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.