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This study was performed to develop and validate a predictive growth model of pathogenic Escherichia coli to ensure the safety of fresh‐cut produce. Samples were inoculated with a cocktail of seven E. coli strains of five pathotypes (EHEC, Enterohemorrhagic E. coli; ETEC, Enterotoxigenic E. coli; EPEC, Enteropathogenic E. coli; EIEC, Enteroinvasive E. coli, and EAEC, Enteroaggregative E. coli) and stored at 4, 10, 12, 15, 25, 30, and 37°C. Growth of pathogenic E. coli was observed above 12°C. The primary growth model for pathogenic E. coli in fresh‐cut produce was developed based on the Baranyi model. The secondary model was developed as a function of temperature for lag phase duration (LPD) and maximum specific growth rate (μmax) based on the polynomial second‐order model. The primary and secondary models for pathogenic E. coli were fitted with a high degree of goodness of fit (R2 ≥ 0.99). The bias factor (Bf), accuracy factor (Af), and root mean square error (RMSE) were 0.995, 1.011, and 0.084, respectively. The growth model we developed can provide useful data for assessing the quantitative microbial risk of pathogenic E. coli in fresh‐cut produce intended for human consumption. In addition, it is thought to be widely available in industries that produce, process, distribute, and sell fresh‐cut produce., Primary model of the growth of pathogenic Escherichia coli in fresh‐cut produce. (A) 4°C, (B) 10°C, (C) 12°C, (D) 15°C, (E) 25°C, (F) 30°C, (G) 37°C.

The diverse microbial communities in kimchi are dependent on fermentation period and temperature. Here, we investigated the effect of enterotoxigenic Escherichia coli (ETEC) during the fermentation of kimchi at two temperatures using high-throughput sequencing. There were no differences in pH between the control group, samples not inoculated with ETEC, and the ETEC group, samples inoculated with ETEC MFDS 1009477. The pH of the two groups, which were fermented at 10 and 25°C, decreased rapidly at the beginning of fermentation and then reached pH 3.96 and pH 3.62. In both groups, the genera Lactobacillus, Leuconostoc, and Weissella were predominant. Our result suggests that microbial communities during kimchi fermentation may be affected by the fermentation parameters, such as temperature and period, and not enterotoxigenic E. coli (ETEC).

Colistin is an important antibiotic currently used to manage infections caused by multidrug-resistant pathogens in both humans and livestock animals. A new mobile colistin-resistance (mcr-9) gene w...

This study was to investigate the prevalence and characteristics of antimicrobial-resistant Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from 4,264 retail meat samples including beef, pork, and chicken in Korea between 2013 and 2018. A broth microdilution antimicrobial susceptibility testing was performed for S. aureus. Molecular typing by multilocus sequence typing (MLST), spa typing, and pulsed-field gel electrophoresis (PFGE), was performed on mecA-positive S. aureus strain. S. aureus was isolated at a rate of 18.2% (777/4,264), of which MRSA comprised 0.7% (29 strains). MLST analysis showed that 11 out of the 29 MRSA isolates were predominantly sequence type (ST) 398 (37.9%). In addition, ST72, ST692, ST188, ST9, and ST630 were identified in the MRSA isolates. The spa typing results were classified into 11 types and showed a high correlation with MLST. The antimicrobial resistance assays revealed that MRSA showed 100% resistance to cefoxitin and penicillin. In addition, resistance to tetracycline (62.1%), clindamycin (55.2%), and erythromycin (55.2%) was relatively high; 27 of the 29 MRSA isolates exhibited multidrug resistance. PFGE analysis of the 18 strains excluding the 11 ST398 strains exhibited a maximum of 100% homology and a minimum of 64.0% homology. Among these, three pairs of isolates showed 100% homology in PFGE; these results were consistent with the MLST and spa typing results. Identification of MRSA at the final consumption stage has potential risks, suggesting that continuous monitoring of retail meat products is required.

