Faculty Profiles - SHIBAYAMA Keigo

Papers - SHIBAYAMA Keigo

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Prevalence and genetic characterization of pertactin-deficient bordetella pertussis in Japan Reviewed

Nao Otsuka, Hyun Ja Han, Hiromi Toyoizumi-Ajisaka, Yukitsugu Nakamura, Yoshichika Arakawa, Keigo Shibayama, Kazunari Kamachi

PLoS ONE Vol. 7 ( 2 ) page： e31985 2012.2

Language：English Publishing type：Research paper (scientific journal) Publisher：2

The adhesin pertactin (Prn) is one of the major virulence factors of Bordetella pertussis, the etiological agent of whooping cough. However, a significant prevalence of Prn-deficient (Prn -) B. pertussis was observed in Japan. The Prn - isolate was first discovered in 1997, and 33 (27%) Prn - isolates were identified among 121 B. pertussis isolates collected from 1990 to 2009. Sequence analysis revealed that all the Prn - isolates harbor exclusively the vaccine-type prn1 allele and that loss of Prn expression is caused by 2 different mutations: an 84-bp deletion of the prn signal sequence (prn1ΔSS, n = 24) and an IS481 insertion in prn1 (prn1::IS481, n = 9). The frequency of Prn - isolates, notably those harboring prn1ΔSS, significantly increased since the early 2000s, and Prn - isolates were subsequently found nationwide. Multilocus variable-number tandem repeat analysis (MLVA) revealed that 24 (73%) of 33 Prn - isolates belong to MLVA-186, and 6 and 3 Prn - isolates belong to MLVA-194 and MLVA-226, respectively. The 3 MLVA types are phylogenetically closely related, suggesting that the 2 Prn - clinical strains (harboring prn1ΔSS and prn1::IS481) have clonally expanded in Japan. Growth competition assays in vitro also demonstrated that Prn - isolates have a higher growth potential than the Prn + back-mutants from which they were derived. Our observations suggested that human host factors (genetic factors and immune status) that select for Prn - strains have arisen and that Prn expression is not essential for fitness under these conditions. © 2012 Otsuka et al.

DOI： 10.1371/journal.pone.0031985

Predominance of sequence type 1 group with serotype VI among group B streptococci with reduced penicillin susceptibility identified in japan Reviewed

Kouji Kimura, Noriyuki Nagano, Yukiko Nagano, Jun ichi Wachino, Satowa Suzuki, Keigo Shibayama, Yoshichika Arakawa

Journal of Antimicrobial Chemotherapy Vol. 66 ( 11 ) page： 2460 - 2464 2011.11

Language：English Publishing type：Research paper (scientific journal) Publisher：11

Background: Although group B Streptococcus (GBS; i.e. Streptococcus agalactiae) has been considered to be uniformly susceptible to β-lactams, GBS isolates with reduced penicillin susceptibility (PRGBS) have been reported from Japan and North America. In this study, PRGBS from Japan were characterized by multilocus sequence typing (MLST) and the results compared with data on PRGBS reported from the USA. Methods: Twenty-eight clinical isolates of PRGBS recovered in Japan (including 22 isolates previously analysed by PFGE) were analysed by MLST and eBURST (http://eburst.mlst.net/). Results: Twenty-three isolates were found to belong to the sequence type 1 (ST1) group (11 ST458, 7 ST1, 3 ST297, 1 ST358 and 1 ST4), while the remaining 5 isolates formed the ST23 group. Among 11 ST458 and 7 ST1 isolates, 9 and 4 were serotype VI, respectively, indicating a probable correlation between the ST1 group and serotype VI for PRGBS in Japan. Conclusions: PRGBS in Japan could be classified into at least two ST groups, ST1 and ST23, which are genetically different from the ST19 PRGBS isolated in the USA, though five allele variations were seen between ST1 and ST19, implying a slight genetic relatedness. © The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

DOI： 10.1093/jac/dkr352

SMB-1, a novel subclass B3 metallo-β-lactamase, associated with ISCR1 and a class 1 integron, from a carbapenem-resistant Serratia marcescens clinical isolate Reviewed

