Papers - SHIBAYAMA Keigo

Evaluation of a Double-Disk Synergy Test with a Common Metallo-beta-Lactamase Inhibitor, Mercaptoacetate, for Detecting NDM-1-Producing Enterobacteriaceae and Acinetobacter baumannii Reviewed Open Access

Jun-ichi Wachino, Mari Matsui, Hoang Huy Tran, Masato Suzuki, Satowa Suzuki, Keigo Shibayama

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 67 ( 1 ) page： 66 - 68   2014.1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL INST INFECTIOUS DISEASES  

DOI： 10.7883/yoken.67.66

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Biochemical characterization of quinolinic acid phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv and inhibition of its activity by pyrazinamide. Reviewed International journal Open Access

Hyun Kim, Keigo Shibayama, Emiko Rimbara, Shigetarou Mori

PloS one   Vol. 9 ( 6 ) page： e100062   2014

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Quinolinic acid phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) is a key enzyme in the de novo pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis and a target for the development of new anti-tuberculosis drugs. QAPRTase catalyzes the synthesis of nicotinic acid mononucleotide from quinolinic acid (QA) and 5-phosphoribosyl-1-pyrophosphate (PRPP) through a phosphoribosyl transfer reaction followed by decarboxylation. The crystal structure of QAPRTase from Mycobacterium tuberculosis H37Rv (MtQAPRTase) has been determined; however, a detailed functional analysis of MtQAPRTase has not been published. Here, we analyzed the enzymatic activities of MtQAPRTase and determined the effect on catalysis of the anti-tuberculosis drug pyrazinamide (PZA). The optimum temperature and pH for MtQAPRTase activity were 60°C and pH 9.2. MtQAPRTase required bivalent metal ions and its activity was highest in the presence of Mg2+. Kinetic analyses revealed that the Km values for QA and PRPP were 0.08 and 0.39 mM, respectively, and the kcat values for QA and PRPP were 0.12 and 0.14 [s-1], respectively. When the amino acid residues of MtQAPRTase, which may interact with QA, were substituted with alanine residues, catalytic activity was undetectable. Further, PZA, which is an anti-tuberculosis drug and a structural analog of QA, markedly inhibited the catalytic activity of MtQAPRTase. The structure of PZA may provide the basis for the design of new inhibitors of MtQAPRTase. These findings provide new insights into the catalytic properties of MtQAPRTase.

DOI： 10.1371/journal.pone.0100062

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Effect of antivenom therapy of Rhabdophis tigrinus (Yamakagashi snake) bites. Reviewed International journal Open Access

Toru Hifumi, Atsushi Sakai, Akihiko Yamamoto, Masahiro Murakawa, Manabu Ato, Keigo Shibayama, Hiroshi Kato, Yuichi Koido, Junichi Inoue, Yuko Abe, Kenya Kawakita, Masanobu Hagiike, Akihiko Ginnaga, Yasuhiro Kuroda

Journal of intensive care   Vol. 2 ( 1 ) page： 44 - 44   2014

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BACKGROUND: Rhabdophis tigrinus (Yamakagashi snake) is a rear-fanged colubrid snake present throughout Russia and Asia. Its venom induces life-threatening hemorrhagic symptoms and severe disseminated intravascular coagulation with a fibrinolytic phenotype. R. tigrinus antivenom manufactured by the immunization of horses to neutralize the venom has the risk of adverse events such as anaphylaxis and serum sickness disease. It should be used when benefit is greater than the risk of adverse effects; however, its efficacy has not been well evaluated. Although our previous survey of nine cases demonstrated that seven of all cases treated with antivenom survived, the clinical characteristics and prognosis without antivenom administration remained unclear. We assumed that R. tigrinus antivenom administration overlaps self-recovery with supportive care. We aimed to determine the association between antivenom administration and outcome with further analyzed cases. METHODS: We retrospectively reviewed the records of the Japan Snake Institute between January 1, 1973 and December 31, 2013. Antivenom and without antivenom groups were compared with regard to baseline demographic features, treatment-related factors, and outcomes. RESULTS: In total, 34 patients were analyzed (97% male, median age 37.5 years). Twenty-five patients were further examined from our previous study. On admission, the median levels of fibrinogen and fibrinogen degradation products were 35 mg/dL and 200 μg/mL, respectively, and platelet counts were 107,000/mm(3). The median disseminated intravascular coagulation score (defined by the Japanese Association of Acute Medicine) was 5. Antivenom was administered to 19 patients, with a median interval of 32 h between bite and antivenom administration. The in-hospital mortality rate was 12%. In univariate analysis, baseline characteristics and laboratory data were not significantly different between the antivenom and without antivenom groups. Hospital mortality in the antivenom group was significantly better than that in the without antivenom group (0% vs. 26.7%, P = 0.03). Moreover, the number of patients developing renal failure requiring hemodialysis was significantly lower in the antivenom group (5.3% vs. 40.0%, P = 0.03). CONCLUSIONS: In our small retrospective study, antivenom administration was likely to be effective in the management of R. tigrinus bites. Apparently, further research is required to confirm its efficacy.

