The prevalence and characteristics of Shiga toxin-producing Escherichia coli isolated by the enteric pathogens active surveillance network (Enter-Net) in the Republic of Korea, 2009-2018.

OBJECTIVE: Shiga toxin-producing Escherichia coli (STEC) is a water- and food-borne pathogenic agent that causes diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS), and end-stage renal disease. As the annual incidence of STEC increases, disease control is also becoming important in Korea. In this study, we aimed to analyze the incidence trends and characteristics of STEC isolated from diarrheal patients over 10 years. METHODS: From 2009 to 2018, STECs were collected by the Enteric Pathogens Active Surveillance Network (Enter-Net) and analyzed according to clinical epidemiological information (month of isolation, age, and sex of patient), O serogroup, and shiga toxin type. Shiga toxin genes (stx1 and stx2) and O serogroups of isolates were determined using multiplex PCR and an agglutination method with the available O antisera, respectively. RESULTS: A total of 418 strains were isolated over 10 years. The isolation rate according to age group and season was highest in children ≤4 years old (38.1%) and in the summer season (June to August). Among the 418 isolates, the major serogroups were divided O157 (20.3%), O103 (13.6%), O26 (7.7%), O111 (5.5%), O91 (4.3%), O108 (2.4%), and O8 (2.2%). The most frequently isolated O157 showed a lower isolation rate compared to that isolated from other developed countries. The profiles of stx genes were distinct among serogroups. In O157 and O91, stx1+stx2 was detected more frequently than either stx1 or stx2 alone. Particularly, most of the O157 (98%) isolates harbored the stx2 gene, which is an important factor in severe diseases, including HUS. In O103, O26, O111, and O108, stx1-only was more frequently present than stx2-only or stx1+stx2. CONCLUSIONS: As a result of analyzing domestic STECs collected through Enter-Net, it was confirmed that patients ≤4 years of age and in the summer months require attention, and that STEC with a serogroup of O157 is highly likely to cause diseases such as HUS. Therefore, the pathogen active surveillance network for characterization and provision of STEC isolates must be operated continuously.

Comparative Genomic Analysis of the 2016 Vibrio cholerae Outbreak in South Korea.

In August 2016, South Korea experienced a cholera outbreak that caused acute watery diarrhea in three patients. This outbreak was the first time in 15 years that an outbreak was not linked to an overseas source. To identify the cause and to study the epidemiological implications of this outbreak, we sequenced the whole genome of Vibrio cholerae isolates; three from each patient and one from a seawater sample. Herein we present comparative genomic data which reveals that the genome sequences of these four isolates are very similar. Interestingly, these isolates form a monophyletic clade with V. cholerae strains that caused an outbreak in the Philippines in 2011. The V. cholerae strains responsible for the Korean and Philippines outbreaks have almost identical genomes in which two unique genomic islands are shared, and they both lack SXT elements. Furthermore, we confirm that seawater is the likely source of this outbreak, which suggests the necessity for future routine surveillance of South Korea's seashore.

Molecular epidemiology of sequence type 33 of Shiga toxin-producing Escherichia coli O91:H14 isolates from human patients and retail meats in Korea.

Sequence type (ST) 33 of Shiga toxin-producing Escherichia coli (STEC) strain O91:H14 has been proposed as a potential domestic clone of STEC in Korea because of its high prevalence among human patients with mild diarrhea or asymptomatic carriers. Herein, the clonal diversity of 17 STEC O91:H14 isolates of ST33 during 2003 to 2014 was analyzed by pulsed-field gel electrophoresis, including 14 isolates from human patients and 3 from retail meats. Their virulence characteristics, acid resistance, and antimicrobial susceptibility were also determined. Our results showed that all isolates were clustered mainly into three different pulsotypes and were likely low pathogenic without antimicrobial resistance.

Cholera Outbreak due to Raw Seafood Consumption in South Korea, 2016.