Jun Ichi Wachino, Hiroyuki Yoshida, Kunikazu Yamane, Satowa Suzuki, Mari Matsui, Takuya Yamagishi, Atsuko Tsutsui, Toshifumi Konda, Keigo Shibayama, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy Vol. 55 ( 11 ) page： 5143 - 5149 2011.11

Language：English Publishing type：Research paper (scientific journal) Publisher：11

A carbapenem-resistant Serratia marcescens strain, 10mdr148, was identified in a Japanese hospital in 2010. The carbapenem resistance of this strain was attributed to the production of a novel metallo-β-lactamase (MBL), named SMB-1 (Serratia metallo-β-lactamase). SMB-1 possessed a zinc binding motif, H(Q)XHXDH (residues 116 to 121), H196, and H263 and was categorized as a member of subclass B3 MBL. SMB-1 has 75% amino acid identity with the most closely related MBL, AMO1, of uncultured bacterium, recently identified through the metagenomic analysis of apple orchard soil. The introduction of bla SMB-1 into Escherichia coli conferred resistance to a variety of β-lactam antibiotics, penicillins, cephalosporins, and carbapenems, but not aztreonam, a resistance pattern consistent with those of other MBLs. SMB-1 demonstrated high k cat values of >500 s -1 for carbapenems, resulting in the highest hydrolyzing efficiency (k cat/K m) among the agents tested. The hydrolyzing activity of SMB-1 was well inhibited by chelating agents. The bla SMB-1 gene was located on the chromosome of S. marcescens strain 10mdr148 and at the 3′ end of the ISCR1 element in complex with a typical class 1 integron carrying aac(6′)-Ib and catB3 gene cassettes. Downstream of bla SMB-1, the second copy of the 3′ conserved segment and ISCR1 were found. To our knowledge, this is the first subclass B3 MBL gene associated with an ISCR1 element identified in an Enterobacteriaceae clinical isolate. A variety of antibiotic resistance genes embedded with ISCR1 have been widely spread among Enterobacteriaceae clinical isolates, thus the further dissemination of bla SMB-1 mediated by ISCR1 transposition activity may become a future concern. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/AAC.05045-11

Structural insights into the novel diadenosine 5′,5‴-P <sup>1</sup>,P<sup>4</sup>-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv Reviewed International journal

Shigetarou Mori, Keigo Shibayama, Jun Ichi Wachino, Yoshichika Arakawa

Journal of Molecular Biology Vol. 410 ( 1 ) page： 93 - 104 2011.7

Language：English Publishing type：Research paper (scientific journal) Publisher：1

Rv2613c is a diadenosine 5′,5‴-P1,P 4-tetraphosphate (Ap4A) phosphorylase from Mycobacterium tuberculosis H37Rv. Sequence analysis suggests that Rv2613c belongs to the histidine triad (HIT) motif superfamily, which includes HIT family diadenosine polyphosphate (ApnA) hydrolases and Ap4A phosphorylases. However, the amino acid sequence of Rv2613c is more similar to that of HIT family ApnA hydrolases than to that of typical Ap4A phosphorylases. Here, we report the crystal structure of Rv2613c, which is the first structure of a protein with ApnA phosphorylase activity, and characterized the structural basis of its catalytic activity. Our results showed that the structure of Rv2613c is similar to those of other HIT superfamily proteins. However, Asn139, Gly146, and Ser147 in the active site of Rv2613c replace the corresponding Gln, Gln, and Thr residues that are normally found in HIT family ApnA hydrolases. Furthermore, analyses of Rv2613c mutants revealed that Asn139, Gly146, and Ser147 are important active-site residues and that Asn139 has a critical role in catalysis. The position of Gly146 might influence the phosphorylase activity. In addition, the tetrameric structure of Rv2613c and the presence of Trp160 might be essential for the formation of the Ap4A binding site. These structural insights into Rv2613c may facilitate the development of novel structure-based inhibitors for treating tuberculosis. © 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2011.04.059

Biochemical and pathophysiological characterization of Helicobacter pylori asparaginase Reviewed International journal

Keigo Shibayama, Hiroaki Takeuchi, Jun ichi Wachino, Shigetarou Mori, Yoshichika Arakawa

