Young-Jo Kim

Rapid Detection of Listeria monocytogenes by Real-Time PCR in Processed Meat and Dairy Products

The objectives of this study were to evaluate the detection of Listeria monocytogenes in different ready-to-eat foods using real-time PCR (RT-PCR). Various concentrations (10(0) to 10(5) CFU/ml) of L. monocytogenes ATCC 19115 were inoculated into ham, sausage, ground meat, processed milk, cheese, and infant formula. L. monocytogenes ATCC 19115 in the samples was then enumerated on Oxford agar, and DNA was extracted from the samples before and after incubation at 36°C for 4 h. A set of primers and hybridization probe designed in this study was then used to detect the pathogen. The standard curve was then prepared by plotting cycle threshold values for each dilution versus L. monocytogenes cell counts (log CFU). The specificity of the set of primers and hybridization probe was appropriate. A 4-h incubation at 36°C before DNA extraction produced optimum standard curves in comparison to the results for a 0-h incubation. Thus, a 4-h incubation at 36°C was applied for monitoring L. monocytogenes in collected food samples. To monitor L. monocytogenes in foods, 533 samples (ham, 129; sausage, 226; ground meat, 72; processed cheese, 54; processed milk, 42; and infant formula, 10) were collected from retail markets and from the step before pasteurization in plants. Of all 533 samples, 4 samples (0.8%) showed positive signals in RT-PCR. Two samples from hams (1.6%) and two samples from sausages (0.9%) were determined to be positive for L. monocytogenes at < 100 CFU/g. The results indicate that the RT-PCR detection method with the set of primers and hybridization probe designed in this study should be useful in monitoring for L. monocytogenes in processed meat and milk products.

Validation of PCR and ELISA Test Kits for Identification of Domestic Animal Species in Raw Meat and Meat Products in Korea

In this study, two commercial PCR and ELISA test kits were examined for identification of eight animal species (beef, pork, chicken, duck, turkey, goat, lamb, and horse) from raw meat and meat products in Korea. The detection limit in RAW meat ELISA kit ® on three types of meat samples blended with beef, pork and chicken, demonstrated that all meat species were differentiable down to 0.2%. RAW meat ELISA kit ® on animal species resulted in differentiation rate of 94.5% for beef, 93.3% for pork, 90% for lamb, and 100% for chicken, duck, turkey, goat, and horse. In contrast, Powercheck Animal Species ID PCR kit TM resulted in 100% specificity at 0.05% limit of detection for all meat species. The detection limit of Cooked Meat ELISA kit ® on mixed meat samples heat-treated with different temperatures and times, resulted in 0.1% for all heat-treated mixed meat except for chicken at 1.0%. Additionally, ELISA kit on sixty meat products resulted in specificity of 31.8% for ham, 13.6% for sausages, and 12.5% for ground processed products, and relatively low rate for more than 2 types of mixed meats. On the contrary, meat species differentiation using PCR kit showed higher percentage than that using ELISA kit ® : 50.0% for ham, 41.7% for sausages, and 28.6% for ground processed meat. Futhermore, PCR kit on 54 dried beef meats detected pork genes in 13 products whereas ELISA kit showed negative results for all products. Hence, the possibility of cross-con- tamination during manufacturing process was investigated, and it was found that identical tumblers, straining trays, cutters and dryers were used in both beef and pork jerky production line, suggesting the inclusion of pork genes in beef products due to cross-contamination. In this study, PCR and ELISA test kits were found to be excellent methods for meat species differentiation in raw meat and heat-processed mixed meat. However, lower differentiation rate demon- strated in case of meat processed products raised the possibility of inclusion of other species due to cross-contamina- tion during manufacturing process.

