Survival of Salmonella Enteritidis on four types of stainless steel surface under a dry condition and recovery by swabbing.

The survival and recovery of Salmonella Enteritidis inoculated on stainless steel surfaces with different metal contents and surface finishes were examined. Two S. Enteritidis strains possessing different levels of biofilm productivity were inoculated with tryptone soya broth (TSB) and egg yolk emulsion (EY) on the surface of stainless steel squares (1 cm × 1 cm) and stored at 22℃ under a dry condition. After storage, cells were recovered from the stainless steel surfaces by swabbing with a cotton swab. The numbers of cells recovered by swabbing and the cells remaining on the stainless steel squares were counted. The survival ratio of the strain possessing high biofilm productivity was greater than that of the strain possessing low biofilm productivity. The survival ratio of S. Enteritidis suspended in TSB was often higher than that in EY. There were no significant differences in the survival and recovery ratios of S. Enteritidis based on stainless steel composition or surface finish. From all except one sample, more than 98% of viable cells of S. Enteritidis were recovered by swabbing with a cotton swab.

Decontamination of Vibrio parahaemolyticus in fish by washing with hygienic seawater and impacts of the high level contamination in the gills and viscera.

The effect of washing in Vibrio parahaemolyticus contaminated and hygienic seawater on fish, and the frequency and level of natural V. parahaemolyticus contamination in fish were investigated. In the first experiment, live horse mackerel was experimentally kept in seawater artificially contaminated with V. parahaemolyticus. After washing in contaminated and hygienic seawater, the contamination in fish was quantitatively analyzed. Washing fish in the seawater contaminated with V. parahaemolyticus increases the contamination level on the surface and in the gills of the fish. Washing in hygienic seawater was effective in reducing the contamination in fish and cutting board surfaces, but not in the gills or viscera. In the second experiment, natural V. parahaemolyticus contamination in various fish caught by us was analyzed. V. parahaemolyticus was detected in 6 of 28 gill samples and 10 of 28 viscera samples of naturally contaminated fish. The means of V. parahaemolyticus level on gills were 3.3 and 3.9 log cfu/g, and those in viscera were 2.6 and 4.4 log cfu/g by culture method and a real-time PCR assay, respectively. These results indicate that the gills and viscera are able to spread the pathogens to fish meat as well as fish surface contamination by washing in the contaminated seawater. Washing with hygienic seawater and control of contamination from gills and viscera are critically important to prevent V. parahaemolyticus infections.

Antibiotic resistance in bacterial pathogens from retail raw meats and food-producing animals in Japan.

To determine the prevalence and antimicrobial susceptibility profiles of Campylobacter, Salmonella, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and vancomycin-resistant enterococci (VRE) in food-producing animals and retail raw meats in Japan, raw meat samples as well as food-producing animal feces, cutaneous swabs, and nasal swabs collected from 2004 to 2006 were analyzed. Isolation rates of Campylobacter jejuni and Campylobacter coli, Salmonella, and S. aureus were 34.6% (363 of 1,050), 2.7% (28 of 1,050), and 32.8% (238 of 725), respectively. MRSA was isolated from 3% (9 of 300) of meat samples. No VRE were isolated in this study. Antibiotic resistance in C. coli was higher than that in C. jejuni. Three C. jejuni isolates from a patient with diarrhea in a hospital of Shizuoka Prefecture and two chicken samples that exhibited resistance to ciprofloxacin had identical pulsed-field gel electrophoresis patterns, suggesting that ciprofloxacin-resistant C. jejuni could have been distributed in meat. S. aureus isolates showed the highest level of resistance to ampicillin and tetracycline. Resistance to tetracycline in S. aureus isolates from beef was lower than that seen in isolates from chicken and pork (P < 0.01). This study revealed that the prevalence of MRSA and VRE were low in food-producing animals and retail domestic meats in Japan, although Campylobacter isolates resistant to fluoroquinolone and erythromycin were detected. The occurrence of antimicrobial-resistant pathogens should be monitored continuously to improve the management of the risks associated with antimicrobial drug resistance transferred from food-producing animals to humans.

Levels of Vibrio parahaemolyticus and thermostable direct hemolysin gene-positive organisms in retail seafood determined by the most probable number-polymerase chain reaction (MPN-PCR) method.

The incidence and levels of Vibrio parahaemolyticus and thermostable direct hemolysin gene (tdh)-positive organisms in retail seafood were determined. The most probable number-polymerase chain reaction (MPN-PCR) method using a PCR procedure targeting the species-specific thermolabile hemolysin gene (tlh) and tdh was used to determine the levels of V. parahaemolyticus and tdh-positive organisms, respectively. In seafood for raw consumption, V. parahaemolyticus was found in four (13.3%) of 30 fish samples, 11 (55.0%) of 20 crustacean samples, and 29 (96.7%) of 30 mollusc samples. Levels of V. parahaemolyticus were below 10(4) MPN/100 g in all fish and crustacean samples tested. However, they were above 10(4) MPN/100 g in 11 (36.7%) of the 30 mollusc samples. In all seafood for raw consumption, the level of tdh-positive organisms was below the limit of detection (< 30 MPN/100 g). In seafood for cooking, V. parahaemolyticus was found in 15 (75.0%) of 20 fish samples, nine (45.0%) of 20 crustacean sample, and 20 (100%) of 20 mollusc samples. Levels of V. parahaemolyticus were above 10(4) MPN/100 g in only three (15.0%) and one (5.0%) of the 20 fish and 20 crustacean samples, respectively. However, they were above 10(4) MPN/100 g in 18 (90.0%) of the 20 mollusc samples. In seven (35.0%) of the 20 mollusc samples, tdh-positive organisms were found and their levels ranged from 3.6x10 to 1.1 x 103 MPN/100 g. From four of seven tdhpositive samples, tdh-positive V. parahaemolyticus was isolated.

