An interlaboratory study on the detection methods for enterotoxigenic Escherichia coli in vegetables using enterotoxin gene screening and selective agars for ETEC-specific isolation.

Enterotoxigenic Escherichia coli (ETEC) causes acute diarrhea and is transmitted through contaminated food and water; however, systematic procedures for its specific detection in foods have not been established. To establish an efficient detection method for ETEC in food, an interlaboratory study using ETEC O148 and O159 as representative serogroups was first conducted with 13 participating laboratories. A series of tests including enrichment, real-time PCR assays, plating on selective agars, and concentration by immunomagnetic separation followed by plating onto selective agar (IMS-plating methods) were employed. This study particularly focused on the detection efficiencies of real-time PCR assays for enterotoxin genes (sth, stp, and lt), IMS-plating methods, and direct plating onto sorbitol MacConkey agar and CHROMagar STEC medium, supplemented with tobramycin, which is a novel modification in the preparation of a selective agar. Cucumber and leek samples inoculated with ETEC O148 and O159, either at 4-7 CFU/25 g (low levels) or at 21-37 CFU/25 g (high levels) were used as samples with uninoculated samples used as controls. At high inoculation levels, the sensitivities of sth, stp, and lt detection, direct-plating, and IMS-plating methods in cucumber inoculated with O148 and in both foods inoculated with O159 were 100%. In leek inoculated with high levels of O148, the sensitivities of sth, stp, and lt detection, direct-plating, and the IMS-plating method were 76.9%, 64.1%, and 74.4%, respectively. At low inoculation levels, the sensitivities of sth, stp, and lt detection, direct plating, and IMS-plating method in cucumber inoculated with O148 and in both foods inoculated with O159 were in the range of 87.2-97.4%. In leek inoculated with low levels of O148, the sensitivities of sth, stp, and lt detection, direct plating, and the IMS-plating method were 59.0%, 33.3%, and 38.5%, respectively. Thus, ETEC in food contaminated with more than 21 CFU/25 g were detected at high rate (over 74%) using real-time PCR assays and IMS-plating onto selective agar. Therefore, screening sth, stp, and lt genes followed by isolation of STEC using the IMS-plating method may be an efficient method for ETEC detection.

Discrimination between edible and poisonous mushrooms among Japanese Entoloma sarcopum and related species based on phylogenetic analysis and insertion/deletion patterns of nucleotide sequences of the cytochrome oxidase 1 gene.

Entoloma sarcopum is widely known as an edible mushroom but appears morphologically similar to the poisonous mushrooms E. rhodopolium sensu lato (s. l.) and E. sinuatum s. l. Many cases of food poisoning caused by eating these poisonous mushrooms occur each year in Japan. Therefore, they were recently reclassified based on both morphological and molecular characteristics as sensu stricto species. In this study, we analyzed the nucleotide sequences of the rRNA gene (rDNA) cluster region, mainly including the internal transcribed spacer regions and mitochondrial cytochrome oxidase 1 (CO1) gene, in E. sarcopum and its related species, to evaluate performances of these genes as genetic markers for identification and molecular phylogenetic analysis. We found that the CO1 gene contained lineage-specific insertion/deletion sequences, and our CO1 tree yielded phylogenetic information that was not supported by analysis of the rDNA cluster region sequence. Our results suggested that the CO1 gene is a better genetic marker than the rDNA cluster region, which is the most widely used marker for fungal identification and classification, for discrimination between edible and poisonous mushrooms among Japanese E. sarcopum and related species. Our study thus reports a new genetic marker that is useful for detection of Japanese poisonous mushrooms, Entoloma.

A New Protocol for the Detection of Sterigmatocystin-producing Aspergillus Section Versicolores Using a High Discrimination Polymerase.

Aspergillus section Versicolores species, except Aspergillus sydowii, produce a carcinogenic mycotoxin sterigmatocystin (STC). Since these fungi are found in varied environmental milieu including indoor dust and food products, our aim was to develop a sensitive and convenient assay to detect STC producing fungal strains. We made use of a high discrimination DNA polymerase (HiDi DNA polymerase), for single nucleotide polymorphism (SNP)-based PCR amplification. Using specific primer pairs based on the SNPs between A. sydowii and other strains of Aspergillus section Versicolores, we succeeded in amplifying the genomic DNA all target strains except A. sydowii. These results confirm that the SNP-based PCR amplification technique, using a high discrimination DNA polymerase, was a reliable and robust screening method for target fungal strains.