This study aimed to monitor microbial contamination levels in a variety of health functional foods and to establish new microbial criteria. Indicator organisms (i.e., aerobic bacteria, coliform bacteria, and Escherichia coli) were monitored in 10 health functional food categories (743 items, 3,715 samples). The mean total aerobic counts of ginseng and Korean red ginseng were -0.35 and -0.74 log CFU/g; and the mean total coliform counts were -1.4 and -1.39 log CFU/g, respectively. In addition, the mean total coliform counts of fiber and protein products were -1.34 and -1.22 log CFU/g, respectively. However, no aerobic or coliform cells were detected in any other health functional food products (vitamins, minerals, probiotics, milk thistle extract, propolis, eicosapentaenoic acid, docosahexaenoic acid, or lutein products), and no E. coli was detected in any of the categories. These results can potentially be used to update the microbial criteria of the Health Functional Food Code.

Owing to convenience, ease of preparation, and price, the consumption of commercial kimchi is gradually rising in South Korea. Here, we estimated the risk level posed by pathogenic Escherichia coli in commercial kimchi products using the quantitative microbial risk assessment (QMRA) approach to develop measures for preventing potential foodborne outbreaks from kimchi consumption. We collected 610 samples of commercial kimchi products produced in Korea, 267 kimchi samples from foreign countries imported to Korea, and 187 raw materials used in kimchi preparation, and analyzed them for contamination with pathogenic E. coli. A Predictive model was developed to observe the survival characteristics of pathogenic E. coli. A dose-response model was selected, and the risk level was estimated using @RISK software. Although a prior epidemiological study indicated the frequent occurrence of foodborne outbreaks arising from contaminated kimchi products consumed in food service facilities, we found a low probability of foodborne illness caused by pathogenic E. coli in commercial kimchi products.

Human noroviruses are major causes of nonbacterial gastroenteritis and are transmitted by both food and water, as well as person-to-person. Asymptomatic norovirus infection of food handlers may play a role in transmission. The outbreak of norovirus infections was recognized in the PyeongChang Winter Olympics, starting with security staff on 3 February 2018. The Ministry of Food and Drug Safety in the Republic of Korea conducted norovirus surveillance from asymptomatic food handlers of food-catering facilities related to the Olympics to prevent the spread of noroviruses. Rectal swab samples (707) from food handlers were collected and examined for noroviruses by using real-time reverse transcription PCR and conventional reverse transcription PCR. Five of 707 samples were identified as noroviruses. Genotypes of the norovirus-positive samples were determined with sequencing analysis. Identified genotypes of norovirus in asymptomatic food handlers included GI.3, GII.4, and GII.17. The GII.17 strain was prevalent among the genotypes, accounting for three of five detections. Food handlers with noroviruses detected in rectal swabs were excluded from cooking, and all food handled by infected food handlers was discarded. Surveillance of norovirus infection for food handlers contributed to preventing norovirus spread. HIGHLIGHTS

Antimicrobial-resistant bacteria in poultry meat pose a threat to public health. This article is the first to report the prevalence of antimicrobial-resistant Escherichia coli in retail poultry meat labeled with various claims of antibiotic use in Korea. A total of 719 E. coli strains were isolated from 1,107 raw poultry (chicken and duck) meat samples purchased from nationwide retail stores between 2017 and 2019. All strains were tested for antimicrobial susceptibility with a broth microdilution method. The prevalence of antimicrobial-resistant E. coli in chicken was significantly higher than that in duck for almost all antibiotics tested, and 87.9% of E. coli strains in chicken samples were multidrug resistant. The most prevalent types of antimicrobial resistance in these E. coli strains from poultry meat were to nalidixic acid (75.7%), ampicillin (69.1%), and tetracycline (64.0%), consistent with national sales data for veterinary antibiotics in the Korean poultry production industry. Organic or antibiotic-free and conventional chicken products were equally likely to be contaminated with antimicrobial-resistant E. coli. Contamination may occur during slaughtering and subsequent processing, and antibiotic use is permitted in certain cases under organic or antibiotic-free poultry standards. Therefore, close surveillance is required throughout the chicken production chain to prevent the spread of antimicrobial-resistant E. coli strains. HIGHLIGHTS

