Masakado Matsumoto

Cloning of a Novel Gene for Quinolone Resistance from a Transferable Plasmid in Shigella flexneri 2b

ABSTRACT 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.

Ability of Shiga Toxin-Producing Escherichia coli and Salmonella spp. To Survive in a Desiccation Model System and in Dry Foods

ABSTRACT In order to determine desiccation tolerances of bacterial strains, the survival of 58 diarrheagenic strains (18 salmonellae, 35 Shiga toxin-producing Escherichia coli [STEC], and 5 shigellae) and of 15 nonpathogenic E. coli strains was determined after drying at 35°C for 24 h in paper disks. At an inoculum level of 107 CFU/disk, most of the salmonellae (14/18) and the STEC strains (31/35) survived with a population of 103 to 104 CFU/disk, whereas all of the shigellae (5/5) and the majority of the nonpathogenic E. coli strains (9/15) did not survive (the population was decreased to less than the detection limit of 102 CFU/disk). After 22 to 24 months of subsequent storage at 4°C, all of the selected salmonellae (4/4) and most of the selected STEC strains (12/15) survived, keeping the original populations (103 to 104 CFU/disk). In contrast to the case for storage at 4°C, all of 15 selected strains (5 strains each of Salmonella spp., STEC O157, and STEC O26) died after 35 to 70 days of storage at 25°C and 35°C. The survival rates of all of these 15 strains in paper disks after the 24 h of drying were substantially increased (10 to 79 times) by the presence of sucrose (12% to 36%). All of these 15 desiccated strains in paper disks survived after exposure to 70°C for 5 h. The populations of these 15 strains inoculated in dried foods containing sucrose and/or fat (e.g., chocolate) were 100 times higher than those in the dried paper disks after drying for 24 h at 25°C.

Characterization of Shiga Toxin-Producing Escherichia coli O26 Strains and Establishment of Selective Isolation Media for These Strains

ABSTRACT We 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. coli strains (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.

Molecular Epidemiology of Salmonella enteritidis. An Outbreak and Sporadic Cases Studied by means of Pulsed-field Gel Electrophoresis

Large outbreaks of diarrhoea due to Salmonella enteritidis in Aichi-ken, Japan, provided the opportunity to investigate aspects of the molecular epidemiology of this and related organisms. This was performed by comparing the plasmid profile types, phage types, antimicrobial resistance, and the restriction fragment length polymorphisms (RFLPs) by pulsed-field gel electrophoresis (PFGE) of S. enteritidis from outbreaks and sporadic cases. Among the isolates studied, 10 distinctive RFLP types were found with XbaI and four with NotI, while 12 combination types were identified among the 68 isolates from 16 Health Centres in Aichi-ken, Japan. A total of 22 isolates from four outbreaks, however, had the same RFLP and phage types. The RFLP type was subdivided by means of the plasmid profile and phage type. Conversely, plasmid profile and phage type were separated by means of RFLP. This PFGE method may prove useful for subclassifying S. enteritidis and differentiating isolates of the same plasmid profile and phage type.

Detection of Clostridium perfringens enterotoxin gene by the polymerase chain reaction amplification procedure

The polymerase chain reaction (PCR) procedure was evaluated to see if it is a simple and rapid method to detect Clostridium perfringens enterotoxin gene. The method, involving the use of two synthesised primers and gene amplification by the PCR procedure, detects a DNA fragment of 364 base pairs of C. perfringens enterotoxin gene by gel electrophoresis. The enterotoxin gene of strains was detected by use of purified chromosomal DNA. The supernatant of sporulating cultures in a sporulating medium was able to be used as template DNA. Template DNA can be obtained by merely culturing the strain in DS medium, a sporulating medium, for 48 h at 37 degrees C. All C. perfringens strains showing positive results in the PCR procedure were demonstrated to produce enterotoxin by a conventional method and all strains showing negative results were enterotoxin negative. To detect the enterotoxin gene in stool specimens by the PCR procedure, the specimen was heat-treated for 10 min at 90 degrees C and cultured in a sporulating medium, the supernatant of which was used as template DNA. From the stool specimens showing positive results in the PCR procedure by this method, enterotoxigenic C. perfringens was isolated from the heat-treated specimens. Thus, it is possible to detect enterotoxigenic C. perfringens in stools without isolation of the organism.

Genetic Features of Clinical Isolates of Streptococcus dysgalactiae subsp. equisimilis Possessing Lancefield's Group A Antigen

ABSTRACT Thirteen Streptococcus dysgalactiae subsp. equisimilis isolates possessing Lancefields 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 emm types were observed within members of this clone.

Detection of invasive protein profile of Streptococcus pyogenes M1 isolates from pharyngitis patients.

Hasegawa T, Okamoto A, Kamimura T, Tatsuno I, Hashikawa S‐N, Yabutani M, Matsumoto M, Yamada K, Isaka M, Minami M, Ohta M. Detection of invasive protein profile of Streptococcus pyogenes M1 isolates from pharyngitis patients. APMIS 2010; 118: 167–78.

Antimicrobial ointments and methicillin-resistant Staphylococcus aureus USA300.

We tested 259 methicillin-resistant Staphylococcus aureus isolates and 2 USA300 ATCC type strains for susceptibility to bacitracin and neomycin contained in over-the-counter antibacterial ointments. Resistance to both bacitracin and neomycin was found only in USA300. The use of over-the counter antimicrobial drugs may select for the USA300 clone.

Phylogenetic Clades 6 and 8 of Enterohemorrhagic Escherichia coli O157:H7 With Particular stx Subtypes are More Frequently Found in Isolates From Hemolytic Uremic Syndrome Patients Than From Asymptomatic Carriers

EHEC O157:H7 clade 6 strains harboring stx2a and/or stx2c and clade 8 strains harboring stx2a or stx2a/stx2c were frequently associated with childhood HUS cases in Japan. Rapid and specific detection of such lineages are required for infection control measures.

Development of a Rapid PCR Method Using the Insertion Sequence IS1203 for Genotyping Shiga Toxin-Producing Escherichia coli O157

ABSTRACT 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 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 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.