Detection of Kudoa hexapunctata and Kudoa neothunni from retail raw tuna in Japan using a novel duplex polymerase chain reaction.

Kudoa hexapunctata was taxonomically separated from Kudoa neothunni, but their main host is tuna. K. hexapunctata has been identified as causative agent of foodborne diseases associated with the ingestion of raw Pacific bluefin tuna (PBT) in Japan, but K. neothunni has not. Therefore, it is clinically and epidemiologically important to detect and distinguish these two species. In the present study, we developed a novel duplex polymerase chain reaction (dPCR) targeting the 28S rRNA gene sequences of K. hexapunctata and K. neothunni. The dPCR amplified the desired genetic regions of each species, and the detection limit was 10 copies/reaction. A total of 36 retail tuna samples from different fishing ports were purchased and tested by dPCR. Thirty-one tested positive for K. hexapunctata and four tested positive for K. neothunni. Several retail PBT samples were examined in some of the fishing ports, and among these samples, the detection rates of K. hexapunctata was higher than 85%, and the rates were similar between wild and farmed PBT. The detection rates of K. hexapunctata in wild and farmed retail PBT were 75% and 71%, respectively, in May. However, the rates in June and July were 100% for both. K. hexapunctata and K. neothunni myxospores were not observed in the dPCR-positive samples, except in juvenile PBT, suggesting that the number of parasites was insufficient to cause foodborne disease. Thus, dPCR is a useful method for detecting and distinguishing K. hexapunctata and K. neothunni, and can be used in epidemiological studies of these parasites.

Yersinia infection in two captive guereza colobus monkeys (Colobus guereza).

Two guereza colobus monkeys (Colobus guereza) reared in a zoological garden in Japan suddenly died of multifocal fibrinonecrotic gastroenteritis and septicemia associated with infection by Yersinia spp. It was necessary to microbiologically differentiate Yersinia frederiksenii and Y. enterocolitica. We described the pathological findings and discuss the causal agent to emphasize the need to revert to using a combination of multiple examinations for diagnosis.

[Major Vehicles and O-Serogroups in Foodborne Enterotoxigenic Escherichia coli Outbreaks in Japan, and Effective Detection Methods of the Pathogen in Food Associated with An Outbreak].

Enterotoxigenic Escherichia coli (ETEC) is a common pathogen in developing countries, and causes foodborne infections through contaminated vegetables and water. ETEC also caused some foodborne infections in developed countries, though the vehicles are often unclear. We analyzed ETEC foodborne outbreaks in Japan based on the National Food Poisoning Statistics. Vegetables and private well water accounted for 50% and 22.2% of vehicles, respectively. The main vehicles were similar to those in developing countries. Serogroups of ETEC were also analyzed, and O6, O25, O27, O148, O153, O159, and O169 were the seven major O-serogroups. We investigated suitable detection methods for the pathogen (O148) in food samples associated with an outbreak of ETEC in Japan in 2011. We show that ETEC O148 could be effectively detected in cut leeks by means of a two-step enrichment and real-time PCR assay targeting heat-stable enterotoxin gene. Our survey of the vehicles and the major O-serogroups of ETEC outbreaks in Japan indicates that ETEC survives in the environment in Japan.

Distribution of Sterigmatocystin-producing Aspergilli in Japan.

Sterigmatocystin is a genotoxic and hepatocarcinogenic mycotoxin that contaminates foods and environments worldwide. Sterigmatocystin is produced as a precursor to aflatoxin B1 or as an end product by certain Aspergilli. Aspergillus section Versicolores is one of the major sections including sterigmatocystin-producing species and is thus a potential health and environmental hazard. Recently, the taxonomy of this section was revised and classified into 14 species on the basis of molecular phylogenetic analysis. However, investigation of the distribution and sterigmatocystin production of each species has been limited; in particular, its distribution in foods has been scarcely reported. In this study, we collected isolates of Aspergillus section Versicolores from various foods and environments in Japan and investigated their distribution and sterigmatocystin production. The isolates were classified into nine species or species groups, which revealed that A. creber, A. puulaauensis/tennesseensis and A. sydowii are the main species/species groups in Japan. In addition, A. versicolor sensu stricto was detected with some frequency, specifically in foods. Furthermore, the two species A. creber and A. versicolor sensu stricto frequently produced sterigmatocystin. It is therefore important for food safety to intensively monitor these two species and distinguish them from other species, especially A. sydowii, which is not considered to produce sterigmatocystin.