Microbiology and Immunology Vol. 55 ( 6 ) page： 408 - 417 2011.6

Language：English Publishing type：Research paper (scientific journal) Publisher：6

Asparaginase was purified from Helicobacter pylori 26695 and its pathophysiological role explored. The Km value of asparagine was 9.75 ± 1.81 μM at pH 7.0, and the optimum pH range was broad and around a neutral pH. H. pylori asparaginase converted extracellular asparagine to aspartate. H. pylori cells were unable to take up extracellular asparagine directly. Instead, aspartate produced by the action of the asparaginase was transported into H. pylori cells, where it was partially converted to β-alanine. Asparaginase exhibited striking cytotoxic activity against histiocytic lymphoma cell line U937 cells via asparagine deprivation. The cytotoxic activity of live H. pylori cells against U937 cells was significantly diminished by deletion of the asparaginase gene, indicating that asparaginase functions as a cytotoxic agent of the bacterium. The cytotoxic effect was negligible for gastric epithelial cell line AGS cells, suggesting that the effect differs across host cell types. An asparaginase-deficient mutant strain was significantly less capable of colonizing Mongolian gerbils. Since asparagine depletion by exogenous asparaginase has been shown to suppress lymphocyte proliferation in vivo, the present results suggest that H. pylori asparaginase may be involved in inhibition of normal lymphocyte function at the gastric niche, allowing H. pylori to evade the host immune system. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

DOI： 10.1111/j.1348-0421.2011.00333.x

Report of an international collaborative study to evaluate the suitability of multiplex PCR as an identity assay for different sub-strains of BCG vaccine Reviewed International journal

Kevin Markey, Mei M. Ho, Babna Choudhury, Masaaki Seki, Liu Ju, Luiz R.R. Castello-Branco, Sunil Gairola, Aihua Zhao, Keigo Shibayama, Murielle Andre, Michael J. Corbel

Vaccine Vol. 28 ( 43 ) page： 6964 - 6969 2010.10

Language：English Publishing type：Research paper (scientific journal) Publisher：43

Current methods for the identification of BCG vaccine in quality control settings involve acid-fast staining with microscopic examination. However, this method is unable to distinguish the many different sub-strains of BCG, or to differentiate BCG strains from virulent members of the Mycobacterium tuberculosis complex. A multiplex PCR (mPCR) which uses six target regions in mycobacteria has been developed to identify specific sub-strains of BCG. This study reports the findings from an international collaborative study to assess the accuracy, robustness and reproducibility of this mPCR method to differentiate BCG sub-strains. The method was found to fulfil these criteria successfully and was able to distinguish BCG sub-strains in vaccine preparations. The majority of the participants in the study generated the expected PCR product profiles indicating the method is also robust. © 2010 Elsevier Ltd.

DOI： 10.1016/j.vaccine.2010.08.045

RmtC introduces G1405 methylation in 16S rRNA and confers high-level aminoglycoside resistance on Gram-positive microorganisms Reviewed

Jun Ichi Wachino, Keigo Shibayama, Kouji Kimura, Kunikazu Yamane, Satowa Suzuki, Yoshichika Arakawa

FEMS Microbiology Letters Vol. 311 ( 1 ) page： 56 - 60 2010.10

Language：English Publishing type：Research paper (scientific journal) Publisher：1

Seven plasmid-mediated 16S rRNA methyltransferases (MTases), RmtA, RmtB, RmtC, RmtD, RmtE, ArmA, and NpmA, conferring aminoglycoside resistance have so far been found in Gram-negative pathogenic microorganisms. In the present study, by performing an RNase protection assay, primer extension, and HPLC, we confirmed that RmtC indeed methylates at the N7 position of nucleotide G1405 in 16S rRNA as found in ArmA and RmtB. RmtC has an MTase activity specific for the bacterial 30S ribosomal subunit consisting of 16S rRNA and several ribosomal proteins, but not for the naked 16S rRNA, as seen in ArmA, RmtB, and NpmA. All seven 16S rRNA MTases have been found exclusively in Gram-negative bacilli to date, and no plasmid-mediated 16S rRNA MTase has been reported in Gram-positive pathogenic microorganisms. Thus, we checked whether or not the RmtC could function in Gram-positive bacilli, and found that RmtC could indeed confer high-level resistance to gentamicin and kanamycin in Bacillus subtilis and Staphylococcus aureus. 16S rRNA MTases seemed to be functional to some extent in any bacterial species, regardless of the provenance of the 16S rRNA MTase gene responsible for aminoglycoside resistance. © 2010 Federation of European Microbiological Societies.