DOI： 10.1186/s40560-014-0044-5

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Clinical characteristics of yamakagashi (Rhabdophis tigrinus) bites: a national survey in Japan, 2000-2013. Reviewed International journal Open Access

Toru Hifumi, Atsushi Sakai, Akihiko Yamamoto, Masahiro Murakawa, Manabu Ato, Keigo Shibayama, Akihiko Ginnaga, Hiroshi Kato, Yuichi Koido, Junichi Inoue, Yuko Abe, Kenya Kawakita, Masanobu Hagiike, Yasuhiro Kuroda

Journal of intensive care   Vol. 2 ( 1 ) page： 19 - 19   2014

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BACKGROUND: Yamakagashi (Rhabdophis tigrinus) is a species of pit viper present throughout Russia and Eastern Asia. Although R. tigrinus venom is known to induce life-threatening hemorrhagic symptoms, the clinical characteristics and effective treatment of R. tigrinus bites remain unknown. The present study aimed to clarify these issues. METHODS: Records in the Japan Snake Institute between 2000 and 2013 were retrospectively investigated. The following were determined: patient characteristics, coagulation and fibrinolytic system abnormalities, effect of antivenom treatment, and outcomes. RESULTS: Nine patients (all males; median age, 38 years) with R. tigrinus bites were identified. On admission, the median levels of fibrinogen and fibrinogen degradation products, and platelet counts were 50 mg/dL, 295 μg/mL, and 107,000/mm(3), respectively. The median (minimum-maximum) disseminated intravascular coagulation (DIC) score defined by the Japanese Association of Acute Medicine was 8 (1-8). Antivenom was administered to seven patients, with a median interval of 35 h between bite and antivenom administration. All patients treated with antivenom survived, and the in-hospital mortality rate was 11%. CONCLUSIONS: Patients with R. tigrinus bites presented with DIC of a fibrinolytic phenotype, which can result in life-threatening injury unless appropriate antivenom and DIC treatment are provided.

DOI： 10.1186/2052-0492-2-19

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Clinical characteristics of redback spider bites. Reviewed International journal Open Access

Toru Hifumi, Satoshi Fujimi, Takuya Yamagishi, Satoru Arai, Kyoko Sawabe, Akihiko Yamamoto, Manabu Ato, Keigo Shibayama, Akihiko Ginnaga, Nobuaki Kiriu, Hiroshi Kato, Yuichi Koido, Junichi Inoue, Masanobu Kishikawa, Yuko Abe, Kenya Kawakita, Masanobu Hagiike, Yasuhiro Kuroda