Improvement of Karmali Agar by Addition of Polymyxin B for the Detection of Campylobacter jejuni and C. coli in Whole‐Chicken Carcass Rinse

The Karmali agar was modified by supplementation with a high concentration of polymyxin B. The goal of the study was to evaluate the effect of a high concentration of polymyxin B on the ability and selectivity of the modified Karmali agar to isolate Campylobacter jejuni and Campylobacter coli from whole chicken carcass rinse. A total of 80 whole chickens were rinsed with 400 mL of buffer peptone water. The rinsed samples were incubated with 2× blood-free modified Bolton enrichment broth for 48 h, and then streaked onto unmodified Karmali agar and modified Karmali agar supplemented with 100000 IU/L polymixin B (P-Karmali agar). The suspected colonies were finally confirmed by colony PCR. The P-Karmali agar exhibited a significantly better (P < 0.05) isolation rate than the unmodified Karmali agar (P-Karmali agar, 73.8%; unmodified Karmali agar, 33.8%). Moreover, the selectivity of the P-Karmali agar was also better (P < 0.05) than that of the other selective agar when comparing the number of contaminated plates (P-Karmali agar, 68.8%; unmodified Karmali agar, 87.5%) and growth index of competing flora (P-Karmali agar, 1.4; unmodified Karmali agar, 2.7). The improved selective agar excluded competing flora resistant to antibiotic agents in unmodified Karmali agar, increasing isolation rate and selectivity for C. jejuni and C. coli.

Traceback Investigation for Salmonella Contamination at Egg Processing Plants in South Korea: Prevalence, Antibiotic Resistance, and Epidemiological Tracing by Rep‐PCR Fingerprinting

We conducted a survey of Salmonella from 8 egg-breaking plants and a farm to determine the prevalence and the source of the bacteria. The contents of 2400 shell eggs (20 eggs per pool), 75 pasteurized liquid egg products, and 120 unpasteurized liquid egg products from 8 egg-breaking plants in South Korea were examined. In liquid egg samples, 4 Salmonella-positive samples from 120 unpasteurized ones (3.3%) and 5 positive samples from 75 pasteurized ones (6.7%) were identified; no eggs were positive for Salmonella among shell egg samples. To trace the source of Salmonella, we revisited the 2 Salmonella-positive plants (plants A and C). We investigated the equipment and environments of the plants and a henhouse (farm A) that supplied shell eggs to plant A, and collected additional liquid eggs and shell eggs from plants A and C. All Salmonella isolates from plant A and the associated farm A, except for a single Typhimurium strain from farm A, were serotyped as Bareilly. Three serovars, including one Bareilly, four Tennessee, and one Richmond, were isolated from plant C. Most Salmonella isolates were susceptible to tested antibiotics. To identify differences between isolates, molecular subtyping by using the automated rep-PCR system was conducted. All Salmonella Bareilly (S. Bareilly) strains from plant A exhibited high similarity, indicating possible contamination by Salmonella strains from the henhouse A. Meanwhile, 2 S. Bareilly strains from plant C, one from liquid egg at the 1st visit and the other from container at the 2nd visit, exhibited identical antibiotic resistance and similar subtyping pattern, but clearly discriminated from the ones of plant A.

Prevalence Analysis and Molecular Characterization of Salmonella at Different Processing Steps in Broiler Slaughter Plants in South Korea

In this study, changes in the prevalence of Salmonella during the processing of broiler chicken carcasses were investigated. A total of 1040 fecal swabs and chicken carcasses samples were collected from 2 processing plants at the 4 stages of broiler processing, which included live birds in slaughter line, postevisceration/prewashing, postwashing/prechilling, and postchilling, respectively. The intraspecific biodiversity of the Salmonella isolates was determined using a DiversiLab automated repetitive sequence-based PCR (rep-PCR) system. In both plants, the prevalence of Salmonella increased considerably after evisceration (from 4.6% to 30.8%, P < 0.05) and decreased after washing (from 30.8% to 25.4%, P < 0.05). However, the chilling step had little effect on Salmonella prevalence (from 25.4% to 22.7%, P > 0.05). The most frequent Salmonella serovar in plant A was Infantis (35.8%), followed by Enteritidis (26.2%) and Montevideo (15.0%), while Montevideo (43.6%) and Enteritidis (35.9%) were most prevalent in plant B. A difference in the rep-PCR banding pattern was found to be related to the processing plant origin and serovar rather than sampling point or sampling day, although there were some exceptional strains.

