鈴木 匡弘

Out of 68 Escherichia coli O157 field isolates tested invitro for Shiga toxin (Six) 2 production, 12 (17.6%) produced no or a limited amount of Stx2 (Six 2 non- or low-producing strain; TNLP) even though all 68 possessed the stx(2) gene. The remaining 56 were Stx2 high-producing strains. The 12 TNLPs carried the q21 gene allele, which encodes a transcription anti terminator Q protein and is highly homologous to that of Phi 21 phage. They also carried nucleotide substitutions and insertions in the promoter region of the stx(2) gene compared with that of O157 EDL933, producing a considerable amount of Stx2. In contrast, the Stx2 high-producing strains carried the q933 gene allele, which was first reported on an stx(2) phage (933W), but not the q21 gene allele, and did not have mutations in the promoter region of the six(2) gene. These 2 genetic characteristics, i.e., replacement of the q gene and mutation in the prornoter region of the stx(2) gene, seemed to determine the amount of Stx2 produced by each strain. The TNLPs were more frequently isolated from healthy carriers than from patients (P < 0.05), suggesting that TNLPs are less virulent than those with normal Stx2 production.

Aims: To develop a rapid genotyping method for investigating outbreaks of methicillin-resistant strains of Staphylococcus aureus (MRSA) isolated in Japan. Methods and Results: Isolates were genotyped by detecting the keeping pattern of 16 open-reading frames (ORFs), a process we call phage ORF typing (POT). Thirteen of the ORFs were selected from phage genomes and one from a genomic island SaGIm in the genome of strain Mu50. The other two ORFs, one from Tn554 and one from staphylococcal cassette chromosome mec (SCCmec) type II, were used as strain markers. Three hundred and sixty-eight isolates from five hospitals were classified into 133 types by POT, whereas they were classified into 139 types by pulsed-field gel electrophoresis (PFGE) subtyping. The discriminatory power of POT (D = 0.989) was equal to that of PFGE subtyping (D = 0.986). Conclusions: MRSA isolates collected in Japan can be genotyped by detecting the keeping pattern of phage-derived ORFs with a discriminatory power equal to that of PFGE subtyping. Significance and Impact of the Study: MRSA isolates can be genotyped rapidly by detecting phage-derived ORFs. As particular pandemic clones can be found in a specific region, a typing method localized to a pandemic clone may be effective for the rapid genotyping of MRSA during outbreaks. © 2006 The Authors.

A novel gene for quinolone resistance was cloned from a transferable plasmid carried by a clinical isolate of Shigella flexneri 2b that was resistant to fluoroquinolones. The plasmid conferred low-level resistance to quinolones on Escherichia coli HB101. The protein encoded by the gene showed 59% amino acid identity with Qnr.

We developed a rapid PCR method utilizing the diversity of the insertion site IS1203 for genotyping Shiga toxin-producing Escherichia coli (STEC) O157 (IS1203 PCR typing). DNA fragments digested by PvuII, which cut IS1203 at one site, were ligated with themselves and detected by PCR with outward-facing primer pairs for IS1203. To minimize nonspecific bands, nested PCR was also performed. Two fingerprinting patterns produced from the upstream or downstream regions of IS1203 were obtained within I or 2 days. By combining the two patterns, 79 STEC O157 isolates were classified into 39 types, which were then classified into 36 subtypes by pulsed-field gel electrophoresis (PFGE). The discriminatory power of IS1203 PCR typing (D = 0.974) is similar to that of PFGE (D = 0.981). This method can be used for rapid and simplified genotyping.

We found two Ship toxin producing Escherichia coli O157:H7 strains isolated from humans carrying the stx(1) gene with an IS1203-like element (designated as IS1203v(1)). The IS1203v(1) was inserted into the coding region of the A subunit 7 bp upstream from the TGA termination codon, resulting in a loss of two amino acid residues (Ser-Ser) from its C terminus. Toxicity of the Stx1 was confirmed by Vero cell assay. IS1203v(1) hardly affected the stx(1) gene in either its expression or the toxicity of its product. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

During the past 20 years, outbreaks of enterohemorrhagic Escherichia coli (EHEC) have been increasing worldwide and have been recognized as a potential health concern. Vero toxins produced by EHEC seem to be the most common cause of hemolytic uremic syndrome. Rapid diagnosis of EHEC infection is important to prevent the expansion of infection. Diagnosis is carried out by both isolation of EHEC and detection of Vero toxins in fecal extracts or fecal cultures. This review describes briefly about the current knowledge of the EHEC and Vero toxins, and about the determination methods for Vero toxins. The attempt to identify Vero toxins by electrospray ionization-liquid chromatography/mass spectrometry is also discussed.

