Among Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of various serotypes, O157:H7 and five major non-O157 STEC (O26:H11, O111:H8, O103:H2, O121:H19 and O145:H28) can be selectively isolated by using tellurite-containing media. While human infections by O165:H25 STEC strains have been reported worldwide, their detection and isolation are not easy, as they are not resistant to tellurite. Systematic whole-genome sequencing (WGS) analyses have not yet been conducted. Here, we defined O165:H25 strains and their close relatives, including O172:H25 strains, as clonal complex 119 (CC119) and performed a global WGS analysis of the major lineage of CC119, called CC119 sensu stricto (CC119ss), by using 202 CC119ss strains, including 90 strains sequenced in this study. Detailed comparisons of 13 closed genomes, including 7 obtained in this study, and systematic analyses of Stx phage genomes in 50 strains covering the entire CC119ss lineage, were also conducted. These analyses revealed that the Stx2a phage, the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS), many prophages encoding T3SS effectors, and the virulence plasmid were acquired by the common ancestor of CC119ss and have been stably maintained in this lineage, while unusual exchanges of Stx1a and Stx2c phages were found at a single integration site. Although the genome sequences of Stx2a phages were highly conserved, CC119ss strains exhibited notable variation in Stx2 production levels. Further analyses revealed the lack of SpLE1-like elements carrying the tellurite resistance genes in CC119ss and defects in rhamnose, sucrose, salicin and dulcitol fermentation. The genetic backgrounds underlying these defects were also clarified.
We describe our laboratory investigation of a massive foodborne outbreak of gastrointestinal illness caused by enterotoxigenic Escherichia coli (ETEC) serotype O169:H41 that occurred during a 2-day traditional festival held in September 2012 in Osaka Prefecture, Japan. Of 126 customers who patronized a particular Japanese restaurant during the event, 102 developed symptoms of gastrointestinal disease. We isolated strains of ETEC serotype O169:H41 from 1 food sample and from fecal samples collected from 19 of 34 patients and 2 of 4 food handlers. Pulsed-field gel electrophoresis analysis of these isolates suggested that the foodborne pathogen that caused the diarrheal outbreak was a specific clone of ETEC serotype O169:H41. Based on these findings and our interviews with the restaurant owner and employees, we concluded that a likely cause of the outbreak was an overwhelmed capacity of the restaurant kitchen in terms of preservation of sanitary procedures during the festival and the inability of the restaurant staff to handle the relatively large quantity of food to ensure a lack of contamination with ETEC. Thus, we reconfirm that ETEC strains of serotype O169:H41 remain important causes of domestic foodborne outbreaks in developed countries, including Japan.
In Osaka Prefecture, Japan, three foodborne outbreaks were caused by Salmonella enterica serotype Montevideo in rapid succession between September 2007 and May 2008. Further, Salmonella Montevideo was also isolated from several sporadic diarrhea patients and asymptomatic carriers examined during approximately the identical period. To investigate the relatedness of the isolates, we performed antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) analysis, and multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) for 29 Salmonella Montevideo isolates obtained in this region between 1991 and 2008. Although antimicrobial susceptibility tests had low discriminatory power, PFGE patterns revealed 17 unique types with <90% similarity in combined analyses involving XbaI and BlnI. Moreover, we detected three VNTR loci that were useful to genotype Salmonella Montevideo isolates, with our method ultimately classifying the isolates into 11 MLVA types based on differences in repeat unit number in each examined locus. Six isolates obtained from patients of two separate foodborne disease outbreaks, one sporadic patient, and three different carriers between 2007 and 2008 had nearly identical PFGE patterns and were classified into the identical MLVA type; further, the isolates with this PFGE and MLVA pattern appeared only at that time between 1991 and 2008. These data strongly suggest that genetically identical Salmonella Montevideo strains may have caused the 2007 and 2008 outbreaks in Osaka Prefecture. Our results demonstrate that PFGE using XbaI and BlnI is useful for discriminating between Salmonella Montevideo isolates, even within a limited area, and reconfirm that continuous epidemiological surveillance for bacterial intestinal infections such as salmonellosis may be useful to not only monitor changes in the genetic diversity of isolates, but to also detect diffuse outbreaks.
