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ABSTRACT: Objectives: Global spread of mobilized colistin resistance gene (mcr)-carrying Escherichia coli poses serious threats to public health. This study aimed to provide insights into different threats posed by two major mcr variants: mcr-1.1 and mcr-3.1. Methods: Genetic backgrounds and characteristics of mobile genetic elements carrying mcr-1.1 or mcr-3.1 in 74 (mcr)-carrying E. coli isolated from swine farms were analysed, and comparative genomic analysis was performed with the public sequence database. Results: The mcr-1.1 showed high horizontal transferability (6.30 logCFU/ml). Genetic background of mcr-1.1, including genetic cassette/plasmid, was transferred without insertion sequences (ISs) and/or multi-drug resistance (MDR) and highly shared across strains. The major mcr-1.1-cassette was “mcr-1.1-pap2”, mainly encoded in IncI2 and IncX4. Mcr-3.1 exhibited relatively lower conjugation frequency (0.97 logCFU/ml). The mcr-3.1-cassette was flanked by IS26 and was highly variable across strains because of the insertion, deletion, or truncation of IS6100, IS4321, or IS5075. Near the mcr-3.1 cassette, MDR regions consisting of antimicrobial/heavy metal resistance genes were identified, which varied across strains. From the MCR3-E13 strain, a mcr-3.1-carrying IncHI2-fragment was integrated into the bacterial chromosome via IS26-mediated co-integration. To our knowledge, this was the first study to describe that a mcr-3.1-carrying plasmid could be inserted into the bacterial chromosome. Conclusions: Based on high horizontal transferability, mcr-1.1 could play a major role on colistin resistance propagation. On the other hand, mcr-3.1 could be transmitted with MDR and have dual pathways mediated by plasmid transfer (horizontal transmission) and chromosomal insertion (vertical transmission), enabling it to proliferate stably despite its lower horizontal transferability.

Campylobacter is a major zoonotic pathogen that causes gastrointestinal and, rarely, immune diseases in humans. The antimicrobial-resistance gene cfr(C) carried by Campylobacter and is a cfr-like gene that targets bacterial 23S rRNA through A2503 methylation. cfr(C) confers cross-resistance to five antimicrobial classes (PhLOPSA), including lincosamide, streptogramin A, and pleuromutilin, which are classified as critically important antimicrobials to human by the World Health Organization. To elucidate the genetic variation and horizontal transfer mechanism of cfr(C), we analyzed the genetic background and horizontal transfer unit of Campylobacter-derived cfr(C) through comparative genomic analysis. We identified nine cfr(C)-positive C. coli strains of 157 strains isolated from swine sources. Three novel cfr(C) gene single nucleotide polymorphism (SNP) sites (19delA, 674C > A, and 890 T > C) were identified from nine cfr(C)-positive strains. Among six identified cfr(C) SNP variant types (SNP-I to -VI), five types of randomly inserted cfr(C)-cassettes on chromosome and one type of plasmid-like element were identified, their gene cassette composition differing depending on the cfr(C) variants. Three of six cfr(C) cassette types contained aminoglycoside-streptothricin resistance cluster “aphA3-sat4-aadE.” The cfr(C) gene cassette with pcp gene (GC-1, GC-4, and GC-5) formed a pcp-mediated circular intermediate “pcp-hp-cfr(C)-aphA3,” which has not been previously reported. Other two cfr(C) cassette-types with ISChh1 formed circular intermediate “ISChh1-aphA3-cfr(C)-lnu (G)-pnp-ant1-hp-ATPase” and “ISChh1-aphA3-cfr(C)-hp.” In conjugation assay, the pcp-mediated circular intermediate was naturally transferred to the plasmid of recipient C. coli wild-type strain from swine source, and comparative genomic analysis revealed that cfr(C) encoded in pcp-mediated circular intermediate was inserted into the plasmid of recipient by homologous recombination with pcp and aphA3. This study revealed that novel multidrug resistance gene cfr(C) carried by C. coli from swine sources can be highly genetically diverse and transferable. Moreover, we suggest that the transferability of chromosomal cfr(C) may contribute to the global spread of multidrug resistance against clinically important antimicrobials.

