Molecular and Genomic Analysis of the Virulence Factors and Potential Transmission of Hybrid Enteropathogenic and Enterotoxigenic Escherichia coli (EPEC/ETEC) Strains Isolated in South Korea.

Hybrid strains Escherichia coli acquires genetic characteristics from multiple pathotypes and is speculated to be more virulent; however, understanding their pathogenicity is elusive. Here, we performed genome-based characterization of the hybrid of enteropathogenic (EPEC) and enterotoxigenic E. coli (ETEC), the strains that cause diarrhea and mortality in children. The virulence genes in the strains isolated from different sources in the South Korea were identified, and their phylogenetic positions were analyzed. The EPEC/ETEC hybrid strains harbored eae and est encoding E. coli attaching and effacing lesions and heat-stable enterotoxins of EPEC and ETEC, respectively. Genome-wide phylogeny revealed that all hybrids (n = 6) were closely related to EPEC strains, implying the potential acquisition of ETEC virulence genes during ETEC/EPEC hybrid emergence. The hybrids represented diverse serotypes (O153:H19 (n = 3), O49:H10 (n = 2), and O71:H19 (n = 1)) and sequence types (ST546, n = 4; ST785, n = 2). Furthermore, heat-stable toxin-encoding plasmids possessing estA and various other virulence genes and transporters, including nleH2, hlyA, hlyB, hlyC, hlyD, espC, espP, phage endopeptidase Rz, and phage holin, were identified. These findings provide insights into understanding the pathogenicity of EPEC/ETEC hybrid strains and may aid in comparative studies, virulence characterization, and understanding evolutionary biology.

Prevalence of pathogenic Arcobacter species in South Korea: Comparison of two protocols for isolating the bacteria from foods and examination of nine putative virulence genes.

Contamination of foodstuffs by potentially enteropathogenic Arcobacter spp. is becoming a concern worldwide. However, few studies have examined virulence-associated genes in isolates of Arcobacter spp. from food. Here, we investigated the prevalence of three pathogenic Arcobacter species, A. butzleri, A. cryaerophilus, and A. skirrowii, in chicken, pork, and leafy green vegetables (n = 323) in South Korea. Samples were examined using two different protocols selected from a literature review: Acrobacter selective broth (ASB) II + Arcobacter selective medium (ASM) II (protocol A), and ASB II + modified charcoal cefoperazone deoxycholate agar supplemented with CAT (protocol B). Overall, Arcobacter spp. were detected in 45.8% of food samples, and the recovery rate of protocol B (37.8%) was significantly higher than that of protocol A (30.7%) (p < 0.05). Refrigerated chicken gizzard samples showed the highest detection rate (100%), followed by refrigerated chicken wing (79.5%), intestine (77.3%), neck skin (63.3%), pork (55.6%), frozen chicken legs (5.0%), and leafy green vegetables (4.4%) (p < 0.05). All isolates from chicken and leafy green vegetables were identified as A. butzleri, whereas A. cryaerophilus and A. skirrowii were mainly detected in pork. Most samples (95.8%) harbored more than one of nine putative virulence factors (cadF, ciaB, cj1349, hecA, hecB, mviN, pldA, irgA, and tlyA), and 91.3% harbored more than two. Isolates harboring all nine putative virulence genes were obtained from 1.9% of samples: five pork and one chicken. This study provides comprehensive and de facto evidence regarding prevalence of an emerging pathogen, Arcobacter spp., in various foods, along with their virulence potential. The results justify further research with respect to their role in food safety.

Novel motB as a potential predictive tool for identification of B. cereus, B. thuringiensis and differentiation from other Bacillus species by triplex real-time PCR.