Journal of intensive care   Vol. 2 ( 1 ) page： 62 - 62   2014

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BACKGROUND: Redback spiders (Latrodectus hasselti) (RBSs) are venomous spiders that have recently spread to Asia from Australia. Since the first case report in 1997 (Osaka), RBS bites have been a clinical and administrative issue in Japan; however, the clinical characteristics and effective treatment of RBS bites, particularly outside Australia remains unclear. This study aimed to elucidate the clinical characteristics of RBS bites and to clarify the effectiveness of the administration of antivenom for treatment. METHODS: We performed a retrospective questionnaire survey from January 2009 to December 2013 to determine the following: patient characteristics, effect of antivenom treatment, and outcomes. To clarify the characteristics of patients who develop systemic symptoms, we compared patients with localized symptoms and those with systemic symptoms. We also examined the efficacy and adverse effects in cases administered antivenom. RESULTS: Over the 5-year study period, 28 patients were identified from 10 hospitals. Of these, 39.3% were male and the median age was 32 years. Bites most commonly occurred on the hand, followed by the forearm. Over 80% of patients developed local pain and erythema, and 35.7% (10 patients) developed systemic symptoms. Baseline characteristics, vital signs, laboratory data, treatment-related factors, and outcome were not significantly different between the localized and systemic symptoms groups. Six patients with systemic symptoms received antivenom, of whom four experienced symptom relief following antivenom administration. Premedication with an antihistamine or epinephrine to prevent the adverse effects of antivenom was administered in four patients, which resulted in no anaphylaxis. One out of two patients who did not receive premedication developed a mild allergic reaction after antivenom administration that subsided without treatment. CONCLUSIONS: Approximately one third of cases developed systemic symptoms, and antivenom was administered effectively and safely in severe cases. Further research is required to identify clinically applicable indications for antivenom use.

DOI： 10.1186/s40560-014-0062-3

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Rhabdophis tigrinus is not a pit viper but its bites result in venom-induced consumptive coagulopathy similar to many viper bites. Reviewed International journal Open Access

Anjana Silva, Toru Hifumi, Atsushi Sakai, Akihiko Yamamoto, Masahiro Murakawa, Manabu Ato, Keigo Shibayama, Akihiko Ginnaga, Hiroshi Kato, Yuichi Koido, Junichi Inoue, Yuko Abe, Kenya Kawakita, Masanobu Hagiike, Yasuhiro Kuroda

Journal of intensive care   Vol. 2 ( 1 ) page： 43 - 43   2014

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As a response to the recent article by Hifumi et al. published in the Journal of Intensive Care, the present correspondence clarifies the family-level taxonomy of the yamakagashi (Rhabdophis tigrinus). Further, the relevance of the term 'venom-induced consumptive coagulopathy,' instead of disseminated intravascular coagulation, in describing the procoagulant coagulopathy of R. tigrinus is highlighted.

DOI： 10.1186/s40560-014-0043-6

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Isolation of Genetically Indistinguishable Carbapenem-Resistant and -Susceptible Acinetobacter baumannii Isolates from a Single Patient Reviewed Open Access

Mari Matsui, Keigo Shibayama, Yasuhiro Tsuji, Hidetoshi Kamimura, Yoshiharu Karube, Mayumi Yoshida, Yoko Masuda, Yoichi Hiraki, Kazutaka Takaki, Fumio Kawano

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY   Vol. 57 ( 11 ) page： 5781 - 5782   2013.11

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

DOI： 10.1128/AAC.01631-13

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Rapid and Reliable Loop-Mediated Isothermal Amplification Method for Detecting Streptococcus agalactiae Reviewed Open Access

Kouji Kimura, Hideji Yanagisawa, Jun-ichi Wachino, Keigo Shibayama, Yoshichika Arakawa

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 66 ( 6 ) page： 546 - 548   2013.11

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Streptococcus agalactiae (group B Streptococcus, GBS) is the leading cause of neonatal sepsis and meningitis and an important pathogen in elderly patients and those with underlying diseases. The diagnosis of GBS infections is primarily based on culture of GBS. Some clinical laboratories perform the Christie-Atkins-Munch-Peterson (CAMP) test for discrimination of GBS from other streptococci. Here, we developed a rapid GBS identification method, i.e., the loop-mediated isothermal amplification (LAMP) method for detecting the cfb gene encoding the CAMP factor. This method detected at least 4 copies of the cfb gene in GBS under isothermal conditions with in a short time (65 degrees C, within 90 min). No inappropriate amplification of nucleotide by this method was observed when the chromosomal DNA of 17 streptococci and enterococci species, other than GBS, were used as templates. In this investigation, we successfully developed a LAMP method for rapid and highly sensitive detection of GBS, which provides a beneficial alternative to the conventional CAMP test.