Fumonisin-production by Aspergillus section Nigri isolates from Japanese Foods and Environments.

Fumonisins are well known as mycotoxins produced by various Fusarium species. Recently Aspergillus niger has been reported to be a fumonisin B2 (FB2) producer. Aspergillus niger is a member of Aspergillus section Nigri. Members of this section are common food contaminants and are also distributed widely in the environment. This study aimed to determine 1) optimum culture conditions of A. niger for fumonisin production including growth medium, temperature and incubation period and 2) fumonisin production among isolates of Aspergillus section Nigri and closely related species isolated from Japanese food and environmental samples. Growth on Czapek yeast extract broth +5% NaCl (CYBS) at 28°C for 7 days resulted in the highest levels of FB2 production as determined by quantitative LC-MS/MS of culture extracts. Sixty-two isolates were collected from various foods in domestic markets as well as from soil and air. The isolates principally separated into two groups; A. niger and A. luchuensis/A. piperis/A. tubingensis, following molecular phylogenetic analysis. ELISA using the tip culture method was shown to be suitable for screening of the fumonisin-producing strains. Phylogenic analysis of Aspergillus section Nigri isolates from food and environmental samples indicated that fumonisin producing strains could be grouped into the A. niger clade. Nineteen of 35 (54%) isolates classified as A. niger were FB2 producers. The current study suggests that FB2-producing A. niger are distributed throughout several regions of Japan.

The Molecular Detection of Cryptosporidium and Giardia in Sika Deer (Cervus Nippon Centralis) in Japan.

Fecal specimens (271 samples) from wild deer, Cervus nippon centralis, were collected from nine different areas in Japan; these samples were subjected to a real-time reverse transcription PCR for Cryptosporidium-and Giardia-specific 18S ribosomal RNA to investigate the prevalence of Cryptosporidium and Giardia infection. The incidence of Cryptosporidium and Giardia in the nine areas ranged from 0% to 20.0% and 0% to 3.4%, respectively. The prevalence of Cryptosporidium among male and female deer was 8.1% and 3.9%, respectively, while that of Giardia was 0.7% and 0.8%. Sequence analysis identified the Cryptosporidium deer genotype, Cryptosporidium bovis, Cryptosporidium ryanae and Cryptosporidium meleagridis from the sequence of Cryptosporidium-specific partial 18S ribosomal RNA and Giardia intestinalis assemblage A from the partial sequence of Giardia-specific 18S rRNA. The variation in regional prevalence indicates that Cryptosporidium infection depends on environmental factors, and that bovine Cryptosporidium was detected more frequently than cervine Cryptosporidium. These data suggest that wild deer might be a healthy carrier of bovine Cryptosporidium.

Allergic Bronchopulmonary Mycosis due to Exposure to Eurotium herbariorum after the Great East Japan Earthquake.

BACKGROUND: Indoor mold levels typically increase after natural disasters, flooding, and water damage. Eurotium herbariorum is the sexual stage of Aspergillus glaucus. Case Presentation A 66-year-old, Japanese male, ex-smoker had been diagnosed with bronchial asthma when he was five years old; he achieved remission at the age of 13 years. He was displaced from his home during the Great East Japan Earthquake on March 11, 2011 and moved to temporary housing in Miyagi Prefecture in June 2011. He experienced the first episode of chest tightness, coughing, and wheezing in February 2012, when he again was diagnosed as having bronchial asthma. Mycofloral surveillance detected high counts of Eurotium in the air of his bedroom, kitchen, and living room, with a maximal fungal count of 163,200 colony-forming units per cubic meter (CFU/m3). Although Cladosporium and Penicillium typically predominate in the indoor air of residential dwellings, only low levels of these organisms were present in the patient's home. Morphologic identification confirmed the isolates as E. herbariorum. The patient had positive reactions to E. herbariorum in skin prick testing and the presence of antigen-specific precipitating antibodies to E. herbariorum. Computed tomography of the chest in August 2013 revealed central bronchiectasis and bronchial wall thickening. The patient experienced late reactions after provocation testing with E. herbariorum. CONCLUSION: This report presents the rare case of a patient who developed allergic bronchopulmonary mycosis (ABPM) due to exposure to E. herbariorum during temporary housing after the Great East Japan Earthquake. Oshikata C , Watanabe M , Saito A , Ishida M , Kobayashi S , Konuma R , Kamata Y , Terajima J , Cho J , Yanai M , Tsurikisawa N . Allergic bronchopulmonary mycosis due to exposure to eurotium herbariorum after the Great East Japan Earthquake. Prehosp Disaster Med. 2017;32(6):688-690.

