[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.

Genomic analysis of the evolutionary lineage of Norovirus GII.4 from archival specimens during 1975-1987 in Tokyo.

This study aimed to analyze NoV GII.4 sequences from archival specimens obtained during 1975-1987 by comparing them with reference sequences. The first NoV GII.P4_GII.4 sequence was identified in 1980. NoV GII.4 collected in 1970 had a GII.P1_GII.4 sequence. These results indicate that the GII.P4_GII.4 sequence may be the result of a recombination that might have occurred around 1980. Amino acid substitutions based on this replacement were mainly accumulated in the NTPase, p22, and RdRp regions. The emergence of GII.P4_GII.4 sequence is considered to have ended the major prevalence of NoV GII.4. J. Med. Virol. 89:363-367, 2017. © 2016 Wiley Periodicals, Inc.

[Two Outbreaks of Yersinia enterocolitica O:8 Infections in Tokyo and the Characterization of Isolates].

Although the number of outbreaks caused by Yersinia enterocolitica has been very small in Japan, 4 outbreaks were occurred during the 2 years between 2012 and 2013. We describe herein 2 outbreaks which were examined in Tokyo in the present study. Outbreak 1: A total of 39 people (37 high school students and 2 staff) stayed at a hotel in mountain area in Japan had experienced abdominal pain, diarrhea and fever in August, 2012. The Y. enterocolitica serogroup O:8 was isolated from 18 (64.3%) out of 28 fecal specimens of 28 patients. The infection roots could not be revealed because Y. enterocolitica was not detected from any meals at the hotel or its environment. Outbreak 2: A total of 52 students at a dormitory had diarrhea and fever in April, 2013. The results of the bacteriological and virological examinations of fecal specimens of patients showed that the Y. enterocolitica serogroup O:8 was isolated from 24 fecal specimens of 21 patients and 3 kitchen staff. We performed bacteriological and virological examination of the stored and preserved foods at the kitchen of the dormitory to reveal the suspect food. For the detection of Y. enterocolitica, food samples. together with phosphate buffered saline (PBS) were incubated at 4 degrees C for 21 days. Then, a screening test for Y. enterocolitica using realtime-PCR targeting the ail gene was performed against the PBS culture. One sample (fresh vegetable salad) tested was positive on realtime-PCR. No Y. enterocolitica was isolated on CIN agar from the PBS culture because many bacteria colonies other than Y. enterocolitica appeared on the CIN agar. After the alkaline-treatments of the culture broth or the immunomagnetic beads concentration method using anti-Y. enterocolitica O:8 antibodies, Y. enterocolitica O:8 which was the same serogroup as the patients' isolates was successfully isolated from the PBS culture. The fresh vegetable salad was confirmed as the incrimination food of this outbreak.

Four foodborne disease outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens.

The epidemiological and bacteriological investigations on four foodborne outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens are described. C. perfringens isolated from patients of these outbreaks did not produce any known enterotoxin and did not carry the C. perfringens enterotoxin gene. However, the culture filtrates of these isolates induced the accumulation of fluid in rabbit ileal loop tests. The molecular weight of the new enterotoxin may be between 50,000 and 100,000, although the known C. perfringens enterotoxin is ca. 35,000. This new enterotoxin was heat labile, and its biological activities were inactivated by heating for 5 min at 60°C. The new enterotoxin was sensitive to pH values higher than 11.0 and protease treatment but was resistant to trypsin treatment. These results suggest that the new enterotoxin may be a protein. Although C. perfringens enterotoxin induced morphological changes in Vero cells, the changes induced by the new enterotoxin differed from those by the known C. perfringens enterotoxin. The new enterotoxin also induced morphological changes in L929 cells, whereas the known C. perfringens enterotoxin did not, because L929 cells lacked an appropriate enterotoxin receptor. Although C. perfringens enterotoxin is recognized as the only diarrheagenic toxin responsible for C. perfringens foodborne outbreaks, the results of the present study indicate that C. perfringens isolated from these four outbreaks produced a new type of enterotoxin.

