Predictive Growth Model of Listeria monocytogenes Under Fluctuating Temperature Conditions in Pasteurized Milk by Using Real-Time Polymerase Chain Reaction.

The aim of this study was to evaluate the application of real-time polymerase chain reaction (PCR)-based quantification as a rapid and accurate tool for the monitoring and prediction of Listeria monocytogenes growth in pasteurized milk under constant and fluctuating temperature conditions. The growth of L. monocytogenes was monitored under constant temperature conditions at 4°C, 10°C, 15°C, 20°C, and 35°C. High correlation was obtained between the bacterial growth rate and incubation temperature, where the R2 of the slope of the square root model was calculated to be 0.993 and 0.996 for real-time PCR and the conventional culture method, respectively. Moreover, the obtained maximum specific growth rate (µmax) data plots were correlated with 188 L. monocytogenes µmax data points from the existing model according to ComBase database, with an R2 of 0.961 for real-time PCR and of 0.931 for the conventional culture method. The growth models were examined under three different patterns of fluctuating temperature conditions ranging from 2°C to 30°C. The prediction results fell within ±20% of the relative error zone, showing that real-time PCR quantification could be used for fast, sensitive, and specific bacterial growth monitoring with high-throughput results. Real-time PCR should be considered a promising option and powerful tool for the construction of a bacterial growth prediction model for safety risk analysis in the dairy industry.

Predictive Modeling for the Growth of Salmonella Enteritidis in Chicken Juice by Real-Time Polymerase Chain Reaction.

The goals of this study were to monitor the growth kinetics of Salmonella Enteritidis in chicken juice using real-time polymerase chain reaction (PCR) and to evaluate its efficacy by comparing the results with an experimental database. Salmonella Enteritidis was inoculated in chicken juice samples at an initial inoculum of 104 CFU/mL with inoculated samples incubated at six different temperatures (10, 15, 20, 25, 30, and 35°C). Sampling was carried out for 36 h to observe the growth of Salmonella Enteritidis. The total DNA was extracted from the samples, and the copy number of the Salmonella invasion gene (invA) was quantified by real-time PCR and converted to Salmonella Enteritidis cell concentration. Growth kinetics data were analyzed by the Baranyi and Roberts model to obtain growth parameters, whereas the Ratkowsky's square-root model was used to describe the effect of the interactions between growth parameters and temperature on the growth of Salmonella Enteritidis. The growth parameters of Salmonella Enteritidis obtained from an experiment conducted at a constant temperature were validated with growth data from chicken juice samples that were incubated under fluctuating temperature conditions between 5°C and 30°C for 30-min periods. A high correlation was observed between maximum growth rate (µmax) and storage temperature, indicating that the real-time PCR-monitoring method provides a precise estimation of Salmonella Enteritidis growth in food material with a microbial flora. Moreover, the µmax data reflected data from microbial responses viewer database and ComBase. The results of this study suggested that real-time PCR monitoring provides a precise estimation of Salmonella Enteritidis growth in food materials with a background microbial flora.

Prevalence of foodborne pathogens in retailed foods in Thailand.

The consumption of foodborne pathogens contaminated in food is one of the major causes of diarrheal diseases in Thailand. The objective of this study was to evaluate the prevalence and types of contaminating bacteria in retailed foodstuffs in Thailand. Food from four categories (137 samples total), including meat (51 samples), vegetables (38 samples), fish or seafood (37 samples), and fermented food (11 samples), was purchased randomly from seven different open-markets and seven supermarkets in Thailand from August 2010 to March 2011. Seven types of major foodborne pathogens were identified using conventional culture methods. Approximately 80% of meat samples tested was contaminated with Salmonella spp. In contrast, the Salmonella spp. contamination rate of vegetable (5%) or fermented food (9%) samples was comparatively low. Six strains of Cronobacter sakazakii and two strains of Yersinia enterocolitica were also isolated. A substantially higher rate of contamination by Bacillus cereus was observed in fermented food (82%) than in samples of meat (2%) and fish or seafood (5%). Seven Listeria spp. isolates were obtained from meat and fish or seafood samples. Approximately 39% of samples tested were found to be contaminated with Staphylococcus spp. (54 isolates). The rate of bacterial contamination of meat did not depend on the type of market. However, the contamination rate of Staphylococcus spp. in vegetables was higher in open markets than in supermarkets, and the contamination rate of Salmonella spp. and Staphylococcus spp. in fish or seafood samples purchased in open markets was likewise higher than in those purchased in supermarkets. Therefore, improvement of hygienic practices throughout the food chain may be required to reduce the risk of food poisoning.