Egg products are widely consumed in Korea and continue to be associated with risks of Staphylococcus aureus-induced food poisoning. This prompted the development of predictive mathematical models to understand growth kinetics of S. aureus in egg products in order to improve the production of domestic food items. Egg products were inoculated with S. aureus and observe S. aureus growth. The growth kinetics of S. aureus was used to calculate lag-phase duration (LPD) and maximum specific growth rate (µ(max)) using Baranyi model as the primary growth model. The secondary models provided predicted values for the temperature changes and were created using the polynomial equation for LPD and a square root model for µ(max). In addition, root mean square errors (RMSE) were analyzed to evaluate the suitability of the mathematical models. The developed models demonstrated 0.16–0.27 RMSE, suggesting that models properly represented the actual growth of S. aureus in egg products.

Norovirus (NoV) is a major pathogenic virus that is responsible for foodborne and waterborne gastroenteritis outbreaks. Groundwater is an important source of drinking water and is used in agriculture and food manufacturing processes. This study investigated norovirus contamination of groundwater treatment systems at 1360 sites in seven metropolitan areas and nine provinces in 2015-2016. Temperature, pH, residual chlorine, and turbidity content were assessed to analyze the water quality. In 2015, six sites were positive for the presence of NoV (0.88%) and in 2016, two sites were positive (0.29%); in total, NoV was detected in 8 of the 1360 sample sites (0.59%) investigated. Identified genotypes of NoV in groundwater included GI.5, 9 and GII.4, 6, 13, 17, and 21. GII.17 was the most prevalent genotype in treated groundwater used in the food industry. This dominance of GII.17 was corroborated by NoV infection outbreak cases and the results of a survey of coastal waters in South Korea in 2014-2015. Although a low detection rate was observed in this study, NoV is a pathogen that can spread extensively. Therefore, it is necessary to periodically monitor levels of norovirus which is responsible for food poisoning in groundwater. This is a first report to reveal epidemic genotype shift of norovirus in groundwater treatment system of food facilities in South Korea. Our results may contribute to the enhancement of public health and sanitary conditions by providing molecular epidemiological information on groundwater NoV.

The aims of this study was to determine the actual levels of microbial contamination in cereal and Saeng-sik products and establish new microbial criteria for cereal and Saeng-sik products for the “Korea Food Code”. Monitoring microbial indicator organisms (i.e., total aerobic, coliform, and Escherichia coli counts) and food-borne pathogens (i.e., Bacillus cereus, Staphylococcus aureus, and Clostridium perfringens) revealed that the total aerobic counts of cereal and Saeng-sik products were not detected (ND)–2.48 and 4.00–7.88 log10 CFU/g, respectively, whereas coliform bacteria were only detected in Saeng-sik (ND–5.24 log10 CFU/g). Cereals and Saeng-sik products were also contaminated with B. cereus (ND–2.95 log10 CFU/g and −0.40–3.30 log10 CFU/g, respectively). However, S. aureus, C. perfringens and E. coli were not detected in any of the cereal or Saeng-sik products. Based on this study, a revision was proposed for microbial criteria in cereal and Saeng-sik products.

The effect of power levels on inactivation of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in tomato paste was investigated using 915-MHz microwave heating (MW) and ohmic heating (OH). Heating uniformity, pathogen inactivation, and quality aspects were determined with 1.8-, 2.1-, 2.4-, and 3.0-kW MW and corresponding OH. GInaFit was used to analyze pathogen inactivation. The heating uniformity of MW-treated samples was inferior to that of OH-treated samples at low power levels of 1.8 to 2.4 kW but improved as the power level increased. Pathogen inactivation of MW-treated samples was significantly higher than that of OH-treated samples at low power levels of 1.8 to 2.4 kW (P0.05) but was not significantly different at the highest power level of 3.0 kW (P0.05). Quality aspects (color, pH, and lycopene content), except for L*, of MW-treated samples were not significantly degraded (P0.05) by increased power levels. Our results indicate that increasing power levels of MW ensures heating uniformity and microbiological safety and preserves quality aspects of tomato paste.