Listeria monocytogenes is the etiologic agent of listeriosis, a foodborne disease that poses a threat to public health globally. Chicken meat exhibits heightened susceptibility to L. monocytogenes contamination during butchery. The persistence of this pathogen in the slaughterhouse environment enables recurring contamination of meat products. This study aimed at identifying the sources and transmission routes of L. monocytogenes contamination within an abattoir where it was consistently detected for three consecutive years (2019–2021). Furthermore, the environmental factors aiding contamination along chicken processing lines were determined by surveying the microbiome within the facility. Samples collected in 2019 to 2021 were subjected to culture-dependent analysis to assess the prevalence, serotypes, and multi-locus sequence typing (MLST) of L. monocytogenes. Additionally, the specimens collected in 2021 underwent culture-independent analysis via real-time quantitative polymerase chain reaction (qPCR) and 16S rRNA gene amplicon sequencing to identify the contamination sources and characterize the entire microbial community within the slaughterhouse. L. monocytogenes was isolated only from the clean zone, where the final slaughtering stage occurs. Most strains isolated from the final carcasses showed the same genetic cluster as the isolate in the chilling water and were assigned to MLST profile ST3. Culture-independent qPCR confirmed L. monocytogenes contamination in all samples, excluding post-scalding carcasses, prewashed post-evisceration carcasses, and the bleeding areas. Consequently, qPCR enabled more comprehensive identification of L. monocytogenes contamination points than culture-dependent approaches. Moreover, 16S rRNA gene amplicon sequencing demonstrated that psychro-tolerant and spoilage-related bacteria with L. monocytogenes-like attributes exhibited enhanced viability in the clean zone and immersion-chilling water. Metagenomics-based source tracking analysis further revealed that the shackles and chilling waters represent predominant sources of cross-contamination between different slaughterhouse zones, whereas the grading and packaging workstations and chilling water in the clean zone were deemed crucial sources affecting final carcass contamination. Collectively, these findings demonstrate through culture-dependent and -independent methods that L. monocytogenes spreads along the slaughter line, contaminating the slaughterhouse. Moreover, by investigating changes in microbial community and bacterial flow along the slaughter line within the facility, the sources influencing carcass contamination can be effectively traced.

ABSTRACT Red ginseng, widely used in traditional medicine for various conditions, imparts health benefits mainly by modulating the gut microbiota in humans. Given the similarities in gut microbiota between humans and dogs, red ginseng-derived dietary fiber may have prebiotic potential in dogs; however, its effects on the gut microbiota in dogs remain elusive. This double-blinded, longitudinal study investigated the impact of red ginseng dietary fiber on the gut microbiota and host response in dogs. A total of 40 healthy household dogs were randomly assigned to low-dose (n = 12), high-dose (n = 16), or control (n = 12) groups and fed a normal diet supplemented with red ginseng dietary fiber (3 g/5 kg body weight per day, 8 g/5 kg per day, or no supplement, respectively) for 8 weeks. The gut microbiota of the dogs was analyzed at 4 weeks and 8 weeks using 16S rRNA gene sequencing of fecal samples. Alpha diversity was significantly increased at 8 and 4 weeks in the low-dose and high-dose groups, respectively. Moreover, biomarker analysis showed that short-chain fatty acid producers such as Sarcina and Proteiniclasticum were significantly enriched, while potential pathogens such as Helicobacter were significantly decreased, indicating the increased gut health and pathogen resistance by red ginseng dietary fiber. Microbial network analysis showed that the complexity of microbial interactions was increased by both doses, indicating the increased stability of the gut microbiota. These findings suggest that red ginseng-derived dietary fiber could be used as a prebiotic to modulate gut microbiota and improve gut health in dogs. IMPORTANCE The canine gut microbiota is an attractive model for translational studies, as it responds to dietary interventions similarly to those in humans. Investigating the gut microbiota of household dogs that share the environment with humans can produce highly generalizable and reproducible results owing to their representativeness of the general canine population. This double-blind and longitudinal study investigated the impact of dietary fiber derived from red ginseng on the gut microbiota of household dogs. Red ginseng dietary fiber altered the canine gut microbiota by increasing diversity, enriching short-chain fatty acid-producing microbes, decreasing potential pathogens, and increasing the complexity of microbial interactions. These findings indicate that red ginseng-derived dietary fiber may promote canine gut health by modulating gut microbiota, suggesting the possibility of its use as a potential prebiotic.

