Different threats posed by two major mobilized colistin resistance genes - mcr-1.1 and mcr-3.1 - revealed through comparative genomic analysis.

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.

Circular intermediate-mediated horizontal transfer of the chromosome-encoded cfr(C) gene in multi-drug resistant Campylobacter coli from swine sources.

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.

Prevalence, Characteristics, and Clonal Distribution of Escherichia coli Carrying Mobilized Colistin Resistance Gene mcr-1.1 in Swine Farms and Their Differences According to Swine Production Stages.

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 ß-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 < 0.01), suggesting a high probability of clonal expansion of MCR1-EC within closed environments such as livestock husbandry. To the best of our knowledge, this is the first study to analyze the differences in the characteristics and clonal distribution of MCR1-EC according to production stages in swine farms, an important reservoir of MCR1-EC. Our results highlight the need to establish MCR1-EC control plans in swine farms based on an in-depth understanding of MCR1-EC characteristics according to swine production stages, focusing especially on the weaning stages.

Hyper-Aerotolerant Campylobacter coli From Swine May Pose a Potential Threat to Public Health Based on Its Quinolone Resistance, Virulence Potential, and Genetic Relatedness.

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.

Prevalence, Characteristics and Clonal Distribution of Extended-Spectrum ß-Lactamase- and AmpC ß-Lactamase-Producing Escherichia coli Following the Swine Production Stages, and Potential Risks to Humans.

The worldwide spread of extended spectrum ß-lactamase (ESBL)- and AmpC ß-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.

Complete genome sequence and comparative genomic analysis of hyper-aerotolerant Campylobacter lari strain SCHS02 isolated from duck for its potential pathogenicity.

Campylobacter lari strain SCHS02, a novel hyper-aerotolerant strain that survives under aerobic conditions, was isolated from retail duck meat. The genome is a single chromosome of 1,520,838 base pairs, with a mean GC content of 29.7%. It harbors 1546 protein-coding sequences and 45 tRNA and 9 rRNA genes. Genes associated with the oxidative stress response, including perR, bcp, ahpC, and sodB, were identified in the genome. Furthermore, 68 virulence-related genes were identified and sorted into 9 classes and 14 subclasses. The virulence gene profile of SCHS02 was similar to those of two human clinical C. lari isolates. Comparative genomic analysis of strain SCHS02 and 18 C. lari strains retrieved from a public database revealed the core and accessory gene profiles of C. lari strains, as well as putative core gene involved in halotolerance. Phylogenetic analysis revealed that strain SCHS02 is genetically related to isolates from bird samples and human clinical isolates, rather than to isolates from other environmental sources. These findings reveal essential genomic information about the newly identified hyper-aerotolerant C. lari strain isolated from a duck source, providing a basis for future studies of the strain considering its potential threat to public health and further research of the pathogenicity of C. lari.

Hyper-Aerotolerant Campylobacter coli from Duck Sources and Its Potential Threat to Public Health: Virulence, Antimicrobial Resistance, and Genetic Relatedness.

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.

Correction to: Metagenomic analysis of isolation methods of a targeted microbe, Campylobacter jejuni, from chicken feces with high microbial contamination.

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

Metagenomic analysis of isolation methods of a targeted microbe, Campylobacter jejuni, from chicken feces with high microbial contamination.

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 Wild Mouse (Micromys minutus): Reservoir of a Novel Campylobacter jejuni Strain.

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.

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.

Differences in the gut microbiota of dogs (Canis lupus familiaris) fed a natural diet or a commercial feed revealed by the Illumina MiSeq platform.

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 < 0.05) in the natural diet group than in the commercial feed group. Based on beta diversity, most samples clustered together according to the diet type (p = 0.004). Additionally, the core microbiota between the two groups was different at the phylum, family, and species levels. Marked differences in the taxonomic composition of the core microbiota of the two groups were observed at the species level; Clostridium perfringens (p = 0.017) and Fusobacterium varium (p = 0.030) were more abundant in the natural diet group. CONCLUSIONS: The gut microbiota of dogs is significantly influenced by diet type (i.e., natural diet and commercial feed). Specifically, dogs fed a natural diet have more diverse and abundant microbial composition in the gut microbiota than dogs fed a commercial feed. In addition, this study suggests that in dogs fed a natural diet, the potential risk of opportunistic infection could be higher, than in dogs fed a commercial feed. The type of diet might therefore play a key role in animal health by affecting the gut microbiota. This study could be the basis for future gut microbiota research in dogs.

Prevalence, virulence potential, and pulsed-field gel electrophoresis profiling of Shiga toxin-producing Escherichia coli strains from cattle.

