Faculty Profiles - SHIBAYAMA Keigo

Quantification of two variant strains contained in freeze-dried Japanese BCG vaccine preparation by real-time PCR Reviewed

Keigo Shibayama, Keiko Mochida, Tetsuya Yagi, Shigetaro Mori, Yoshichika Arakawa, Saburo Yamamoto

Biologicals Vol. 35 ( 2 ) page： 139 - 143 2007.4

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

Japanese bacillus Calmette-Guerin (BCG) vaccine preparation contains two types of variant strains, Type I, which has a 22-base-pair deletion in the RD16 region, and Type II, which has an identical sequence to those of other BCG strains. In this study, we established a method to quantify the percentage of variant strain Type II contained in freeze-dried BCG product with real-time PCR. With this method we examined the master seed lot Tokyo 172, two secondary seed lots, Tokyo 172-1 and Tokyo 172-2, which were produced from Tokyo 172, and four commercial lots produced form Tokyo 172-1. Tokyo 172, Tokyo 172-1, and Tokyo 172-2 contained 55.1%, 19.5%, and 3.6% of Type II variant strain, respectively. Commercial lots contained 1.5%, 4.5%, 7.4%, and 4.3% of Type II variant strain, respectively. These results indicated that the two variant strains contained in the master seed lot continued to coexist in subsequently produced lots with a decrease in population of variant strain Type II. This method would be useful for quality control of commercial Japanese BCG vaccine preparations. © 2006 The International Association for Biologicals.

DOI： 10.1016/j.biologicals.2006.07.005

PCR classification of CTX-M-type β-lactamase genes identified in clinically isolated gram-negative bacilli in Japan Reviewed

Usefulness of adult bovine serum for Helicobacter pylori culture media Reviewed

Keigo Shibayama, Mitsuaki Nagasawa, Takafumi Ando, Masaaki Minami, Jun Ichi Wachino, Satowa Suzuki, Yoshichika Arakawa

Journal of Clinical Microbiology Vol. 44 ( 11 ) page： 4255 - 4257 2006.11

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

Fetal bovine serum (FBS) and adult bovine serum (BS) exhibited bactericidal activity against Helicobacter pylori at various levels, which were higher in BS than in FBS. The bactericidal activity was inactivated by heat treatment at 56°C for 30 min. Our results demonstrated that heat-treated BS is a useful serum source of H. pylori culture medium. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/JCM.00477-06

Naohiro Shibata, Hiroshi Kurokawa, Yohei Doi, Tetsuya Yagi, Kunikazu Yamane, Jun Ichi Wachino, Satowa Suzuki, Kouji Kimura, Satoshi Ishikawa, Haru Kato, Yoshiyuki Ozawa, Keigo Shibayama, Kumiko Kai, Toshifumi Konda, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy Vol. 50 ( 2 ) page： 791 - 795 2006.2

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

Of 1,456 strains isolated from 2001 to 2003 demonstrating resistance to either oxyimino-cephalosporin, 317 strains, isolated in 57 of 132 clinical facilities, were found to harbor bla/CTX-M genes by PCR. Fifty-seven, 161, and 99 strains harbored bla/CTX-M genes belonging to the blaCTX-M-1, bla/CTX-M-2, and bla/CTX-M-9 clusters, respectively. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/AAC.50.2.791-795.2006

Novel plasmid-mediated 16S rRNA methylase, RmtC, found in a Proteus mirabilis isolate demonstrating extraordinary high-level resistance against various aminoglycosides Reviewed