[Serovars and Drug-Resistance of Salmonella Strains Isolated from Domestic Chicken Meat in Tokyo (1992-2012)].

A total of 477 Salmonella strains isolated from retail domestic chicken meat during 1992-2012 in Tokyo, were examined regarding their serovars and drug-resistance. These strains were detected in 469 (29.8%) of 1,576 samples. The detection rate in every two years was 10.1% to 46.3% of the range. Serological typing results showed that 477 strains were classified into 22 serovars excepting 2 untypable strains. Among them, S. Infantis (312 strains) was the most prevalent, followed by II O4: b: [e, n, x] (S. II Sofia) (71 strains), S. Hadar (20 strains), S. Typhimurium (20 strains), S. Manhattan (12 strains), S. Schwarzengrund (9 strains), S. Agona (7 strains), and other 15 serovars (24 strains). Results of the antibacterial drug susceptibility test for 477 strains revealed that 89.9% was resistant to some of the 12 drugs tested, and multidrug-resistant strains accounted for 90.2% among them. The frequencies of resistance to each drug were 81.8%; 77.8%, 45.5%, 33.3%, 11.3%, 9.6%, 2.9%, 0.6%, 0.6% and 0.2%, in order with high frequency, for SM, TC, KM, ST, NA, ABPC, CP, FOM, CTX and CAZ, respectively. None of the strains was resistant to NFLX or IPM. Three CTX-resistant strains were CTX-M type extended-spectrum ß-lactamase (ESBL) producers, and the group of CTX-M type ESBL genes were CTX-M-2 group (2 strains) and CTX-M-9 group (1 strain). CAZ-resistant 1 strain was an ESBL producer, but the ESBL gene was not determined.

[Isolation of Neisseria elongata subsp. elongata Isolated from an Acute Myelogenous Leukemia Patient].

Gram-negative cocci with a rod-like shape were isolated from a blood sample of a patient with acute myelogenous leukemia (AML). The 16S rRNA sequence of the isolate was similar to that of Neisseria elongata. Because previous reports about N. elongata as a pathogen have been extremely rare, more reliable identification seemed to be needed. We thus additionally performed a Multilocus Sequencing Analysis (MLSA) based on another four regions (argF, rho, recA, glnA), and confirmed the identification of N. elongata. The results from the MLSA identified the species; however, we could not identify the isolates into subspecies from the sequences. Three subspecies of N. elongata (N. elongata subsp. elongata, N. elongata subsp. glycolytica and N. elongata subsp. nitroreducens) were classified based on three definitive characteristics (catalase possession, nitrite reducibility, and acid from glucose). The results of the tests of three characteristics supported the identification of the isolate as N. elongata subsp. elongata. Therefore we determined the isolate from the AML patient to be N. elongata subsp. elongata.

[An autochthonous outbreak of dengue type 1 in Tokyo, Japan 2014].