Inhibitory Activities of Blasticidin S Derivatives on Aflatoxin Production by Aspergillus Flavus.

Blasticidin S (BcS) is a protein synthesis inhibitor which shows strong growth inhibitory activity against a number of microorganisms. However, BcS inhibited aflatoxin production by Aspergillus flavus without affecting its growth. In order to obtain information about the structure-activity relationship of BcS as an aflatoxin production inhibitor, BcS derivatives were prepared and their aflatoxin production inhibitory activities were evaluated. Among five derivatives, blasticidin S carboxymethyl ester, deaminohydroxyblasticidin S, and pyrimidinoblasticidin S showed inhibitory activity, while the others did not. The IC50 value for aflatoxin production of the carboxymethyl ester derivative was one-fifth of that of BcS although their antimicrobial activities were almost the same. These results indicate that the inhibitory activity of BcS against aflatoxin production was enhanced by esterification of its carboxyl group and that the carboxymethyl ester derivative might be more suitable for practical use than BcS because of the specificity of the carboxymethyl ester derivative, which inhibited aflatoxin production more than BcS.

Characterization of Shiga toxin-producing Escherichia coli from feces of sika deer (Cervus nippon) in Japan using PCR binary typing analysis to evaluate their potential human pathogenicity.

This study examined the potential pathogenicity of Shiga toxin-producing Escherichia coli (STEC) in feces of sika deer by PCR binary typing (P-BIT), using 24 selected STEC genes. A total of 31 STEC strains derived from sika deer in 6 prefectures of Japan were O-serotyped and found to be O93 (n=12), O146 (n=5), O176 (n=3), O130 (n=3), O5 (n=2), O7 (n=1), O96 (n=1), O116 (n=1), O141 (n=1), O157 (n=1) and O-untypable (n=1). Of the 31 STEC strains, 13 carried both stx1 and stx2, 5 carried only stx1, and 13 carried one or two variants of stx2. However, no Stx2 production was observed in 3 strains that carried only stx2: the other 28 strains produced the appropriate Stx. P-BIT analysis showed that the 5 O5 strains from two wild deer formed a cluster with human STEC strains, suggesting that the profiles of the presence of the 24 P-BIT genes in the deer strains were significantly similar to those in human strains. All of the other non-O157 STEC strains in this study were classified with strains from food, domestic animals and humans in another cluster. Good sanitary conditions should be used for deer meat processing to avoid STEC contamination, because STEC is prevalent in deer and deer may be a potential source of STEC causing human infections.

Shiga Toxin (Verotoxin)-producing Escherichia coli and Foodborne Disease: A Review.

Shiga toxin (verotoxin)-producing Escherichia coli (STEC) is an important cause of foodborne disease. Since outcomes of the infections with STEC have a broad range of manifestation from asymptomatic infection or mild intestinal discomfort, to bloody diarrhea, hemolytic uremic syndrome (HUS), end-stage renal disease (ESRD), and death, the disease is a serious burden in public health and classified as a notifiable infectious disease in many countries. Cattle and other ruminants are considered to be the major reservoirs of STEC though isolation of STEC from other animals have been reported. Hence, the source of contamination extends to a wide range of foods, not only beef products but also fresh produce, water, and environment contaminated by excretes from the animals, mainly cattle. A low- infectious dose of STEC makes the disease relatively contagious, and causes outbreaks with unknown contamination sources and, therefore, as a preventive measure against STEC infection, it is important to obtain characteristics of prevailing STEC isolates in the region through robust surveillance. Analysis of the isolates by pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) could help finding unrecognized foodborne outbreaks due to consumption of respective contaminated sources. However, though the results of molecular analysis of the isolates could indicate linkage of sporadic cases of STEC infection, it is hardly concluded that the cases are related via contaminated food source if it were not for epidemiological information. Therefore, it is essential to combine the results of strain analysis and epidemiological investigation rapidly to detect rapidly foodborne outbreaks caused by bacteria. This article reviews STEC infection as foodborne disease and further discusses key characteristics of STEC including pathogenesis, clinical manifestation, prevention and control of STEC infection. We also present the recent situation of the disease in Japan based on the surveillance of STEC infection.