ABSTRACT Wild migratory birds are essential for sustaining healthy ecosystems, but the effects of a rehabilitation period on their gut microbiomes are still unclear. Here, we performed longitudinal sampling, 16S rRNA sequencing, and antibiotic resistance monitoring of the gut microbiome of six species of wild migratory birds protected as natural monuments in South Korea that are subject to short- or long-term rehabilitation periods. Overall, gut microbiome diversity was significantly decreased in the early stages of rehabilitation, and it did not recover to a level comparable to that of wild birds. Moreover, while the abundance of short-chain fatty acid-producing bacteria decreased, that of zoonotic pathogens increased, indicating rehabilitation-induced dysbiosis. The metabolic pathways involved in the degradation of aromatic pollutants were significantly downregulated, suggesting the depletion of pollutant-degrading microorganisms. Antibiotic resistance of Escherichia coli significantly increased during rehabilitation, particularly ciprofloxacin and tetracycline resistance, and seven of the rehabilitated wild birds acquired multidrug resistance. The diet and habitat changes experienced by wild migratory birds during rehabilitation may have induced the observed gut microbiome dysbiosis and acquisition of antibiotic resistance. These rehabilitation-induced alterations might affect the adaptability of wild birds to their natural environments and contribute to the spread of antibiotic resistance after their release. IMPORTANCE Wild migratory birds are key for ecosystem health but highly sensitive to anthropogenic activities. Therefore, wild migratory birds often undergo rehabilitation to prevent species extinction or biodiversity monitoring. However, the impact of rehabilitation on the gut microbiome of wild migratory birds, which is closely associated with host fitness, remains unclear. For the migratory bird species considered natural monuments in South Korea evaluated here, such impacts could include rehabilitation-induced gut microbiome dysbiosis and acquisition of antibiotic resistance, with possible repercussions on the adaptability of wild birds and spread of antibiotic resistance in the environment after their release. Therefore, the dynamics of the gut microbiome and antibiotic resistance should be considered for implementing sustainable rehabilitation strategies.

Abstract Are predators of rodents beneficial for public health? This question focuses on whether predators regulate the spillover transmission of rodent-borne diseases. No clear answer has emerged because of the complex linkages across multiple trophic levels and the lack of accessible data. Although previous empirical findings have suggested ecological mechanisms, such as resource partitioning, which implies protective effects from predator species richness, epidemiological evidence is needed to bolster these arguments. Thus, we investigated the association between predator species richness and incidence of rodent-borne haemorrhagic fever with renal syndrome in the human population using district-level longitudinal data of 13 years for South Korea. With the exception of districts with low species richness, we found a significant negative association between the incidence of haemorrhagic fever with renal syndrome and the species richness of both avian and mammalian predators; the trends for both predator types were similar. Thus, biodiversity conservation may benefit public health.

Global spread of Escherichia coli strains carrying the mobilized colistin resistance gene mcr-1.1 (MCR1-EC) poses serious threats to public health. Colistin has been generally prescribed for swine colibacillosis, having made swine farms as major reservoirs of MCR1-EC. The present study aimed to understand characteristic differences of MCR1-EC, including prevalence, antimicrobial resistance, and virulence, according to swine production stages. In addition, genetic relatedness was evaluated between MCR1-EC isolated from this study as well as pig-, human-, and chicken-derived strains published in the National Center for Biotechnology Information (NCBI), based on the multi-locus sequence types (MLSTs) and whole-genome sequences (WGS). Individual fecal samples (n = 331) were collected from asymptomatic weaning-piglets, growers, finishers, and sows from 10 farrow-to-finishing farms in South Korea between 2017 and 2019. The weighted prevalence of MCR1-EC was 11.6% (95% CI: 8.9%–15.0%, 55/331), with the highest prevalence at weaning stage. The 96.2% of MCR1-EC showed multi-drug resistance. Notably, weaning stage-derived MCR1-EC showed higher resistance rates (e.g., against extended-spectrum β-lactams or quinolones) than those from other stages. MCR1-EC with virulence advantages (e.g., intestinal/extraintestinal pathogenic E. coli or robust biofilm formation) were identified from all pig stages, accounting for nearly half of the total strains. WGS-based in-depth characterization showed that intestinal pathogenic MCR1-EC harbored multi-drug resistance and multiple virulence factors, which were highly shared between strains isolated from pigs of different stages. The clonal distribution of MCR1-EC was shared within swine farms but rarely across farms. The major clonal type of MCR1-EC from swine farms and NCBI database was ST10-A. Core genomes of MCR1-EC isolated from individuals within closed environments (same farms or human hospitals) were highly shared (genetic distance