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

Antimicrobial Agents and Chemotherapy Vol. 50 ( 1 ) page： 178 - 184 2006.1

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

Proteus mirabilis ARS68, which demonstrated a very high level of resistance to various aminoglycosides, was isolated in 2003 from an inpatient in Japan. The aminoglycoside resistance of this strain could not be transferred to recipient strains Escherichia coli CSH-2 and E. coli HB101 by a general conjugation experiment, but E. coli DHSα was successfully transformed by electroporation with the plasmid of the parent strain, ARS68, and acquired an unusually high degree of resistance against aminoglycosides. Cloning and sequencing analyses revealed that the presence of a novel 16S rRNA methylase gene, designated rmtC, was responsible for resistance in strain ARS68 and its transformant. The G+C content of rmtC was 41.1%, and the deduced amino acid sequences of the newly identified 16S rRNA methylase, RmtC, shared a relatively low level of identity (£29%) to other plasmid-mediated 16S rRNA methylases, RmtA, RmtB, and ArmA, which have also been identified in pathogenic gram-negative bacilli. Also, RmtC shared a low level of identity (≤28%) with the other 16S rRNA methylases found in aminoglycoside-producing actinomycetes. The purified histidine-tagged RmtC clearly showed niethyltransferase activity against E. coli 16S rRNA in vitro. rmtC was located downstream of an ISEcp1-like element containing trip A. Several plasmid-mediated 16S rRNA methylases have been identified in pathogenic gram-negative bacilli belonging to the family Enterobacteriaceae, and some of them are dispersing worldwide. The acceleration of aminoglycoside resistance among gram-negative bacilli by producing plasmid-mediated 16S rRNA methylases, such as RmtC, RmtB, and RmtA, may indeed become an actual clinical hazard in the near future. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/AAC.50.1.178-184.2006

Pathophysiological role of Helicobacter pylori gamma-glutamyltranspeptidase

Shibayama K, Wachino J, Arakawa Y, Saidijam M, Rutherford N, Henderson P

Helicobacter Vol. 11 ( 4 ) page： 355-356 2006

Active membrane transport and receptor proteins from bacteria Reviewed

M. Saidijam, K. E. Bettaney, G. Szakonyi, G. Psakis, K. Shibayama, S. Suzuki, J. L. Clough, V. Blessie, A. Abu-Bakr, S. Baumberg, J. Meuller, C. K. Hoyle, S. L. Palmer, P. Butaye, K. Walravens, S. G. Patching, J. O'Reilly, N. G. Rutherford, R. M. Bill, D. I. Roper, M. K. Phillips-Jones, P. J.F. Henderson

Biochemical Society Transactions Vol. 33 ( 4 ) page： 867 - 872 2005.8

Language：English Publishing type：Research paper (international conference proceedings)

A general strategy for the expression of bacterial membrane transport and receptor genes in Escherichia coli is described. Expression is amplified so that the encoded proteins comprise 5-35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGSH6 or a Strep tag at the C-terminus. These enable purification in mg quantities for crystallization and NMR studies. Examples of one nutrient uptake and one multidrug extrusion protein from Helicobacter pylori are described. This strategy is successful for membrane proteins from H. pylori, E. coli, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Microbacterium liquefaciens, Brucella abortus, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides, Streptomyces coelicolor and Rhodobacter sphaeroides. ©2005 Biochemical Society.

DOI： 10.1042/BST0330867

Practical methods using boronic acid compounds for identification of class C β-lactamase-producing Klebsiella pneumoniae and Escherichia coli Reviewed

Tetsuya Yagi, Jun Ichi Wachino, Hiroshi Kurokawa, Satowa Suzuki, Kunikazu Yamane, Yohei Doi, Naohiro Shibata, Haru Kato, Keigo Shibayama, Yoshichika Arakawa