Three cases of cholera occurred in South Korea during a period of three weeks in August 2016. All the cases were associated with the consumption of raw seafood in southern coastal area of South Korea. Epidemiologic investigations were performed to track the spread of cholera, including persons in contact with the cholera patients, seafood, and seawater from the fish tank and marine environments. A microbiological investigation demonstrated that cholera isolated from the three patients and a seawater sample at the Korea Strait showed identical serotype (O1 Ogawa), biotype (El tor), and toxin (ctx-positive). Pulsed-field gel electrophoresis analysis showed that the three clinical strains are identical (100%) and shared 97% identity with the seawater sample.

Dairy Cattle, a Potential Reservoir of Human Campylobacteriosis: Epidemiological and Molecular Characterization of Campylobacter jejuni From Cattle Farms.

Campylobacter jejuni is a major foodborne pathogen that is increasingly found worldwide and that is transmitted to humans through meat or dairy products. A detailed understanding of the prevalence and characteristics of C. jejuni in dairy cattle farms, which are likely to become sources of contamination, is imperative and is currently lacking. In this study, a total of 295 dairy cattle farm samples from 15 farms (24 visits) in Korea were collected. C. jejuni prevalence at the farm level was 60% (9/15) and at the animal level was 23.8% (68/266). Using the multivariable generalized estimating equation (GEE) model based on farm-environmental factors, we estimated that a high density of cattle and average environmental temperature (7 days prior to sampling) below 24°C affects the presence and survival of C. jejuni in the farm environment. Cattle isolates, together with C. jejuni from other sources (chicken and human), were genetically characterized based on analysis of 10 virulence and survival genes. A total of 19 virulence profile types were identified, with type 01 carrying eight genes (all except hcp and virB11) being the most prevalent. The prevalence of virB11 and hcp was significantly higher in isolates from cattle than in those from other sources (p < 0.05). Multilocus sequence typing (MLST) of C. jejuni isolates from three different sources mainly clustered in the CC-21 and CC-48. Within the CC-21 and CC-48 clusters, cattle isolates shared an indistinguishable pattern with human isolates according to pulsed-field gel electrophoresis (PFGE) and flaA-restriction fragment length polymorphism (RFLP) typing. This suggests that CC-21 and CC-48 C. jejuni from dairy cattle are genetically related to clinical campylobacteriosis isolates. In conclusion, the farm environment influences the presence and survival of C. jejuni, which may play an important role in cycles of cattle re-infection, and dairy cattle represent potential reservoirs of human campylobacteriosis. Thus, environmental management practices could be implemented on cattle farms to reduce the shedding of C. jejuni from cattle, subsequently reducing the potential risk of the spread of cattle-derived C. jejuni to humans through the food chain.

Surveillance of Bacillus cereus Isolates in Korea from 2012 to 2014.

OBJECTIVES: To investigate the prevalence and toxin production characteristics of non-emetic and emetic Bacillus cereus strains isolated via the laboratory surveillance system in Korea. METHODS: A total of 667 B. cereus strains were collected by the Korea National Research Institute of Health laboratory surveillance system from 2012 to 2014. The collected strains were analyzed by geographical region, season, patient age, and patient sex. Additionally, the prevalence rates of enterotoxin and emetic toxin genes were evaluated. RESULTS: The isolation rate of B. cereus strains increased during the summer, but the isolation rate was evenly distributed among patient age groups. Emetic toxin was produced by 20.2% of the isolated strains. The prevalence rates of five enterotoxin genes (entFM, nheA, cytK2, hblC, and bceT) were 85.0, 78.6, 44.5, 36.6, and 29.7%, respectively, among non-emetic strains and 77.8, 59.3, 17.8, 11.9 and 12.6%, respectively, among emetic strains. Thus, the prevalence rates of all five enterotoxin genes were lower in emetic B. cereus. CONCLUSION: The prevalence of enterotoxin genes differed between non-emetic and emetic B. cereus strains. Among emetic B. cereus strains, the prevalence rates of two enterotoxin genes (cytK2 and hblC) were lower than those among the non-emetic strains. In both the emetic and non-emetic strains isolated in Korea, nheA and entFM were the most prevalent enterotoxin genes.