Abstract Background Originating from poultry, particularly chickens, Campylobacter jejuni is the leading foodborne pathogen worldwide and a major cause of campylobacteriosis. Isolating C. jejuni is difficult due to its specific growth requirements, the presence of viable but non-culturable bacteria, and because it is often masked by competing flora. Currently, there is no optimized method for isolating C. jejuni from chicken feces. Here, we evaluated the method for isolating C. jejuni from chicken feces using culture-independent sequence-based metagenomics and culture-dependent tools. Further, we assessed changes in microbial communities during microbe isolation to determine how the process can be improved. Results Fourteen different variations of C. jejuni isolation procedures were applied to all 35 chicken fecal samples. These variations included using different enrichment broths (without enrichment or enrichment in Bolton or Preston broth), different ratios of sample-to-enrichment broth (1:101, 1:102, and 1:103), and different selective agars (modified charcoal-cefoperazone-deoxycholate agar (mCCDA) or Preston agar). Enrichment during isolation of C. jejuni was evaluated on the basis of microbial diversity and taxonomic composition using metagenomics tools. The effect of selective media was evaluated using a combination of metagenomics and culture-dependent tools. Microbial diversity significantly decreased during the enrichment process, regardless of the type of enrichment broth, with the most significant decrease observed at a feces-to-broth ratio of 1:103. Particularly, in 103-Preston broth, the relative abundance of Campylobacter increased, while extended-spectrum beta-lactamase-producing Escherichia coli, which interfere with Campylobacter isolation, decreased. Metagenomics results were validated by quantitative PCR and culture-dependent analysis. Additionally, selective media affected the isolation results, although microbes with high relative abundance during enrichment were also frequently isolated using culture-dependent methods. Significantly more C. jejuni was isolated from mCCDA than from Preston agar enriched in 103 Preston broth. Conclusions Enrichment in Preston broth at a ratio of 1:103 followed by spreading onto mCCDA was the most effective method for isolating C. jejuni. This is the first study to apply metagenomics to evaluate a method for isolating a targeted microbe, C. jejuni, from chicken feces, a source with high microbial contamination. Thus, metagenomics can be applied to improve methods for isolating bacteria that are difficult to separate.

The worldwide spread of extended spectrum β-lactamase (ESBL)- and AmpC β-lactamase (AmpC)-producing Escherichia coli poses serious threats to public health. Swine farms have been regarded as important reservoirs of ESBL/AmpC-EC. This study aimed to determine the prevalence, ESBL/AmpC types, and clonal distribution of ESBL/AmpC-EC from swine farms and analyze the difference according to the swine production stages. In addition, we evaluated the potential risks of swine ESBL/AmpC-EC clones to humans. Individual fecal samples (n = 292) were collected from weaning, growing, finishing, and pregnant pigs in nine swine farms of South Korea between July 2017 and March 2020. In total, 161 ESBL/AmpC-EC isolates were identified (55.1%), with the highest prevalence detected in the weaning stage (86.3%). The dominant ESBL and AmpC types were CTX-M-55 (69.6%) and CMY-2 (4.3%), respectively. CTX-M found in all production stages, while CMY was only found in growing and finishing stages. In the conjugation assay, the high transferability of CTX-M gene (55.8%) was identified, while the transfer of CMY gene was not identified. The major clonal complexes (CCs) were CC101-B1 (26.8%), CC10-A (8.7%), and CC648-F (2.9%). There was similarity in clonal distribution between different swine production stages within swine farms, estimated using the k-means analysis, which suggested a clonal transmission between the different swine stages. Among swine ESBL/AmpC-EC sequence types (STs), seven STs (ST101, ST10, ST648, ST457, ST410, ST617, and ST744) were common with the human ESBL/AmpC-EC, which registered in National Center for Biotechnology Information database. The clonal population structure analysis based on the virulence factor (VF) presented that swine ESBL/AmpC-EC clones, especially ST101-B1, harbored a highly virulent profile. In conclusion, ESBL/AmpC-EC was distributed throughout the swine production stages, with the highest prevalence in the weaning stage. The CTX-M was present in all stages, while CMY was mostly found in growing-finishing stages. The swine ESBL/AmpC-EC was identified to harbor shared clone types with human ESBL/AmpC-EC and a virulent profile posing potential risk to humans. Considering the possibility of genetic and clonal distribution of ESBL/AmpC-EC among swine production stages, this study suggests the need for strategies considering the production system to control the prevalence of ESBL/AmpC-EC in swine farms.

Campylobacter, a major foodborne pathogen, is susceptible to oxygen. Recently, aerotolerant Campylobacter with enhanced tolerance to aerobic stress has become a major concern in food safety. However, the aerotolerance of Campylobacter coli from pigs has not been studied extensively. Here, we sought to investigate the prevalence of C. coli across multiple swine groups in farms, including weaning, growing, and fattening pigs in production stages and pregnant sows. Additionally, we analyzed C. coli aerotolerance, quinolone resistance, virulence potential, and multilocus sequence typing (MLST) genotypes. Finally, we compared the characteristics of C. coli according to the aerotolerance levels. In total, we obtained 124 (66.3%) C. coli isolates from 187 swine fecal samples across six swine farms. The pathogen was prevalent in weaning (45.5%), growing (68.3%), and fattening (75.4%) pigs, and pregnant sows (66.7%). Hyper-aerotolerant HAT C. coli (13.7% of 124 isolates) was present in all swine groups, with the highest proportion in the pregnant sows (27.3%). All HAT isolates possessed diverse virulence-related genes such as flaA, cadF, pldA, ceuE, and cdtA. All C. coli isolates were resistant to quinolones, and 12 (10%) presented high-level ciprofloxacin resistance (MIC ≥ 32 μg/mL). The proportion of C. coli isolates with a high-level ciprofloxacin resistance was the highest in HAT C. coli (18.8%). Furthermore, six MLST sequence types (STs) (ST827, ST830, ST854, ST1016, ST1068, and ST1096) of swine-derived C. coli were in common with human-derived C. coli (PubMLST). The proportion of C. coli belonging to such shared STs at each aerotolerance level was the highest in HAT C. coli (HAT vs. oxygen-sensitive; OR = 3.13). In conclusion, quinolone resistance of C. coli may be distributed throughout in all swine groups in farms. HAT C. coli is likely to remain in pig farms and re-infect other pigs in the farms. Furthermore, swine-derived HAT C. coli could be transmitted to humans easily through the food chain owing to its aerotolerance, and it could pose a threat to public health owing to its high-level ciprofloxacin resistance and virulence. This study highlights the need to develop management practices that prevent the transmission of swine-derived HAT C. coli to humans.