Journal of Clinical Microbiology Vol. 43 ( 6 ) page： 2551 - 2558 2005.6

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

Detection of the resistance mediated by class C β-lactamases remains a challenging issue, considering that transferable plasmid-mediated class C β-lactamases are of worldwide concern. Methods for the identification of strains that produce extended-spectrum β-lactamases (ESBLs) or metallo-β-lactamases (MBLs) have been developed and applied for routine use in clinical microbiology laboratories, but no practical methods for identification of plasmid-mediated class C producers have been established to date. We therefore developed three simple methods for clinical microbiology laboratories that allow identification of plasmid-mediated class C β-lactamase-producing bacteria using a boronic acid derivative, 3-aminophenylboronic acid (APB), one of the specific inhibitors of class C β-lactamases. Detection by the disk potentiation test was based on the enlargement of the growth-inhibitory zone diameter (by greater than or equal to 5 mm) around a Kirby-Bauer disk containing a ceftazidime (CAZ) or a cefotaxime (CTX) disk in combination with APB. In a double-disk synergy test, the discernible expansion of the growth-inhibitory zone around the CAZ or the CTX disk toward a disk containing APB was indicative of class C β-lactamase production. A greater than or equal to eightfold decrease in the MIC of CAZ or CTX in the presence of APB was the criterion for detection in the microdilution test. By using these methods, Escherichia coli and Klebsiella pneumoniae isolates producing plasmid-mediated class C β-lactamases, ACT-1, CMY-2, CMY-9, FOX-5, LAT-1, and MOX-1, were successfully distinguished from those producing other classes of β-lactamases, such as ESBLs and MBLs. These methods will provide useful information needed for targeted antimicrobial therapy and better infection control. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/JCM.43.6.2551-2558.2005

Inhibitor-sensitive AmpC β-lactamase variant produced by an Escherichia coli clinical isolate resistant to oxyiminocephalosporins and cephamycins Reviewed

Yohei Doi, Jun Ichi Wachino, Masaji Ishiguro, Hiroshi Kurokawa, Kunikazu Yamane, Naohiro Shibata, Keigo Shibayama, Keiko Yokoyama, Haru Kato, Tetsuya Yagi, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy Vol. 48 ( 7 ) page： 2652 - 2658 2004.7

Language：English Publishing type：Research paper (scientific journal) Publisher：7

Escherichia coli HKY28, a ceftazidime-resistant strain isolated from a urine specimen in Japan, produced an inhibitor-sensitive AmpC β-lactamase variant. The deduced amino acid sequence of the enzyme contained a number of substitutions and a tripeptide deletion (Gly286-Ser287-Asp288) compared with the sequence of native AmpC of E. coli. When the deletion was reverted by a 9-base insertion at the relevant site of ampC in the clone, the typical inhibitor-resistant phenotype of AmpC was restored, while at the same time the levels of resistance to ceftazidime, cefpirome, and cefepime were reduced eightfold or more. Molecular modeling studies indicated that a structural change took place in the H-10 helix as a result of the deletion, and this change caused an alteration of the substrate binding site, leading to a unique phenotype analogous to that of inhibitor-sensitive class A extended-spectrum β-lactamases. The degree of inhibition was greater with sulbactam and tazobactam than with clavulanic acid. To our knowledge, this is the first report to have characterized an E. coli ampC that encodes chromosomal AmpC β-lactamase sensitive to the available β-lactamase inhibitors.

DOI： 10.1128/AAC.48.7.2652-2658.2004

Genetic environments of the rmtA gene in Pseudomonas aeruginosa clinical isolates Reviewed

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

Antimicrobial Agents and Chemotherapy Vol. 48 ( 6 ) page： 2069 - 2074 2004.6

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

Nine Pseudomonas aeruginosa strains showing very high levels of resistance to various aminoglycosides have been isolated from clinical specimens in seven separate Japanese hospitals in five prefectures since 1997. These strains harbor the newly identified 16S rRNA methylase gene (rmtA). When an rmtA gene probe was hybridized with genomic DNAs of the nine strains digested with EcoRI, two distinct patterns were observed. The 11.1- and 15.8-kb regions containing the rmtA genes of strains AR-2 and AR-11, respectively, were sequenced and compared. In strain AR-2, a transposase gene-like sequence (sequence 1) and a probable tRNA ribosyltransferase gene (orfA) were located upstream of rmtA, and a Na+/H+ antiporter gene-like sequence (sequence 2) was identified downstream of rmtA. This 6.2-kbp insert (the rmtA locus) was flanked by 262-bp κγ elements. Part of the orfQ gene adjacent to an inverted repeat was found outside of the rmtA locus. In strain AR-11, the rmtA gene and sequence 2 were found, but the 5′ end of the orfA gene was truncated and replaced with IS6100. An orfQ-orfI region was present on each side of the rmtA gene in strain AR-11. The G+C content of the rmt4 gene was about 55%, and since the newly identified rmtA gene may well be mediated by some mobile genetic elements such as Tn5041, further dissemination of the rmtA gene could become an actual clinical problem in the near future.