Mathematical Models to Predict Staphylococcus aureus Growth on Processed Cheeses

This study developed predictive models for the kinetic behavior of Staphylococcus aureus on pro- cessed cheeses. Mozzarella slice cheese and cheddar slice cheese were inoculated with 0.1 ml of a S. aureus strain mixture (ATCC13565, ATCC14458, ATCC23235, ATCC27664, and NCCP10826). The inoculated samples were then stored at 4 o C (1440 h), 15 o C (288 h), 25 o C (72 h), and 30 o C (48 h), and the growth of all bacteria and of S. aureus were enumerated on tryptic soy agar and mannitol salt agar, respectively. The Baranyi model was fitted to the growth data of S. aureus to calculate growth rate (µmax; log CFU·g �1 ·h �1 ), lag phase duration (LPD; h), lower asymptote (log CFU/ g), and upper asymptote (log CFU/g). The growth parameters were further analyzed using the square root model as a function of temperature. The model performance was validated with observed data, and the root mean square error (RMSE) was calculated. At 4 o C, S. aureus cell growth was not observed on either processed cheese, but S. aureus growth on the mozzarella and cheddar cheeses was observed at 15 o C, 25 o C, and 30 o C. The µmax values increased, but LPD values decreased as storage temperature increased. In addition, the developed models showed acceptable perfor- mance (RMSE = 0.3500-0.5344). This result indicates that the developed kinetic model should be useful in describing the growth pattern of S. aureus in processed cheeses.

Comparison of an Automated Most-Probable-Number Technique TEMPO ® TVC with Traditional Plating Methods Petrifilm TM for Estimating Populations of Total Aerobic Bacteria with Livestock Products

We compared between an automated most-probable-number technique TEMPO ® TVC and tradi- tional plating methods Petrifilm TM for estimating populations of total aerobic bacteria in various livestock products. 257 samples randomly selected in local retail stores and 87 samples inoculated with E. coli ATCC 25922, Staphylo- coccus aureus ATCC 12868 were tested in this study. The degree of agreement was estimated according to the CCFRA (Campden and Chorleywood Food Research Association Group) Guideline 29 and the agreement indicates the differ- ence of two kinds methods is lower than 1 log base 10(log10 ). The samples of hams, jerky products, ground meat prod- ucts, milks, ice creams, infant formulas, and egg heat formed products were showed above 95% in the agreement of methods. In contrast, proportion of agreement on meat extract products, cheeses and sausages were 93.1%, 92.1%, 89.1%, respectively. One press ham and five sausages containing spice and seasoning, two pork cutlets containing spice and bread crumbs, two meat extract product and two natural cheeses and one processing cheese with a high fat content, and one ice cream containing chocolate of all samples showed the discrepancy. Our result suggest that TEMPO ® TVC system is efficient to analyses total aerobic bacteria to compare manual method in time-consuming and

Development and Validation of Predictive Model for Salmonella Growth in Unpasteurized Liquid Eggs