Quantitative triplex real-time PCR (qPCR) offers an alternative method for detection of bacterial contamination. It provides quantitation of the number of gene copies. In our study, we established a qPCR assay to detect and quantify the specificity towards Bacillus cereus and B. thuringiensis. The assay was designed to detect a 280 bp fragment of motB gene encoding the flagellar motor protein, specific for detection of B. cereus and B. thuringiensis, excluding other group species B. pseudomycoides, B. mycoides and B. weihenstephanensis. Specificity of the assay was confirmed with 111 strains belonging to Bacillus cereus group and performed against 58 B. cereus, 50 B. thuringiensis, 3 other Bacillus bacteria and 9 non-Bacillus bacteria. Detection limit was determined for each assay. Direct analysis of samples revealed the specificity towards identification and characterization of B. cereus group cultured in nutrient media. Based on results, it was observed that motB showed 97% specificity towards B. cereus strains, 98% for B. thuringiensis but other B. cereus group showed less sensitivity (0%), thus, provides an efficient tool to identify B. cereus and B. thuringiensis. Further, environmental and food samples do not require band isolation, re-amplification or sequence identification. Thus, reducing the time and cost of analysis.

Changes in microbial composition and the prevalence of foodborne pathogens in crab marinated in soy sauce produced by six manufacturing plants.

BACKGROUND: The present study examined the changes in microbiological composition during the production process of crab marinated in soy sauce, potential microbial hazards, potential contamination routes and effective critical control points. Crab and soy sauce samples were obtained from six different manufacturing plants at different stages, and their microbiological content was comprehensively assessed by quantitative and qualitative analyses. RESULTS: The results revealed the following: (1) the final products contained 4.0 log colony-forming units (CFU) g-1 aerobic plate counts (APCs) and 1.1 log CFU g-1 coliforms, which may have been introduced from the raw materials (the level of APCs in raw crab and soy sauce mixed with other ingredients was 3.8 log CFU g-1 and 4.0 log CFU mL-1 respectively); (2) marination of crab in soy sauce may allow cross-contamination by coliforms; (3) only Bacillus cereus and Staphylococcus aureus were qualitatively detected in samples at different stages of manufacture (detection rate of 28 and 5.6% respectively), and these bacteria may impact the microbiological quality and safety of crab marinated in soy sauce; and (4) bacterial counts were either maintained or increased during the manufacturing process (suggesting that no particular step can be targeted to reduce bacterial counts). CONCLUSION: Proper management of raw materials and the marination process are effective critical control points, and alternative interventions may be needed to control bacterial quantity. The results provide important basic information about the production of crab marinated in soy sauce and may facilitate effective implementation of sanitary management practices in related industries and research fields. © 2016 Society of Chemical Industry.

Estimating the burden of foodborne disease, South Korea, 2008-2012.

Estimating the actual occurrence of foodborne illness is challenging because only a small proportion of foodborne illnesses are confirmed and reported. Many studies have attempted to accurately estimate the overall number of cases of foodborne illness, but none have attempted to estimate the burden of foodborne disease in South Korea. This study used data from the Health Insurance Review and Assessment Service (HIRA), a public health surveillance system in South Korea, to calculate the number of cases and hospitalizations due to 18 specific pathogens and unspecified agents commonly transmitted through contaminated food between 2008 and 2012 in South Korea while accounting for uncertainty in the estimate. The estimated annual occurrences of foodborne illness were 336,138 (90% credible interval [CrI]: 258,379-430,740), with inpatient stays (hospitalizations), outpatient visits (foodborne disease infections), and patients' experiences (without visiting physicians) accounting for 2.3% (n=7809 [90% CrI: 7016-8616]), 14.4% (n=48,267 [90% CrI: 45,883-50,695]) and 83.3% (n=280,062 [90% CrI: 201,795-374,091]), respectively. Escherichia coli, including enterohemorrhagic E. coli, caused most illnesses, followed by nontyphoidal Salmonella spp., Staphylococcus aureus, hepatitis A virus, and norovirus. These results will be useful to food safety policymakers for the prevention and control of foodborne pathogens in South Korea.

Enhanced protection of pathogenic Escherichia coli ingested by a soil nematode Caenorhabditis elegans against sanitizer treatments.

We employed Caenorhabditis elegans as a model to study the effectiveness of sanitizers in killing pathogenic Escherichia coli strains ingested by free-living nematodes. Adult worms that had fed on six pathogenic E. coli strains (highly persistent in the nematode intestine) were treated with three chemical solutions. In planktonic cells, none of the H2O2 and acetic acid treatments influenced the survival of the pathogenic E. coli strains, whereas sodium hypochlorite critically decreased the viability of the strains. Importantly, the survival of the E. coli strains was dramatically increased by persistence in the C. elegans gut under 0.1% sodium hypochlorite, and several strains could survive at a concentration of 0.5%. In addition, all pathogenic E. coli strains in the C. elegans gut survived on the lettuce for 5 days even though they were washed with 0.1% sodium hypochlorite. Taken together, our results indicate that pathogenic E. coli ingested by C. elegans may be protected against washing treatment with commercial sanitizers on raw food materials.

