Susumu Kawasaki

Multiplex PCR for Simultaneous Detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in Meat Samples

A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. DNA detection sensitivity for this method was 10(3) CFU/ml for each pathogen. When this protocol was used for the detection of each of the above pathogenic bacteria in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 30 h. In the samples of naturally contaminated meat, Salmonella spp., L. monocytogenes, and E. coli O157:H7 were detected over the same time period. Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method, which suggests that the multiplex PCR is a reliable and useful method for rapid screening of meat products for Salmonella spp., L. monocytogenes, and E. coli O157:H7 contamination.

Efficacy of Acidified Sodium Chlorite Treatments in Reducing Escherichia coli O157:H7 on Chinese Cabbage

Efficacy of acidified sodium chlorite for reducing the population of Escherichia coli O157:H7 pathogens on Chinese cabbage leaves was evaluated. Washing leaves with distilled water could reduce the population of E. coli O157:H7 by approximately 1.0 log CFU/g, whereas treating with acidified chlorite solution could reduce the population by 3.0 log CFU/g without changing the leaf color. A similar level of reduction was achieved by washing with sodium chlorite solution containing various organic acids. However, acidified sodium chlorite in combination with a mild heat treatment reduced the population by approximately 4.0 log CFU/g without affecting the color, but it softened the leaves. Moreover, the efficacy of the washing treatment was similar at low (4 degrees C) and room (25 degrees C) temperatures, indicating that acidified sodium chloride solution could be useful as a sanitizer for surface washing of fresh produce.

Evaluation of a multiplex PCR system for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in foods and in food subjected to freezing.

Conventional culture methods were compared to a multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 from enrichment cultures of various types of artificially inoculated and naturally contaminated foods. The multiplex PCR assay was evaluated in 44 types of spiked food samples, including meat, produce, fish, and dairy products targeting genes specific for each pathogen for simultaneous detection. The sensitivity of the assay was <or=5 CFU/25 g of inoculated sample after 20 hours of enrichment. The PCR assay was also evaluated in inoculated food samples stored at -20 degrees C for 2 weeks or 2 months. Out of 28 food samples tested, 27, 27, and 26 samples were positive for Salmonella Enteritidis, L. monocytogenes, and E. coli O157:H7, respectively, using the multiplex PCR assay, whereas only 13, 26, and 20 samples were positive, respectively, using the culture method after 2 weeks of storage at -20 degrees C. Similar results were obtained for samples stored at -20 degrees C for 2 months. The multiplex PCR assay method was capable of detecting 5 colony-forming units of each of the three pathogens per 25 g of more than 40 types of food, and the detection rate of the PCR assay was higher than that of conventional culture methods. As a result, the multiplex PCR assay is a valuable method for simultaneous rapid screening for the three pathogens in food, even after frozen storage.

Multiplex Real-Time Polymerase Chain Reaction Assay for Simultaneous Detection and Quantification of Salmonella Species, Listeria monocytogenes, and Escherichia coli O157:H7 in Ground Pork Samples

Salmonella sp., Listeria monocytogenes, and Escherichia coli O157:H7 are foodborne pathogens capable of causing serious gastrointestinal illness. We previously described simultaneous detection of these pathogens by multiplex polymerase chain reaction (PCR) in 44 types of spiked food samples, including meat, produce, fish, and dairy products, targeting genes specific for each pathogen. Based on the previous work, a multiplex real-time PCR assay using fluorescent probes was developed to detect and accurately quantify Salmonella sp., L. monocytogenes, and E. coli O157:H7 in ground pork samples. The detection sensitivity for this method was 2.0 x 10(2) CFU/mL for each pathogen, and the quantification range was 10(2)-10(7) CFU/mL with a high correlation coefficient (R(2) > 0.99) and high PCR efficiency (84.2% to 99.2%). When this protocol was used for the detection of each of the pathogens in spiked pork samples, one cell per 25 g of inoculated sample after enrichment for 20 h could be detected within 24 h. As a result, this multiplex real-time PCR assay will be valuable as a screening method for foods contaminated with these pathogens.

Survival of Escherichia coli O157:H7, Salmonella Enteritidis, Staphylococcus aureus, and Listeria monocytogenes in Kimchi

The survival of gram-positive and gram-negative foodborne pathogens in both commercial and laboratory-prepared kimchi (a traditional fermented food widely consumed in Japan) was investigated. It was found that Escherichia coli O157:H7, Salmonella Enteritidis, Staphylococcus aureus, and Listeria monocytogenes could survive in both commercial and laboratory-prepared kimchi inoculated with these pathogens and incubated at 10 degrees C for 7 days. However, when incubation was prolonged, the S. aureus level decreased rapidly from the initial inoculum level to the minimum detectable level within 12 days, whereas Salmonella Enteritidis and L. monocytogenes took 16 days to reach similar levels in commercial kimchi. On the other hand, E. coli O157:H7 remained at high levels throughout the incubation period. For laboratory-prepared kimchi, the S. aureus level decreased rapidly from the initial inoculum level to the minimum detectable level within 12 days, and L. monocytogenes took 20 days to reach a similar level. E. coli O157:H7 and Salmonella Enteritidis remained at high levels throughout the incubation period. The results of this study suggest that the contamination of kimchi with E. coli O157:H7, Salmonella Enteritidis, S. aureus, or L. monocytogenes at any stage of production or marketing could pose a potential risk.

Calcinated calcium killing of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on the surface of tomatoes.