The multilocus variable-number tandem repeat analysis (MLVA) method to target eight variable-number tandem repeat loci, based on agarose gel electrophoresis separation of multiplexed PCR products, and the PFGE method were applied to clinical isolates of Escherichia coli O157 : H7 with the aim of comparing their performance as methods of typing this bacterium. Using MLVA, a total of 57 isolates from patients in Shizuoka prefecture, Japan, were divided into 20 types and classified into 23 PFGE types. Twenty-four isolates from four sporadic infections, four household contact infections and one outbreak that occurred in central parts of Shizuoka prefecture during August to November in 2005 were shown to be the same MLVA type, and most of the isolates had identical PFGE banding patterns, suggesting the diffuse outbreak in these parts of Japan. Thus, there was a good correlation between MLVA types and PFGE types, with both methods displaying broadly similar discriminatory powers. However, the MLVA typing proved to be a much easier and more rapid method for the analysis of E. coli O157 : H7 strain relatedness to identify transmission routes. Hence, our MLVA method would be a suitable technique for routine typing in many laboratories, including public health agencies, and even in hospitals.
SUMMARY: We surveyed β-lactamase-producing Escherichia coli from farm animals (chickens, pigs, and cattle) and raw retail meat in Shizuoka Prefecture, Japan. In total 305 E. coli isolates, 15 isolates collected from broilers, beef cattle, chicken meat, and pork meat, were found to contain β-lactamase genes encoding CTX-M-2, CTX-M-14, CMY-2, SHV-2, and/or TEM-1, whereas 7 possessed mutations in the ampC promoter region. The findings suggest that broilers are more important than other farm animals with regards to the surveillance of β-lactamase-producing E. coli in this region. Enterobacteriaceae that harbor β-lactamases, especially extended-spectrum β-lactamase (ESBL), are a significant pathogen in nosocomial infections and have become an international topic of concern in both farm animals and humans (1–4). Numerous hypotheses have suggested that resistant bacteria and/or resistant genes could spread between farm animals and humans. In Japan, ESBL- and class C βlactamase-producing Escherichia coli, Klebsiella spp., and Proteus mirabilis are often detected in humans (5–9). In farm animals, Shiraki et al. (10) first reported the isolation of CTXM-2-producing E. coli from cattle in Japan. Kojima et al. (11) reported the isolation of CTX-M-type-producing E. coli from broilers but not from cattle or pigs. Moreover, there has only been one report regarding the antibiotic resistance of E. coli isolates from raw chicken meat (12). The distribution of β-lactamase-producing E. coli in farm animals and food products therefore remains unclear in Japan. In this study we surveyed β-lactamase-producing E. coli in food-producing animals and raw retail meat obtained from farms and markets located in Shizuoka Prefecture in Japan. Livestock feces (from broilers, layers, pigs, cows, and beef cattle) were collected in farms, and raw meat (chicken, pork, and beef) was obtained from supermarkets or meat markets in Shizuoka Prefecture in the period 2004–2006. The fecal samples were placed onto DHL agar (Nissui, Tokoy, Japan) plates and incubated aerobically at 37°C overnight to allow E. coli growth. Meat samples (25 g of each) were suspended in Bacto buffered peptone water (Difco, Detroit, Mich., USA) and incubated at 37°C for 18 h. The cultures were then spread onto Chromocult Coliform Agar ES (Merck, Darmstadt, Germany) plates and further incubated at 37°C for 24 h. The antimicrobial susceptibilities of the E. coli isolates were determined by the broth microdilution method using Dry Plate Eiken (Eiken, Tokyo, Japan). The Clinical Laboratory Standards Institute (CLSI) guidelines were followed with regard to inoculum standardization, medium and incubation conditions, and the selection of internal qualitycontrol organisms (E. coli ATCC 25922). All E. coli isolates from the samples were tested for resistance against cefotaxime (CTX), ampicillin (ABPC), gentamicin (GM), tetracycline (TC), chloramphenicol (CP), ciprofloxacin (CPFX), fosfomycin (FOM), and imipenem (IPM). Potential ESBLproducing E. coli with low susceptibility to CTX (MIC ≥2 μg/ ml) were selected and a phenotypic confirmatory test was conducted using CTX and ceftazidime, with or without clavulanic acid, according to the CLSI recommendations. The blaTEM, blaSHV, blaCTX-M-1, blaCTX-M-2, blaCTX-M-9, blaPSE-1,