DOI： 10.7883/yoken.66.546

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Rapid and Reliable Loop-Mediated Isothermal Amplification Method for Detecting Streptococcus agalactiae Reviewed Open Access

Kimura Kouji, Yanagisawa Hideji, Wachino Jun-ichi, Shibayama Keigo, Arakawa Yoshichika

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 66 ( 6 ) page： 546 - 548   2013.11

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DOI： 10.7883/yoken.66.546

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Genome Sequence of a Strain of the Human Pathogenic Bacterium Pseudomonas alcaligenes That Caused Bloodstream Infection. Reviewed International journal

Masato Suzuki, Satowa Suzuki, Mari Matsui, Yoichi Hiraki, Fumio Kawano, Keigo Shibayama

Genome announcements   Vol. 1 ( 5 )   2013.10

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Pseudomonas alcaligenes, a Gram-negative aerobic bacterium, is a rare opportunistic human pathogen. Here, we report the whole-genome sequence of P. alcaligenes strain MRY13-0052, which was isolated from a bloodstream infection in a medical institution in Japan and is resistant to antimicrobial agents, including broad-spectrum cephalosporins and monobactams.

DOI： 10.1128/genomeA.00919-13

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Genetic Analysis of Bordetella pertussis Isolates from the 2008-2010 Pertussis Epidemic in Japan Reviewed Open Access

Yusuke Miyaji, Nao Otsuka, Hiromi Toyoizumi-Ajisaka, Keigo Shibayama, Kazunari Kamachi

PLoS ONE   Vol. 8 ( 10 ) page： e77165   2013.10

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A large pertussis epidemic occurred between 2008 and 2010 in Japan. To investigate epidemic strains, we analyzed 33 Bordetella pertussis isolates from the epidemic period by sequencing virulence-associated genes (fim3, ptxP, ptxA, and prn) and performing multilocus variable-number tandem repeat analysis (MLVA), and compared these results with those of 101 isolates from non-epidemic, earlier and later time periods. DNA sequencing of the fim3 allele revealed that the frequency of fim3B was 4.3%, 12.8%, 30.3%, and 5.1% within isolates in 2002-2004, 2005-2007, 2008-2010, and 2011-2012, respectively. The isolation rate of the fim3B strain therefore temporarily increased during the epidemic period 2008-2010. In contrast, the frequencies of the virulence-associated allelic variants, ptxP3, ptxA1, and prn2, increased with time during overall study period, indicating that these variants were not directly involved in the occurrence of the 2008-2010 epidemic. MLVA genotyping in combination with analysis of allele types showed that the prevalence of an MT27d strain temporarily increased in the epidemic period, and that this strain carried virulence-associated allelic variants (fim3B, ptxP3, ptxA1, and prn2) also identified in recent epidemic strains of Australia, Europe, and the US. Phenotypic analyses revealed that the serotype Fim3 strain was predominant (≥87%) during all the periods studied, and that the frequency of adhesion pertactin (Prn) non-expressing B. pertussis decreased by half in the epidemic period. All MT27d strains expressed Prn and Fim3 proteins, suggesting that B. pertussis MT27d strains expressing Prn and Fim3B have the potential to cause large epidemics worldwide. © 2013 Miyaji et al.

DOI： 10.1371/journal.pone.0077165

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Role of γ-glutamyltranspeptidase in the pathogenesis of Helicobacter pylori infection. Reviewed International journal