Enterococci have been used as starter cultures and probiotics. They also have been considered as indicator organisms for antibiotic resistance due to their ability to harbor and to easily acquire antibiotic resistance. This study aimed to show the antimicrobial resistance profiles and genotyping of Enterococcus faecalis in retail pork meat products in Korea. Enterococcus spp. were analyzed for 124 collected samples, which included minced pork meat, marinated pork meat with soy sauce or kochujang (fermented hot pepper-soybean paste), and frozen processed pork meat products. The isolates of E. faecalis (n=36) were resistant to tetracycline (58.3%), erythromycin (11.1%), and nitrofurantoin (2.8%). No vancomycin resistant enterococci were observed in the present study. Most of the E. faecalis isolates were sensitive to all antibiotics or resistant to single antibiotics. As a result of the automated repetitive-sequence-based PCR (rep-PCR), which was used as an approach for genotyping enterococci, 7 out of 36 isolates of E. faecalis were assigned to one cluster with a similarity >95%, and all isolates were found to have originated from minced pork meat, suggesting that this clone might circulate in minced pork meat products. Given the importance of antimicrobial resistance of enterococci in food safety as well as in public health, our results on the occurrence, antimicrobial resistance, and genotyping could provide useful information to derive risk management options.

Division of Food Chemical Residues, National Institute of Food and Drug Safety EvaluationAbstract The purpose of this study was to compare a conventional culture method and real-time PCR for the detectionof Yersinia enterocolitica (Y. enterocolitica) in sausage and in vegetable salad. Food samples inoculated with Y.enterocolitica were enriched in peptone-sorbitol bile-broth, and swabs were then streaked onto cefsulodin-irgasan-novobiocin agar. Biochemical tests for suspected colonies were performed with an API 20E strip. In parallel, real-timePCR was performed, targeting the 16S rRNA gene using 1 mL of enrichment broth. In sausage, the number of positivesamples detected by culture method (49 out of 60) was similar (p>0.05) with that of real-time PCR (50 out of 60).However, the number of positive samples of real-time PCR (26 out of 60) was significantly higher (p

In this study, a total of 256 samples of retail raw meats (beef, pork and chicken) and sashimi were investigated for the presence of Enterococcus faecalis and Enterococcus faecium. We isolated a total of 117 E. faecalis and E. faecium from the samples, with contamination rates ranging from 18.8% for sashimi samples to 68.8% of chicken samples. E. faecalis was the predominant species recovered from all of the retail raw meats beef (42.2%), pork (42.2%), chicken (65.6%) and sashimi (12.5%). Among 117 isolates, 61 isolates (52.1%) were resistant to tetracycline, 32 isolates (27.4%) were resistant to erythromycin, 23 isolates (19.7%) were resistant to chloramphenicol, 16 isolates (13.7%) were resistant to ripampin, 10 isolates (8.5%) were resistant to gentamycin, 9 isolates (7.7%) were resistant to ciprofloxacin and 1 isolate (0.9%) was resistant to ampicillin and penicillin G. No resistance to amoxicillin + clavulanic acid and vancomycin was observed. Although no strain was resistant to vancomycin, the vanB gene was observed in 9 of 117 of Enterococcus (7.7%) demonstrating potential risk of vancomycin-resistant Enterococcus (VRE). Our results indicate that E. faecalis and E. faecium were highly prevalent in retail raw meats, but most strains were sensitive to tested antibiotics.

A real-time PCR assay was developed and validated inhouse specifically for the detection of Clostridium perfringens (Cl. perfringens) in meats and vegetables by comparing with the culture method. The detection limit of the real-time PCR assay in phosphate-buffered saline was 10² CFU/ml. When the two methods were compared in food samples inoculated with Cl. perfringens, the culture method detected 52 positives, whereas real-time PCR detected 51 positives out of 160 samples. The difference was without statistical significance (p>0.05). Real-time PCR assay is an option for quality assurance laboratories to perform standard diagnostic tests, considering its detection ability and time-saving efficiency.