Disinfection of radish and alfalfa seeds inoculated with Escherichia coli O157:H7 and Salmonella by a gaseous acetic acid treatment.

Abstract The majority of seed sprout-related outbreaks have been associated with Escherichia coli O157:H7 and Salmonella. Therefore, we aimed to find an effective method to inactivate these organisms on seeds before sprouting. Treatment with 8.7% (v/v) acetic acid at 55°C for 2-3 h reduced the population of E. coli O157:H7 and Salmonella inoculated on alfalfa (Medicago sativa L.) and radish seeds (Raphanus sativus L.) by more than 5.0 log CFU/g, and a longer treatment time completely eliminated the E. coli O157:H7 population. The E. coli O157:H7 populations were reduced to an undetectable level with a gaseous acetic acid treatment for 48 h. After enrichment, no E. coli O157:H7 were found in the alfalfa and radish seeds (25 g). However, these treatments were unable to eliminate Salmonella in both seed types. No significant difference between the germination rates of treated alfalfa seeds and control seeds was found, and germination rates greater than 95% were obtained for the radish seeds. Although chlorine washing is commonly used for seed decontamination, chlorine washing at 200 and 20,000 ppm resulted in a reduction of pathogens by less than or equal to 3 log CFU/g. Therefore, these results suggested that gaseous acetic acid is more effective than chlorine washing in controlling pathogenic bacteria on sprout seeds.

Effectiveness of acidified sodium chlorite and other sanitizers to control Escherichia coli O157:H7 on tomato surfaces.

The use of a suitable sanitizer can reduce the risk of produce-related foodborne illnesses. We evaluated the effectiveness of several sanitizers to reduce inoculated Escherichia coli O157:H7 on the surface of cherry tomatoes (Solanum lycopersicum var. cerasiform). Depending on the method of inoculation (dipping/spotting), each of 80 g (eight tomatoes) of inoculated cherry tomatoes was washed in 400 mL of sanitizer solutions or 400 mL distilled water for 5 minutes. The effectiveness of sanitizers on spot-inoculated E. coli O157:H7 on tomato surfaces was found higher than on dip-inoculated tomatoes. Washing with water or chlorine water (0.1 g/L as free chlorine) could reduce 1.3 log CFU/g of E. coli O157:H7 in dip-inoculated (6.8 log CFU/g) tomatoes. Washing with lactic acid (LA) solution (1.0 g/L), phytic acid solution (1.0 g/L), calcinated seashells (oyster/sakhalin surf clam), and 1.0 g/L chitosan in 0.5 g/L LA (Chito) did not exhibit a significant higher effectiveness than that of water wash alone (1.0 log CFU/g). Acidified sodium chlorite (ASC) solution prepared from 0.5 g/L of sodium chlorite and 1.0 g/L LA or phytic acid reduced 3.5 log CFU/g of E. coli O157:H7 in dip-inoculated tomato surfaces. ASC (0.5 g/L of sodium chlorite and 1.0 g/L of LA) wash followed by a second wash with LA exhibited an additional sanitary effectiveness compared to a single wash with ASC. However, washing with ASC followed by a second wash with Chito exhibited an additional 1.0 log CFU/g reduction compared to a secondary wash with water. No significant difference of color, taste, and texture was observed among the washed cherry tomatoes.

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.

Combined effect of low-dose irradiation and acidified sodium chlorite washing on Escherichia coli O157:H7 inoculated on mung bean seeds.

