Background: Campylobacter jejuni (C. jejuni), a major global cause of foodborne bacterial diseases, accounts for more than 90% of all reported cases. Poultry is considered a major reservoir for the transmission of Campylobacter to humans. While extensive research has been conducted abroad on the occurrence and epidemiology of C. jejuni in laying hens, there are scant reports on its prevalence in layer chickens in China. The present study was designed to isolate C. jejuni from 482 cloacal swabs collected from seven laying hen farms located in Hubei Province between January and March 2024. Furthermore, the study aimed to explore the genetic diversity, antibiotic resistance, and virulence gene profiles of the isolated strains.

Results: The overall prevalence of C. jejuni amounted to 4.36% (21/482). Whole-genome sequencing of these 21 isolates revealed 11 distinct sequence types (STs) and eight clonal complexes (CCs), with ST-6522 and CC-443 emerging as the predominant genotypes. Antimicrobial susceptibility testing against 11 antibiotics revealed high resistance rates among C. jejuni isolates, particularly towards ceftriaxone and enrofloxacin, where resistance was universal (100%). Similarly, high resistance levels were also observed for doxycycline (95.24%), ceftiofur (80.95%), tilmicosin (76.19%), and amoxicillin-clavulanic acid (57.14%). Through genomic resistance gene prediction, a total of eighteen resistance genes were identified within the 21 C. jejuni isolates. The most frequently occurring resistance genes were the gyrA (T86I) point mutation (95.14%), cmeR (95.14%), and tet(O) (95.14%). Notably, a robust correlation was discernibled between enrofloxacin resistance and the gyrA (T86I) point mutation, as well as between resistance to ceftriaxone and tilmicosin and the presence of the cmeR gene. Conversely, the correlations between other antibiotic resistance phenotypes and their corresponding resistance genes were less robust. A comprehensive analysis of virulence genes isolated from C. jejuni strains revealed a total of 117 virulence genes, categorized according to their functional roles. These categories encompass adhesion (cadF, jlpA, porA, pebA), invasion (ciaB), motility (flaA, flgB, flhB), toxin production (cdtA, cdtB, cdtC), and immune modulation (htrB, wlaN).

Conclusions: Our results revealed the high resistance rate of C. jejuni from laying-hens in hubei Province, China, which will help the farms take the necessary action to develop effective mitigation strategies for reducing Campylobacter infection in poultry.

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