Evaluation of the removal and destruction effect of a chlorine and thiamine dilaurylsulfate combined treatment on L. monocytogenes biofilm.

The present study investigated the efficacy of single and combined treatment of both chlorine and thiamine dilaurylsulfate (TDS) on the reduction of Listeria monocytogenes biofilms in microtiter plate. The disinfectants used in this study were 50, 100, and 200 mg/L chlorine and 100, 500, and 1000 mg/L of TDS. Biofilm-forming index (BFI) and culturable cell count were used to evaluate the disinfectant assay. The highest BFI reduction was 0.80, achieved by the combination of 200 mg/L chlorine and 1000 mg/L TDS. In contrast, the highest culturable cell count reduction was 4.80 log colony-forming units/well by the combination of 200 mg/L chlorine and 100 mg/L TDS. The BFI was reduced in a concentration-dependent manner while culturable cell count was significantly reduced only when all chlorine concentration was combined with 100 mg/L TDS. However, when chlorine was combined with a higher concentration of TDS, the reduction decreased significantly. The result in this study showed that the combination of the 200 mg/L chlorine and 1000 mg/L TDS could be a practical application in removing L. monocytogenes biofilms from surfaces in food industry, and for the 200 mg/L chlorine and 100 mg/L, it can be used for killing the pathogen biofilms. However, more studies are still needed in order to show its efficacy on foods surfaces as well as to develop an even more effective treatment in both killing and removing biofilms.

Genetic Characteristics of Extended-Spectrum Beta-Lactamase-Producing Salmonella Isolated from Retail Meats in South Korea.

Earlier studies have validated the isolation of extended-spectrum beta-lactamase-producing Salmonella (ESBL-Sal) strains from food. While poultry is recognized as a reservoir for Salmonella contamination, pertinent data regarding ESBL-Sal remains limited. Consequently, the Ministry of Food and Drug Safety has isolated Salmonella spp. from retail meat and evaluated their antibiotic susceptibility and genetic characteristics via whole-genome sequencing. To further elucidate these aspects, this study investigates the prevalence, antibiotic resistance profiles, genomic characteristics, and homology of ESBL-Sal spp. obtained from livestock-derived products in South Korean retail outlets. A total of 653 Salmonella spp. were isolated from 1,876 meat samples, including 509 beef, 503 pork, 555 chicken, and 309 duck samples. The prevalence rates of Salmonella were 0.0%, 1.4%, 17.5%, and 28.2% in the beef, pork, chicken, and duck samples, respectively. ESBL-Sal was exclusively identified in poultry meat, with a prevalence of 1.4% in the chicken samples (8/555) and 0.3% in the duck samples (1/309). All ESBL-Sal strains carried the blaCTX-M-1 gene and exhibited resistance to ampicillin, ceftiofur, ceftazidime, nalidixic acid, and tetracycline. Eight ESBL-Sal isolates were identified as S. Enteritidis with sequence type (ST) 11. The major plasmid replicons of the Enteritidis-ST11 strains were IncFIB(S) and IncFII(S), carrying antimicrobial resistance genes (ß-lactam, tetracycline, and aminoglycoside) and 166 virulence factor genes. The results of this study provide valuable insights for the surveillance and monitoring of ESBL-Sal in South Korean food chain.

Evaluation of crAssphages as a potential marker of human viral contamination in environmental water and fresh leafy greens.