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 < 0.001). The O157 serogroup was most prevalent, followed by O108, O8, O84, O15, and O119. In the stx variant test, high prevalence of stx2a and stx2c (known to be associated with high STEC virulence) were observed, and stx2g, a bovine STEC variant, was detected in STEC O15 and O109. Additionally, stx1c was detected in eae-positive STEC, suggesting genetic dynamics among the virulence genes in the STEC isolates. STEC non-O157 strains were resistant to tetracycline (17.9%), ampicillin (14.3%), and cefotaxime (3.6%), while STEC O157 was susceptible to all tested antimicrobials, except cefotaxime. The antimicrobial resistance genes, blaTEM (17.5%), tetB (6.3%), and tetC (4.8%), were only detected in STEC non-O157, whereas tetE (54.0%) was detected in STEC O157. AmpC was detected in all STEC isolates. Clustering was performed based on the virulence gene profiles, which grouped STEC O84, O108, O111, and O157 together as potentially pathogenic STEC strains. Finally, PFGE suggested the presence of a prototype STEC that continues to evolve by genetic mutation and causes within- and between-farm transmission within the Gyeonggi province. CONCLUSIONS: Considerable numbers of STEC non-O157 were isolated from cattle farms, and the virulence and antimicrobial resistance features were different between the STEC O157 and non-O157 strains. STEC from cattle with virulence or antimicrobial resistance genes might represent a threat to public health and therefore, continual surveillance of both STEC O157 and non-O157 would be beneficial for controlling and preventing STEC-related illness.

Antibiotic resistance patterns and genetic relatedness of Enterococcus faecalis and Enterococcus faecium isolated from military working dogs in Korea.

Enterococcus spp. are normally present in the gastrointestinal tracts of animals and humans, but can cause opportunistic infections that can be transmitted to other animals or humans with integrated antibiotic resistance. To investigate if this is a potential risk in military working dogs (MWDs), we analyzed antibiotic resistance patterns and genetic relatedness of Enterococcus spp. isolated from fecal samples of MWDs of four different age groups. Isolation rates of Enterococcus spp., Enterococcus (E.) faecalis, and E. faecium, were 87.7% (57/65), 59.6% (34/57), and 56.1% (32/57), respectively, as determined by bacterial culture and multiplex PCR. The isolation rate of E. faecalis gradually decreased with age (puppy, 100%; adolescent, 91.7%; adult, 36.4%; and senior, 14.3%). Rates of resistance to the antibiotics ciprofloxacin, gentamicin, streptomycin, sulfamethoxazole/trimethoprim, imipenem, and kanamycin among Enterococcus spp. increased in adolescents and adults and decreased in senior dogs, with some isolates having three different antibiotic resistance patterns. There were indistinguishable pulsed-field gel electrophoresis patterns among the age groups. The results suggest that Enterococcus is horizontally transferred, regardless of age. As such, periodic surveillance studies should be undertaken to monitor changes in antibiotic resistance, which may necessitate modification of antibiotic regimens to manage antibiotic resistance transmission.

The Fecal Microbial Communities of Dairy Cattle Shedding Shiga Toxin-Producing Escherichia coli or Campylobacter jejuni.

Cattle are a natural reservoir of Shiga toxin-producing Escherichia coli (STEC) and have recently been recognized as a major source of Campylobacter jejuni contamination. While several factors are known to be associated with bacterial colonization, the underlying microbial factors have not been clarified. In this study, we characterized the fecal microbiota of dairy cattle (n = 24) using next-generation sequencing to elucidate the intestinal bacterial communities and the microbial diversity in relation to the presence of the foodborne pathogens STEC and C. jejuni (STEC-positive samples, n = 9; STEC-negative samples, n = 15; C. jejuni-positive samples, n = 9; and C. jejuni-negative samples, n = 15). While no significant differences were observed in alpha diversity between STEC-positive and STEC-negative samples, a high diversity index was observed in C. jejuni-positive samples compared to C. jejuni-negative samples. Nine phyla, 13 classes, 18 orders, 47 families, 148 genera, and 261 species were found to be the core microbiota in dairy cattle, covering 80.0-100.0% of the fecal microbial community. Diverse microbial communities were observed between cattle shedding foodborne pathogens and nonshedding cattle. C. jejuni-positive cattle had a higher relative abundance of Bacteroidetes (p = 0.035) and a lower relative abundance of Firmicutes (p = 0.035) compared to C. jejuni-negative cattle. In addition, while the relative abundance of 2 and 6 genera was significantly higher in cattle-shedding STEC and C. jejuni, respectively, the relative abundance of 3 genera was lower in both STEC- and C. jejuni-negative cattle. Our findings provide fundamental information on the bacterial ecology in cattle feces and might be useful in developing strategies to reduce STEC or C. jejuni shedding in dairy cattle, thereby reducing the incidence of STEC infection and campylobacteriosis in humans.