This study was conducted to evaluate the efficacy of calcinated calcium, 200 ppm chlorine water (1% active chlorine), and sterile distilled water in killing Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on the surfaces of spot-inoculated tomatoes. Inoculated tomatoes were sprayed with calcinated calcium, chlorinated water, or sterile distilled water (control) and hand rubbed for 30 s. Populations of E coli O157:H7, Salmonella, and L. monocytogenes in the rinse water and in the residual (0.1% peptone) wash solution were determined. Treatment with 200 ppm chlorine and calcinated calcium resulted in 3.40- and 7.85-log10 reductions of E. coli O157:H7, respectively, and 2.07- and 7.36-log10 reductions of Salmonella, respectively. Treatment with 200 ppm chlorine and calcinated calcium reduced L monocytogenes numbers by 2.27 and 7.59 log10 CFU per tomato, respectively. The findings of this study suggest that calcinated calcium could be useful in controlling pathogenic microorganisms in fresh produce.

A survey of iceberg lettuce for the presence of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in Japan.

No information has been available on the prevalence of pathogens in fresh produce in Japan. In the present study, information was collected on the occurrence of contamination by Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in iceberg lettuce in a Japanese retail store. A total of 419 samples of lettuce that had been harvested in different districts and/or by different producers from July 2008 to March 2009 were examined. A multiplex PCR method was used to simultaneously identify the three bacterial pathogens. No pathogenic bacteria, including Salmonella, E. coli O157:H7, and L. monocytogenes, were detected from any of the samples with this highly sensitive and validated procedure. The aerobic bacteria plate counts and coliform bacteria counts in lettuce throughout the examination period did not show any seasonal trends, and the numbers were comparable to those reported by others from around the world. Based on the results of this study, we concluded that none of the three major pathogens were present in this limited survey of iceberg lettuce sold by a retailer in Japan.

Prewashing with Acidified Sodium Chlorite Reduces Pathogenic Bacteria in Lightly Fermented Chinese Cabbage

Efficacy of prewashing with acidified sodium chlorite (ASC) for the sanitation of lightly fermented Chinese cabbage was evaluated. The population of the natural microflora on the cabbage leaves was reduced about 2.0 log CFU/g just after washing with ASC, a significant reduction compared with the control distilled water wash (P < or = 0.05). In the control experiment, viable aerobic bacteria increased gradually when incubated at 10 degrees C; however, ASC-washed cabbage maintained a lower microbial concentration. The treatment of Chinese cabbage with ASC reduced the population of artificially inoculated Escherichia coli O157:H7, Salmonella Enteritidis, Staphylococcus aureus, and Listeria monocytogenes by 2.4 log CFU/g. The sanitation efficacy of ASC was 1.6 log CFU/g higher than that of distilled water washing. The viable cell counts of all pathogenic bacteria tested remained constant during 8 days of storage at 10 degrees C for both washing treatments, with the exception of L. monocytogenes, whose viable cell counts increased gradually with time for both treatments. No significant differences in color, odor, taste, and texture in raw leaves were observed after the ASC wash compared with after the distilled water wash. These results indicate that prewashing with ASC could control bacterial growth in lightly fermented Chinese cabbage without changing the product quality.

Species-Specific Identification of Campylobacters by PCR-Restriction Fragment Length Polymorphism and PCR Targeting of the Gyrase B Gene

ABSTRACT PCR-restriction fragment length polymorphism (RFLP) analysis of a 960-bp fragment of the Campylobacter gyrB gene with either DdeI or XspI restriction enzymes generated unique digestion patterns for 12 different Campylobacter species. In addition, PCR assays using species-specific primer sets targeting gyrB were specific for the respective Campylobacter species. Therefore, PCR-RFLP analysis and species-specific PCR assays based on the gyrB gene provide valuable tools for rapid and unambiguous identification of the majority of Campylobacter species.

Comparison of the effectiveness of acidified sodium chlorite and sodium hypochlorite in reducing Escherichia coli.

This study was designed to compare the effectiveness of acidified sodium chlorite (ASC) and sodium hypochlorite (NaClO) in reducing several Escherichia coli strains isolated from different retail meat and fresh produce. Forty nonpathogenic E. coli strains were isolated and used in this study. A type strain of E. coli (JCM 1649) and four O157:H7 serotypes of E. coli (CR-3, MN-28, MY-29, and DT-66) were used as reference. In vitro assay results revealed that the viable cell counts of each isolated E. coli strain and control strains exhibited a reduction of ∼ 4.3 ± 0.9 log and 7.8 ± 1.7 log CFU/mL after a 3-minute exposure to 100 mg/L NaClO and 20 mg/L ASC (pH 4.6), respectively, at 25°C, when compared with the viable bacterial counts obtained from phosphate-buffered saline. The one exception was the flocs-forming strain, which showed a reduction of only 1.0 log CFU/mL with both disinfectants. However, reductions of only 1.7 ± 0.3 log and 1.9 ± 0.4 log CFU/g were observed in lettuce after 5 minutes of washing with NaClO and ASC, respectively. On the other hand, reductions of 1.6 ± 0.2 log and 1.6 ± 0.4 log CFU/g were observed in spinach after 5 minutes of washing with NaClO and ASC, respectively. No reduction in the population was observed after washing the inoculated, fresh-cut vegetables with distilled water only. No significant difference in the reduction of E. coli was observed among all the tested strains with both sanitizers in the in vivo assay. These data suggest that the tested sanitizers exhibit a similar reduction of the surface-attached E. coli on leafy vegetables irrespective of the strain source.