Prevalence and contamination levels of listeria monocytogenes in ready-to-eat foods in Tokyo, Japan.

We surveyed prevalence and contamination levels of Listeria monocytogenes in ready-to-eat foods between 2000 and 2012 in Tokyo. L. monocytogenes was isolated from 52 (1.7%) out of 2,980 samples. Comparing the prevalence in the study period, 2.2% were positive in the former period (2000-2005) and 1.2% in the latter (2006-2012). Using the most probable number (MPN) technique, 32 samples were contaminated with fewer than 0.3 L. monocytogenes/g, 10 samples with 0.3-1.0/g and 4 samples with more than 1.0/g (the maximum was 2.3/g). The most common serovar was 1/2a, followed by 1/2b, 4b and 1/2c. We revealed that ready-to-eat foods in Tokyo were contaminated with L. monocytogenes, although the contamination levels were low.

Multiplex PCR serogrouping of Listeria monocytogenes isolated in Japan.

PCR serogrouping methods were used to examine strains of L. monocytogenes isolated in Japan. Among 187 strains, 99.5% were classified into 4 PCR serogroups corresponding to conventional serotypes. Only one isolate had a new PCR profile, which may be a variant of serogroup IVb.

Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks.

There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins' gene(s) among the Genus Clostridium.

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.

[Discrimination of Aspergillus flavus group fungi using phylogenetic tree analysis and multiplex PCR].

We describe a simple method for discrimination of Aspergillus flavus group fungi, including aflatoxin (AF) producers, by means of molecular-biological analysis of 45 strains of A. flavus. First, 20 strains of A. flavus were compared using phylogenetic tree analysis based on the nucleotide sequences of ITS 1-5.8S-ITS 2 (ITS-1/2) and aflR-aflJ intergenic spacer (aflR/J-IGS). In this analysis, the tested strains were discriminated into 4 groups at the aflR/J-IGS region. Although ITS-1/2 region analysis could not discriminate between A. flavus (AF producers) and A. oryzae/A. flavus (AF nonproducers), aflR/J-IGS region analysis could discriminate between these groups. Moreover, 45 strains of A. flavus were compared by means of both phylogenetic tree analysis based on the aflR/J-IGS region and the conventional aflatoxin production test (culture method). The phylogenetic tree analysis of the tested strains was consistent with the findings of the culture method. In addition, 49 strains of A. flavus and related species (Aspergillus spp.) were tested by multiplex PCR with primers designed on the basis of the phylogenetic tree analysis. These results were consistent with phylogenetic tree analysis based on the aflR/J-IGS region for 41 strains.

Multiplex real-time PCR assays for the detection of group C rotavirus, astrovirus, and Subgenus F adenovirus in stool specimens.

Group C rotavirus (GCRV), astrovirus (AstV), and adenovirus (subgenus F AdenoV) are etiologic agents of acute nonbacterial gastroenteritis, which often represents community outbreaks. For the efficient detection of GCRV, AstV, and subgenus F AdenoV in stool specimens, a multiplex real-time PCR assay was developed to detect these three viruses simultaneously, with high sensitivity and specificity. In total, 8404 clinical specimens were collected between April 2008 and March 2011 and tested for GCRV, AstV, and subgenus F AdenoV by the multiplex real-time PCR, as well as for norovirus (NoV), sapovirus (SaV), and group A rotavirus (GARV) by non-multiplex real-time PCR. Forty-one specimens were positive for GCRV, AstV, or subgenus F AdenoV, including 15 specimens that were also positive for NoV, SaV, or GARV. Multiple viruses were detected simultaneously in 29 out of 4596 (0.63%) specimens infected with at least one virus. The association rates of AstV and subgenus F AdenoV with other viruses were significantly higher than those of NoV, SaV, GARV, or GCRV.

Development of a method for detecting Coxiella burnetii in cheese samples.

Coxiella burnetii is the causative agent of Q fever, and the main route of infection in humans is inhalation of contaminated aerosols. Although oral transmission by contaminated raw milk or dairy products is also a possible route of human infection, there have been few studies investigating the presence of C. burnetii in dairy products. We developed a new method of extracting DNA from cheese and detecting C. burnetii DNA in cheese samples with a nested PCR assay. The limit of detection was 6.0 × 10(2) C. burnetii particles per gram. We subsequently used this method to examine the presence of C. burnetii in cheese at commercial markets in Tokyo from June 2005 to December 2008. Twenty-eight of 147 cheese samples were found to be positive for C. burnetii DNA. However, when we assessed the viability of C. burnetii by inoculating mice with DNA-positive samples, all of the samples were found to be negative. Thus, the viability of C. burnetii appears to have been lost in these cheese samples.