Genome Sequencing Analysis of Atypical Shigella flexneri Isolated in Korea.

OBJECTIVES: An atypical Shigella flexneri strain with a plural agglutination pattern [i.e., reacting not only with serum samples containing type antigen II but also with serum samples containing group antigens (3)4 and 7(8)] was selected for genome sequencing, with the aim of obtaining additional comparative information about such strains. METHODS: The genomic DNA of atypical S. flexneri strain NCCP 15744 was sequenced using an Ion Torrent PGM sequencing machine (Life Technologies, USA). The raw sequence data were preprocessed and reference-assembled in the CLC Assembly Cell software (version 4.0.6; CLC bio, USA). RESULTS: Ion Torrent sequencing produced 1,450,025 single reads with an average length of 144 bp, totaling ~209 Mbp. The NCCP 15744 genome is composed of one chromosome and four plasmids and contains a gtrX gene. Among the published genome sequences of S. flexneri strains, including 2457T, Sf301, and 2002017, strain NCCP 15744 showed high similarity with strain 2002017. The differences between NCCP 15744 and 2002017 are as follows: i) NCCP 15744 carries four plasmids whereas 2002017 carries five; ii) 19 genes (including CI, CII, and cro) were lost in the SHI-O genomic island of NCCP 15744 and six genes were gained as compared with strain 2002017. CONCLUSION: Strain NCCP 15744 is genetically similar to 2002017, but these two strains have different multilocus sequence types and serotypes. The exact reason is unclear, but the 19 lost genes may be responsible for the atypical seroconversion of strain NCCP 15744.

Enteric Bacteria Isolated from Diarrheal Patients in Korea in 2014.

OBJECTIVES: The aim of this study was to characterize the pathogens responsible for causing diarrhea according to season, region of isolation, patient age, and sex as well as to provide useful data for the prevention of diarrheal disease. METHODS: Stool specimens from 14,886 patients with diarrhea were collected to identify pathogenic bacteria from January 2014 to December 2014 in Korea. A total of 3,526 pathogenic bacteria were isolated and analyzed according to season, region of isolation, and the age and sex of the patient. RESULTS: The breakdown of the isolated pathogenic bacteria were as follows: Salmonella spp. 476 (13.5%), pathogenic Escherichia coli 777 (22.0%), Vibrio parahaemolyticus 26 (0.74%), Shigella spp. 13 (0.37%), Campylobacter spp. 215 (6.10%), Clostridium perfringens 508 (14.4%), Staphylococcus aureus 1,144 (32.4%), Bacillus cereus 356 (10.1%), Listeria monocytogenes 1 (0.03%), and Yersinia enterocolitica 10 (0.3%). The isolation rate trend showed the highest ratio in the summer season from June to September for most of the pathogenic bacteria except the Gram-positive bacteria. The isolation rate of most of the pathogenic bacteria by patient age showed highest ratio in the 0-19 year age range. For isolation rate by region, 56.2% were isolated from cities and 43.8% were isolated from provinces. CONCLUSION: Hygiene education should be addressed for diarrheal disease-susceptible groups, such as those younger than 10 years, aged 10-19 years, and older than 70 years, and monitoring for the pathogens is still required. In addition, an efficient laboratory surveillance system for infection control should be continued.

Outbreak of Ciprofloxacin-Resistant Shigella sonnei Associated with Travel to Vietnam, Republic of Korea.

We investigated an October 2014 outbreak of illness caused by Shigella sonnei in a daycare center in the Republic of Korea (South Korea). The outbreak strain was resistant to extended-spectrum cephalosporins and fluoroquinolones and was traced to a child who had traveled to Vietnam. Improved hygiene and infection control practices are needed for prevention of shigellosis.