DOI： 10.1111/j.1574-6968.2010.02068.x

Profile of Expression of Helicobacter pylori gamma-glutamyltranspeptidase. Reviewed International journal

Jun-Ichi Wachino, Keigo Shibayama, Satowa Suzuki, Kunikazu Yamane, Shigetarou Mori, Yoshichika Arakawa

Helicobacter Vol. 15 ( 3 ) page： 184 - 92 2010.6

Language：English Publishing type：Research paper (scientific journal) Publisher：3

BACKGROUND: Helicobacter pylori produces gamma-glutamyltranspeptidase (GGT), a potential virulence factor involved in induction of host cell apoptosis. Regulation of the production of this protein is not known. METHODS: The transcription start sites were determined by primer extension analysis. Transcription level of the GGT gene was examined by measuring the mRNA by RT-PCR and expression level of GGT protein was examined by Western blot analysis under different conditions. RESULTS: Two transcription start sites were identified; thymine at 78-bp upstream and adenine at 79-bp upstream from the ATG codon of the GGT gene. There was a possible -10 consensus promoter sequence (ATTAAT), but no apparent -35 consensus sequence was found. The transcription of the mRNA and the expression of the protein were at almost constant level during the course of culture. The mRNA level increased by exposure to low pH; however, the actual protein expression level remained almost constant. Addition of glutamine or glutamate did not affect the mRNA level and the protein expression level to a remarkable degree, nor did co-culture with AGS cells affect the GGT activity level. CONCLUSION: It was suggested that H. pylori GGT is constitutively expressed under various conditions.

DOI： 10.1111/j.1523-5378.2010.00755.x

Virulence genes, quinolone and fluoroquinolone resistance, and phylogenetic background of uropathogenic Escherichia coli strains isolated in Japan Reviewed

Kawamura-Sato K.

Japanese Journal of Infectious Diseases Vol. 63 ( 2 ) page： 113 - 115 2010.4

Virulence Genes, Quinolone and Fluoroquinolone Resistance, and Phylogenetic Background of Uropathogenic Escherichia coli Strains Isolated in Japan Reviewed

Kawamura-Sato Kumiko, Yoshida Risa, Shibayama Keigo, Ohta Michio

JAPANESE JOURNAL OF INFECTIOUS DISEASES Vol. 63 ( 2 ) page： 113 - 115 2010.3

Virulence genes, quinolone and fluoroquinolone resistance, and phylogenetic background of uropathogenic Escherichia coli strains isolated in Japan. Reviewed

Kawamura-Sato K, Yoshida R, Shibayama K, Ohta M

Japanese journal of infectious diseases Vol. 63 ( 2 ) page： 113 - 5 2010.3

Purification and molecular characterization of a novel diadenosine 5′,5′′′-P<sup>1</sup>,P<sup>4</sup>-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv Reviewed International journal

