Masaaki Shinagawa

Sapporo Medical University, Sapporo, Hokkaidō, Japan

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Publications (6)11.6 Total impact

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    ABSTRACT: The precise identities of the virulence factors of Pseudomonas aeruginosa after bacteriolysis are still unknown. In the present study, we identified PA0423 protein, which was isolated from the Pseudomonas PAO-1 strain, as the factor responsible for cytotoxicity in lung epithelial cells. Whole bacterial cell lysate of P. aeruginosa PAO-1 caused cytotoxicity in A549 lung epithelial cells. This cytotoxic factor could be partially purified via gel-filtration and anion-exchange column chromatography, and its activity was attenuated by proteinase K treatment. The cytotoxic fraction increased caspase-3/7 activity in A549 cells, suggesting the induction of apoptosis. This fraction was then subjected to amino-acid sequence analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, resulting in the identification of 7 matches, 4 of which were with known proteins (PA0122, PA2687, PA3406, and PA0423). Deletion mutant analysis of these 7 candidates revealed that only the PA0423 mutation led to reduced cytotoxicity, indicating that this protein is the virulence factor. Furthermore, PA0423 recombinant protein was constructed, purified, and refolded. Transduction of recombinant PA0423, but not PA0122, into A549 cells engendered a dose-dependent cytotoxic effect. These results show the first evidence that specific bacteriolysis-induced virulence factor PA0423 from Pseudomonas is toxic to lung epithelial cells.
    No preview · Article · Aug 2014 · Microbial Pathogenesis
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    ABSTRACT: Arginine catabolic mobile element (ACME), a genomic island consisting of the arc and/or opp3 gene clusters found in staphylococcal species, is related to increased bacterial adaptability to hosts. Staphylococcus epidermidis is considered a major ACME reservoir; however, prevalence and genetic diversity of ACME in coagulase-negative staphylococci (CNS) have not yet been well characterized for clinical isolates in Japan. A total of 271 clinical isolates of CNS in a Japanese hospital were investigated for the presence and genotype of ACME and SCCmec. The prevalence of ACME-arcA was significantly higher (p<0.001) in S. epidermidis (45.8%) than in other CNS species (3.7%). ACME in S. epidermidis isolates (n=87) were differentiated into type I (n=33), variant forms of type I (ΔI, n=26) newly identified in this study, type II (n=6), and type ΔII (n=19). ACME-type ΔI, which were further classified into three subtypes, lacked some genetic components between the arc and opp3 clusters in archetypal type I, whereas the arc and opp3 clusters were intact. The arc cluster exhibited high sequence identity (95.8-100 %) to that of type I ACME; in contrast, the opp3 cluster was highly diverse, and showed relatively lower identities (94.8- 98.7%) to the identical regions in type I ACME. Twenty-one isolates of ΔI ACME-carrying S. epidermidis possessed SCCmec IVa and belonged to ST5 (clonal complex 2). Phylogenetic analysis revealed that isolaets harbouring ACME ΔI in this study clustered with previously reported S. epidermidis strains with other lineges, suggesting that S. epidermidis originally had some genetic variations in the opp3 cluster. In summary, ACME type ΔI, a truncated variant of ACME-I, was first identified in S. epidermidis, and revealed to be prevalent in ST5 MRSE clinical isolates with SCCmec IVa.
    No preview · Article · Oct 2013 · Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases
  • M. Onishi · N. Urushibara · M. Shinagawa · N. Watanabe · N. Kobayashi