Emiko Rimbara, Shigetarou Mori, Hyun Kim, Keigo Shibayama

Microbiology and immunology   Vol. 57 ( 10 ) page： 665 - 73   2013.10

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γ-Glutamyltranspeptidase and asparaginase have been shown to play important roles in Helicobacter pylori colonization and cell death induced by H. pylori infection. In this study, the association of γ-glutamyltranspeptidase and asparaginase was elucidated by comparing activities of both deamidases in H. pylori strains from patients with chronic gastritis, gastric and duodenal ulcers, and gastric cancer. γ-Glutamyltranspeptidase activities in H. pylori strains from patients with gastric cancer were significantly higher than in those from patients with chronic gastritis or gastric ulcers. There was a wide range of asparaginase activities in H. pylori strains from patients with gastric cancer and these were not significantly than those from patients with other diseases. To identify the contributions of γ-glutamyltranspeptidase and asparaginase to gastric cell inflammation, human gastric epithelial cells (AGS line) were infected with H. pylori wild-type and knockout strains and inflammatory responses evaluated by induction of interleukin-8 (IL-8). IL-8 response was significantly decreased by knockout of the γ-glutamyltranspeptidase-encoding gene but not by knockout of the asparaginase-encoding gene. Additionally, IL-8 induction by infection with the H. pylori wild-type strain was significantly decreased by adding glutamine during infection. These findings indicate that IL-8 induction caused by γ-glutamyltranspeptidase activity in H. pylori is mainly attributable to depletion of glutamine. These data suggest that γ-glutamyltranspeptidase plays a significant role in the chronic inflammation caused by H. pylori infection.

DOI： 10.1111/1348-0421.12089

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Draft Genome Sequence of Helicobacter fennelliae Strain MRY12-0050, Isolated from a Bacteremia Patient. Reviewed International journal

Emiko Rimbara, Mari Matsui, Shigetarou Mori, Satowa Suzuki, Masato Suzuki, Hyun Kim, Tsuyoshi Sekizuka, Makoto Kuroda, Keigo Shibayama

Genome announcements   Vol. 1 ( 4 )   2013.8

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Helicobacter fennelliae, a human enterohepatic pathogen, causes bacteremia and colitis. We isolated H. fennelliae strain MRY12-0050 from a female patient; this strain was isolated from 2 other patients from the same hospital during the same period, suggesting human-to-human transmission. This is the first report of an H. fennelliae genome sequence.

DOI： 10.1128/genomeA.00512-13

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Rapid discrimination of Acinetobacter baumannii international clone II lineage by pyrosequencing SNP analyses of bla(OXA-51-like) genes Reviewed

Mari Matsui, Satowa Suzuki, Masato Suzuki, Yoshichika Arakawa, Keigo Shibayama

JOURNAL OF MICROBIOLOGICAL METHODS   Vol. 94 ( 2 ) page： 121 - 124   2013.8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We found that Acinetobacter baumannii international clone II generally possesses unique GTA sequence at nucleotide positions 106-108 in the bla(OXA-51-like) genes. We exploited this to develop an easy and rapid method for discrimination of international clone II from other A. baumannii by employing pyrosequencing analyses of single nucleotide polymorphisms. (C) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.mimet.2013.05.014

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Genome Sequences of Multidrug-Resistant Acinetobacter baumannii Strains from Nosocomial Outbreaks in Japan. Reviewed International journal

Masato Suzuki, Mari Matsui, Satowa Suzuki, Emiko Rimbara, Satomi Asai, Hayato Miyachi, Tohru Takata, Yoichi Hiraki, Fumio Kawano, Keigo Shibayama

Genome announcements   Vol. 1 ( 4 )   2013.7

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Acinetobacter baumannii has emerged worldwide as an important nosocomial pathogen in medical institutions. Here, we present the draft genome sequences of A. baumannii strains MRY09-0642, MRY10-0558, and MRY12-0277 that were isolated from nosocomial outbreaks in Japan between 2008 and 2012 and that are resistant to antimicrobial agents, including carbapenems, fluoroquinolones, and aminoglycosides.

DOI： 10.1128/genomeA.00476-13

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Helicobacter cinaedi and Helicobacter fennelliae transmission in a hospital from 2008 to 2012. Reviewed International journal Open Access

Emiko Rimbara, Shigetarou Mori, Hyun Kim, Mari Matsui, Satowa Suzuki, Shunji Takahashi, Satoshi Yamamoto, Masaya Mukai, Keigo Shibayama

Journal of clinical microbiology   Vol. 51 ( 7 ) page： 2439 - 42   2013.7

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Forty-six Helicobacter cinaedi isolates from the same hospital were analyzed by multilocus sequence typing. Most H. cinaedi isolates exhibited clonal complex 9 and were mainly isolated from immunocompromised patients in the same ward. Three Helicobacter fennelliae isolates were obtained from the same ward and exhibited the same pulsed-field gel electrophoresis patterns. All isolates were resistant to clarithromycin and ciprofloxacin. H. cinaedi and H. fennelliae must be carefully monitored to prevent nosocomial infection.

