Koichi Takeshi

Distribution of the Secondary Type III Secretion System Locus Found in Enterohemorrhagic Escherichia coli O157:H7 Isolates among Shiga Toxin-Producing E. coli Strains

ABSTRACT The ability of the complete genome sequence of enterohemorrhagic Escherichia coli O157 led to the identification of a 17-kb chromosomal region which contained a type III secretion system gene cluster at min 64.5. This locus contains open reading frames whose amino acid sequences show high degrees of similarity with those of proteins that make up the type III secretion apparatus, which is encoded by the inv-spa-prg locus on a Salmonella SPI-1 pathogenicity island. This locus was designated ETT2 (E. coli type III secretion 2) and consisted of the epr, epa, and eiv genes. ETT2 was found in enteropathogenic E. coli strains and also in some non-O157 Shiga toxin-producing E. coli (STEC) strains, but most of them contained a truncated portion of ETT2. Most O157 isolates had a complete collection of toxin-encoding genes eae and hlyA and the ETT2 locus, while most O26 strains had toxin-encoding genes eae and hlyA genes but an incomplete ETT2 locus. Thus, an intact copy of ETT2 might mark a pathogenic distinction for particular STEC strains. Therefore, the presence of the ETT2 locus can be used for identification of truly pathogenic STEC strains and for molecular fingerprinting of the epidemic strains in humans and animals.

Molecular Epidemiological Study of a Mass Outbreak Caused by Enteropathogenic Escherichia coli O157:H45

We made a molecular analysis of O157:H45 Escherichia coli isolated from a mass outbreak that occurred in Obihiro City. Using DNA analysis, we confirmed this infection case as a mass outbreak. Although the isolates expressed O157 antigen, they did not produce Vero toxin. We concluded they were enteropathogenic E. coli (EPEC) because they had a bfp gene and an EAF plasmid, and further they exhibited local adherence to HEp‐2 cells. We believe this is the first report of a mass outbreak by O157 EPEC, and we suggest that PCR using eae‐ and bfp‐specific primers and HEp‐2 adherence assay are useful to identify EPEC.

Therapeutic Effect of Anti-TNF-α Antibody and Levofloxacin(LVFX)in a Mouse Model of Enterohemorrhagic Escherichia coli O157 Infection

The ability of an anti-TNF-alpha antibody to confer protection against enterohaemorrhagic Escherichia coli (EHEC) O157 was investigated in germfree IQI mice. The use of an antibiotic levofloxacin (LVFX) alone or with the antibody was also studied. Protection included an increase in survival rate. Treatment with the anti-TNF-alpha antibody inhibited the histological signs associated with EHEC infection but did not prevent the colonization of EHEC or production of Shiga toxin (Stx). No clinical signs were observed and EHEC was completely eliminated in the mouse model receiving both anti-TNF-alpha antibody and LVFX. Anti-TNF-alpha antibody suppressed inflammatory cytokine response in the mouse kidney and brain by EHEC infection.

Isolation and characterization of Listeria monocytogenes from commercial asazuke (Japanese light pickles).

Asazuke is a ready-to-eat Japanese light pickle, mainly made of vegetables which are known to be one of the sources of Listeria monocytogenes contamination. Although asazuke is a popular side-dish in Japan, the hazard of bacterial contamination has not been evaluated yet. In this study, we investigated the prevalence of L. monocytogenes, Salmonella spp., verotoxigenic E. coli (VTEC) and coliforms in 108 asazuke samples that randomly collected from supermarkets in Obihiro (Hokkaido prefecture, Japan) during the period of June to November 2007. Twelve (11.11%) L. monocytogenes were isolated with predominant serotype 4b (seven isolates) followed by 1/2a (two isolates), 1/2b, 3b and 4c (one isolate each) while Salmonella spp., VTEC and coliforms were not detected. All L. monocytogenes isolates demonstrated hemolytic activity by CAMP test and possessed all the virulence-associated genes (prfA, actA, mpl, inlA, inlC, plcA, plcB, hly, iap, clpC and opuCA) as resulted in PCR, thus revealed their potential pathogenicity. Moreover, 7 out of 12 isolates were from asazuke samples produced by the same factory and their pulsed-field gel electrophoresis (PFGE) profiles suggested that 6 of them were indistinguishable and one was different. L. monocytogenes contamination in the asazuke factory environment was further investigated and 23 out of 60 environmental swabs (38.33%) contained the bacterium. Comparison of PFGE profiles showed relatedness between food and environmental isolates indicating that contamination probably occurred in the asazuke factory during manufacturing. Interestingly, after HACCP training course conducted to the factory workers, 20 samples collected during the period of November to December 2008 were negative to L. monocytogenes revealing that the hygienic status has improved.