It is important to understand pathogen transmissibility in a population to establish an effective disease prevention policy. The basic reproduction number (R0) is an epidemiologic parameter for understanding the characterization of disease and its dynamics in a population. We aimed to estimate the R0 of the highly pathogenic avian influenza (HPAI) subtypes H5N1, H5N8, and H5N6, which were associated with nine outbreaks in Korea between 2003 and 2018, to understand the epidemic transmission of each subtype. According to HPAI outbreak reports of the Animal and Plant Quarantine Agency, we estimated the generation time by calculating the time of infection between confirmed HPAI-positive farms. We constructed exponential growth and maximum likelihood (ML) models to estimate the basic reproduction number, which assumes the number of secondary cases infected by the index case. The Kruskal-Wallis test was used to analyze the epidemic statistics between subtypes. The estimated generation time of H5N1, H5N8, and H5N6 were 4.80 days [95% confidence interval (CI) 4.23–5.38] days, 7.58 (95% CI 6.63–8.46), and 5.09 days (95% CI 4.44–5.74), respectively. A pairwise comparison showed that the generation time of H5N8 was significantly longer than that of the subtype H5N1 (P = 0.04). Based on the ML model, R0 was estimated as 1.69 (95% CI 1.48–2.39) for subtype H5N1, 1.60 (95%CI 0.97–2.23) for subtype H5N8, and 1.49 (95%CI 0.94–2.04) for subtype H5N6. We concluded that R0 estimates may be associated with the poultry product system, climate, species specificity based on the HPAI virus subtype, and prevention policy. This study provides an insight on the transmission and dynamics patterns of various subtypes of HPAI occurring worldwide. Furthermore, the results are useful as scientific evidence for establishing a disease control policy.

Campylobacter, the most common etiologic agent of zoonotic gastroenteritis in humans, is present in many reservoirs including livestock animals, wildlife, soil, and water. Previously, we reported a novel Campylobacter jejuni strain SCJK02 (MLST ST-8388) from the gut of wild mice (Micromys minutus) using culture-dependent methods. However, due to fastidious growth conditions and the presence of viable but non-culturable Campylobacter spp., it is unclear whether M. minutus is a Campylobacter reservoir. This study aimed to: 1) determine the distribution and proportion of Campylobacter spp. in the gut microbiota of wild mice using culture-independent methods and 2) investigate the gut microbiota of wild mice and the relationship of Campylobacter spp. with other gut microbes. The gut microbiota of 38 wild mice captured from perilla fields in Korea and without any clinical symptoms (18 M. minutus and 20 Mus musculus) were analyzed. Metagenomic analysis showed that 77.8% (14 of 18) of the captured M. minutus harbored Campylobacter spp. (0.24–32.92%) in the gut metagenome, whereas none of the captured M. musculus carried Campylobacter spp. in their guts. Notably, 75% (6 of 8) of M. minutus determined to be Campylobacter-negative using culture-dependent methods showed a high proportion of Campylobacter through metagenome analysis. The results of metagenome analysis and the absence of clinical symptoms suggest that Campylobacter may be a component of the normal gut flora of wild M. minutus. Furthermore, linear discriminant analysis (LDA) showed that Campylobacter was the most enriched genus in the gut microbiota of M. minutus (LDA score, 5.37), whereas Lactobacillus was the most enriched genus in M. musculus (LDA score, −5.96). The differences in the presence of Campylobacter between the two species of wild mice may be attributed to the differential abundance of Campylobacter and Lactobacillus in their respective gut microbiota. In conclusion, the results indicate that wild M. minutus may serve as a potential Campylobacter reservoir. This study presents the first metagenomics analysis of the M. minutus gut microbiota to explore its possible role as an environmental Campylobacter reservoir and provides a basis for future studies using culture-independent methods to determine the role of environmental reservoirs in Campylobacter transmission.

