Faculty Profiles - SHIBAYAMA Keigo

Growth Competition of Macrolide-Resistant and -Susceptible Helicobacter pylori Strains Reviewed

[H. pylori infection and epithelial cell apoptosis]. Invited

Keigo Shibayama, Yoshichika Arakawa

Nihon rinsho. Japanese journal of clinical medicine Vol. 63 Suppl 11 page： 156 - 60 2005.11

H. pylori infection and epithelial cell apoptosis Invited

Shibayama K., Arakawa Y.

Nippon rinsho. Japanese journal of clinical medicine Vol. 63 Suppl 11 page： 156 - 160 2005.11

H.pylori感染胃上皮細胞におけるアポトーシス

柴山恵吾

日本臨床 (in press) 2005

Helicobacter pyloriにおけるアモキシシリン耐性について

柴山恵吾

日本ヘリコバクター学会雑誌 (in press) 2005

KANAI Kyoko, SHIBAYAMA Keigo, SUZUKI Satowa, WACHINO Jun-ichi, ARAKAWA Yoshichika

Microbiology and Immunology Vol. 48 ( 12 ) page： 977 - 980 2004.12

Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa. Reviewed International journal

Keiko Yokoyama, Yohei Doi, Kunikazu Yamane, Hiroshi Kurokawa, Naohiro Shibata, Keigo Shibayama, Tetsuya Yagi, Haru Kato, Yoshichika Arakawa

Lancet (London, England) Vol. 362 ( 9399 ) page： 1888 - 93 2003.12

Language：English Publishing type：Research paper (scientific journal) Publisher：9399

BACKGROUND: Bacteria develop resistance to aminoglycosides by producing aminoglycoside-modifying enzymes such as acetyltransferase, phosphorylase, and adenyltransferase. These enzymes, however, cannot confer consistent resistance to various aminoglycosides because of their substrate specificity. Notwithstanding, a Pseudomonas aeruginosa strain AR-2 showing high-level resistance (minimum inhibitory concentration >1024 mg/L) to various aminoglycosides was isolated clinically. We aimed to clone and characterise the genetic determinant of this resistance. METHODS: We used conventional methods for DNA manipulation, susceptibility testing, and gene analyses to clone and characterise the genetic determinant of the resistance seen. PCR detection of the gene was also done on a stock of P aeruginosa strains that were isolated clinically since 1997. FINDINGS: An aminoglycoside-resistance gene, designated rmtA, was identified in P aeruginosa AR-2. The Escherichia coli transformant and transconjugant harbouring the rmtA gene showed very high-level resistance to various aminoglycosides, including amikacin, tobramycin, isepamicin, arbekacin, kanamycin, and gentamicin. The 756-bp nucleotide rmtA gene encoded a protein, RmtA. This protein showed considerable similarity to the 16S rRNA methylases of aminoglycoside-producing actinomycetes, which protect bacterial 16S rRNA from intrinsic aminoglycosides by methylation. Incorporation of radiolabelled methyl groups into the 30S ribosome was detected in the presence of RmtA. Of 1113 clinically isolated P aeruginosa strains, nine carried the rmtA gene, as shown by PCR analyses. INTERPRETATION: Our findings strongly suggest intergeneric lateral gene transfer of 16S rRNA methylase gene from some aminoglycoside-producing microorganisms to P aeruginosa. Further dissemination of the rmtA gene in nosocomial bacteria could be a matter of concern in the future.

DOI： 10.1016/S0140-6736(03)14959-8

PCR Typing of Genetic Determinants for Metallo-β-Lactamases and Integrases Carried by Gram-Negative Bacteria Isolated in Japan, with Focus on the Class 3 Integron Reviewed

Naohiro Shibata, Yohei Doi, Kunikazu Yamane, Tetsuya Yagi, Hiroshi Kurokawa, Keigo Shibayama, Haru Kato, Kumiko Kai, Yoshichika Arakawa

