 15098T=ATCC 13524T) and Terrimonas lutea sp. nov. (type strain DYT=IAM 15284T=CCTCC AB205006T), respectively.

A nested polymerase chain reaction (PCR) amplification technique was used to detect Flavobacterium psychrophilum from washings of fish gill surfaces and benthic diatoms as environmental samples. Gill washing samples were prepared from kawamutsu, Zacco temminckii (Temminck & Schlegel) and oikawa, Z. platypus (Temminck & Schlegel). Benthic diatom samples were collected from stone surfaces. All samples were collected from rivers in Wakayama Prefecture, Japan from November 2003 to January 2004. Following simple DNA extraction using a chelating resin, nested PCR techniques targeting 16S-rDNA and gyrB regions were performed, and PCR products were cloned and sequenced. With nested PCR amplification for the 16S-rDNA gene, ambiguous PCR products were detected from two of six samples, and by cloning and sequencing analysis were found not to be DNA fragments amplified from F. psychrophilum. Using nested PCR for the gyrB gene, however, five of six samples were clearly positive for F. psychrophilum in agarose gel electrophoresis, and were found to be identical with nucleotide sequences of F. psychrophilumgyrB deposited in DNA databases by sequencing analysis. Results indicate that nested PCR for the gyrB region is a useful technique to detect low levels of F. psychrophilum from environmental samples contaminated with many other organisms.

A novel Ferrimonas species is described on the basis of phenotypic, chemotaxonomic and phylogenetic studies. Four halophilic organisms were isolated from marine sand and marine macroalgae samples by using high-pH marine agar 2216. An analysis of the nearly complete 16S rRNA gene sequences of these new isolates indicated that they were phylogenetically close (16S rRNA gene sequence similarity >99.5%, gyrB gene sequence similarity >97.8%), and were most closely related to Ferrimonas balearica (16S rRNA gene sequence similarity 97.1-97.3%, gyrB gene sequence similarity 84.4-85.0%). Chemotaxonomic data (major menaquinone MK7; major fatty acids C(16:0) and C(18:1)omega9c) supported the affiliation of the new isolates to the genus Ferrimonas. The results of physiological and biochemical tests allowed phenotypic differentiation of the isolates from F. balearica. It is therefore proposed that the new isolates represent a novel species with the name Ferrimonas marina sp. nov. and type strain A4D-4(T) (=MBIC06480(T)=DSM 16917(T)).

Quinolone-resistant strains of the fish-pathogenic bacterium, Photobacterium damselae subsp. piscicida are distributed widely in cultured yellowtail, Seriola quinqueradiata (Temminck & Schlegel), in Japan. The quinolone resistance-determining region (QRDR) was amplified with degenerate primers, followed by cassette ligation-mediated PCR. Open reading frames encoding proteins of 875 and 755 amino acid residues were detected in the gyrA and parC genes, respectively. Resistant strains of P. damselae subsp. piscicida carried a point mutation only in the gyrA QRDR leading to a Ser-to-Ile substitution at residue position 83. No amino acid alterations were discovered in the ParC sequence. A mutation in the gyrA gene was also detected in nalidixic acid-resistant mutants of strain SP96002 obtained from agar medium containing increased levels of quinolone. These results suggest that GyrA, as in other Gram-negative bacteria, is a target of quinolone in P. damselae subsp. piscicida. Furthermore, we attempted to detect a point mutation using targeting-induced local lesions in genomes (TILLING), which is a general strategy used for the detection of a variety of induced point mutations and naturally occurring polymorphisms. We developed a new detection method for the rapid and large-scale identification of quinolone-resistant strains of P. damselae subsp. piscicida using TILLING.