CrAssphages are human gut bacteriophages with potential use as an indicator of human fecal contamination in water and other environmental systems. We determined the prevalence and abundance of crAssphages in water, food, and fecal samples and compared these estimates with the prevalence of norovirus. Samples were tested using two crAssphage-specific qPCR assays (CPQ056 and TN201-203) and for norovirus using TaqMan realtime RT-PCR. CrAssphage was detected in 40% of human fecal specimens, 61% of irrigation water samples, 58.5% of stream water samples, and 68.5% of fresh leafy greens samples. Interestingly, across all sample categories, crAssphage concentrations were 2-3 log10 higher than norovirus concentrations. The correlation of detection of crAssphage and norovirus was significant for the irrigation water samples (r = 0.74, p = 7.4e-06). Sequences obtained from crAssphage positive samples from human fecal and stream water samples phylogenetically clustered with genotype I crAssphages, whereas sequences derived from irrigation water samples clustered differently from other genotypes. Our data show that crAssphages were prevalent in norovirus-positive water samples and in fresh leafy green samples, there was a strong correlation between the presence of crAssphage and norovirus. CrAssphage genomic copies were consistently higher than norovirus copies in all sample types. Overall, our findings suggest that crAssphages could be used as reliable indicators to monitor fecal-borne virus contamination within the food safety chain.

Genetic and virulence characteristics of hybrid Shiga toxin-producing and atypical enteropathogenic Escherichia coli strains isolated in South Korea.

Introduction: The predominant hybrid pathogenic E. coli, enterohemorrhagic E. coli (EHEC), combines characteristics of Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC), contributing to global outbreaks with severe symptoms including fatal consequences. Since EHEC infection was designated as a notifiable disease in 2000 in South Korea, around 2000 cases have been reported, averaging approximately 90 cases annually. Aim: In this work, genome-based characteristic analysis and cell-based assay of hybrid STEC/aEPEC strains isolated from livestock feces, animal source foods, and water in South Korea was performed. Methods: To identify the virulence and antimicrobial resistance genes, determining the phylogenetic position of hybrid STEC/aEPEC strains isolated in South Korea, a combination of real-time PCR and whole-genome sequencing (WGS) was used. Additionally, to assess the virulence of the hybrid strains and compare them with genomic characterization, we performed a cell cytotoxicity and invasion assays. Results: The hybrid STEC/aEPEC strains harbored stx and eae genes, encoding Shiga toxins and E. coli attachment/effacement related protein of STEC and EPEC, respectively. Furthermore, all hybrid strains harbored plasmid-carried enterohemolysin(ehxCABD), a key virulence factor in prevalent pathogenic E. coli infections, such as diarrheal disease and hemolytic-uremic syndrome (HUS). Genome-wide phylogenetic analysis revealed a close association between all hybrid strains and specific EPEC strains, suggesting the potential acquisition of Stx phages during STEC/aEPEC hybrid formation. Some hybrid strains showed cytotoxic activity against HeLa cells and invasive properties against epithelial cells. Notably, all STEC/aEPEC hybrids with sequence type (ST) 1,034 (n = 11) exhibited higher invasiveness than those with E2348/69. This highlights the importance of investigating potential correlations between STs and virulence characteristics of E. coli hybrid strains. Conclusion: Through genome-based characterization, we confirmed that the hybrid STEC/aEPEC strains are likely EPEC strains that have acquired STEC virulence genes via phage. Furthermore, our results emphasize the potential increased danger to humans posed by hybrid STEC/aEPEC strains isolated in South Korea, containing both stx and eaeA, compared to STEC or EPEC alone.

Genome-Based Characterization of Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains Isolated in South Korea, 2016-2020.

The global emergence of hybrid diarrheagenic E. coli strains incorporating genetic markers from different pathotypes is a public health concern. Hybrids of Shiga toxin-producing and enterotoxigenic E. coli (STEC/ETEC) are associated with diarrhea and hemolytic uremic syndrome (HUS) in humans. In this study, we identified and characterized STEC/ETEC hybrid strains isolated from livestock feces (cattle and pigs) and animal food sources (beef, pork, and meat patties) in South Korea between 2016 and 2020. The strains were positive for genes from STEC and ETEC, such as stx (encodes Shiga toxins, Stxs) and est (encodes heat-stable enterotoxins, ST), respectively. The strains belong to diverse serogroups (O100, O168, O8, O155, O2, O141, O148, and O174) and sequence types (ST446, ST1021, ST21, ST74, ST785, ST670, ST1780, ST1782, ST10, and ST726). Genome-wide phylogenetic analysis revealed that these hybrids were closely related to certain ETEC and STEC strains, implying the potential acquisition of Stx-phage and/or ETEC virulence genes during the emergence of STEC/ETEC hybrids. Particularly, STEC/ETEC strains isolated from livestock feces and animal source foods mostly exhibited close relatedness with ETEC strains. These findings allow further exploration of the pathogenicity and virulence of STEC/ETEC hybrid strains and may serve as a data source for future comparative studies in evolutionary biology.