We developed a rapid and reliable identification method for Shiga toxins in Shiga toxin-producing Escherichia coli (STEC) using immunoprecipitation and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). Polyclonal antisera specific for Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) were raised in rabbits so as to be used for the immunoprecipitation. The immunoprecipitaion was carried out by mixing sample solutions with 50 mul each of the antisera to Stx1 and Stx2 followed by allowing the mixed solutions to stand for 30 min. The quantity required to obtain the immunoprecipitate was more than 0.5 mug of Shiga toxins. HPLC-ESI-MS analysis of the resulting immunoprecipitates provided accurate molecular weight information on Shiga toxins, leading to direct evidence for the presence of these toxins. It requires at most two days to perform our procedure from toxin extraction to measurement of HPLC-ESI-MS whereas the previous method using isolation procedures required about two weeks to complete. The usefulness of the present method has been demonstrated by identifying Stx1, Stx2 and a variant of Stx2 (Stx2e) in the immunoprecipitates prepared from STEC strains.

A fatal case of cholera caused by Vibrio cholerae O1 El Tor serotype Ogawa occurred in Aichi Prefecture, Japan in 1995. The patient was identified locally, but the route of the infection was unknown. The causative isolate and 38 other domestic and imported V. cholerae 01 isolates, obtained between 1984 and 1997, were analysed by prophage typing, antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). This was done to determine whether the isolate from this case differed from others associated with either mild cholera infections or asymptomatic carriage, and to elucidate the route of infection. Cholera toxin (CT) from 37 toxigenic isolates was assayed semiquantitatively. The 39 isolates were divided into 12 temporary types in accordance with the results of the three typing techniques. The isolate from the fatal infection and nine other isolates were classified as temporary type IV. No difference in CT production was found between the isolate from the fatal case and the other 36 toxigenic isolates. Taken together, it is unlikely that a V cholerae 01 isolate of distinguishable type was responsible for the fatal illness. Temporary type IV isolates were frequently present in both domestic and imported cases from 1994 to 1997 in Aichi, but they did not emerge before 1993. These results suggest that a new clone was introduced after 1993 from overseas and then disseminated into Aichi, and this may have been an important step in triggering the fatal case of cholera.

To investigate the prevalence of drug-resistant isolates of Salmonella Typhimurium in Aichi, Japan, we performed antimicrobial susceptibility tests for 148 isolates from healthy carriers, and from sporadic and outbreak cases of salmonellosis from 1980 to 1999. We found an increase in drug-resistant isolates from 56% (37/66) in the 1980s to 74% (61/82) in the 1990s due to increasing examples of four-, five-, and six-drug resistances. Of 98 resistant isolates in 1980 - 1999, 12 were identified as ampicillin (A)-, chloramphenicol (C)-, streptomycin (S)-, sulfonamide (Su)-, and tetracycline (T)-resistant S. Typhimurium (4 in the 1980s, 8 in the 1990s), whose pattern was identical to that of multi-drug-resistant S. Typhimurium definitive phage type 104 (DT104) which has been recently detected in various developed countries. Six-drug-resistance ACSSuTP (piperacillin), in which P was added to the core pattern of the ACSSuT, was also found in four isolates in the 1980s and seven in the 1990s. Another six-drug-resistant pattern, ACSSuTN (nalidixic acid), appeared in five isolates in the 1990s. These multi-drug-resistant isolates were predominately found in healthy carriers (21/28), suggesting that in Aichi the multi- (five- or six-) drug-resistant isolates of S. Typhimurium have existed in healthy carriers as well as in diarrhea patients in 1980 to 1999.