Complete nucleotide sequence of the IncI1 plasmid pSH4469 encoding CTX-M-15 extended-spectrum ß-lactamase in a clinical isolate of Shigella sonnei from an outbreak in the Republic of Korea.

An outbreak of extended-spectrum ß-lactamase (ESBL)-producing Shigella sonnei infections occurred in a school for disabled children in Gyeongbuk Province, Republic of Korea, in 2008. Five students were affected. Pulsed-field gel electrophoresis (PFGE) analysis revealed that all of the ESBL-producing S. sonnei isolates belonged to the same clone, and nucleotide sequence analysis of ESBL genes revealed that they harboured bla(CTX-M-15). This is the first identification of bla(CTX-M-15) in Shigella spp. in South Korea. In this study, a plasmid carrying the bla(CTX-M-15) gene, designated pSH4469, recovered from a S. sonnei isolate responsible for the outbreak was characterised. Replicon typing and plasmid multilocus sequence typing (pMLST) analysis of plasmids in the outbreak strain identified that the bla(CTX-M-15) gene was located on an IncI1 incompatibility group plasmid of sequence type 16 (ST16). The complete nucleotide sequence of pSH4469 revealed that this plasmid is 91109bp and harbours 119 putative genes, including another antibiotic resistance gene (bla(TEM-1b)) that is often associated with the ISEcp1-bla(CTX-M-15)-orf477delta transposable unit. The plasmid consists of a large backbone with considerable homology to the pEK204 plasmid isolated from Escherichia coli in the UK, except for insertion of an IS66 element found in pEK204. These data demonstrate that IncI1 plasmids are used as a successful platform for efficient horizontal gene transfer, thereby resulting in the dissemination of CTX-M-type ß-lactamases among Enterobacteriaceae.

Genetic diversity of Campylobacter jejuni isolates from Korea and travel-associated cases from east and southeast Asian countries.

Forty domestic and travel-associated Campylobacter jejuni isolates were analyzed by profiling 7 pathogenic genes (cdtB, cadF, Cj0131, ciaB, racR, wlaN, and virB11) along with multilocus sequence typing (MLST) and antimicrobial susceptibility testing. cdtB, cadF, and Cj0131 were present in all isolates, whereas virB11 was not detected in either domestic or travel-associated isolates. ciaB was present in all domestic isolates and 94% of travel-associated isolates. The respective detection rates of racR and wlaN in domestic and travel-associated isolates were 94% and 71% and 35.3% and 23%, respectively. MLST analyses of the 40 isolates generated 25 different sequence types (STs). ST-443 (12 isolates) and ST-21 (8 isolates) were dominant among the domestic isolates; however, STs varied among travel-associated isolates. Nalidixic acid, tetracycline, and ciprofloxacin resistance rates of the 40 isolates were 100% (40/40), 95% (38/40), and 88% (35/40), respectively. Domestic isolates exhibited 2-fold higher ciprofloxacin, telithromycin, and chloramphenicol resistance rates than travel-associated isolates. These results indicate a diverse genetic background for travel-associated C. jejuni and suggest that this pathogen may be an important emerging public health threat to travelers.

Comparative proteomic label-free analysis of Campylobacter jejuni NCTC 11168 cultured with porcine mucin.

Campylobacter jejuni is a major gastrointestinal pathogen in humans. Poultry is a primary reservoir for C. jejuni, and C. jejuni appears to be highly adapted to the gastrointestinal tracts of avian species. We determined the protein expression profiles of C. jejuni NCTC 11168 cultured in medium containing porcine mucin. Differentially expressed proteins in the presence and absence of porcine mucin were identified using the label-free method. We identified 52 proteins with expression that was either upregulated (32 proteins) or downregulated (20 proteins) by porcine mucin. These proteins are involved in diverse cellular functions, such as motility, cell wall synthesis, iron transport, energy production, and amino acid metabolism. In particular, the upregulated proteins were involved in chemotaxis (CheV and CetA), motility (FlaA), colonization and adherence (CadF, FrdA, CfrA, MapA, and HydA), and stress tolerance (TrxB and ClpB). These results suggest that C. jejuni changes its protein expression in response to porcine mucin and that this change in expression may contribute to host adaptation of C. jejuni NCTC 11168.