Optimization of RT-PCR methods for enterovirus detection in groundwater.

Enteroviruses (EVs), which belong to the Picornaviridae family, infect individuals asymptomatically or cause mild symptoms (fever, runny nose, cough, skin rash, sneezing, mouth blister). Severe cases can cause various diseases, such as acute hemorrhagic conjunctivitis, aseptic meningitis, or myocarditis, especially in infants. These viruses can be transmitted via the fecal-oral route via contaminated water. In this study, we established a polymerase chain reaction (PCR) method for detecting EVs in water sample using Coxsackievirus B5 (CV-B5) and Echovirus 30 (E-30), which belong to species B of the four species of EVs (EV-A to D). Several methods have been investigated and compared for the detection of EVs, including real-time reverse transcription (RT) polymerase chain reaction and conventional RT-PCR. The most sensitive primer sets were selected, and the PCR conditions were modified to increase sensitivity. We also quantified the detection limits of real-time and conventional RT-PCR. The detection limits of conventional RT-PCR were detected in 105-106 copy/mL for CV-B5 and 106-107 copy/mL for E-30, respectively. This optimized method for detecting EVs is expected to contribute substantially to the investigation of EV outbreaks in water samples.

Novel next generation sequencing panel method for the multiple detection and identification of foodborne pathogens in agricultural wastewater.

Detecting and identifying the origins of foodborne pathogen outbreaks is a challenging. The Next-Generation Sequencing (NGS) panel method offers a potential solution by enabling efficient screening and identification of various bacteria in one reaction. In this study, new NGS panel primer sets that target 18 specific virulence factor genes from six target pathogens (Bacillus cereus, Yersinia enterocolitica, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus) were developed and optimized. The primer sets were validated for specificity and selectivity through singleplex PCR, confirming the expected amplicon size. Crosscheck and multiplex PCR showed no interference in the primer set or pathogenic DNA mixture. The NGS panel analysis of spiked water samples detected all 18 target genes in a single reaction, with pathogen concentrations ranging from 108 to 105 colony-forming units (CFUs) per target pathogen. Notably, the total sequence read counts from the virulence factor genes showed a positive association with the CFUs per target pathogen. However, the method exhibited relatively low sensitivity and occasional false positive results at low pathogen concentrations of 105 CFUs. To validate the detection and identification results, two sets of quantitative real-time PCR (qPCR) analyses were independently performed on the same spiked water samples, yielding almost the same efficiency and specificity compared to the NGS panel analysis. Comparative statistical analysis and Spearman correlation analysis further supported the similarity of the results by showing a negative association between the NGS panel sequence read counts and qPCR cycle threshold (Ct) values. To enhance NGS panel analysis for better detection, optimization of primer sets and real-time NGS sequencing technology are essential. Nonetheless, this study provides valuable insights into applying NGS panel analysis for multiple foodborne pathogen detection, emphasizing its potential in ensuring food safety.

Development and Evaluation of a Next-Generation Sequencing Panel for the Multiple Detection and Identification of Pathogens in Fermented Foods.

These days, bacterial detection methods have some limitations in sensitivity, specificity, and multiple detection. To overcome these, novel detection and identification method is necessary to be developed. Recently, NGS panel method has been suggested to screen, detect, and even identify specific foodborne pathogens in one reaction. In this study, new NGS panel primer sets were developed to target 13 specific virulence factor genes from five types of pathogenic Escherichia coli, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium, respectively. Evaluation of the primer sets using singleplex PCR, crosscheck PCR and multiplex PCR revealed high specificity and selectivity without interference of primers or genomic DNAs. Subsequent NGS panel analysis with six artificially contaminated food samples using those primer sets showed that all target genes were multi-detected in one reaction at 108-105 CFU of target strains. However, a few false-positive results were shown at 106-105 CFU. To validate this NGS panel analysis, three sets of qPCR analyses were independently performed with the same contaminated food samples, showing the similar specificity and selectivity for detection and identification. While this NGS panel still has some issues for detection and identification of specific foodborne pathogens, it has much more advantages, especially multiple detection and identification in one reaction, and it could be improved by further optimized NGS panel primer sets and even by application of a new real-time NGS sequencing technology. Therefore, this study suggests the efficiency and usability of NGS panel for rapid determination of origin strain in various foodborne outbreaks in one reaction.