Shigetarou Mori, Keigo Shibayama, Jun ichi Wachino, Yoshichika Arakawa

Protein Expression and Purification Vol. 69 ( 1 ) page： 99 - 105 2010.1

Language：English Publishing type：Research paper (scientific journal) Publisher：1

In this study, Rv2613c, a protein that is encoded by the open reading frame Rv2613c in Mycobacterium tuberculosis H37Rv, was expressed, purified, and characterized for the first time. The amino acid sequence of Rv2613c contained a histidine triad (HIT) motif consisting of H-phi-H-phi-H-phi-phi, where phi is a hydrophobic amino acid. This motif has been reported to be the characteristic feature of several diadenosine 5′,5′′′-P1,P4-tetraphosphate (Ap4A) hydrolases that catalyze Ap4A to adenosine 5′-triphosphate (ATP) and adenosine monophosphate (AMP) or 2 adenosine 5′-diphosphate (ADP). However, enzymatic activity analyses for Rv2613c revealed that Ap4A was converted to ATP and ADP, but not AMP, indicating that Rv2613c has Ap4A phosphorylase activity rather than Ap4A hydrolase activity. The Ap4A phosphorylase activity has been reported for proteins containing a characteristic H-X-H-X-Q-phi-phi motif. However, no such motif was found in Rv2613c. In addition, the amino acid sequence of Rv2613c was significantly shorter compared to other proteins with Ap4A phosphorylase activity, indicating that the primary structure of Rv2613c differs from those of previously reported Ap4A phosphorylases. Kinetic analysis revealed that the Km values for Ap4A and phosphate were 0.10 and 0.94 mM, respectively. Some enzymatic properties of Rv2613c, such as optimum pH and temperature, and bivalent metal ion requirement, were similar to those of previously reported yeast Ap4A phosphorylases. Unlike yeast Ap4A phosphorylases, Rv2613c did not catalyze the reverse phosphorolysis reaction. Taken together, it is suggested that Rv2613c is a unique protein, which has Ap4A phosphorylase activity with an HIT motif. © 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.pep.2009.09.010

Crystallization and preliminary X-ray analysis of the diadenosine 5′,5′′′-P <sup>1</sup>,P <sup>4</sup>-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv Reviewed International journal

Shigetarou Mori, Keigo Shibayama, Jun Ichi Wachino, Yoshichika Arakawa

Acta Crystallographica Section F: Structural Biology and Crystallization Communications Vol. 66 ( 3 ) page： 279 - 281 2010

Language：English Publishing type：Research paper (scientific journal) Publisher：Pt 3

A novel diadenosine 5′,5′′′-P 1,P 4-tetraphosphate (Ap4A) phosphorylase (Rv2613c) from Mycobacterium tuberculosis H37Rv has been crystallized by the sitting-drop vapour-diffusion method. The crystal belonged to space group C2, with unit-cell parameters a = 101.5, b = 63.6, c = 79.1 Å, Β = 110.9°. The diffraction of the crystals extended to 1.9 Å resolution. The asymmetric unit is expected to contain two molecules of Rv2613c, with a corresponding crystal volume per protein weight (VM) of 2.41 Å3 Da-1 and a solvent content of 49.1%. This is the first report of a crystal of Ap4A phosphorylase. © 2010 International Union of Crystallography All rights reserved.

DOI： 10.1107/S174430910905444X

The nosocomial transmission of Helicobacter cinaedi infections in immunocompromised patients Reviewed

Koichiro Minauchi, Shunji Takahashi, Toshiya Sakai, Makoto Kondo, Keigo Shibayama, Yoshichika Arakawa, Masaya Mukai

Internal Medicine Vol. 49 ( 16 ) page： 1733 - 1739 2010

Language：English Publishing type：Research paper (scientific journal) Publisher：16

Background We encountered 15 cases of Helicobacter cinaedi (H. cinaedi) infection between March and July 2008. Patient, Method, and Result The underlying diseases were hematological malignancies in a majority of cases, many of which received chemotherapy. All patients had a fever. The fever was followed by cellulitis in three, a skin rash in six, pain in the lower limbs in three, and diarrhea in three cases. We analyzed the bacterial 23S rRNA genes. The fifteen strains were divided according to base sequence into Groups A, B, and C, respectively. All four cases in Group A were women and all ten in Group C were men, indicating that the gender of the patient corresponded precisely to the genotypes of the separated bacilli in these two groups. These findings also suggested the strong possibility of nosocomial spread. Conclusion It is highly likely that H. cinaedi infections have been overlooked due to the difficulties encountered in culturing the bacterium. The possibility of septicemia caused by H. cinaedi should be suspected especially in immunocompromised patients such as those undergoing chemotherapy, with symptoms such as fever, rash, arthritis, cellulitis, leg pain, and other systemic or local symptoms. © 2010 The Japanese Society of Internal Medicine.