Journal of Clinical Microbiology Vol. 41 ( 12 ) page： 5407 - 13 2003.12

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

From January 2001 to December 2002, 587 strains of gram-negative bacterial isolates demonstrating resistance to ceftazidime and a combination of sulbactam and cefoperazone were subjected to a disk diffusion screening test using sodium mercaptoacetic acid; 431 strains (73.4%) appeared to produce metallo-β-lactamase (MBL). Of these 431 strains, 357 were found by PCR to carry genes for IMP-1 type MBL (blaIMP-1), while only 7 and 67 strains carried the IMP-2 gene (blaIMP-2) and the VIM-2 gene (blaVIM-2), respectively. Neither VIM-1 nor SPM-1 type MBL genes were found among the strains tested. Of 431 strains, 427 carried the intI1 gene, and 4 strains carrying both the intI1 and intI3 genes were reidentified as Pseudomonas putida harboring blaIMP-1. Of these four P. putida strains, three strains and one strain, respectively, were separately isolated from two hospitals located in the same prefecture, and the three strains showed very similar pulsed-field gel electrophoresis patterns. Of 357 bla IMP-1 carriers, 116, 53, 51, 47, and 30 strains were identified as Pseudomonas aeruginosa, Alcaligenes xylosoxidans, P. putida/fluorescens, Serratia marcescens, and Acinetobacter baumannii, respectively. Four strains carrying blaIMP-2 were reidentified as P. putida. Sixty-three P. aeruginosa strains and four P. putida strains carried blaVIM-2. Of 427 intI1-positive strains, 180, 53, 51, 47, and 35 were identified as P. aeruginosa, A. xylosoxidans, P. putida/fluorescens, S. marcescens, and A. baumannii, respectively. In the present study, it was confirmed that strains carrying blaIMP-1 with a class 1 integron are the most prevalent type in Japan, although several intI3 carriers have also been identified sporadically in this country.

DOI： 10.1128/JCM.41.12.5407-5413.2003

A new TEM-derived extended-spectrum β-lactamase (TEM-91) with an R164C substitution at the ω-loop confers ceftazidime resistance Reviewed

Hiroshi Kurokawa, Naohiro Shibata, Yohei Doi, Keigo Shibayama, Kazunari Kamachi, Tetsuya Yagi, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy Vol. 47 ( 9 ) page： 2981 - 3 2003.9

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

A new plasmid-mediated TEM-derived extended-spectrum β-lactamase, TEM-91, was identified in a ceftazidime-resistant (MIC, >128 μg per ml) Escherichia coli strain isolated in 1996 in Japan. TEM-91 has three amino acid substitutions, R164C, M184T, and E240K, compared with TEM-1 penicillinase. The isoelectric point (pI), Km, and kcat of TEM-91 for ceftazidime were 5.7, 179 μM, and 29.0 s-1, respectively. The Ki of clavulanic acid for ceftazidime hydrolysis was 30.3 nM.

DOI： 10.1128/AAC.47.9.2981-2983.2003

A novel apoptosis-inducing protein from Helicobacter pylori Reviewed

Keigo Shibayama, Kazunari Kamachi, Noriyo Nagata, Tetsuya Yagi, Toshi Nada, Yohei Doi, Naohiro Shibata, Keiko Yokoyama, Kunikazu Yamane, Haru Kato, Yoshitsugu Iinuma, Yoshichika Arakawa

Molecular Microbiology Vol. 47 ( 2 ) page： 443 - 51 2003.1

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

Helicobacter pylori infection induces apoptosis in gastric epithelial cells. Here, we report a novel apoptosis-inducing protein that functions as a leading factor in H. pylori-mediated apoptosis induction. We purified the protein from H. pylori by separating fractions that showed apoptosis-inducing activity. This protein induced apoptosis of AGS cells in a dose-dependent manner. The purified protein consisted of two protein fragments with molecular masses of about 40 and 22 kDa, which combined to constitute a single complex in their natural form. N-terminal sequencing indicated that both these protein fragments were encoded by the HP1118 gene. The purified protein exhibited γ-glutamyl transpeptidase activity, the inhibition of which by 6-diazo-5-oxo-L-norleucine resulted in a complete loss of apoptosis-inducing activity. To the best of our knowledge, the apoptosis-inducing function is a newly identified physiological role for bacterial γ-glutamyl transpeptidase. The apoptosis-inducing activity of the isogenic mutant γ-glutamyl transpeptidase-deficient strain was significantly lower compared with that of the parent strain, demonstrating that γ-glutamyl transpeptidase plays a significant role in H. pylori-mediated apoptosis. Our findings provide new insights into H. pylori pathogenicity and reveal a novel aspect of the bacterial γ-glutamyl transpeptidase function.