Seasonal Changes in the Microbial Communities on Lettuce (Lactuca sativa L.) in Chungcheong-do, South Korea.

Lettuce is one of the most consumed vegetables worldwide. However, it has potential risks associated with pathogenic bacterial contamination because it is usually consumed raw. In this study, we investigated the changes in the bacterial community on lettuce (Lactuca sativa L.) in Chungcheong-do, South Korea, and the prevalence of foodborne pathogens on lettuce in different seasons using 16S rRNA gene-based sequencing. Our data revealed that the Shannon diversity index showed the same tendency in term of the number of OTUs, with the index being greatest for summer samples in comparison to other seasons. Moreover, the microbial communities were significantly different between the four seasons. The relative abundance of Actinobacteriota varied according to the season. Family Micrococcaceae was most dominant in all samples except summer, and Rhizobiaceae was predominant in the microbiome of the summer sample. At the genus level, the relative abundance of Bacillus was greatest in spring samples, whereas Pseudomonas was greatest in winter samples. Potential pathogens, such as Staphylococcus and Clostridium, were detected with low relative abundance in all lettuce samples. We also performed metagenome shotgun sequencing analysis on the selected summer and winter samples, which were expected to be contaminated with foodborne pathogens, to support 16S rRNA gene-based sequencing dataset. Moreover, we could detect seasonal biomarkers and microbial association networks of microbiota on lettuce samples. Our results suggest that seasonal characteristics of lettuce microbial communities, which include diverse potential pathogens, can be used as basic data for food safety management to predict and prevent future outbreaks.

Prevalence and Characteristics of Salmonella spp. Isolated from Raw Chicken Meat in the Republic of Korea.

In this study, we sought to investigate the various characteristics of Salmonella spp. isolated from raw chicken meats available in Korean markets. The data collected, such as food source of isolation, sampling information, serotype, virulence, and genetic profile including sequence type, were registered in the database for further comparative analysis of the strains isolated from the traceback investigation samples. To characterize serotype, virulence and gene sequences, we examined 113 domestically distributed chicken meat samples for contamination with Salmonella spp. Phylogenetic analysis was conducted on 24 strains (21.2%) of Salmonella isolated from 113 commercially available chicken meats and by-products, using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Serotyping of the isolated Salmonella spp. revealed S. Enteritidis in 11 strains (45.8%), S. Virchow in 6 strains (25%), S. Montevideo in 2 strains (8.3%), S. Bsilla in 2 strains (8.3%), S. Bareilly in 1 strain (4.2%), S. Dessau in 1 strain (4.2%), and S. Albany in 1 strain (4.2%). The genetic correlation indicated that 24 isolated strains were classified into 18 clusters with a genetic similarity of 64.4-100% between them. Eleven isolated S. Enteritidis strains were classified into 9 genotypes with a sequence identity of 74.4%, whereas the most distantly related S. Virchow was divided into five genotypes with 85.9% identity. Here, the MLST analysis indicated that the major Sequence Type (ST) of the Salmonella spp. isolated from domestic chicken sold in Chungcheong Province belongs to the ST 11 and 16, which differs from the genotype of Salmonella isolated from imported chicken. The differential sequence characteristics can be a genetic marker for identifying causative bacteria for epidemiological investigations of food poisoning.