The effect of low-dose irradiation (0.75 and 1.5 kGy) in combination with acidified sodium chlorite (ASC) on the reduction of Escherichia coli O157:H7 on mung bean seeds was examined. Washing with ASC (0.2, 0.5, 0.8, and 1.2 g/L sodium chlorite and 1.0 g/L citric acid) for 2 h reduced the E. coli O157:H7 population from 5.2 to 2.3-3.3 log CFU/g, depending on the concentrations of sodium chlorite. Gamma ray irradiation at 0.75 and 1.5 kGy resulted in reductions of about 1.8 and 2.8 log CFU/g, respectively. Therefore, a single treatment with ASC washing or gamma ray irradiation at 0.75 or 1.5 kGy could not achieve the complete elimination of E. coli O157:H7 on mung bean seeds. Conversely, low-dose irradiation (0.75 and 1.5 kGy) followed by washing with ASC (0.5-1.2 g/L) reduced the population of E. coli O157:H7 to below the detection limit (<1 log CFU/g). However, E. coli O157:H7 was detected in most samples in the enrichment and germination studies. When the treatment order was reversed (ASC washing followed by low-dose irradiation), the E. coli O157:H7 population was also observed to be below the detection limit. Under this treatment, fewer samples (16.7%) were shown to be positive in the enrichment and germination studies, and complete elimination was not achieved. The germination rates of mung bean seeds were not affected by ASC washing and gamma irradiation; however, the yield and length of sprouts were decreased by gamma irradiation.

Efficacy of chlorine and acidified sodium chlorite on microbial population and quality changes of spinach leaves.

Efficacy of washing with distilled water, chlorine solution, and acidified sodium chlorite (ASC) solution on populations of microorganisms on spinach leaves was evaluated. Washing with chlorine (100 mg/L) and ASC (sodium chlorite, 15 mg/L; citric acid, 200 mg/L) resulted in significant population reduction (1.1-1.9 log CFU/g) of aerobic microflora, coliform, and Escherichia coli O157:H7 (p < 0.05). There was no remarkable difference in decontamination efficacy between chlorine and ASC solution. In recent years, several sodium chlorite chemicals have been commercially available, and no difference in decontamination efficacy among the chemicals was observed when same concentration of sodium chlorite and citric acid were used. In addition, the reduction of E. coli O157:H7 population was influenced depending on the inoculation method and type of washing. It has been seen that dip-inoculated spinach leaves showed lower reduction than that of spot-inoculated spinach. After washing, populations of aerobic microflora, coliform, and E. coli O157:H7 were increased during storage at 10 degrees C, and washing condition before storage did not affect the subsequent increases in microbial population. Color of spinach leaves washed with ASC solution was not different from the color of those washed with water or chlorine solution, and washing with ASC solution was concluded to has no effect on appearance of spinach leaves.

Evaluation and control of the risk of foodborne pathogens and spoilage bacteria present in Awa-Uirou, a sticky rice cake containing sweet red bean paste.

The risk of food poisoning and growth of spoilage bacteria in Awa-Uirou, a sticky rice cake containing sweet red bean paste, was evaluated. Toxin-producing bacteria such as Staphylococcus aureus and Bacillus cereus are the main causes of food poisoning linked to this kind of food. The water activity in this product is in the range suitable for growth of S. aureus, B. cereus, and B. subtilis. The viable count of S. aureus or B. cereus spore cocktail was significantly reduced to 2.3 log colony-forming units (CFU)/g after 70 minutes steaming treatment at 100 degrees C. However, the heat-resistant endospores of B. subtilis germinated during storage at 30 degrees C to cause appreciable syneresis of the starch gel matrix in 4 days. The addition of 0.5% glycine before steaming treatment was found to effectively suppress the growth of B. cereus but was not effective in controlling S. aureus throughout the 7 days incubation period at 30 degrees C. On the other hand, S. aureus and B. cereus could grow > 5.0 log CFU/g in an inoculated sample without glycine within 3 days when stored at 30 degrees C. Moreover, addition of 0.5% glycine before the steaming process did not have any significant effect on color, texture, or taste of sticky rice cake. Therefore, results of this study demonstrated that the addition of 0.5% glycine before the steaming process could inhibit B. cereus and B. subtilis multiplication in the steamed rice confection which in turn may help reduce the risk of food poisoning or quality loss.

Microbioligical Hazard Contamination in Fermented Vegetables Sold in Local Markets in Cambodia.