DOI： 10.1046/j.1365-2958.2003.03305.x

Characterization of a novel plasmid-mediated cephalosporinase (CMY-9) and its genetic environment in an Escherichia coli clinical isolate Reviewed

Yohei Doi, Naohiro Shibata, Keigo Shibayama, Kazunari Kamachi, Hiroshi Kurokawa, Keiko Yokoyama, Tetsuya Yagi, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy Vol. 46 ( 8 ) page： 2427 - 34 2002.8

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

An Escherichia coli strain, HKYM68, which showed resistance to broad-spectrum cephalosporins was isolated from a sputum specimen in Japan. The high-level resistance of the strain to ceftazidime, cefpirome, and moxalactam was carried by a self-transferable plasmid. The β-lactamase gene responsible for the resistance was cloned and sequenced. The deduced amino acid sequence of this gene product, CMY-9, had a single amino acid substitution (E85D), the residue reported to be part of the recognition site for the R1 side chain of β-lactams, compared with the amino acid sequence of CMY-8 and also had 78% identity with the amino acid sequence of CepH, a chromosomal cephalosporinase of Aeromonas hydrophila. A sull-type class 1 integron containing an aacA1-orfG gene cassette was identified upstream of blaCMY-9 and ended with a truncated 3′ conserved segment. The following 2.1 kb was almost identical to the common region of integrons In6 and In7 and the integron of pSAL-1, except that orf513 encoding a putative transposase was identified instead of orf341 due to addition of a single nucleotide. blaCMY-9 was closely located downstream of the end of the common region. These observations are indicative of the exogenous derivation of blaCMY-9 from some environmental microorganisms such as aeromonads.

DOI： 10.1128/AAC.46.8.2427-2434.2002

Trends in antimicrobial-drug resistance in Japan Reviewed

Yoshichika Arakawa, Yasuyoshi Ike, Mitsuaki Nagasawa, Naohiro Shibata, Yohei Doi, Keigo Shibayama, Tetsuya Yagi, Takeshi Kurata

Emerging Infectious Diseases Vol. 6 ( 6 ) page： 572 - 575 2000

Language：Japanese Publishing type：Research paper (scientific journal)

Multidrug resistance in gram-positive bacteria has become common worldwide. In Japan until recently, gram-negative bacteria such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Serratia marcescens were controlled by carbapenems, fluoroquinolones, and aminoglycosides. However, several of these microorganisms have recently developed resistance against many antimicrobial drugs.

DOI： 10.3201/eid0606.000604

β-Lactamase inhibitors have antibacterial activities against Helicobacter pylori Reviewed

HORII Toshinobu, KIMURA Taku, SATO-KAWAMURA Kumiko, NADA Tochi, SHIBAYAMA Keigo, OHTA Michio

J Infect Chemother. Vol. 5 ( 4 ) page： 206 - 207 1999.12

β-Lactamase inhibitors have antibacterial activities against Helicobacter pylori Reviewed

Horii T., Kimura T., Sato-Kawamura K., Nada T., Shibayama K., Ohta M.

Journal of Infection and Chemotherapy Vol. 5 ( 4 ) page： 206 - 207 1999.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Infection and Chemotherapy

Recently, it was reported that amoxicillin-clavulanate has slightly higher activity than amoxicillin against Helicobacter pylori. In this study, we evaluated the in-vitro antibacterial activity of β-lactamase inhibitors against H. pylori. We investigated the susceptibility of 30 H. pylori strains to β-lactamase inhibitors, including clavulanate, sulbactam, and tazobactam. In short-term bactericidal studies, a clinical isolate of H. pylori NU27 was exposed to 1 x minimum inhibitory concentration (MIC) of the β-lactamase inhibitors, amoxicillin, clarithromycin, and amoxicillin-clavulanate for 3 and 6h. The MICs90 for these β-lactamase inhibitors were 2, 4, and 2 mg/l, respectively. The short-term bactericidal studies showed that these β- lactamase inhibitors decreased viable counts of H. pylori during 6-h exposure at 1 x MIC. Our results suggest that β-lactamase inhibitors have in-vitro antibacterial activity against H. pylori. Amoxicillin and clavulanate used in combination resulted in increased antibacterial activity.