DOI： 10.2169/internalmedicine.49.3649

A novel insertion sequence, IS1642, of Mycobacterium avium, which forms long direct repeats of variable length. Reviewed International journal

Zhenyu Piao, Keigo Shibayama, Shigetarou Mori, Jun-ichi Wachino, Yoshichika Arakawa

FEMS microbiology letters Vol. 291 ( 2 ) page： 216 - 21 2009.2

Language：English Publishing type：Research paper (scientific journal) Publisher：2

A new insertion sequence (IS), IS1642, was identified in a Mycobacterium avium strain isolated from a human patient. IS1642 had a size of 1642 bp and contained a single ORF encoding a probable transposase of 503 amino acid residues homologous (79% identity) to that of IS1549 found in Mycobacterium smegmatis. The IS1642 included imperfect inverted repeats (5'-cctgacttttatca-3', 5'-tgataaaagtcggg-3') on its ends, and was flanked by direct repeats of variable length ranging from 5 to 161 bp. It was suggested that the IS1642 was widely distributed in many M. avium strains of human patients, and the Southern blot profile of IS1642 was very diverse among the strains examined. The transposition event of IS1642 was observed by in vitro repeated passages, showing that the IS1642 is actually a transposable element. In light of these characteristics, IS1642 could be a new useful marker when genotyping with high discrimination is required.

DOI： 10.1111/j.1574-6968.2008.01459.x

The sodium-dependent D-glucose transport protein of Helicobacter pylori Reviewed International journal

Georgios Psakis, Massoud Saidijam, Keigo Shibayama, Julia Polaczek, Kim E. Bettaney, Jocelyn M. Baldwin, Stephen A. Baldwin, Ryan Hope, Lars Oliver Essen, Richard C. Essenberg, Peter J.F. Henderson

Molecular Microbiology Vol. 71 ( 2 ) page： 391 - 403 2009.1

Language：English Publishing type：Research paper (scientific journal) Publisher：2

Helicobacter pylori is a Gram-negative pathogenic microaerophile with a particular tropism for the mucosal surface of the gastric epithelium. Despite its obligatory microaerophilic character, it can metabolize d-glucose and/or d-galactose in both oxidative and fermentative pathways via a Na +-dependent secondary active transport, a glucokinase and enzymes of the pentose phosphate pathway. We have assigned the Na+-dependent transport of glucose to the protein product of the H. pylori 1174 gene. The gene was heterologously expressed in a glucose transport-deficient Escherichia coli strain, where transport activities of radiolabelled d-glucose, d-galactose and 2-deoxy-d-glucose were restored, consistent with the expected specificity of the hexose uptake system in H. pylori. d-Mannose was also identified as a substrate. The HP1174 transport protein was purified and reconstituted into proteoliposomes, where sodium dependence of sugar transport activity was demonstrated. Additionally the tryptophan/tyrosine fluorescence of the purified protein showed quenching by 2-deoxy-d-glucose, d-mannose, d-glucose or d-galactose in the presence of sodium ions. This is the first reported purification and characterization of an active glucose transport protein member of the TC 2.1.7 subgroup of the Major Facilitator Superfamily, constituting the route for entry of sugar nutrients into H. pylori. A model is derived of its three-dimensional structure as a paradigm of the family. © 2008 The Authors.

DOI： 10.1111/j.1365-2958.2008.06535.x

[Molecular mechanism of Helicobacter pylori-mediated pathogenesis]. Reviewed

Keigo Shibayama

Nihon saikingaku zasshi. Japanese journal of bacteriology Vol. 63 ( 2-4 ) page： 387 - 90 2008.12

First molecular characterization of group B streptococci with reduced penicillin susceptibility Reviewed International journal

Kouji Kimura, Satowa Suzuki, Jun Ichi Wachino, Hiroshi Kurokawa, Kunikazu Yamane, Naohiro Shibata, Noriyuki Nagano, Haru Kato, Keigo Shibayama, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy Vol. 52 ( 8 ) page： 2890 - 2897 2008.8

