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ABSTRACTThirteen Streptococcus dysgalactiaesubsp. equisimilisisolates possessing Lancefield's group A antigen recovered from people in Japan during 2000 to 2004 were genotyped. The results indicate that a conserved clone has persisted and spread within Japan, and two different emmtypes were observed within members of this clone.

DNase B is a major nuclease and a possible virulence factor in Streptococcus pyogenes. The allelic diversity of streptococcal DNase B (sdaB) gene was investigated in 83 strains with 14 emmgenotypes. Of the 15 alleles identified, 11 alleles carried only synonymous nucleotide substitutions. On the other hand, 4 alleles had a non‐synonymous substitution other than synonymous substitutions, resulting in the substitution of a single amino acid. The distribution of each allele was generally emmgenotype‐specific. Only sdaB7was found in both emm2and emm4. The promoter region was highly conserved and DNase B protein was similarly expressed in all alleles.

ABSTRACTWe developed a rapid PCR method utilizing the diversity of the insertion site IS1203for genotyping Shiga toxin-producing Escherichia coli(STEC) O157 (IS1203PCR typing). DNA fragments digested by PvuII, which cut IS1203at 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 IS1203were obtained within 1 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 IS1203PCR typing (D= 0.974) is similar to that of PFGE (D= 0.981). This method can be used for rapid and simplified genotyping.

We developed a rapid and reliable identification method for Shiga toxins in Shiga toxin-producing Escherichia coliSTEC 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 µl 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 µg 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.

ABSTRACTWe characterized the carbohydrate-fermenting ability of 31 strains of Shiga toxin-producing Escherichia coli(STEC) O26 isolated from diarrhea patients in Aichi Prefecture, Japan, in order to establish selective isolation media for these strains. None of the 31 STEC O26 strains (24 O26:H11, 7 O26:H-) fermented rhamnose, whereas all of the other 108 STEC strains (100 O157, 8 O111) and all of the non-STEC strains except one (i.e., 58 of 59) fermented rhamnose. The great majority of the STEC O26 strains (96.8% [30 of 31]) showed very high resistance to potassium tellurite (MIC = 50 µg/ml), whereas the majority of the non-STEC strains (72.9% [43 of 59]) showed very high sensitivity (MIC = 1.56 µg/ml) to this compound. Accordingly, we developed a rhamnose-MacConkey (RMAC) medium in which the lactose in MacConkey medium was replaced by rhamnose, and cefixime-tellurite-RMAC (CT-RMAC) medium in which potassium tellurite (2.5 mg/liter) and cefixime (0.05 mg/liter) were added to RMAC. All of the STEC O26 strains generated colorless (rhamnose-nonfermented) colonies on both media; the vast majority of selected E. colistrains (95.7% [89 of 93; including 26 STEC O157, 8 STEC O111]), other than STEC O26, generated red colonies on RMAC, and most of the non-STEC strains (84.7% [50 of 59]) did not grow on CT-RMAC. We demonstrate that both the RMAC and the CT-RMAC media can be used for the isolation of STEC O26 and that CT-RMAC has better specificity for the routine isolation for STEC O26 in a laboratory.

ABSTRACTAcute respiration distress syndrome (ARDS) is a typical complication in toxic shock-like syndrome (TSLS) caused byStreptococcus pyogenes. An isolated perfused rat lung model was used to identify the causative agent of ARDS in TSLS in this study. Some crude preparations separated from the culture supernatants ofS. pyogenesisolates caused rapid increases in the weight of perfused lungs, indicating vascular permeabilization. Six samples from M type 1 and 3 isolates from TSLS and pharyngitis patients showed strong permeabilization activity, whereas preparations from isolates of other M types (although the number of isolates examined was limited) were negative. The active substance was purified to a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using various columns, and the N-terminal amino acid sequence was determined. The resultant sequence of eight amino acids was identical to SpeF (mitogenic factor). Moreover, the vascular permeabilization activity of the purified band was abolished with anti-SpeF antiserum prepared by immunizing with the purified SpeF. This activity was also neutralized by the antiserum prepared by immunizing with a synthetic peptide derived from the published SpeF sequence. These results suggested that streptococcal SpeF is a major cause of permeabilization of lung blood vessels and sufficient for the pathogenesis of ARDS under the conditions of TSLS caused by S. pyogenes.

We noted that eight outbreaks of Shigella sonneifrom an unknown source occurred sequentially in Aichi Prefecture, Japan, between October 1992‐June 1993. For comparative purposes we analyzed 53 outbreak‐related isolates of Shigella sonneiusing different subtyping methods and studied the epidemiology of the outbreaks. It appeared from our study that DNA‐based techniques such as plasmid typing and pulsed‐field gel electrophoresis (PFGE) were more useful tools for subtyping Shigella sonneithan colicin typing and the antimicrobial susceptibility test. Moreover, according to PFGE analysis, four genetically related isolates of Shigella sonneiwere responsible for the eight sequential outbreaks. To further investigate the epidemiology of outbreaks, 58 sporadic isolates of Shigella sonneifrom overseas travelers with shigellosis during the same period were also examined. We found that some sporadic isolates from travelers in Asia were genetically related to those of the outbreak‐related isolates, indicating that genetically related isolates prevailed in Asia during this period, probably because of the extensive movement of people or food.

More than 100 cases have been identified as streptococcal toxic shock‐like syndrome (TSLS) in Japan, since the first case was reported in 1993. Of 26 S. pyogenesisolates associated with TSLS, 69% were M‐types 1 and 3. This study focused on speAalleles which were carried by all M‐type 1 and 3 isolates and isolates of some other M‐types, irrespective of sources. We sequenced the nucleotides of the relevant region of the speAallele in all of these isolates. Consistent with other reports, these M‐type 1 and 3 isolates carried the speA2and speA3alleles, respectively. Our results indicate that these two speAalleles, which were prevalent worldwidely have been disseminating in Japan.

ABSTRACTA novel gene for quinolone resistance was cloned from a transferable plasmid carried by a clinical isolate of Shigella flexneri2b that was resistant to fluoroquinolones. The plasmid conferred low-level resistance to quinolones on Escherichia coliHB101. The protein encoded by the gene showed 59% amino acid identity with Qnr.

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