Bacillus cereus sometimes causes food poisoning, known as the diarrhoeal type associated with enterotoxin, and the emetic type associated with emetic toxin “Cereulide”. Standard methods for detection of cereulide are oral challenge in mammals, microscopic assessment of vacuole formation in HEp-2 cells and liquid chromatography-mass spectrometry assay; however, each method has some difficulties requiring specific facilities, skilled techniques or an expensive analyzer, respectively. We previously reported a colorimetric cell metabolic assay in HEp-2 cells using 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) - 2 H -tetrazolium, monosodium salt (WST-8), as an simple way to detect cereulide. In the present study, we compared and evaluated four methods: vacuolation, MTT, WST-8, and LC/MS usingB. cereus strains isolated from retail foods. Colorimetric cell metabolic assay in HEp-2 cells using WST-8 was confirmed as sensitive and useful for a general laboratory.

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Between April and October 2005, 120 raw minced meat samples comprising 40 pork, 40 beef, and 40 chicken samples purchased from 40 supermarkets in Hyogo (n=20) and Osaka (n=20) prefectures were examined for contamination with Staphylococcus aureus. The rate of isolation was 75.0% (30/40) for pork, 65.0% (26/40) for beef and 80.0% (32/40) for chicken meat. The most probable number (MPN) of the samples contaminated was widely distributed from 0.3 to>110/g. Seventy-nine (89.8%) of the positive samples had MPN values of less than 46/g. The majority of pork and beef isolates were classified as belonging to both human and K-β+CV: Abiotypes. Also, most chicken isolates belonged to both poultry and human biotypes. Seven (23.3%) of 30 pork isolates, 4 (15.4%) of 26 beef isolates and 9 (28.1%) of 32 chicken isolates produced one or two enterotoxins . Overall, the 20 enterotoxigenic isolates produced B (n=10), C (n=5), A (n=4) and AD (n=1). Enterotoxintype B was dominant in isolates from chicken meat. Eighty-two (93.2%) of the 88 isolates tested were differentiated into 8 coagulase types. All isolates except one (type I) were widely distributed from types II to VIII. Samples positive for S. aureus were detected in 38 of the 40 surpermarkets investigated. Interestingly, identical coagulase types, biotypes or enterotoxin types were found in isolates from all 2 or 3 kinds of meat samples retailed at te same supermarkets, indicating clonal spread of the same strain within meat-processing plants by cross-contamination.

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By using 181 Listeria spp. strains except for L. ivanovii, we evaluated the three distinct PCR methods targeting the iap genes encoding p60 proteins for genus or species identification, and the PCR-RFLP analysis for genotyping. Each of the two species-specific PCR methods yielded the expected amplicons from all of the 126 L. monocytogenes and 43 L. innocua strains tested. The genus-specific PCR method amplified the target regions (about 1.4 kb) from 178 Listeria strains other than3 L. grayi strains tested. The PCR-RFLP analysis with either HhaI or HindIII showed distinct several patterns: 97L. monocytogenes strains divided into 2 genotypes (types I and II) with Hind III and 5 genotypes (types A, B, C, D and E) withHhaI. On the other hand, 43 L. innocua strains showed an identical genotype (type I with Hind III and type F withHhal). Bot h L. seeligeri and L. welshimeri strains showed a distinct genotype (type I withHind III and type G withHhal). The L. monocytogenes genotypes correlated with the serovars. All 4b strains exhibited one genotype (type I and type A). 1/2b strains exhibited type I and types A or B. In contrast, 1/2a strains exhibited type II and types C, D, or E. 1/2c strains exhibited only type II and type C. Thus, these results suggest that the PCR and PCR-RFLP methods can identify or estimate the genus and species of listerial strains rapidly and are useful tools to genotype Listeria spp. strains phylogenetically.

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The practicality and validity of the protein detection method were tested for selfinspection in an actual food process plant. The relationship between the results of the protein detection method based on the principle of protein error and microbial contamination causes were investigated. The protein detection method detected 40-80μg of protein pollution. The results of hygienic evaluation on a food processing line (omelet, Japanese-style confectionery, chicken, side dishes, vegetables, etc.) by using the protein detection method and microbial contamination causes (total viable number, coliform number, Escherichia coli, and Staphylococcus aureus) were compared. A total of 152 samples was tested; the agreement rate with each result was 94.7%, showing that the results of the protein detection method were similar to microbial contamination. Only 2.0% (3 samples) gave false-negative results; however, low-level microbial contamination of these samples was observed (<10² CFU/100 cm²). This study showed a high relationship between protein pollution and microbial contamination in an actual food processing plant. Also, the protein detection method is simple, rapid, requires no skill and is a technique to detect pollution in an actual food processing plant.

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With the newly designed LAMP primers for C. jejuni and C. coli, we examined Campylobacter in 134 samples of retail chickens, and the usefulness of the LAMP method in comparison with a culture method. Twenty-four samples (17.9%) were positive, and 99 samples (73.9%) were negative in both test methods; the consistency of both tests was high (91.8%). Among the samples showing inconsistent results, 10 samples (7.5%) were positive in the LAMP method and negative in the culture method; in contrast, only 1 sample (0.7%) was negative in the LAMP method and positive in the culture method. No significant difference was noted when the detection status was compared by chi-square test. Analysis of Campylobacter according to the types of materials showed that 5 of 23 chicken wing samples (21.7%) were positive by the LAMP method, while none by the culture method. For others, no marked difference was noted for thigh, liver, ground meat, breast, and chicken fillet by both test methods. In this study, the LAMP method showed excellent results comparable to those by the culture method and thus was a very useful test method for food sanitation, considering its characteristics such as convenience and rapidity.

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[in Japanese]

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