Abstract Liquid egg products can be contaminated with Salmonella spp. during processing. A predictive model for the growth of Salmonella spp. in unpasteurized liquid eggs was developed and validated. Liquid whole egg, liquid yolk, and liquid egg white samples were prepared and inoculated with Salmonella mixture (approximately 3 Log CFU/mL) containing five serovars (S. Bareilly, S. Richmond, S. Typhimurium monophasic, S. Enteritidis, and S. Gallinarum). Salmonella growth data at isothermal temperatures (5, 10, 15, 20, 25, 30, 35, and 40°C) was collected by 960 h. The population of Salmonella in liquid whole egg and egg yolk increased at above 10°C, while Salmonella in egg white did not proliferate at all temperature. These results demonstrate that there is a difference in the growth of Salmonella depending on the types of liquid eggs (egg yolk, egg white, liquid whole egg) and storage temperature. To fit the growth data of Salmonella in liquid whole egg and egg yolk, Baranyi model was used as the primary model and the maximum growth rate and lag phase duration for each temperature were determined. A secondary model was developed with maximum growth rate as a function of temperature. The model performance measures, bias factor (Bf, 0.96-0.99) and r2 (0.96-0.99) indicated good fit for both primary and secondary models. In conclusion, it is thought that the growth model can be used usefully to predict Salmonella spp. growth in various types of unpasteurized liquid eggs when those are exposed to various temperature and time conditions during the processing.

Addition of Rifampicin to Bolton Broth to Inhibit Extended‐Spectrum β‐Lactamase‐Producing Escherichia coli for the Detection of Campylobacter

Exponential growth of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in Campylobacter media has become a common problem for the detection of Campylobacter in chicken meats. We investigated the minimum inhibitory concentration of 40 ESBL-producing E. coli isolates from meats obtained from various countries against antibacterial agents in Bolton broth (cefoperazone, vancomycin, and trimethoprim). All ESBL-producing E. coli strains were resistant to cefoperazone and vancomycin, whereas 50% of them were resistant to trimethoprim and grew in Bolton broth. We found that 20 μg/mL of rifampicin inhibited the growth of trimethoprim-resistant E. coli strains. Hence, we added 20 μg/mL of rifampicin to Bolton broth to improve the isolation of Campylobacter from chicken carcass rinses. The isolation rate of Campylobacter was significantly higher in the modified broth (44 out of 58, 75.9%, P < 0.05) than in the normal broth (0 out of 58, 0%). Furthermore, the number of agar plates with non-Campylobacter spp. was much lower after enrichment in the modified broth (4 out of 58, 6.9%, P < 0.05) than in the normal broth (58 out of 58, 100%).

Genotypic characterization of ESBL-producing E. coli from imported meat in South Korea

Twenty extended-spectrum β-lactamase (ESBL)-producing E. coli strains were isolated from imported meat in South Korea. ESBL strains of E. coli were detected in chicken (14/20) more often than in pork (6/20) and beef (0/20); the highest number (12/20) was detected in Brazilian meats. The blaCTX-M genes were predominant in meats from many countries. E. coli from pork imported from France produced the blaCTX-M-58 enzyme, which has never been documented previously in ESBL-producing bacteria from clinical or environmental sources. Additionally, the coexistence of the blaCTX-M-2 and blaOXA-1 enzymes in EC12-5 isolate was found for the first time in an ESBL E. coli isolate. A rare blaCTX-M type, blaCTX-M-25, was found in 40% of ESBL E. coli isolates. Phenotypic susceptibility testing showed that E. coli isolates were resistant to up to eleven antibiotics, including ciprofloxacin. For the first time, a new combination in an integron gene cassette, aacA4-cmlA6-qacEΔ1, was found in an E. coli isolate from poultry imported from Brazil. Three E. coli ST117 isolates, from an avian pathogenic lineage producing CTX-M-94, harbored fimH, fyuA, iutA, papC, rfc, and traT virulence genes and were not susceptible to quinolones. For the first time, rfc and papG virulence factors were detected in ESBL E. coli strains isolated from meat products. Even though E. coli CC21 and CC22 were obtained from meats from the USA and Brazil, respectively, they had a similarity coefficient higher than 99% in rep-PCR and the same MLST type (ST117), phenotypic antibiotic resistance pattern, integron gene (qacEΔ1), and plasmid DNA profile. This study indicates that imported meat products may be a source of ESBL-producing E. coli strains in South Korea.