Enhanced Type III Secretion System Expression of Atypical Shigella flexneri II:(3)4,7(8).

OBJECTIVES: We aimed at evaluating the virulence of atypical Shigella flexneri II:(3)4,7(8) by DNA microarray and invasion assay. METHODS: We used a customized S. flexneri DNA microarray to analyze an atypical S. flexneri II:(3)4,7(8) gene expression profile and compared it with that of the S. flexneri 2b strain. RESULTS: Approximately one-quarter of the atypical S. flexneri II:(3)4,7(8) strain genes showed significantly altered expression profiles; 344 genes were more than two-fold upregulated, and 442 genes were more than 0.5-fold downregulated. The upregulated genes were divided into the category of 21 clusters of orthologous groups (COGs), and the "not in COGs" category included 170 genes. This category had virulence plasmid genes, including the ipa-mxi-spa genes required for invasion of colorectal epithelium (type III secretion system). Quantitative reverse-transcription polymerase chain reaction results also showed the same pattern in two more atypical S. flexneri II:(3)4,7(8) strains. Atypical S. flexneri II:(3)4,7(8) showed four times increased invasion activity in Caco-2 cells than that of typical strains. CONCLUSION: Our results provide the intracellularly regulated genes that may be important for adaptation and growth strategies of this atypical S. flexneri.

Anti-Shiga toxin immunoglobulin G antibodies in healthy South Korean slaughterhouse workers.

BACKGROUND: Slaughterhouse workers are in direct contact with cattle nearly every day. The purpose of this study was to survey the presence and distribution of anti-Shiga toxin 1 (Stx1) immunoglobulin G (IgG) in slaughterhouse workers, enabling a study of the serologic response to this toxin while working in an area at high-risk of Stx-producing Escherichia coli (STEC) infection. METHODS: One thousand seven hundred and twenty-nine serum samples from healthy slaughterhouse employees were collected and surveyed by indirect enzyme-linked immunosorbent assay (ELISA). RESULTS: Among the 5 slaughterhouse positions, slaughterers had the highest distribution of anti-Stx1 IgG values by an ELISA. Based on the ELISA values, 25% (433/1729) of the workers had anti-Stx1 IgG. Slaughterers, residual products handlers, inspectors, livestock hygiene controllers, and grading testers had anti-Stx1 IgG-positive rates of 28%, 25%, 20%, 19%, and 17%, respectively. The ELISA values of anti-Stx1 IgG increased with increases in the number of years worked by slaughterers, but not by residual products handlers, inspectors, livestock hygiene controllers, or grading testers. CONCLUSIONS: From these results, slaughterhouse workers are healthy and asymptomatic; slaughterers in particular are at high-risk for STEC exposure.

Prevalence of Shiga toxin-encoding genes and risk factors among dairy farmers in Gyeonggi Province, Korea.

BACKGROUND: Dairy farmers perform various types of work and are in direct contact with dairy cattle nearly every day. The purpose of this study was to assess the prevalence of Shiga toxin-encoding genes (stx) among dairy farmers and to evaluate the relationship between stx and risk factors. METHODS: A questionnaire developed in-house was sent to dairy farmers in Gyeonggi Province, Korea by registered mail. Researchers obtained stool samples and identified or administered the questionnaires by interview. The stool samples were examined for stx genes by polymerase chain reaction. RESULTS: Twenty (3.4%) of 589 stool samples from dairy farmers were stx-positive. The distribution of stx-positive stool samples revealed an increase in Shiga toxin-producing Escherichia coli infection with age, duration of work, and herd size. There was no association between stx-positive stool samples and type of work. For artificial insemination, taking a shower after work was significant, and the proportion of stx-positive dairy farmers increased as taking a shower after work decreased. CONCLUSIONS: Hygiene-related education to include taking a shower after sessions of artificial insemination should be considered. However, the stx-positive dairy farmers were small in number and the results should be interpreted with caution.