In Korea, there is a need for research on human–animal relationships because of an increase in the number of companion animals and the positive changes in public perception toward them. Few studies have examined these changes. This epidemiological study investigates the characteristics of Korean dog owners and their pet dogs and identifies the owner- and dog-dependent factors that influence the owners' attitudes toward pets. We conducted a cross-sectional study of dog owners by asking them to complete a Pet Attitude Scale-based questionnaire about their dogs and themselves. The participants included 654 young adults between 19 and 39 years of age who lived in Seoul and owned dogs. We found that most dogs were owned by single, educated, high-income men who preferred small purebred dogs. Most were also likely to underestimate their dog's body condition score (BCS). The multivariable logistic regression (odds ratio, OR) and the multiple linear regression (unstandardized coefficients, B) models suggested that positive pet attitudes were associated with nine factors: overweight (OR = 2.68, B = 5.28) or a normal BCS (OR = 2.09, B = 5.58), having a medical history of related diseases (OR = 2.36, B = 6.38) and vaccination (OR = 2.10, B = 6.22), buying the pet dog (OR = 0.60, B = −3.85), having a small dog (≤10 kg) (OR = 1.66), visiting the veterinarian frequently (OR = 1.08, B = 0.39), spending more time with the dog (OR = 1.23, B =1.32), and keeping other species in the house (B = −4.27). This study is the first to identify the relationships between owner- and dog-dependent factors and pet owner attitude toward pets, all within a Korean cultural context. This study highlights the factors associated with the development of relationships between pet dogs and their owners. The exploratory study is novel because it examines pet ownership in the context of the Korean culture; previous pet ownership studies were set in the West and are analyzed with Western cultural values in mind.

Abstract Background Recent advances in next-generation sequencing technologies have enabled comprehensive analysis of the gut microbiota, which is closely linked to the health of the host. Consequently, several studies have explored the factors affecting gut microbiota composition. In recent years, increasing number of dog owners are feeding their pets a natural diet i.e., one consisting of bones, raw meat (such as chicken and beef), and vegetables, instead of commercial feed. However, the effect of these diets on the microbiota of dogs (Canis lupus familiaris) is unclear. Methods and results Six dogs fed a natural diet and five dogs fed a commercial feed were selected; dog fecal metagenomic DNA samples were analyzed using the Illumina MiSeq platform. Pronounced differences in alpha and beta diversities, and taxonomic composition of the core gut microbiota were observed between the two groups. According to alpha diversity, the number of operational taxonomic units, the richness estimates, and diversity indices of microbiota were significantly higher (p

Abstract Background In 2007, a nationwide Salmonella Tennessee outbreak occurred via contaminated peanut butter. Here, we developed a single-nucleotide polymorphism (SNP)-typing method for S. Tennessee to determine the clonal subtypes of S. Tennessee that were associated with the peanut butter outbreak. Methods and results One seventy-six S. Tennessee isolates from various sources, including humans, animals, food, and the environment, were analyzed by using the SNP technique. Eighty-four representative SNP markers were selected by comparing the sequences of three representative S. Tennessee strains with different multi-locus sequence typing and variable number tandem repeats from our collection. The set of eighty-four SNP markers showed 100% typeability for the 176 strains, with the nucleotide diversity ranging from 0.011 to 0.107 (mean = 0.049 ± 0.018, median = 0.044) for each marker. Among the four clades and nine subtypes generated by the SNP typing, subtype 1, which comprised 142 S. Tennessee strains, was the most predominant. The dominance of single-strain clones in subtype 1 revealed that S. Tennessee is highly clonal regardless of outbreak-association, source, or period of isolation, suggesting the presence of an S. Tennessee strain prototype. Notably, a minimum 18 SNP set was able to determine clonal S. Tennessee strains with similar discrimination power, potentially allowing more rapid and economic strain genotyping for both outbreaks and sporadic cases. Conclusions The SNP-typing method described here might aid the investigation of the epidemiology and microevolution of pathogenic bacteria by discriminating between outbreak-related and sporadic clinical cases. In addition, this approach enables us to understand the population structure of the bacterial subtypes involved in the outbreak.