DOI： 10.1128/JCM.01035-13

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High cephalosporin resistance due to amino acid substitutions in PBP1A and PBP2X in a clinical isolate of group B Streptococcus Reviewed

Kouji Kimura, Jun-ichi Wachino, Hiroshi Kurokawa, Mari Matsui, Satowa Suzuki, Kunikazu Yamane, Noriyuki Nagano, Keigo Shibayama, Yoshichika Arakawa

JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY   Vol. 68 ( 7 ) page： 1533 - 1536   2013.7

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Group B Streptococcus (GBS; Streptococcus agalactiae) has been regarded as uniformly susceptible to penicillins. However, we recently reported the existence of GBS with reduced penicillin susceptibility (PRGBS), with amino acid substitutions in penicillin-binding protein (PBP) 2X. Although most PRGBS show high MICs of ceftizoxime (464 mg/L) and cefotaxime (0.121 mg/L), those for strain B1 are exceptionally high (ceftizoxime MIC 256 mg/L and cefotaxime MIC 2 mg/L). We previously found an amino acid substitution (G539S) neighbouring the conserved K(540)TG motif in PBP1A in addition to the PRGBS-specific amino acid substitution Q557E in PBP2X of B1. The aim of this study was to reveal the effect of the amino acid substitutions in PBP1A and PBP2X of B1 on the high cephalosporin resistance.
A ceftizoxime competition assay was performed to reveal the PBPs that are the main targets of ceftizoxime. We generated two allelic exchange mutants from -lactam-susceptible GBS BAA-611. BAA-611 (B1PBP2X) contained the PBP2X gene derived from B1 and BAA-611 (B1PBP2X, B1PBP1A) contained both the PBP2X and the PBP1A gene derived from B1. These allelic exchange mutants and strain B1 were subjected to susceptibility testing.
The ceftizoxime competition assay revealed that PBP1A and PBP2X were the main targets of ceftizoxime. Although the MICs of ceftizoxime and cefotaxime for BAA-611 (B1PBP2X) were 64 and 0.5 mg/L, respectively, BAA-611 (B1PBP2X, B1PBP1A) showed high cephalosporin resistance (ceftizoxime MIC 256 mg/L and cefotaxime MIC 2 mg/L) comparable to B1.
The high cephalosporin resistance of GBS was caused by amino acid substitutions in PBP1A and PBP2X.

DOI： 10.1093/jac/dkt060

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Ability of the VITEK 2 system to detect group B streptococci with reduced penicillin susceptibility (PRGBS) Reviewed

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

JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY   Vol. 68 ( 6 ) page： 1442 - 1444   2013.6

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DOI： 10.1093/jac/dkt008

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Detection of Tripoli metallo--lactamase 2 (TMB-2), a variant of bla(TMB-1), in clinical isolates of Acinetobacter spp. in Japan Reviewed Open Access

Satowa Suzuki, Mari Matsui, Masato Suzuki, Akira Sugita, Yoko Kosuge, Nobuhiro Kodama, Yasuko Ichise, Keigo Shibayama

JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY   Vol. 68 ( 6 ) page： 1441 - 1442   2013.6

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DOI： 10.1093/jac/dkt031

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Screening for Group B Streptococci with Reduced Penicillin Susceptibility in Clinical Isolates Obtained between 1977 and 2005 Reviewed Open Access

Kimura Kouji, Nishiyama Yasunobu, Shimizu Seiichi, Wachino Jun-ichi, Matsui Mari, Suzuki Satowa, Yamane Kunikazu, Shibayama Keigo, Arakawa Yoshichika

JAPANESE JOURNAL OF INFECTIOUS DISEASES   Vol. 66 ( 3 ) page： 222 - 225   2013.5

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DOI： 10.7883/yoken.66.222

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