OBJECTIVES: An outbreak of autochthonous dengue fever was reported in August 2014, with cases suspected mainly from Yoyogi Park in Tokyo. This is the first epidemic of dengue fever in Japan since 1945. METHODS: From August to October 2014, the following measures were taken to control the outbreak: 1) risk communication and information sharing; 2) active case finding; 3) vector surveillance in affected sites; and 4) laboratory testing. We also reviewed the surveillance data as reported to the National Epidemiological Surveillance of Infectious Diseases during the 44 epidemiological weeks. results: An official dengue fever call center was set up temporarily for the general public and 3,005 calls were received. The Tokyo Metropolitan Government issued 39 press releases regarding patients and nine related to dengue virus (DENV) detection and vector control activities for the media. Confirmed autochthonous dengue fever cases were reported between the 35th and 44th epidemiological weeks. Out of 160 cases of outbreak, 108 (67.5%) confirmed cases were reported in Tokyo. The estimated illness onset dates were between August 9 and October 7, and estimated dates of infections were between August 3 and October 3, 2014. The data suggest that the infective mosquitoes had already been present in Yoyogi Park at the end of July 2014. During the weekly vector surveillance at Yoyogi Park, a total of 1,152 adult mosquitoes, of which 856 (73.3%) were Aedes mosquitoes, were collected over 11 weeks by a light trap with dry ice. DENV was detected from adult Aedes mosquito samples collected on the 2nd, 9th, and 16th of September, 2014. Serum samples from 240 suspected cases were examined at the Tokyo Metropolitan Institute of Public Health, and 78 were positive for the DENV NS1 antigen, DENV-specific IgM antibody, or DENV nucleic acid with reverse transcriptase polymerase chain reaction (RT-PCR) (NS1: 66 cases; IgM: 50 cases; PCR: 57 cases). Genetic analysis of DENV-positive serum and mosquito samples found all to be categorized as DENV-serotype 1 (gene type I). Phylogenetic analysis of the envelope protein genome sequence from patients and mosquitoes in Tokyo revealed more than 99% similarity with each other and with the strain from the first outbreak-associated patient in Saitama. CONCLUSION: Measures important for control of infectious disease epidemic were learned during this recent indigenous dengue outbreak in Tokyo. It also highlighted the importance of preparedness for epidemics of indigenous or imported infectious diseases, especially in light of the fact that Tokyo is in preparation for the Olympic and Paralympic Games in 2020.

Molecular epidemiological characterization of Staphylococcus aureus isolates originating from food poisoning outbreaks that occurred in Tokyo, Japan.

Staphylococcal food poisoning (SFP), one of the commonest food-borne diseases, results from the ingestion of one or more staphylococcal enterotoxins (SEs) produced in foods by Staphylococcus aureus. In the present study, 203 S. aureus strains originating from 83 outbreaks that had occurred in Tokyo were examined for their coagulase type and genotype of SEs to analyze their molecular epidemiological characteristics. The representative subsets of the 83 S. aureus isolates were analyzed by multilocus sequence typing (MLST) and S. aureus pathogenicity island (SaPI) scanning. The isolates were integrated into eight specific clonal complexes (CC) s; CC81, CC8, CC6, CC5, CC508, CC59, CC20 and CC30. The profiles of the coagulase type, SE/SEl genotype and the suspected type of enterotoxin-encoding mobile genetic element (MGE) indicated a correlation with each CC. SaPI scanning showed fixed regularity between the distributions of genomic islands, including SaPIs, and the phylogenetic lineage based on MLST. These results indicate that the S. aureus isolates, which classified into eight CCs, have distinguishable properties concerning specific coagulase type, enterotoxin genotype and MGE type. Strains of S. aureus harboring these particular elements possess the potential to cause SFP.

The 2011 measles outbreak in Tokyo. An analysis of surveillance data.

OBJECTIVES: The study was conducted with the intention of establishing a strategy to eliminate measles on the basis of an analysis of the epidemiological profile of measles cases reported in Tokyo during the year 2011. METHODS: We investigated measles cases reported to the Tokyo Metropolitan Government in 2011, recorded as part of the National Epidemiological Surveillance of Infectious Diseases. Factors analyzed included age, vaccination status for each patient, cases for which records were discarded after laboratory confirmation, genotype of the measles virus and relationships between dates of specimen collection and results of polymerase chain reaction (PCR) and IgM antibody tests. RESULTS: A total of 178 measles cases were reported in Tokyo during 2011, and the majority of cases (128, 71.9%) were reported during the peak period from epiweeks 13 to 24. The largest age group reported was one to four years of age (40, 22.5%) followed by groups of 20-29 and 30-39 years of age (both 34, 19.1%). Most cases were sporadic, with only six outbreaks occurring. Even then, the numbers of cases for each outbreak was less than five. More than half of the patients in all age groups, except for the 1-4-year-old group, had not been vaccinated or did not have a record of vaccination. Genotypes D4 and D9 of measles virus were detected in most cases. However, genotype D5, which had been circulating in Japan before 2008, was not detected. CONCLUSION: Imported viruses were the cause of measles cases reported in Tokyo during 2011. The disease control was better than that in 2007 and 2008 because of the swift and appropriate responses to the occurrences. It is also possible that there has been an increase in the proportion of people with immunity to measles. Increasing the rate of immunization, performing effective surveillance, and confirming suspicious measles cases by using molecular methods are important for achieving the elimination of measles.