Comparative Analysis of Salmonella enterica subsp. enterica Serovar Thompson Isolates associated with Outbreaks Using PFGE and wgMLST

The strains associated with foodborne Salmonella enterica Thompson outbreaks in Korea have not been identified. Therefore, we characterized S. Thompson strains isolated from chocolate cakes linked to foodborne outbreaks in Korea. A total of 56 strains were isolated from preserved cake products, products in the supply chain distribution, the manufacturer's apparatus, and egg white liquid products used for cream preparation. Subsequently, serological typing, pathogenic gene-targeted polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE), and whole-genome multi-locus sequence typing (wgMLST) were performed to characterize these isolates. The antigen formula of all isolates was 7:k:1,5, namely Salmonella enterica subsp. enterica Serovar Thompson. All 56 isolates harbored invA, his, hin, and stn, and were negative for sefA and spvC based on gene-targeted PCR analyses. Based on PFGE results, these isolates were classified into one group based on the same SP6X01.011 pattern with 100% similarity. We selected 19 strains based on the region and sample type, which were subjected to wgMLST. Although the examined strains showed 100% similarity, they were classified into seven clusters based on allelic differences. According to our findings, the cause of these outbreaks was chocolate cake manufactured with egg white liquid contaminated with the same Salmonella Thompson. Additionally, comparative analysis of wgMLST on domestic isolates of S. Thompson from the three outbreaks showed genetic similarities of over 99.6%. Based on the results, the PFGE and wgMLST combination can provide highly resolved phylogeny and reliable evidence during Salmonella outbreak investigations.

Comparison of enrichment methods for isolating Enterohemorrhagic Escherichia coli in kimchi.

This study was conducted to compare the efficiency of four enrichment methods of Enterohemorrhagic Escherichia coli by using the 16S rRNA amplicon sequencing and a predictive model. Four different methods (US FDA, ISO, Japan Food Hygiene Association and Korea Ministry of Food and Drug Safety) were used to enrich EHEC in kimchi inoculated with cocktails of EHEC strains (NCCP 13720, NCCP 13721, and NCCP 14134). The maximum growth rate (µmax) and lag phase duration (LPD) were compared using the Baranyi model, and 16S rRNA targeted sequencing was performed with samples at the end of the exponential phase. As a result, the µmax and LPD values of Baranyi model developed for the four enriched media ranged from 0.82 to 0.92 and from 2.35 to 2.68, respectively, suggesting that the growth of EHEC was similar in all four enrichment media. As for the relative abundance of the bacterial composition at the family level, Enterobacteriaceae was identified as the major component (>50%) in all four enriched media. The relative abundance of Enterobacteriaceae was highest (>90%) in the two enriched media with 20 mg/L novobiocin, demonstrating that significant growth of non-targeted bacteria takes place in enrichment broths utilizing <20 mg/L novobiocin or different antibiotics. In conclusion, this study suggests that all four enrichment broth are suitable for growing EHEC in kimchi and the use and concentration of antibiotics such as novobiocin in enrichment media may have a critical role in species diversity.

Evaluation of Hygiene Indicators and Sampling Plan for Detecting Microbial Contamination in Health Functional Foods.

ABSTRACT: 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.

Genomic characteristics and comparative genomics analysis of Salmonella enterica subsp. enterica serovar Thompson isolated from an outbreak in South Korea.

Salmonella infections represent an important public health problem. In 2018, a multistate outbreak of S. enterica subsp. enterica serovar Thompson infection associated with contaminated chocolate cakes in schools was reported in South Korea. In this study, we sequenced the 37 S. Thompson strains isolated from chocolate cakes, egg whites, preserves, and cookware associated with the outbreak. In addition, we analyze the genomic sequences of 61 S. Thompson strains (37 chocolate cake-related outbreak strains, 4 strains isolated from outbreaks in South Korea and 20 strains available in the National Center for Biotechnology Information) to assess the genomic characteristics of outbreak-related strains by comparative genomics and phylogenetic analysis. The results showed that identically classified clusters divided strains into two clusters, sub-clusters A & I (with strains from 2018 in South Korea) and sub-clusters B & II (with strains from 2014 to 2015 in South Korea). S. Thompson isolated from South Korea were accurately distinguished from publicly-available strains. Unlike other S. Thompson genomes, those of chocolate cake outbreak-related strains had three Salmonella phages (SEN8, vB SosS Oslo, and SI7) integrated into their chromosome. Comparative genomics revealed several genes responsible for the specific genomic features of chocolate cake outbreak-related strains and three bacteriophages that may contribute to the pathogenicity of other S. Thompson strains.