Occurrence of beauvericin and enniatins in wheat flour and corn grits on the Japanese market, and their co-contamination with type B trichothecene mycotoxins.

The contamination levels of beauvericin and four enniatins, A, A1, B and B1, in 207 samples of wheat flour and corn grits on the Japanese market were determined by an analytical method based on LC-MS/MS. The toxins were extracted from samples with acetonitrile-water (85:15, v/v) and then purified with C18 cartridges. The method was validated in a single laboratory using spiked samples at two levels; the recovery of the five toxins ranged from 91.1% to 113.8%. Enniatin B was frequently detected in imported wheat flour (81.8%) and domestic wheat flour (85.6%), and the highest concentration of enniatin B was present in a domestic wheat sample (633 µg kg-1). In corn grits, beauvericin was found in 34% of the samples, but enniatins were not detected at all. The maximum concentration of beauvericin in corn grits was 26.1 µg kg-1. Deoxynivalenol and nivalenol in the same samples were determined by a method using an immunoaffinity column. Co-contamination of deoxynivalenol and enniatins was observed in 61% of the imported wheat samples and in 58% of the domestic wheat samples. These results suggest the need for a risk assessment for cyclic depsipeptide mycotoxins in Japan and a study on the synergistic effect of deoxynivalenol and enniatins.

Evaluation of Four Commercial Kits Based on Immunochromatography for Screening Aflatoxin M1 in Milk.

In Japan, a regulatory limit of 0.5 µg/kg was set in 2015 for aflatoxin M1 (AFM1) in milk. A method using an immunochromatographic kit has been adopted as the official screening method, and criteria for the kit have been set. In order to confirm whether commercial immunochromatographic kits for detecting AFM1 satisfy these criteria, the performance of four kits was evaluated by performing spike-and-recovery experiments using AFM1-free milk samples and milk samples spiked at seven levels (100-700 ng/kg). With the two qualitative kits, determinations of blank samples were all negative and those of the samples spiked at 500 ng/kg were all positive. With the two quantitative kits, the measured values of the blank samples were all less than 100 ng/kg and the recoveries of the samples spiked at 500 ng/kg were all more than 70%. The detection limits of the two quantitative kits were both less than 100 ng/kg. These results indicate that all four immunochromatographic kits meet the criteria and can be used to screen AFM1 in milk.

An interlaboratory study on efficient detection of Shiga toxin-producing Escherichia coli O26, O103, O111, O121, O145, and O157 in food using real-time PCR assay and chromogenic agar.

To establish an efficient detection method for Shiga toxin (Stx)-producing Escherichia coli (STEC) O26, O103, O111, O121, O145, and O157 in food, an interlaboratory study using all the serogroups of detection targets was firstly conducted. We employed a series of tests including enrichment, real-time PCR assays, and concentration by immunomagnetic separation, followed by plating onto selective agar media (IMS-plating methods). This study was particularly focused on the efficiencies of real-time PCR assays in detecting stx and O-antigen genes of the six serogroups and of IMS-plating methods onto selective agar media including chromogenic agar. Ground beef and radish sprouts samples were inoculated with the six STEC serogroups either at 4-6CFU/25g (low levels) or at 22-29CFU/25g (high levels). The sensitivity of stx detection in ground beef at both levels of inoculation with all six STEC serogroups was 100%. The sensitivity of stx detection was also 100% in radish sprouts at high levels of inoculation with all six STEC serogroups, and 66.7%-91.7% at low levels of inoculation. The sensitivity of detection of O-antigen genes was 100% in both ground beef and radish sprouts at high inoculation levels, while at low inoculation levels, it was 95.8%-100% in ground beef and 66.7%-91.7% in radish sprouts. The sensitivity of detection with IMS-plating was either the same or lower than those of the real-time PCR assays targeting stx and O-antigen genes. The relationship between the results of IMS-plating methods and Ct values of real-time PCR assays were firstly analyzed in detail. Ct values in most samples that tested negative in the IMS-plating method were higher than the maximum Ct values in samples that tested positive in the IMS-plating method. This study indicates that all six STEC serogroups in food contaminated with more than 29CFU/25g were detected by real-time PCR assays targeting stx and O-antigen genes and IMS-plating onto selective agar media. Therefore, screening of stx and O-antigen genes followed by isolation of STECs by IMS-plating methods may be an efficient method to detect the six STEC serogroups.