Growth and Survival of Escherichia albertii in Food and Environmental Water at Various Temperatures.

Escherichia albertii is an emerging foodborne pathogen that causes diarrhea. E. albertii has been isolated from various foods, including pork and chicken meat, and environmental waters, such as river water. Although many food poisoning cases have been reported, there have been insufficient analyses of bacterial population behaviors in food and environmental water. In this study, we inoculated 2-5 log CFU of E. albertii into 25 g of pork, chicken meat, Japanese rock oyster, Pacific oyster, and 300 mL of well water and seawater at 4°C, 10°C, 20°C, and 30°C, and analyzed the bacterial population behavior in food and environmental water. After 3 days at 4°C, the population of E. albertii strain EA21 and EA24 in foods maintained approximately 4 log CFU/25 g. After 3 days at 10°C, the population of E. albertii strains in pork and oysters maintained approximately 4 log CFU/25 g, and that in chicken meat increased to approximately 5-6 log CFU/25 g. After 2 days at 20°C, E. albertii strains grew to approximately 6-7 log CFU/25 g in pork and chicken meat, and E. albertii strain EA21 but not EA24 grew to 4.5 log CFU/25 g in Japanese rock oyster, E. albertii strain EA21 but not EA24 slightly grew to 3.1 log CFU/25 g in Pacific oyster. After 1 day at 30°C, E. albertii strains grew to approximately 7-8 log CFU/25 g in chicken meat and pork, grew to approximately 4-6 log CFU/25 g in Japanese rock oyster, and 6-7 log CFU/25 g in Pacific oyster. These results suggest that E. albertii survives without growth below 4°C and grew rapidly at 20°C and 30°C in foods, especially in meat. E. albertii strains did not grow in well water and seawater at 4°C, 10°C, 20°C, and 30°C. The population of E. albertii strains in well water and seawater decreased faster at 30°C than at 4°C, 10°C, and 20°C, suggesting that E. albertii has low viability at 30°C in environmental water.

Evaluation of the culture method NIHSJ-02 alternative to ISO 10272-1:2006 for the detection of campylobacter jejuni and campylobacter coli in chicken: collaborative study.

For the surveillance of the prevalence of Campylobacter jejuni and Campylobacter coli in raw chicken products in Japan, a qualitative method, National Institute of Health Sciences Japan (NIHSJ)-02, was developed as an alternative to International Organization for Standardization (ISO) 10272-1:2006. In the NIHSJ-02 culture method, the enrichment step is carried out in a reduced volume of Preston broth at 42 +/- 1 degrees C to reduce cost and space, and to prevent the overgrowth of background bacteria. To evaluate the performance of NIHSJ-02, a collaborative study was conducted, and the results obtained by NIHSJ-02 were compared with those obtained using the reference method, ISO 10272-1:2006. Fifteen laboratories participated; each examined 48 minced chicken samples consisting of test samples uninoculated, inoculated with C. jejuni at a low or high level, and inoculated with C. coli at a low level. The average probabilities of detection by NIHSJ-02 across laboratories were 0.033, 0.222, 0.678, and 0.267 in samples uninoculated, inoculated with C. jejuni at a low and high level, and with C. coli at a low level, respectively. Those by ISO 10272-1:2006 were 0.051, 0.128, 0.551, and 0.090. Significantly higher probabilities of detection were determined by NIHSJ-02 compared to ISO 10272-1:2006, except for uninoculated samples. On the other hand, significantly lower frequency of occurrence of background bacteria was observed by NIHSJ-02 (43.1%) compared with ISO 10272-1:2006 (92.6%). NIHSJ-02 showed better performance than ISO 10272-1:2006 with regard to the selective detection of C. jejuni and C. coli in chicken.

