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. 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. 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.

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.

Campylobacter jejuni is a major cause of foodborne illnesses, and its increasing antimicrobial resistance (AMR) poses serious public health risks. Owing to their high genetic diversity and frequent intraspecific recombination, understanding the virulence traits of Campylobacter remains challenging. We elucidated the resistance and virulence mechanisms of C. jejuni in livestock using comparative genomic and phenotypic analyses. We analyzed C. jejuni strains isolated from chicken meat, chicken slaughterhouses, and dairy cattle farms. High resistance rates were observed for nalidixic acid, ciprofloxacin, and tetracycline. The chicken-derived strains showed significantly higher tetracycline resistance and marginally higher nalidixic acid resistance, whereas the cattle-derived strains showed marginally higher ciprofloxacin resistance. The key AMR determinants included gyrA and tet(O), which were correlated with resistance phenotypes. Ten virulence factor families were identified with prevalences exceeding 90%. Biofilm formation was observed in 31.9% of strains and correlated with flagella-associated virulence factors. Eighteen plasmid types were detected, primarily in the pTet family, which carried various AMR genes and components of the Type IV secretion system, potentially facilitating the co-transfer of resistance and virulence traits. Conjugation experiments confirmed the horizontal transfer of two pTet plasmid types into the wild-type C. jejuni strain. Further, our analyses revealed over 95% genetic similarity with European C. jejuni strains in a public database-supporting the hypothesis of zoonotic transmission via global food chains-and the zoonotic risks of livestock-derived Campylobacter jejuni. These findings emphasize the need for extended global surveillance to mitigate the risk of zoonotic transmission.

Extended-spectrum beta-lactamase (ESBL)-producing Salmonella enterica serovar Enteritidis ( S . Enteritidis) is emerging as a significant threat to food safety via its limitation of therapeutic options and potential transmission through poultry products. However, the structural and genetic characteristics of mobile genetic elements (MGEs) associated with horizontal transfer of the ESBL gene in S . Enteritidis isolates from poultry remain insufficiently characterized. The present study aimed to identify and characterize the ESBL gene and its associated MGEs and to assess their distribution. Whole-genome sequencing was applied to ESBL-producing and non-ESBL-producing isolates in combination with pan-genome analysis, conjugation assays, and comparative genomics using publicly available genomes. Among 17 isolates, 9 were ESBL-producing and all carried bla _CTX-M-15 . We observed co-transfer of bla _CTX-M-15 , tetA , and the IncF plasmid at relatively high frequencies (2.0–5.3 × 10⁻²) in ESBL-producing isolates. Moreover, we identified a hybrid transposon (Tn 1721 /Tn 21 ) inserted into IncF plasmids that comprised elements of Tn 21 ( merRTPCADE , tniA , and urf2 ) and Tn 1721 ( tetA , tetR , a DMT-family efflux gene, and a partial tnpA ), with IS Ecp1 and bla _CTX-M-15 adjacent to the hybrid transposon. Tn 1721 /Tn 21 was prevalent among bla _CTX-M-15 -carrying S . Enteritidis isolates from South Korea (19/20) but absent in those from other countries ( n = 9), suggesting geographical variation. This study identified a unique hybrid Tn 1721 /Tn 21 transposon as the dominant MGE in bla _CTX-M-15 -carrying S . Enteritidis from South Korean poultry, highlighting its potential role in the regional dissemination of antimicrobial resistance. Continued surveillance and targeted intervention in poultry production are warranted to mitigate the spread of ESBL-producing S . Enteritidis. IMPORTANCE: Extended-spectrum beta-lactamase (ESBL)-producing Salmonella enterica serovar Enteritidis from poultry represents a growing public health threat due to limited treatment options and the potential for transmission through the food chain. Despite this concern, the mobile genetic elements underlying ESBL gene dissemination remain insufficiently characterized in South Korean poultry-associated S . Enteritidis isolates. In this study, we identified a hybrid transposon, Tn 1721 /Tn 21 , embedded within IncF plasmids and linked to bla _CTX-M-15 in S . Enteritidis isolates. This association between Tn 1721 /Tn 21 and bla _CTX-M-15 suggests a region-specific mechanism of resistance dissemination that may reflect antimicrobial selective pressure within poultry production systems. These findings highlight the importance of integrated One Health surveillance to mitigate the emergence and spread of antimicrobial resistance across animal and human populations.