Language：English Publishing type：Research paper (scientific journal) Publisher：8

Group B Streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for adults. Penicillins are the drugs of first choice for the treatment of GBS infections, since GBS have been regarded to be uniformly susceptible to penicillins so far. Here we characterize the first strains of GBS with reduced penicillin susceptibility (PRGBS) identified in Japan. Fourteen PRGBS strains were clinically isolated from the sputa of elderly patients from 1995 to 2005; and the MICs of penicillin, oxacillin, and ceftizoxime ranged from 0.25 to 1 μg/ml, 2 to 8 μg/ml, and 4 to 128 μg/ml, respectively. Moreover, some strains were also insusceptible to ampicillin, cefazolin, cefepime, and cefotaxime. All the PRGBS isolates tested possessed a few amino acid substitutions adjacent to the conserved SSN and KSG motifs (amino acids 402 to 404 and 552 to 554, respectively) of PBP 2X, and the amino acid substitutions could be classified into two types, Q557E and V405A. Western blotting analysis of the 14 clinical isolates with anti-PBP 2X-specific serum suggested that the amount of PBP 2X among the 14 PRGBS isolates was reduced, although the 2 ATCC strains produced a significant amount of PBP 2X. The introduction of PRGBS-derived PBP 2X genes into penicillin-susceptible strains through allelic exchange elevated their penicillin insusceptibility, suggesting that these altered PBP 2X genes are responsible for the penicillin insusceptibility in PRGBS strains. In this study, we characterized for the first time PRGBS strains on a molecular basis, although several reports have so far mentioned the existence of β-lactam-insusceptible GBS from a phenotypic standpoint. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/AAC.00185-08

Novel plasmid-mediated 16S rRNA m<sup>1</sup>A1408 methyltransferase, NpmA, found in a clinically isolated Escherichia coli strain resistant to structurally diverse aminoglycosides Reviewed

Jun Ichi Wachino, Keigo Shibayama, Hiroshi Kurokawa, Kouji Kimura, Kunikazu Yamane, Satowa Suzuki, Naohiro Shibata, Yasuyoshi Ike, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy Vol. 51 ( 12 ) page： 4401 - 4409 2007.12

Language：English Publishing type：Research paper (scientific journal) Publisher：12

We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/AAC.00926-07

New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate Reviewed

Kunikazu Yamane, Jun Ichi Wachino, Satowa Suzuki, Kouji Kimura, Naohiro Shibata, Haru Kato, Keigo Shibayama, Toshifumi Konda, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy Vol. 51 ( 9 ) page： 3354 - 3360 2007.9

Language：English Publishing type：Research paper (scientific journal) Publisher：9

Plasmid-mediated Qnr and AAC(6′)-Ib-cr have been recognized as new molecular mechanisms affecting fluoroquinolone (FQ) resistance. C316, an Escherichia coli strain demonstrating resistance to various FQs, was isolated in Japan. Resistance to FQs was augmented in an E. coli CSH2 transconjugant, but PCR failed to detect qnr genes, suggesting the presence of novel plasmid-mediated FQ resistance mechanisms. Susceptibility tests, DNA manipulation, and analyses of the gene and its product were performed to characterize the genetic determinant. A novel FQ-resistant gene, qepA, was identified in a plasmid, pHPA, of E. coli C316, and both qepA and rmtB genes were mediated by a probable transposable element flanked by two copies of IS26. Levels of resistance to norfloxacin, ciprofloxacin, and enrofloxacin were significantly elevated in E. coli transformants harboring qepA under AcrB-TolC-deficient conditions. QepA showed considerable similarities to transporters belonging to the 14-transmembrane-segment family of environmental actinomycetes. The effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on accumulation of norfloxacin was assayed in a qepA-harboring E. coli transformant. The intracellular accumulation of norfloxacin was decreased in a qepA-expressing E. coli transformant, but this phenomenon was canceled by CCCP. The augmented FQ resistance level acquired by the probable intergeneric transfer of a gene encoding a major facilitator superfamily-type efflux pump from some environmental microbes to E. coli was first identified. Surveillance of the qepA-harboring clinical isolates should be encouraged to minimize further dissemination of the kind of plasmid-dependent FQ resistance determinants among pathogenic microbes. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/AAC.00339-07

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