Association of Cell-adhesion Activities with Virulence in Shiga toxin-producing Escherichia coli O103：H2.

The characteristics of 11 strains of Stx1-producing and Stx2-non-producing STEC O103：H2 were analyzed to investigate the differences in virulence in a single serotype of Shiga toxin (Stx) -producing Escherichia coli (STEC). Differences in the cell-adhesion activity to Caco-2 cells were observed among the strains. The activity of the one strain, isolated from a patient with hemolytic uremic syndrome was 4-20-fold higher than those of the other strains. Although the strains with high cell-adhesion activity showed high expressions of eae, espB, espD, and tir in the locus of enterocyte effacement related with cell-adhesion, those were not specific for this strain. In addition, the Stx1 production level of the strain was not particularly high. It was indicated that the high adhesion activity might be a potential factor to associate serious symptom.

Characterization of enterohemorrhagic Escherichia coli O111 and O157 strains isolated from outbreak patients in Japan.

In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx2 and stx-negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx-negative E. coli O111 isolates were closely related to EHEC O111 stx2 isolates. Although the EHEC O157 strains had diverse stx gene profiles (stx1, stx2, and stx1 stx2), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx2 isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx2 to an stx-negative strain may have occurred during infection.

Structural determination of a nivalenol glucoside and development of an analytical method for the simultaneous determination of nivalenol and deoxynivalenol, and their glucosides, in wheat.

Trichothecene mycotoxins such as nivalenol and deoxynivalenol frequently contaminate foodstuffs. Recently, several trichothecene glucosides have been found in trichothecene-contaminated foods, and information about their chemistry, toxicity, and occurrence is required. In this study, a glucoside of nivalenol was isolated from nivalenol-contaminated wheat and was identified as nivalenol-3-O-ß-D-glucopyranoside. Analytical methods using a multifunctional column or an immunoaffinity column have been developed for the simultaneous determination of nivalenol, nivalenol-3-O-ß-D-glucopyranoside, deoxynivalenol, and deoxynivalenol-3-O-ß-D-glucopyranoside in wheat. The methods were validated in a single laboratory, and recovery from wheat samples spiked at four levels ranged between 86.4 and 103.5% for the immunoaffinity column cleanup. These mycotoxins in contaminated wheat samples were quantitated by the validated method. Nivalenol-3-O-ß-D-glucopyranoside was detected in the nivalenol-contaminated wheat, and the percentage of nivalenol-3-O-ß-D-glucopyranoside to nivalenol ranged from 12 to 27%. This result indicates that the analytical method developed in this study is useful for obtaining data concerning the state and level of food contamination by nivalenol, deoxynivalenol, and their glucosides.

Shiga Toxin (Verotoxin)-Producing Escherichia coli in Japan.

A series of outbreaks of infection with Shiga toxin (verocytotoxin)-producing Escherichia coli or enterohemorrhagic E. coli (EHEC) O157:H7 occurred in Japan in 1996, the largest outbreak occurring in primary schools in Sakai City, Osaka Prefecture, where more than 7,500 cases were reported. Although the reason for the sudden increase in the number of reports of EHEC isolates in 1996 is not known, the number of reports has grown to more than 3,000 cases per year since 1996, from an average of 105 reports each year during the previous 5-year period (1991-1995). Despite control measures instituted since 1996, including designating Shiga toxin-producing E. coli infection as a notifiable disease, and nationwide surveillance effectively monitoring the disease, the number of reports remains high, around 3,800 cases per year. Serogroup O157 predominates over other EHEC serogroups, but isolation frequency of non-O157 EHEC has gone up slightly over the past few years. Non-O157 EHEC has recently caused outbreaks where consumption of a raw beef dish was the source of the infection, and some fatal cases occurred. Laboratory surveillance comprised prefectural and municipal public health institutes, and the National Institute of Infectious Diseases has contributed to finding not only multiprefectural outbreaks but recognizing sporadic cases that could have been missed as an outbreak without the aid of molecular subtyping of EHEC isolates. This short overview presents recent information on the surveillance of EHEC infections in Japan.