Antimicrobial susceptibilities of Listeria monocytogenes isolated in Japan.

The antimicrobial susceptibility of 201 Listeria monocytogenes isolates from foods, environments, animals and human patients in Japan was determined. All isolates were susceptible to ampicillin, the first choice of drug for listeriosis treatment, chloramphenicol, dihydrostreptomycin, erythromycin, enrofloxacin, gentamicin, kanamycin, lincomycin, nosiheptide, salinomycin, vancomycin, and virginiamycin. A human strain was resistant to oxytetracycline. The Minimum Inhibitory Concentration (MIC) for 50% of the strains and the MIC for 90% of the strains were comparable in all the isolates. This is the first investigation to compare antibiotic resistances between isolates from foods and isolates from human patients in Japan. The result showed that most of the isolates were susceptible to antibiotics used in this study.

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.

[Detection of norovirus RNA in bivalve molluscs by using bacteria-culture-employed method (A3T method)].

Norovirus (NV) RNA has rarely been detected in foods despite the use of highly sensitive methods such as RT-PCR and real-time RT-PCR. In the modified method (A3T method) reported previously, a bacterial culture process was introduced into the standard protocol for NV detection to remove some inhibitor(s) present in food ingredients. To confirm the efficiency of the A3T method and to examine NV contamination in bivalve molluscs, we tried to detect NV RNA in bivalve molluscs on the market and in oyster samples associated with foodborne outbreaks by using the standard method and the A3T method. NV RNAs were detected in 20 samples (18.0%) of 111 bivalve molluscs, including oysters, on the market by use of the A3T method, while only one sample (0.9%) was positive according to the standard method. NV RNA was also detected in 10 of 35 oyster samples related to foodborne outbreaks by the A3T method. Those results show that the A3T method is suitable for the detection of NV in bivalve molluscs in general laboratories.

[Development of effective detection method for Coxiella burnetii in mayonnaise by real-time PCR and investigation of C. burnetii contamination in commercial mayonnaise in Tokyo].

A PCR method for the effective detection of Coxiella burnetii in commercially available mayonnaise was developed. Sample preparations were isolated from 50 g portions of each mayonnaise product by four successive extraction steps in phosphate buffer with 2.0 M NaCl. These extracts were then centrifuged at 20,000 x g for 60 min. DNA was isolated from the solution containing the precipitate with a commercial kit, and amplified quantitatively using real-time PCR that targeted the com1 region of C. burnetii. The recoveries of C. burnetii from 2 kinds of commercial mayonnaise specimens, with a baseline control of 1 x 10(7) particles of the Nine Mile phase II strain, were 85.0 +/- 6.0% and 72.0 +/- 0.4%, respectively. The determination limit of this method was 500 C. burnetii particles per 50 g of mayonnaise. The DNA specimens isolated from 50 different commercial mayonnaise samples sold in Tokyo using this method were amplified using both nested PCR and real-time PCR. No contamination by C. burnetii was detected in any of the mayonnaise samples.

[Investigation of Coxiella burnetii contamination in commercial milk and PCR method for the detection of C. burnetii in egg].

A total of 244 milk samples collected from supermarkets in Tokyo were examined for contamination with Coxiella burnetii. C. burnetii DNA was detected in 131 (53.7%) of the samples by nested PCR. PCR-positive samples were injected into immunosuppressed A/J strain mice. Of the 22 PCR-positive milk samples tested, none resulted in isolation of C. burnetii from the mice. Heat-treatment was sufficient to inactivate C. burnetii in commercial milk. In addition, a PCR detection method for C. burnetii in chicken egg was developed. Egg yolk was added to an equal volume of 1 mol/L of NaCl phosphate buffer and homogenized for removal of protein and lipid. After centrifugal separation, the supernatant was removed, and template DNA in the precipitate was extracted using SDS, proteinase K and NaI. Using such prepared samples, 3.2 x 10(1) C. burnetii particles in 1 g of egg yolk could be detected by nested PCR. All of 200 chicken egg samples collected from supermarkets in Tokyo were negative for C. burnetii by the nested PCR method.