　Fermented vegetables are common part of Cambodian diet. The food safety status for these foods has not been investigated. This study was conducted to evaluate the microbiological hazards that contaminated fermented vegetables. A total of 68 samples of fermented vegetables were purchased randomly from five wet markets in Phnom Penh. The conventional culture methods for microbiological analysis were used. Coliform bacteria (Escherichia coli, Cronobactersakazakii, and Enterobacter spp.), opportunistic non-Entrobacteriaceae, Enterococcus spp., Staphylococcus spp., and Listeria spp. were found in these fermented foods. The highest contamination rate of Enterococcus spp. was 34% of total fermented vegetable samples, followed by Bacillus spp. coliform bacteria and E. coli (31%, 24% and 10%, respectively). The potential foodborne pathogen, C. sakazakii, was identified in one sample. Fermented mixed vegetables showed higher contamination rate of coliform bacteria (50%) than fermented single-type vegetables (13%). The results showed that fermented vegetables sold in wet market are poor in hygiene. The stage in the processing chain where contamination occurred should be identified and basic sanitary practice should be enforced to improve the food safety of fermented vegetables in Cambodia.

Bacterial Contamination into Ready-to-Eat Foods Sold in Middle Thailand.

　Bacterial contamination in ready-to-eat (RTE) foods prepared and sold by small businesses in middle Thailand was surveyed. The 135 samples were randomly purchased from open markets and supermarkets in Bangkok and Pathum Thani provinces during May 2013 to February 2015. The results indicated that the rate of contamination in cooked pork, chicken meat and cooked aquatic items were 13/39 (33%), 18/45 (40%) and 14/57 (25%), respectively and the rate of bacterial contamination of collected samples from open market and supermarket were 26/67 (39%) and 19/68 (27%), respectively. Therefore, no statistically significant difference of contamination rate between two kinds of market or among three categories of food. The most contaminated pathogenic bacteria was Staphylococcus aureus 27/135 (20%) while that of Salmonella spp. was the lowest 5/135 (4%) in each categories of collected food samples. Implementation of suitable hygienic practices in the small food businesses are thought to be required to reduce the risk of foodborne illnesses caused by the consumption of RTE foods sold in middle Thailand.

Fate of Escherichia coli O157 Cells Inoculated into Lightly Pickled Chinese Cabbage during Processing, Storage and Incubation in Artificial Gastric Juice.

Fate of Escherichia coli O157 cells was evaluated when inoculated into each step after production of lightly pickled Chinese cabbage. The efficacy of surface sterilization by 100 mg/L of chlorine water for 10 min on raw leaves (6.0 log CFU/g) was 2.2 log CFU/g reduction. No meaningful change of the population of E. coli O157 (3.5 log CFU/g to 1.5 log MPN/g) contaminated into 19 kinds of products was observed. These results indicated the difficulty of estimating the viable count of the cells between contaminated on farms and further processing and storage steps. The population of E. coli O157 (3 log CFU/g to 1 log MPN/g) inoculated into the Chinese cabbage products was reduced less than 0.6 log CFU/g after 2 h-incubation at 37℃ in artificial gastric juice. Prevention from initial contamination of E. coli O157 on the ingredients of Chinese cabbage products is important to reduce the risk of food poisoning because the reduction of the bacterial counts after processing and consumption are limited.

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.

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.

Survival of Inoculated Escherichia coli O157:H7 in Japanese Sweet Dumplings during Storage.

An outbreak of Escherichia coli O157:H7 occurred due to the consumption of sweet dumplings in Japan. We examined the survival of E. coli O157:H7 inoculated into several types of sweet dumplings to evaluate the progress of the residual contaminating pathogens after the production or packing processes. For all 4 types of tested typical sweet dumplings, no significant reduction in the viable cell counts of inoculated E. coli O157:H7 (3 log CFU/g) was observed during storage at -20 ℃ and 5 ℃ for 5 weeks. Approximately 1 log CFU/g of reduction was observed after storage for 5 weeks at 10 ℃ and 15 ℃, which corresponded to the growth of the naturally contaminating fungi. Similar results were obtained when we used several types of commercially distributed sweet dumplings. It is of vital importance to prevent the cross contamination of sweet dumplings after the heating process in order to reduce the risk of foodborne illness.

Frequency of the insertion sequence IS4Bsu1 among Bacillus subtilis strains isolated from fermented soybean foods in Southeast Asia.

Among 45 Bacillus subtilis strains isolated from non-salted types of fermented soybeans produced in several Southeast Asian countries, 20 had the insertion sequence IS4Bsu1 in the chromosome. In contrast, none of 49 B. subtilis strains of non-food origin contained IS4Bsu1. Frequent occurrence of this mobile DNA element in the soybean-fermenting B. subtilis would reflect the fact that few strains flourish on soybeans and thereby contribute to soybean fermentation.