Comparison of the Serovars and Characteristics of Salmonella Isolated from Human Feces and Foods in the 1990s and 2010s in Tokyo.

Salmonella foodborne disease outbreaks have markedly decreased in recent years, and different Salmonella serovars have been isolated. To clarify the characteristics of Salmonella strains causing annual epidemics and to estimate the source, we conducted a serotyping test on 1,132 human-derived Salmonella isolates in the 1990s and 2010s, and 1,061 food-derived Salmonella isolates in the 2010s in Tokyo. The serovars commonly isolated from human feces in the 1990s and after 2012 were S. Enteritidis, S. Typhimurium, S. Infantis, S. Thompson, and S. Agona. The new main serovars isolated after 2012 were S. Schwarzengrund, S. Enterica serovar 4:i:-, and S. Chester. In contrast, the main serovars detected from foods after 2012 were S. Infantis, S. Schwarzengrund, S. Agona, S. Manhattan, S. Typhimurium, and S. enterica serovar UT: r:1,5. S. Schwarzengrund has recently been frequently isolated. These strains were mainly isolated from chicken meat and offal. It was suggested that the same serovars of human-derived isolates were also isolated from foods, especially chicken meat and offal, and that these were recently an important causative food of Salmonellosis.

[Current status of food-borne diseases in Japan].

The current status of food-borne diseases in Japan was described. Although the number of outbreaks caused by Salmonella, Vibrio parahaemolyticus and Escherichia coli(except enterohaemorrhagic E. coli) is decreasing, outbreaks by Campylobacter or Norovirus are increasing.

Detection of Escherichia albertii in Retail Oysters.

ABSTRACT: Escherichia albertii is an emerging foodborne pathogen. Owing to its distribution in river water, it is important to determine the presence of E. albertii in aquaculture-related foods. In this study, we investigated the distribution of E. albertii in retail oyster samples. A total of 427 raw oyster samples (385 Pacific oysters and 42 Japanese rock oysters) were enriched in modified Escherichia coli broth (mEC) or mEC supplemented with novobiocin (NmEC) at 42°C. The cultures were used for E. albertii-specific nested PCR assay, as well as for E. albertii isolation using deoxycholate hydrogen sulfide lactose agar (DHL), DHL supplemented with rhamnose and xylose, and MacConkey agar supplemented with rhamnose and xylose. The population of E. albertii in nested PCR-positive samples was determined using the most-probable-number (MPN) method. E. albertii isolates were subjected to biochemical and genetic characterization. E. albertii was detected in 5 (1.6%) of 315 Pacific oyster samples (one piece each), 2 (2.9%) of 70 Pacific oyster samples (25 g each), and 2 (4.8%) of 42 Japanese rock oyster samples procured from four geographically distinct regions. A total of 64 E. albertii strains were isolated from eight of the nine nested PCR assay-positive oyster samples, and the MPN value was under the detection limit (<3 MPN/10 g). A specific season or month for detecting E. albertii was not observed in this study, suggesting that the pathogen is present in seawater. All the E. albertii isolates, except one, were positive for the virulence factor eae, indicating that these isolates have the potential to infect humans.

Bacteriological and epidemiological characteristics of enterotoxigenic Escherichia coli isolated in Tokyo, Japan, between 1966 and 2009.

Enterotoxigenic Escherichia coli (ETEC) caused 131 outbreaks in Tokyo, Japan, between 1966 and 2009. The major serogroups were O6, O27, O148, and O159. The incidence of serogroups O25 and O169 recently increased. Heat-stable enterotoxin (ST) subtyping revealed that E. coli of serogroups O6, O15, O25, and O159 possessed the STh gene, whereas those serotyped as O27 and O169 possessed the STp gene.

[Phylogenetic analysis of rabies viruses isolated from animals in Tokyo in the 1950s].