Prevalence of Farm and Slaughterhouse Workers Carrying Shiga Toxin-Producing Escherichia coli in Korea.

OBJECTIVES: The aim of this study was to investigate the distribution of Shiga toxin (Stx) gene-positive stool samples from dairy farmer and slaughterhouse workers in Gyeonggi-Do province. METHODS: A total of 621 samples from healthy farmers and 198 samples from slaughterhouse workers were screened by polymerase chain reaction (PCR) for Shiga toxigenic Escherichia coli (STEC) infection on stool samples. RESULTS: The PCR product of Stx-encoding genes was detected in 21 (3.4%) of 621 farmers and 15 (7.6%) of 198 slaughterhouse workers' stool samples. Distribution of the Stx PCR positive workers by age increment revealed an increase in STEC infection with age increment in both workers. Distribution of the Stx PCR positive workers by working years revealed an increase in STEC infection with working years in farmers. CONCLUSION: These results of the study show that slaughterhouse workers are at higher risk of STEC infection than farmers. In addition, slaughterhouse workers have a more potential source of food contamination of STEC and transmission.

Genetic characterization of atypical Shigella flexneri isolated in Korea.

Three types of serotypically atypical Shigella flexneri strains were isolated from 2007 to 2008 in patients at the Korea National Institute of Health (NIH). These strains were characterized and compared with serologically typical S.flexneri. One type of strain either displayed nonreacting typing or grouping sera, reacting strongly only with polyB antisera, which indicates this strain is S. flexneri (polyB:un). The second type displayed reactions with one of the typing sera (IV) and did not bind any grouping sera (IV:un). The remaining type of strain displayed a plural agglutination pattern, reacted with one typing sera (II), and bound with two grouping sera (II:(3)4,7(8)). Among these atypical strains IV:un and II:(3)4,7(8) strains showed higher multi-antibiotic resistance in ampicillin, streptomycin, and trimethoprim-sulfamethoxazole than typical strains. Furthermore, all II:(3)4,7(8) strains harbored integrons. This study suggests that these multiple antibiotic-resistant atypical S. flexneri are new subserotypes of S.flexneri that await further serological classification.

Asymptomatic healthy slaughterhouse workers in South Korea carrying Shiga toxin-producing Escherichia coli.

A total of 1602 stool samples from healthy employees in a slaughter company were screened by PCR for Shiga toxin (Stx)-producing Escherichia coli (STEC). The PCR product of Stx-encoding genes was detected in 90 (5.6%) of 1602 stool samples. Among the 90 stx-positive workers, the Residual Products Handlers and Slaughterers had rates of 8.0% and 6.0%--higher than Inspectors, Grading Testers and Livestock Hygiene Controllers at 3.3%, 2.0% and 3.5%, respectively. Forty-nine (54.4%) were shown to have stx2; 25 (27.7%) carried stx1 and 16 (17.7%) had both stx1 and 2. Distribution of the stx PCR-positive workers by age revealed an increase in STEC infection with age (P<0.05). Phenotypic and genotypic traits of nine STEC strains isolated from eight slaughter plant workers were characterized. A variety of serotypes, five O serogroups (O8, O54, O59, O103 and O153) and two H serogroups (H7 and H32) were found, but none of the strains belonged to the serogroup O157. Eight Vero cell cytotoxicity assay-positive strains were isolated from the workers and these workers were asymptomatic and healthy. The results of the study show that slaughter plant workers are at high risk of STEC infection.