Abstract Background As a primary source of Shiga-toxin-producing Escherichia coli (STEC) infection, cattle are often targeted to develop strategies for reducing STEC contamination. Monitoring the virulence potentials of STEC isolates from cattle is important for tracing contamination sources, managing outbreaks or sporadic cases, and reducing the risks for human infection. This study aimed to investigate the prevalence of STEC in cattle farm samples in South Korea and to assess their virulence potentials. Results In total, 63 STEC were isolated from 496 cattle farm samples, and temperature and rainfall affected STEC prevalence (p

Campylobacter jejuni is one of the most common zoonotic pathogens worldwide. Although the main sources of human C. jejuni infection are livestock, wildlife can also affect C. jejuni transmission in humans. However, it remains unclear whether wild mice harbor C. jejuni and are involved in the “environment–wildlife–livestock–human” transmission cycle of C. jejuni in humans. Here, we characterized C. jejuni from wild mice and identified genetic traces of wild mouse-derived C. jejuni in other hosts using a traditional approach, along with comparative genomics. We captured 115 wild mice (49 Mus musculus and 66 Micromys minutus) without any clinical symptoms from 22 sesame fields in Korea over 2 years. Among them, M. minutus were typically caught in remote areas of human houses and C. jejuni was solely isolated from M. minutus (42/66, 63.6%). We identified a single clone (MLST ST-8388) in all 42 C. jejuni isolates, which had not been previously reported, and all isolates had the same virulence/survival-factor profile, except for the plasmid-mediated virB11 gene. No isolates exhibited antibiotic resistance, either in phenotypic and genetic terms. Comparative-genomic analysis and MST revealed that C. jejuni derived from M. minutus (strain SCJK2) was not genetically related to those derived from other sources (registered in the NCBI genome database and PubMLST database). Therefore, we hypothesize that C. jejuni from M. minutus is a normal component of the gut flora following adaptation to the gastro-intestinal tract. Furthermore, M. minutus-derived C. jejuni had different ancestral lineages from those derived from other sources, and there was a low chance of C. jejuni transmission from M. minutus to humans/livestock because of their habitat. In conclusion, M. minutus may be a potential reservoir for a novel C. jejuni, which is genetically different from those of other sources, but may not be involved in the transmission of C. jejuni to other hosts, including humans and livestock. This study could form the basis for further studies focused on understanding the transmission cycle of C. jejuni, as well as other zoonotic pathogens originating from wild mice.

Since contaminated poultry meat is the major source of transmitting Campylobacter jejuni to humans, the isolation of Campylobacter from poultry carcasses is frequently performed in many countries as a baseline survey to ensure food safety. However, existing isolation methods have technical limitations in isolating this fastidious bacterium, such as a growth competition with indigenous bacteria in food samples. In this study, we compared the differences in microbiota compositions between Bolton and Preston selective media, two most common selective media to isolate Campylobacter, and investigated how different microbiota compositions resulting from different enrichment methods may affect isolation frequencies. A next-generation sequencing (NGS) analysis of 16S rRNA demonstrated that Bolton and Preston-selective enrichments generated different microbiota structures that shared only 31.57% of Operating Taxonomic Unit (OTU) types. Particularly, Escherichia was highly prevalent in Bolton selective media, and the enrichment cultures that increase Escherichia negatively affected the efficacy of Campylobacter isolation. Furthermore, the combination of the selective media made a significant difference in the isolation frequency. The Bolton broth and Preston agar combination exhibited the highest (60.0%) frequencies of Campylobacter isolation, whereas the Bolton broth and Bolton agar combination showed the lowest (2.5%). These results show that each selective medium generates a unique microbiota structure and that the sequence of combining the selective media also critically affects the isolation frequency by altering microbiota compositions. In this study, we demonstrated how a microbiota analysis using NGS can be utilized to optimize a protocol for bacterial isolation from food samples.

Highly pathogenic avian influenza (HPAI), a zoonotic disease, is a major threat to humans and poultry health worldwide. In January 2014, HPAI virus subtype H5N8 first infected poultry farms in South Korea, and 393 outbreaks, overall, were reported with enormous economic damage in the poultry industry. We analyzed the spatiotemporal distribution of HPAI H5N8 outbreaks in poultry farms using the global and local spatiotemporal interaction analyses in the first (January to July 2014) and second (September 2014 to June 2015) outbreak waves. The space–time K-function analyses revealed significant interactions within three days and in an over-40 km space–time window between the two study periods. The excess risk attributable value (D0) was maintained despite the distance in the case of HPAI H5N8 in South Korea. Eleven spatiotemporal clusters were identified, and the results showed that the HPAI introduction was from the southwestern region, and spread to the middle region, in South Korea. This spatiotemporal interaction indicates that the HPAI epidemic in South Korea was mostly characterized by short period transmission, regardless of the distance. This finding supports strict control strategies such as preemptive depopulation, and poultry movement tracking. Further studies are needed to understand HPAI disease transmission patterns.

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.

Following publication of the original article [1], the authors reported an error in Fig. 2. The correct figure is shown below.