    No preview · Article · Jun 2013 · International Journal of Antimicrobial Agents
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    ABSTRACT: The ST8 methicillin-resistant Staphylococcus aureus (MRSA) with Staphylococcal cassette chromosome mec (SCCmec) type IVa, known as USA300, is a prevalent community-acquired MRSA (CA-MRSA) clone in the United States and has been spreading worldwide. The USA300 characteristically harbors Panton-Valentine Leukocidin (PVL) genes and the arginine catabolic mobile element (ACME, type I). Prevalence and molecular characteristics of PVL(+) and/or ACME(+) S. aureus were investigated in a university hospital located in northern Japan, for 1,366 S. aureus isolates, including 601 MRSA strains derived from clinical specimens collected from 2008 to 2010. The PVL gene was identified in three MRSA strains with SCCmec IV, which belonged to ST8, spa type t008, coagulase type III, and agr type I. Two PVL-positive MRSA strains had also type I ACME, and were isolated from skin abscess of outpatients who have not travelled abroad recently. One of these PVL(+)/ACME(+) strains carried tet(K), msrA, and aph(3')-IIIa, showing resistance to kanamycin, tetracycline, erythromycin, and ciprofloxacin, suggesting acquisition of more resistance than ST8 CA-MRSA reported in Japan previously. In contrast, another PVL(+)/ACME(+) strain and a PVL(+)/ACME(-) strain were susceptible to more antimicrobials and had less virulence factors than PVL(-)/ACME(+) MRSA strains. Besides the two PVL(+) MRSA strains, ACME (type-ΔII) was identified into seven MRSA strains with SCCmec II belonging to ST5, one of the three spa types (t002, t067, and t071), coagulase type II, and agr type II. These PVL(-)/ACME(+) MRSA strains showed multiple drug resistance and harbored various toxin genes as observed for ST5 PVL(-)/ACME(-) MRSA-II. The present study suggested the spread of ST8-MRSA-IV in northern Japan, and a potential significance of ACME-positive ST5-MRSA-II as an emerging MRSA clone in a hospital.
    Full-text · Article · Oct 2012 · Microbial drug resistance (Larchmont, N.Y.)
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    ABSTRACT: The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 2655 strains including 810 strains of Gram-positive bacteria, 1635 strains of Gram-negative bacteria, and 210 strains of anaerobic bacteria obtained from 30 medical institutions during 2009 was examined. The results were as follows; (1) MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multidrug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA). (2) MEPM maintained potent and stable antibacterial activity against Pseudomonas aeruginosa. The proportion of MEPM-resistant strains to ciprofloxacin-resistant strains or imipenem-resistant strains were 53.1% and 58.0% respectively. (3) The proportion of extended-spectrum beta-lactamase (ESBL) strains was 3.1% (26 strains) in enterobacteriaceae. And the proportion of metallo-beta-lactamase strains was 2.0% (6 strains) in P. aeruginosa. (4) Of all species tested, there were no species except for Bacteroides fragilis group, which MIC90 of MEPM was more than 4-fold higher than those in our previous study. Therefore, there is almost no significant decrease in susceptibility of clinical isolates to meropenem. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem for serious infections treatment at present, 14 years passed after available for commercial use in Japan.
    No preview · Article · Apr 2011 · The Japanese journal of antibiotics
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    ABSTRACT: Staphylocoagulase (SC) is a major phenotypic determinant of Staphylococcus aureus. Antigenic specificity of SC (SC serotype) has been classified into at least 10 types and employed as an epidemiological marker. In the present study, from the sequence information of SC genes, a novel multiplex PCR assay was developed to determine SC genotypes (SC types) I-X corresponding to SC serotypes I-X, respectively, and to discriminate two subtypes of SC types IV and V. Two PCR reactions (Sc-R1 and Sc-R2) for a single isolate were designed for assigning common SC types in S. aureus isolates from humans (I-VIII and X). When amplicon was not produced, an additional PCR (Sc-R3) was performed to assign SC type IX or subtype Vb. Subtypes IVa and IVb were discriminated by an additional PCR (Sc-R4). SC types were discriminated successfully for the S. aureus strains with established SC types I-X including two subtypes of IV and V. The multiplex PCR assay established in this study could assign SC types for S. aureus clinical isolates at a high determination rate, providing more accurate information on incidence of SC types, and was considered to be useful for epidemiologic characterization of S. aureus.
    Full-text · Article · Jul 2010