Antimicrobial Resistance Profiles, Virulence Genes, and Genetic Diversity of Thermophilic Campylobacter Species Isolated From a Layer Poultry Farm in Korea

Abstract

Thermophilic Campylobacter species are among the major etiologies of bacterial enteritis globally. This study aimed at assessing the antimicrobial resistance (AMR) profiles, virulence genes, and genetic diversity of thermophilic Campylobacter species isolated from a layer poultry farm in South Korea. One hundred fifty-three chicken feces were collected from two layer poultry farms in Gangneung, South Korea. The Campylobacter species were isolated by cultural techniques, while PCR and sequencing were used for species confirmation. Antimicrobial susceptibility testing for six antimicrobials [ciprofloxacin (CIP), nalidixic acid (NAL), sitafloxacin (SIT), erythromycin (ERY), tetracycline (TET), and gentamicin (GEN)] was carried out by broth microdilution. Three AMR and nine virulence genes were screened by PCR. Genotyping was performed by flaA-restriction fragment length polymorphism (RFLP) and multilocus sequence typing (MLST). Of the 153 samples, Campylobacter spp. were detected in 55 (35.9%), with Campylobacter jejuni and Campylobacter coli being 49 (89.1%) and six (10.9%), respectively. High-level resistance was observed for CIP (100%), NAL (100%), and TET (C. jejuni, 93.9%; C. coli: 83.3%). No resistance was observed for SIT. The missense mutation (C257T) in gyrA gene was confirmed by sequencing, while the tet(O) gene was similar to known sequences in GenBank. The rate of multidrug-resistant (MDR) strains was 8.2%, and they all belonged to C. jejuni. All Campylobacter isolates possessed five virulence genes (cdtB, cstII, flaA, cadF, and dnaJ), but none possessed ggt, while the rates for other genes (csrA, ciaB, and pldA) ranged between 33.3 and 95.9%. The flaA-RFLP yielded 26 flaA types (C. jejuni: 21 and C. coli: five), while the MLST showed 10 sequence types (STs) for C. jejuni and three STs for C. coli, with CC-607 (STs 3611) and CC-460 (ST-460) being predominant. Among the 10 STs of C. jejuni, three were newly assigned. The findings of this study highlight the increased resistance to quinolones and TET, the virulence potential, and the diverse genotypes among Campylobacter strains isolated from the layer poultry farm.

Keywords: Campylobacter; Korea; antimicrobial resistance; flaA RFLP; multilocus sequence typing; poultry; quinolones.

FIGURE 1

PCR detection of antimicrobial resistance and virulence genes. M, marker; 1, gyrA; 2, tet(O); 3, cstII; 4, cdt B; 5, flaA; 6, csrA; 7, cadF; 8, ciaB; 9, pldA; and 10, dnaJ.

FIGURE 2

Distribution of virulence genes in Campylobacter jejuni and Campylobacter coli.

FIGURE 3

PCR–restriction fragment length polymorphism profiles of flaA gene digested with HpyF31 from strains of Campylobacter species. Numbers represent the laboratory code for isolates.

FIGURE 4

Minimum spanning tree of Campylobacter jejuni and Campylobacter coli sequence types (STs) created from multilocus sequence typing allelic profiles. Each ST is represented as a circle, with the size of the circle proportional to the number of isolates of that ST for the isolates obtained in this study (STs colored in yellow and green). The figure also includes STs commonly reported in Korea and elsewhere from poultry and humans which were retrieved from pubMLST. The branch length and thickness represent the allelic distance: a thick short line connects single-locus variants, a thin longer line connects double-locus variants, and a dashed line connects STs separated by three or more allelic differences. Background shading highlights clonal complexes at a distance of two alleles in this dataset.