[Quantitative detection of Vibrio vulnificus in seafood].

To quantify the number of Vibrio vulnificus in shellfish, we compared the most probable number (MPN) combined with a culture (MPN-culture) or polymerase-chain reaction (PCR) assay (MPN-PCR) to a quantitative PCR assay. Enrichment in alkaline peptone water by MPN was conducted at 25 and 35 degrees C. Enrichment at 35 degrees C was superior or similar to enrichment at 25 degrees C in over 65% of samples by MPNculture and in more than 75% of samples by MPN-PCR assay. V. vulnificus was more easily isolated on chromogenic agar medium during culture, MPN-PCR assay was superior or similar to MPNculture in over 90% of samples by enrichment at 25 degrees C and to over 88% of samples by enrichment at 35 degrees C. The number of V. vulnificus by quantitative PCR assay was similar to that of MPN-PCR assay in 6 of 8 samples but not from MPNculture. V. vulnificus contamination was frequently detected in samples from Kyushu Island.

Campylobacter jejuni contamination on broiler carcasses of C. jejuni-negative flocks during processing in a Japanese slaughterhouse.

At a slaughterhouse, we determined the Campylobacter jejuni status of each broiler flock coming from different farms by culturing cecal contents of representative birds, and examined the prevalence of C. jejuni on carcasses during processing. Then, C. jejuni isolates from the carcasses were typed with RAPD PCR and compared with those from the cecal contents of a C. jejuni-positive flock. C. jejuni was not isolated from the carcasses of C. jejuni-negative flocks processed before the C. jejuni-positive flock, and C. jejuni was isolated from the carcasses of C. jejuni-negative flocks processed after the C. jejuni-positive flock. RAPD PCR type of the isolates from the carcasses of C. jejuni-negative flocks was the same as those from the cecal contents of the previously processed C. jejuni-positive flock. Our results suggest that the carcasses of the C. jejuni-negative flocks were contaminated with C. jejuni strains originating from the intestines of the previously processed C. jejuni-positive flock.

Evaluation of MPN method combined with PCR procedure for detection and enumeration of Vibrio parahaemolyticus in seafood.

Vibrio parahaemolyticus densities in spiked and naturally contaminated seafood samples were enumerated by the MPN method combined with a PCR procedure (MPN-PCR method) targeting the species-specific thermolabile hemolysin gene (tlh), and by the MPN method using subcultivation of alkaline-peptone-water (APW) enrichment culture on thiosulfate-citrate-bile-sucrose (TCBS) agar (MPN-TCBS method). In the samples spiked with both V. parahaemolyticus and V. alginolyticus, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were similar to, or higher than the numbers of spiked cells, whereas those enumerated by the MPN-TCBS method were below the numbers of spiked cells. In naturally contaminated seafood samples, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were higher than those by the MPN-TCBS method. In the case of the MPN-TCBS method, isolation of V. parahaemolyticus from some APW cultures was difficult because of the overgrowth of many colonies other than V. parahaemolyticus (e.g., V. alginolyticus) on TCBS agar. In contrast, the PCR technique could detect tlh from APW culture without isolation of V. parahaemolyticus, so the possibility of failing to obtain a positive result in APW culture by the MPN-PCR method was considered to be lower than that by the MPN-TCBS method. Furthermore, utilization of the PCR technique reduces the time and labor required for the biochemical identification tests used in the MPN-TCBS method. For the detection and enumeration of V. parahaemolyticus in seafood, especially for samples that show many colonies other than V. parahaemolyticus on TCBS agar, the MPN-PCR method may be more convenient and reliable than the MPN-TCBS method.

Survival and growth of enterotoxin-positive and enterotoxin-negative Clostridium perfringens in laboratory media.

The survival and growth characteristics of enterotoxin-positive and enterotoxin-negative Clostridium perfringens were compared. Spores of C. perfringens were heated and incubated in laboratory media to simulate the cooking and inadequate storage of cooked food. In our experiment, enterotoxin-positive and enterotoxin-negative spores were heated and incubated individually and it was found that spores of enterotoxin-positive strains were more heat-resistant than spores of the enterotoxin-negative strains. In another experiment, cocktails of enterotoxin-positive and enterotoxin-negative spores were heated and incubated. At the time of inoculation, the ratio of enterotoxin-positive spores to enterotoxin-negative spores was about 1:100. In the case of high-temperature heat treatment, the number of enterotoxin-negative spores decreased to below that of enterotoxin-positive spores after heating, and enterotoxin-positive strains grew to high levels during incubation. In the case of low-temperature heat treatment, the number of enterotoxin-negative spores remained almost unchanged after heating and enterotoxin-positive strains did not grow to high CFU levels during incubation.