DOI： 10.1128/AAC.48.6.2069-2074.2004

Spread of novel aminoglycoside resistance gene aac(6′)-Iad among Acinetobacter clinical isolates in Japan Reviewed

Yohei Doi, Jun Ichi Wachino, Kunikazu Yamane, Naohiro Shibata, Tetsuya Yagi, Keigo Shibayama, Haru Kato, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy Vol. 48 ( 6 ) page： 2075 - 2080 2004.6

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

A novel aminoglycoside resistance gene, aac(6′)-Iad, encoding aminoglycoside 6′-N-acetyltransferase, was identified in Acinetobacter genospecies 3 strain A.51. The gene encoded a 144-amino-acid protein, which shared modest identity (up to 36.7%) with some of the aminoglycoside 6′-N-acetyltransferases. The results of high-pressure liquid chromatography assays confirmed that the protein is a functional aminoglycoside 6′-N-acetyltransferase. The enzyme conferred resistance to amikacin, tobramycin, sisomicin, and isepamicin but not to gentamicin. The prevalence of this gene among Acinetobacter clinical isolates in Japan was then investigated. Of 264 Acinetobacter sp. strains isolated from geographically diverse areas in Japan in 2002, 16 were not susceptible to amikacin, and aac(6′)-Iad was detected in 7. Five of the producers of aminoglycoside 6′ -N-acetyltransferase type Iad were identified as Acinetobacter baumannii, and two were identified as Acinetobacter genospecies 3. These results suggest that aac(6′)-Iad plays a substantial role in amikacin resistance among Acinetobacter spp. in Japan.

DOI： 10.1128/AAC.48.6.2075-2080.2004

[Helicobacter pylori infection and epithelial cell signaling in gastric pathogenesis]. Reviewed

Keigo Shibayama, Yoshichika Arakawa

Nihon saikingaku zasshi. Japanese journal of bacteriology Vol. 59 ( 2 ) page： 415 - 24 2004.5

Plasmid-Mediated 16S rRNA Methylase in Serratia marcescens Conferring High-Level Resistance to Aminoglycosides Reviewed

Yohei Doi, Keiko Yokoyama, Kunikazu Yamane, Jun Ichi Wachino, Naohiro Shibata, Tetsuya Yagi, Keigo Shibayama, Haru Kato, Yoshichika Arakawa

Antimicrobial Agents and Chemotherapy Vol. 48 ( 2 ) page： 491 - 496 2004.2

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

Serratia marcescens S-95, which displayed an unusually high degree of resistance to aminoglycosides, including kanamycins and gentamicins, was isolated in 2002 from a patient in Japan. The resistance was mediated by a large plasmid which was nonconjugative but transferable to an Escherichia coli recipient by transformation. The gene responsible for the aminoglycoside resistance was cloned and sequenced. The deduced amino acid sequence of the resistance gene shared 82% identity with RmtA, which was recently identified as 16S rRNA methylase conferring high-level aminoglycoside resistance in Pseudomonas aeruginosa. Histidine-tagged recombinant protein showed methylation activity against E. coli 16S rRNA. The novel aminoglycoside resistance gene was therefore designated rmtB. The genetic environment of rmtB was further investigated. The sequence immediately upstream of rmtB contained the right end of transposon Tn3, including blaTEM, while an open reading frame possibly encoding a transposase was identified downstream of the gene. This is the first report describing 16S rRNA methylase production in S. marcescens. The aminoglycoside resistance mechanism mediated by production of 16S rRNA methylase and subsequent ribosomal protection used to be confined to aminoglycoside-producing actinomycetes. However, it is now identified among pathogenic bacteria, including Enterobacteriaceae and P. aeruginosa in Japan. This is a cause for concern since other treatment options are often limited in patients requiring highly potent aminoglycosides such as amikacin and tobramycin.