Molecular epidemiological analysis of 96 rabies viruses isolated from animals in Tokyo in the 1950s involves Japanese fixed virus, Komatsugawa, Takamen, and Nishigahara strains. Strains isolated in Tokyo were divided into Tokyo 1 and Tokyo 2, and grouped into a worldwide distribution cluster differing from Takamen and Nishigahara. Tokyo 1 was grouped into the same cluster as viruses isolated from United States west coast dogs in the 1930s and 1940s. Tokyo 2 was grouped into the same cluster as the Komatsugawa strain, also known as a cluster of viruses from the Khabarovsk raccoon dog, and the Lake Baikal stepped fox in Russia. These findings suggest that 1950s Tokyo rabies viruses were related to those in Russia and the USA.

A collaborative study on a method to detect Salmonella in food.

Fourteen laboratories with expertise in Salmonella detection in food joined in a collaborative study. The laboratories performed qualitative analyses of ground pork samples using the proposed detection method. Salmonella Typhimurium (hydrogen sulfide-producing strain) and Salmonella Senftenberg (hydrogen sulfide-nonproducing strain) were used for inoculation. Three levels of Salmonella contamination were used for the study (0, 1-10, and 11-100 cfu/25 g). We evaluated the presence of Salmonella in each sample and the serological O group. Unmarked samples delivered to the laboratories were accurately judged to be inoculated or not inoculated with Salmonella at a 99.8% (419/420) detection rate in this collaborative study. The proposed method is suitable as a standard method to detect Salmonella in food.

[Colony-sweep PCR efficacy in food-borne outbreak associated with six types of enterotoxigenic Escherichia coli].

A box-lunch-associated food-borne outbreak occurred in Tokyo and Chiba Prefecture in June 2003 involved six types of enterotoxigenic Escherichia coli (ETEC). Fecal specimens from patients were screened for ETEC using colony-sweep polymerase chain reaction (PCR). Of the 84 fecal specimens examined, 56 (66.7%) were PCR-positive, i.e. 35 (41.7%) LT-gene-positive, 21 (25.0%) STp-gene-positive and 11 (13.1%) STh-gene-positive. Both of toxin-genes, i.e. LT and STp, LT and STh, STh and STp were positive in 11 patients. ETEC was isolated in confirmation testing from 48 (57.1%) fecal specimens. A single type of ETEC was isolated from 43 fecal samples. Serotype and toxin type of the isolates were O25:NM (LT) (21 samples), O27:H20 (STp) (12 samples), O148:H28 (STh) (8 samples), O25:NM (STh) (1 sample), and O27:7 (STp) (1 sample). Two types of ETEC were isolated from 5 fecal samples, i.e. O25:NM (LT) and O27:H20 (STp) (3 samples), O27:H20 (STp) and O148:H28 (STh) (1 sample), and O25:NM (LT) and O78:NM (STh) (1 sample).

Multiple-locus variable-number of tandem-repeats analysis distinguishes Vibrio parahaemolyticus pandemic O3:K6 strains.

A specific serotype of Vibrio parahaemolyticus, O3:K6, has recently been linked to epidemics of gastroenteritis in Southeast Asia, Japan, and North America. These pandemic O3:K6 strains appear to have recently spread across continents from a single origin to reach global coverage, based on profiling of strains by several molecular typing methods. In this study, variable-number tandem repeats (VNTR)-based fingerprinting was applied to clinical and environmental V. parahaemolyticus O3:K6 strains in an attempt to develop a molecular method with increased sensitivity for discriminating strains; the relative discriminatory powers were compared with ribotyping and pulsed-field gel electrophoresis (PFGE). All clinical strains tested were independent human isolates obtained from different outbreaks or from sporadic cases in Tokyo during the period from 1996 to 2003. Multiple-locus VNTR analysis (MLVA) was shown to have high resolution and reproducibility for typing of V. parahaemolyticus clones. MLVA analysis of 28 pandemic V. parahaemolyticus O3:K6 strains isolated from human cases produced 28 distinct VNTR patterns. The VNTR loci displayed between 2 and 15 alleles at each of eight loci with Nei's diversity index ranging from 0.35 and 0.91. These data demonstrated that MLVA is useful for individual strain typing of new O3:K6 strains, which appear to be closely related by other molecular methods.