DOI： 10.1007/s101560050036

Role of multiple efflux pumps in Escherichia coli in indole expulsion Reviewed

Kumiko Kawamura-Sato, Keigo Shibayama, Toshinobu Horii, Yoshitsugu Iimuma, Yoshichika Arakawa, Michio Ohta

FEMS Microbiology Letters Vol. 179 ( 2 ) page： 345 - 352 1999.10

Language：Japanese Publishing type：Research paper (scientific journal)

Escherichia coli chromosome encodes several multidrug transporters. Despite their protective function against antibacterial agents, the specific physiological actions of these transporters are not fully understood. E. coli produces indole, a metabolite of tryptophan, under physiological conditions. Defined inactivation of the acrEF gene, the product of which is known as an energy-dependent multiple drug efflux pump, decreased indole excretion while reintroduction of the acrEF gene restored it. A ΔacrEF mutant accumulated more intracellular indole than the parent. This mutant was more susceptible to the growth-inhibitory effect of indole than the parent. These results indicate that the AcrEF system plays a significant role in indole efflux. Copyright (C) 1999 Federation of European Microbiological Societies.

DOI： 10.1016/S0378-1097(99)00433-4

Are Autoantibodies against Lewis Antigens Involved in the Pathogenesis of Helicobacter pylori-Induced Peptic Ulcers? Reviewed

KAMIYA Kenji, ARISAWA Tomiyasu, GOTO Hidemi, SHIBAYAMA Keigo, HORII Toshinobu, HAYAKAWA Tetsuo, OHTA Michio

MICROBIOLOGY and IMMUNOLOGY Vol. 43 ( 5 ) page： 403 - 408 1999.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Center For Academic Publications Japan

We examined whether anti-Lewis x (Lex) and y (Ley) autoantibodies affect the pathogenesis of Helicobacter pylori-induced peptic ulcers. Of 11 patients with peptic ulcers, 10 patients had both anti-Lex and -Ley immunoglobulin G (IgG) antibodies, and 1 patient had only anti-Lex antibody. After successful eradication, we measured the serum titer of anti-Lex and -Ley antibodies. Six patients had a reduction of the titers of anti-Lex and/or -Ley antibodies, whereas no notable changes were detected in 5 patients in the follow-up. This result suggests that anti-Lex and -Ley autoantibodies had no critical role in the development of H. pylori-induced peptic ulcer.

CiNii Research

Four critical aspartic acid residues potentially involved in the catalytic mechanism of Escherichia coli K-12 WaaR Reviewed

Shibayama K., Ohsuka S., Sato K., Yokoyama K., Horii T., Ohta M.

FEMS Microbiology Letters Vol. 174 ( 1 ) page： 105 - 109 1999.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：FEMS Microbiology Letters

Escherichia coli K-12 WaaR is a non-processive α-1,2 glucosyltransferase, involved in the synthesis of the R-core of lipopolysaccharide. WaaR possesses the four conserved structural regions I, II, III and IV, each presumably involved in the mechanistic function in catalysis. Regions I and III contain the pair of strictly conserved Asp residues. Asp-129, 131 (region I) and 215, 217 (region III) of WaaR were individually converted to Asn by the site-directed mutagenesis of the waaR gene. All mutated enzymes were inactive, supporting the model for an α-glycosyl transfer reaction where the pair of strictly conserved aspartic acid residues in regions I and III play a critical role in the catalytic function. Copyright (C) 1999 Federation of European Microbiological Societies.

DOI： 10.1016/S0378-1097(99)00127-5