This study is to develop mathematical models to predict the growth of Listeria monocytogenes in kimbab as a function of storage temperature. Kimbab which was inoculated with L. monocytogenes were incubated at 4, 10, 15, and 30°C. The primary model showed a good fit (R2=0.9845 to 0.9967) to a Gompertz equation to obtain specific growth rates (SGR) and lag time (LT) at each temperature. The SGR of L. monocytogenes in the kimbab increased and LT decreased by increasing temperature. Secondary polynomial model was developed using PRISM general nonlinear analysis software for SGR and LT. The secondary models were 0.1479−(0.02457×Temp)+(0.001296 ×Temp2) for SGR and 312.8−(30.21×Temp)+(0.6654 ×Temp2) for LT. This secondary polynomial model was judged as appropriate based on the coefficient of determination (R2 of the SGR and LT model=0.9995, 0.9556), the bias factor (B f of the SGR and LT model=0.97, 0.94), and the accuracy factor (A f of the SGR and LT model=1.10, 1.68). Reliable predictions of L. monocytogenes SGR and LT in kimbab were based on temperature.

In this study, the contamination levels of total aerobic plate counts, Escherichia coli and various pathogenic bacteria in ready-to-eat (RTE) foods were investigated in Korea. A total of 634 samples were purchased randomly from 47 stores located in 3 different provinces. The aerobic plate count levels of RTE occurred in a relatively wide range, of 1.0–7.9 log CFU/g. E. coli and Listeria monocytogenes were detected in only 2 samples in the qualitative test. Staphylococcus aureus and Bacillus cereus were detected in 12.3 and 12.6% of the samples at levels up to 1 log CFU/g. These results demonstrate that RTE foods in Korea can be contaminated by a variety of pathogenic bacteria. Therefore, precautionary measures are necessary for consumer protection, including the improvement of sanitary conditions in the processing plants and sales locations for RTE products in Korea.

In this study, performances of culture methods using two selective media and real-time PCR were evaluated for detection of Campylobacter jejuni (C. jejuni) in various food samples. Sausage, ground beef, and radish sprouts inoculated with C. jejuni were enriched in Hunt broth and then streaked onto modified cefoperazone charcoal deoxycholate agar and Preston agar, followed by incubation under microaerobic conditions. The enriched Hunt broth (1 mL) was used in real-time PCR assay. No statistical differences were observed in sensitivity among the two selective media and real-time PCR for sausage and ground beef. However, the number of positives by real-time PCR in radish sprouts was much higher than the two selective media (p

The prevalence of Salmonella was determined in chicken meat (n = 26), beef (n = 49), and pork (n = 56) collected from wholesale markets, retail stores, and traditional markets in Seoul, South Korea, in 2009. Antibiotic resistance was assessed, and the molecular subtypes of Salmonella isolates were ascertained using an automated repetitive sequence-based PCR (rep-PCR) system (DiversiLab). A total of 18 Salmonella strains were isolated from 17 of 131 samples: 16 strains from each of 16 samples and 2 strains from the same pork sample. The prevalence of Salmonella from the retail meats was 2.0% in beef, 8.9% in pork, and 42.3% in chicken meat. Among 10 different serotypes, Salmonella enterica Panama was recovered from a beef sample, and Salmonella London and Salmonella Montevideo were the predominant serotypes from pork and chicken meat, respectively. The highest antibiotic resistance observed was to erythromycin (100%) followed by streptomycin (22.2%) and tetracycline and chloramphenicol (16.7%). Of the 18 isolates, 5 (27.8%) were resistant to two or more antibiotics, and 1 isolate from chicken meat was resistant to eight antibiotics, including cephalosporins. Differentiation between all of the Salmonella isolates except between Salmonella Montevideo and Salmonella London was successfully performed with the automated rep-PCR system, indicating that it can be added to the toolbox for source tracking of foodborne pathogens associated with outbreaks.

