We applied the polymerase chain reaction (PCR) to the detection of Escherichia coli and coliform bacteria in water and food. Primer sets targeting the transferase gene (wecF), the β-galactosidase gene (lacZ) and the 16S rRNA gene (E. coli 16S rRNA) of E. coli were used in the PCR, and the following results were obtained. 1) The frequencies of preservation of the wecF, lacZ and E. coli 16S rRNA genes were 12/12 (100%), 99/99 (100%) and 99/99 (100%), respectively, for E. coli from food and human feces, and 28/63 (44%), 70/177 (39%) and 0/177 (0%), respectively, for coliform bacteria including Klebsiella spp., Citrobacter spp. and others from food. 2) The detection frequencies of the wecF, lac and E. coli 16S rRNA genes from the BGLB culture of 69 samples of food (23 samples of meat and 46 samples of vegetables) were 73%, 65% and 37%, respectively. A significant correlation was found between the isolation of E. coli and the detection of the E. coli 16S rRNA gene, but no correlation was found between the isolation of coliform bacteria and the detection of the E . coli 16S rRNA gene. Also, no significant correlation was found between the isolation of coliform bacteria and the detection of the wecF or lacZgene. 3) A significant correlation between the detection of E. coli 16S rRNA and the isolation of E. coli was observed, when fecal contamination of 153 water samples from various sources of water supply were examined by the defined substrate method using colilert. These results show that PCR targeting the E. coli 16S rRNA gene is useful for rapid and specific detection of E. coli in water and food.
Sixty-four Staphylococcus aureus isolates from the hands of study participants, foodstuff, prepared food, cooking utensils, and cooking equipment at food preparation facilities were genotyped by Randomly Amplified Polymorphic DNA (RAPD) analysis, Biased Sinusoidal Field Gel Electrophoresis (BSFGE) and Pulsed-Field Gel Electrophoresis (PFGE). The results of the genotyping revealed that diffusion of S. aureus had occurred. S. aureus originating from the hands of a study participant was transmitted to a cooking knife and to prepared food (salad). Furthermore, another transmission was found by an unknown person between a frying pan and a faucet. The strains were divided into 11 different genetic types by RAPD analysis based on two primers, and 12 different genetic types by BSFGE. The PFGE types were completely consistent with the BSFGE types. The types obtained by both techniques were similar for all strains examined. However, in RAPD typing, 8 types (73%) were consistent with the PFGE types, but the other 3 types were not consistent. RAPD provided less discrimination than PFGE.
A ready-to-use and self-diffusible dry medium sheet culture system for coliform bacteria, the Compact Dry CF (CDCF) for food, was evaluated . The CDCF was shown to enumerate 26 different strains of coliform bacteria, and to discriminate coliform bacteria from noncoliform bacteria. Of the 26 coliform bacteria that grew on the CDCF, the 26 strains (100%) were blue-green colonies, whereas 8 Gram-negative bacteria other than coliform bacteria formed non-color colonies. Gram-positive bacteria and yeasts did not grow on the CDCF. The CDCF for coliform bacteria from 80 food samples was compared with the pourplate method using violet red bile agar (VRBA), desoxycholate agar (Deso), X-GAL agar (X-GAL), and the 3M PetrifilmTM CC (PCC) plate method. The correlation coefficients between the CDCF and VRBA, the CDCF and Deso, the CDCF and X-GAL, and the CDCF and the PCC were 0.97, 0.97, 0.97 and 0.99, respectively. The CDCF is useful for the enumeration of coliform bacteria in food and may be a possible suitable alternative to the conventional pour-plate or PetrifilmTM plate methods
To decide how to store food samples for a bacterial test of Shigella sonnei, its survival rate was assessed in saline and various foods such as canned young corn, raw oyster, raw ark shell, raw shrimp and raw cuttlefish at 4°C and -20°C for 13 days . The survival rate of S.sonnei in saline and in food uncontaminated with other bacteria at 4°C storage was better than that at -20°C. On the other hand, the survival of the organisms at -20°C storage was better than that at 4°C in the case of contaminated foods. We investigated the influence on S. sonnei by Escherichia coli using two new methods for S. sonnei. Briefly, they are methods of direct anaerobic culture and of anaerobic culture after aerobic enrichment culture established by the Japanese Ministry of Health and Welfare in 2002. In addition, we performed recovery experiments for S. sonnei using various foods such as canned young corn, raw oyster, raw ark shell and raw shrimp. The sensitivity of both methods was influenced by E. coli and/or other bacteria. Furthermore, the PCR method showed better sensitivity than the traditional incubation method in both culture methods. Although we were able to detect S. sonnei up to ≤10-1 CFU/g in the cases of uncontaminated food, the sensitivity was changed by the kind of foods in the cases of contaminated foods using both methods. These results suggest that either of the two methods and two storage conditions could be chosen depending on the kind of foods.
Since chicken and egg products are widely exported and imported, there is a distinct possibility that Avian Influenza Virus (AIV) may be transmitted and cause epidemics among poultry. There is the additional fear that AIV may become infective to humans. From the point of food safety and risk, we attempted to determine AIV survivability in egg products. We investigated the inactivation of AIV in mayonnaise, which is a typical and widely consumed egg product. A model mayonnaise was made of salad oil, vinegar, egg yolk and salt, similar to commercial products. The following isolates of AIV virus were used in this experiment: A / whistling swan/Shimane/499/83 (H5N3) (H5 AIV), A/whistling swan/Shimane/42/80 (H7N7) (H7 AIV) and A/duck/Hokkaido/26/99 (H9N2) (H9 AIV). After the model mayonnaise and AIV were mixed, H7 AIV and H9 AIV were inactivated immediately, and their infectivity titers decreased to under the detection limit . In the case of H5 AIV, the infectivity titer decreased from 105.0EID50/0.1 ml to under the detection limit after 30 min. These results demonstrated that different isolates of AIV were inactivated in mayonnaise. The inactivation of AIV may be caused by the chemical properties of mayonnaise, such as salt concentration, acid concentration and /or pH.
Halophilic histamine-producing bacteria (halophilic HPB) were isolated from 7 fresh fish and 5 processed fish, and enteric HPB from 8 fresh fish and 13 processed fish, out of 59 fish samples (20 fresh fish and 39 processed fish). Photobacterium phosphoreum was isolated from 6 of 10 fresh tuna samples, while Photobacterium damselae was isolated from only 1 of 10 other fresh fish samples. There was little difference in the isolate rate of the enteric HPB between the fresh and processed fish samples; however, the processed samples had the larger bacterial counts of enteric HPB than the fresh samples, on average. We suggest that histamine fish poisoning from fresh fish is mainly caused by P. phosphoreum, and that from processed fish is mainly caused by enteric HPB.