Campylobacter, a common foodborne human pathogen, is considered sensitive to oxygen. Recently, aerotolerant (AT) Campylobacter jejuni with the ability to survive under aerobic stress has been reported. Here, we investigated the prevalence of hyper-aerotolerant (HAT) Campylobacter coli from duck sources (118 carcasses and meat) and its characteristics to assess potential impacts on public health. Half of 56 C. coli isolates were HAT and most harbored various virulence genes including flaA, cadF, cdtA, ceuB, and wlaN. Moreover, 98.2% of C. coli isolates showed resistance to quinolones, including ciprofloxacin (CIP), and nine (16.1%) showed high-level resistance to ciprofloxacin (Minimum Inhibitory Concentration, MIC ≥ 32 μg/mL) and most of these were HAT. Based on genetic relatedness between C. coli from duck sources and those from human sources (PubMLST and NCBI), HAT isolates sharing the same MLST sequence types were significantly more prevalent than those not sharing the same sequence types as those from human sources. Therefore, HAT C. coli is prevalent in duck sources, and is most likely transmitted to humans through the food chain given its aerotolerance. This being so, it might pose a threat to public health given its virulence and antimicrobial resistance (AMR). This study will assist in improving control strategies to reduce farm-to-table HAT C. coli transmission to humans.

Human infections with Campylobacter are primarily associated with the consumption of contaminated poultry meat. In this study, we isolated Campylobacter jejuni from retail raw chicken and duck meat in Korea and compared their aerotolerance, antibiotic resistance, and virulence gene prevalence. Whereas C. jejuni isolates from chicken dominantly belonged to multilocus sequence typing (MLST) clonal complex (CC)-21, CC-45 is the common MLST sequence type in duck meat isolates. C. jejuni strains from both chicken and duck meat were highly tolerant to aerobic stress. The prevalence of virulence genes was higher in C. jejuni strains from chicken than those from duck meat. However, antibiotic resistance was higher in duck meat isolates than chicken isolates. Based on the prevalence of virulence genes and antibiotic resistance, fluoroquinolone-resistant C. jejuni strains harboring all tested virulence genes except virB11 were predominant on retail poultry. Fluoroquinolone-resistant C. jejuni strains carrying most virulence genes were more frequently isolated in summer than in winter. The comparative profiling analysis in this study successfully demonstrated that antibiotic-resistant and pathogenic strains of C. jejuni are highly prevalent on retail poultry and that retail duck meat is an important vehicle potentially transmitting C. jejuni to humans in Korea.

The emergence of Salmonella enterica serovar Enteritidis (S. Enteritidis) during the past three decades as major contaminant of eggs and other poultry products caused a surge in human infections. This could have been mediated in part by emerging S. Enteritidis strains with enhanced virulence. The overall pathogenicity of Salmonella is controlled by numerous genes. To assess the role of a few specific genes thought to contribute to the pathogenicity of S. Enteritidis in egg laying hens, we conducted an experimental infection of egg-laying hens, with a wild type (WT) S. Enteritidis phage type 4 strain and three mutants (M1, M2, M3). These mutants were produced from the wild type by the inactivation of the prgH, SEN4287, and tyrR genes, respectively. We observed that the WT and the M1 mutant had shorter durations of fecal shedding and faster clearance from internal organs of the infected hens than the M2 and M3 mutants. The isolation rates of the wild type S. Enteritidis and the mutants were highest from the ceca, moderately high from the liver and spleen, and lowest from the ovaries of the infected hens. Hep-2 cells attachment assay revealed attenuated attachment for the M1 mutant while the M3 mutant seemed to have enhanced attachment. Colonization of tissues of the infected hens by M1 mutant appears to have been attenuated.

Background: Gastric ulcer is one of the prevalent diseases in racehorses. However, it has not been recognized as important in Korea, and drugs used to treat gastric ulcers are included in the doping test list, so they are not allowed to be administered to racehorses in training. Objectives: This study was performed 1) to investigate the prevalence and the severity of gastric ulcers in Thoroughbred racehorses in Korea, 2) to confirm the therapeutic effect of ranitidine and omeprazole, and 3) to compare the efficacy between ranitidine and omeprazole. Methods: Forty-nine horses were randomly recruited, and gastroscopy was performed within two days after racing. Twelve horses with a sum grade of five or higher were randomly assigned to two treatment groups. Seven horses were administered ranitidine, and five horses were administered omeprazole. Follow-up gastroscopy was scheduled within one to five days after finishing the treatment. Results: The prevalence of gastric ulcer in Korean Thoroughbred racehorses after racing was 100%, and the grade was more severe in the non-glandular region than in the pyloric region. There was no correlation between the severity of gastric ulcer in the two regions. Omeprazole had a greater therapeutic effect than ranitidine. Conclusions: This study shows the importance of recognizing gastric ulcers as an important factor, and omeprazole as a possible treatment option in Korea, as it has been removed from the list of prohibited substances for racehorses. Thus, the use of omeprazole is currently recommended until one day before the race. [ABSTRACT FROM AUTHOR]