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method.

Escherichia coli O157:H7 is a food-borne pathogen that causes bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). We compared three selective media and evaluated the performance of immunomagnetic separation (IMS) for the detection of low levels of E. coli O157:H7 in ground beef and radish sprouts with different levels of background flora. Bulk food samples (500 g for each trial) were artificially inoculated with nalidixic acid-resistant E. coli O157:H7 at the lowest dose that would generate 20 partial-positive samples of 25 g each. All samples were homogenized in mTSB (225 mL) and incubated overnight at 37 C. IMS was performed using the enriched mTSB samples (1 mL) along with conventional spreads plated onto three different selective media: Sorbitol MacConkey agar (SMAC), Sorbitol MacConkey agar with cefixime and tellulite (CT-SMAC), and Sorbitol MacConkey agar with nalidixic acid (NAL-SMAC) as the gold standard. Two suspicious colonies from each medium were selected and confirmed using a serological test after transfer to tryptic soy broth with yeast extract (TSAYE). CT-SMAC was better than SMAC for detecting E. coli O157:H7 in all food types. Although there was no statistical difference in the number of positive samples when using IMS vs. non-IMS techniques, more positive samples were detected when IMS was used in both ground beef and radish sprouts. It appears that the improvement was more significant in radish sprouts, which had a higher level of background flora than ground beef. The results also suggest that the combination of CT-SMAC and IMS is sufficient to recover low levels of E. coli O157:H7 in high background flora food samples.

본 연구에서는 편육과 브로콜리 싹에 Salmonella 농도를 단계별로 접종하고 배지법, $VIDAS^{(R)}$ , real-time PCR의 양성 검출률을 비교함으로써 $VIDAS^{(R)}$ , real-time PCR의 Salmonella 검출력을 검증하였으며, 또한 각 식품별로 검출률에 차이가 있는가를 알아보았다. 편육과 브로콜리 싹(500 g)에 3단계 농도( $VIDAS^{(R)}$ Salmonella ( $VIDAS^{(R)}$ SLM), real-time PCR법을 시행하여 검출능력을 비교하였다. 편육에서 배지법, $VIDAS^{(R)}$ , real-time PCR를 이용하여 Salmonella를 검출한 결과, 낮은 접종 농도( $VIDAS^{(가)}$ O real-time PCR 모두 배지법과 통계학적 유의차가 나타나지 않았다. 반면에 브로콜리 싹을 이용하였을 때 낮은 접종 농도에서는 Salmonella가 검출되지 않았으며 $VIDAS^{(가)}$ O real-time PCR 모두 배지법보다 더 많은 샘플을 검출하였다. 따라서 정상 세균총이 많은 야채 식품의 경우 배지법은 경쟁 세균에 의해 검출이 저해되므로 이보다 영향을 덜 받는 $VIDAS^{(R)}$ 법이나 real-time PCR법을 이용하여 검출하는 것이 더 효과적이라고 할 수 있겠다. 또한 이러한 신속 진단법을 이용하면 $VIDAS^{(R)}$ 는 3일, real-time PCR은 1일 이내에 배지법과 같거나 보다 높은 검출률로 Salmonella를 검출할 수 있는 것으로 나타났다. 【Salmonellosis is an important worldwide foodborne infectious disease that is transmitted by many food vehicles including raw and processed animal products and fresh produce. In this study, the effectiveness of automated ELISA ( $VIDAS^{(R)}$ ) and realtime PCR in the detection of Salmonella spp. in steamed pork and raw broccoli sprouts was evaluated by comparing their results with those of a conventional culture method. Bulk samples (500 g) of steamed pork and raw broccoli sprouts were inoculated with various levels of Salmonella and divided into 20 samples (25 g each). All the samples, including the controls, were analyzed using a conventional culture method, $VIDAS^{(R)}$ , and real-time PCR to detect the presence of Salmonella. In addition, the levels of background flora in the steamed pork and the raw broccoli sprouts were determined. In the steamed pork that contained less than 100 CFU/g of aerobic bacteria, all three methods detected low levels of Salmonella without a statistical difference in their performance. In the broccoli sprouts with high quantities of background flora (ca. $6.7{\times}10^7$ CFU/g), however, all three methods were unable to detect low levels of Salmonella, and real-time PCR and $VIDAS^{(R)}$ more sensitively detected Salmonella than the culture method, with significant statistical differences. In conclusion, $VIDAS^{(R)}$ and real-time PCR could be superior to conventional culture methods in detecting Salmonella in food with high levels of background flora.】