DOI： 10.1128/AAC.48.2.491-496.2004

Growth competition of macrolide-resistant and -susceptible Helicobacter pylori strains Reviewed

Kyoto Kanai, Keigo Shibayama, Satowa Suzuki, Jun Ichi Wachino, Yoshichika Arakawa

Microbiology and Immunology Vol. 48 ( 12 ) page： 977 - 980 2004

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

We examined population dynamics in a mixed culture of clonally related macrolide-resistant and -susceptible Helicobacter pylori strains isolated from a single patient. The resistant strain had a macrolide resistance-conferring A2143G mutation in the 23S rRNA gene. The growth rate of these two strains did not apparently differ when cultured separately. On the other hand, by conducting sequential passage of a mixed culture of the resistant and the susceptible strains, the ratio of the resistant strain to the susceptible strain in the culture typically decreased per passage, indicating that the resistance imposed a significant disadvantage on bacterial fitness in the population.

DOI： 10.1111/j.1348-0421.2004.tb03628.x

A novel apoptosis-inducing protein from Helicobacter pylori Reviewed

Shibayama K, Kamachi K, Nagata N, Yagi T, Nada T, Doi YH, Shibata N, Yokoyama K, Yamane K, Kato H, Iinuma Y, Arakawa Y

MOLECULAR MICROBIOLOGY Vol. 47 ( 2 ) page： 443 - 451 2003.1

A novel apoptosis-inducing protein from Helicobacter pylori.

Shibayama K, Kamachi K, Yagi T, Yamane K, Doi Y, Shibata N, Kato H, Arakawa, Y

Helicobacter Vol. 8 ( 4 ) page： 354 2003

Apoptotic signaling pathway activated by Helicobacter pylori infection and increase of apoptosis-inducing activity under serum-starved conditions Reviewed

Shibayama K, Doi Y, Shibata N, Yagi T, Nada T, Iinuma Y, Arakawa Y

INFECTION AND IMMUNITY Vol. 69 ( 5 ) page： 3181 - 3189 2001.5

Apoptotic signaling pathway activated by Helicobacter pylori infection and increase of apoptosis-inducing activity under serum-starved conditions Reviewed

K. Shibayama, Y. Doi, N. Shibata, T. Yagi, T. Nada, Y. Iinuma, Y. Arakawa

Infection and Immunity Vol. 69 ( 5 ) page： 3181 - 3189 2001.5

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

The enhanced gastric epithelial cell apoptosis observed during infection with Helicobacter pylori has been suggested to be of significance in the etiology of gastritis, peptic ulcers, and neoplasia. To investigate the cell death signaling induced by H. pylori infection, human gastric epithelial cells were incubated with H. pylori for up to 72 h. H. pylori infection induced the activation of caspase -8, -9, and -3 and the expression of the proapoptotic Bcl-2 family proteins Bad and Bid. The peak of the activity of the caspases occurred at 24 h. At this time, the inhibition of caspase-8 or -9 almost completely suppressed H. pylori-induced apoptosis. Inhibition of caspase-8 suppressed the expression of Bad and Bid and the subsequent activation of caspase-9 and -3. These observations indicate that H. pylori induces apoptosis through a pathway involving the sequential induction of apical caspase-8 activity, the proapoptotic proteins Bad and Bid, caspase-9 activity, and effector caspase-3 activity. Activation of the pathway was independent of CagA or vacuolating toxin. A membrane fraction of H. pylori was sufficient to activate this pathway, and treatment with proteinase K eliminated the activity. Apoptotic activity of the membrane fraction was significantly increased by incubating the bacteria under serum-starved conditions for 24 h. These observations suggest that environmental conditions in the human stomach could induce H. pylori-mediated pathogenesis, leading to a variety of clinical outcomes.

DOI： 10.1128/IAI.69.5.3181-3189.2001