The occurrence of Vibrio parahaemolyticus in 72 samples of raw Korean oysters obtained monthly from April to December from retail outlets was monitored, and the virulence of the strains isolated from 48 oyster samples investigated. V. parahaemolyticus was isolated in oysters from May to November with the highest level in August and September. Multiplex PCR showed that none of the V. parahaemolyticus strains isolated contained the gene for thermostable direct haemolysin (tdh), although one strain was positive for TDH-related haemolysin (trh). These data can be utilized for a risk-management plan to establish Food Safety Objectives (FSO) for V. parahaemolyticus in oysters at retail outlets in Korea.

Rapid and sensitive detection techniques for foodborne pathogens are important to the food industry. However, traditional detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 4 to 7 days to complete. Thus, this study was conducted to address the issue of time lag inherent in traditional methods by developing a novel PCR assay for each of five foodborne pathogenic bacteria. This new system consists of a simultaneous screening method using multiplex PCR in a single reaction tube for the rapid and sensitive detection of each of the five bacteria. Specific primers for multiplex PCR amplification of the Shiga-like toxin (verotoxin type II), femA (cytoplasmic protein), toxR (transmembrane DNA binding protein), iap (invasive associative protein), and invA (invasion protein A) genes were designed to allow simultaneous detection of Escherichia coli O157:H7, Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella, respectively. To confirm the specificity of each primer pair for the respective target gene, three types of experiments were carried out using boiled cell lysates and their DNAs. In the multiplex PCR with mixed DNA samples, specific bands for corresponding genes were simultaneously detected from a single reaction. The detection of all five foodborne pathogenic bacteria could be completed in less than 24 h with this novel PCR method.

Sixteen Salmonella strains resistant to nalidixic acid isolated from kimbab, the most popular ready-to-eat (RTE) food in Korea, and chicken meat were selected for this study. The resistant strains were shown to have high minimal inhibitory concentrations (MICs) against nalidixic acid (512 ~ 4096 μg/mL). Among them, 4 Salmonella enterica serovar Haardt isolates showed multi-drug resistance (MDR) patterns with reduced susceptibility to fluoroquinolone (0.5 μg/mL of ciprofloxacin MICs). The mechanisms of quinolone resistance in the nalidixic acid resistant strains were characterized by PCR and sequence analysis. The presence of plasmid-mediated quinolone resistance (PMQR) genes and amino acid changes in the quinolone resistance determining region (QRDR) were investigated by PCR-based detection and sequencing, and the efflux pump inhibition test was also done using phe-arg-β-naphthylamide (PAβN). Although PMQR genes were not detected in any of the tested strains, the QRDR mutations were found in this study: single mutation in gyrA (Asp87Tyr, Asp87Gly, and Asp87Asn), double mutations in gyrA (Ser83Thr) and parC (Thr57Ser), and single mutation in parC (Thr57Ser). MICs of nalidixic acid were reduced by 2- to 32-folds by the efflux pump inhibitor, PAβN. Pulsed-field gel electrophoresis (PFGE) was carried out to confirm the epidemiological relationship between the nalidixic acid resistant strains. The PFGE patterns were classified into 6 groups at cutoff level of 70 ~ 100% correlation on the dendrogram. Some strains of serotype Haardt and Enteritidis showed several values of genomic identity in accordance with strains, sources, and isolation year. We suggest that point mutation on QRDR and efflux pump systems involved in antimicrobials had independent effects on drug-resistance regardless of bacterial genomic variation.