Characterization of Campylobacter spp. Strains Isolated From Wild Birds in Turkey

Abstract

Turkey is an important stopover site for many migrating birds between Europe, Asia and Africa. Campylobacter spp. are frequently found in wildlife, in particular waterfowl, and distinct strains are disseminated within this reservoir. In this study, 183 wild birds of hunting areas in Turkey were collected and thermophilic Campylobacter spp. from cloacal swabs were isolated at a prevalence of 5.2% from song thrushes (6/116) and 93% from Eurasian coots (41/44). After PCR species differentiation and flaA restriction profiles determination, C. jejuni and C. coli strains were further investigated by whole genome sequencing. PCR target amplification of the ceuE gene, commonly used for C. coli species-identification was inefficient and even hampered in one isolate. A close look on the ceuE sequence revealed that various mismatches in the ceuE oligo annealing sites caused less efficient diagnostic detection. All C. coli isolates belonged to the environmental clade II and clade III, for which thirty-six novel MLST types were identified. Further single nucleotide polymorphism (SNP) analysis showed a high genomic divergence between the C. coli isolates. High variability was also implicated for putative plasmid-located genes detected in 51% of the C. coli isolates. Distinct gene variants in clades II and III C. coli were identified by a k-mer analysis. After substracting k-mers in common with C. coli clade I database, 11 and 35 distinct genes were identified in clades II and III isolates, mainly involved in surface structures and modifications as well as signal transduction, suggesting niche adaptation of C. coli strains in wild birds. All strains were susceptible against (fluoro-)quinolones, erythromycin, tetracycline, gentamicin and only one isolate was resistant against streptomycin, suggesting that the sensitive phenotype was due to absence of selective pressure and niche separation in wild birds in Turkey. We conclude that Campylobacter spp. isolates from wildlife and environmental sources are still scarce in the databases and that there is a need for more studies on thermophilic Campylobacter spp. from different places all over the world in order to complement our understanding on dissemination and adaptation to distinct niches of this global food-borne pathogen.

Keywords: C. coli clade; Eurasian coots; diagnostics; environmental Campylobacter; whole-genome sequencing.

FIGURE 1

The geographic location of sampling sites in Turkey. The sampling areas are shown in green. Gray lines indicate two main bird migration routes on Turkey, adapted from Özkazanç and Özay (2019).

FIGURE 2

Maximum likelihood phylogenetic tree of 42 thermophilic Campylobacter spp. isolates from wild birds in the context of C. jejuni and C. coli complete genomes from NCBI (Supplementary Table 2) constructed on the basis of the core genome. The scale bar represents the distance between the sequences determined by the number of SNP differences in core genes. Dotted line indicates shortened distance to C. jejuni cluster, which is out of scale. *, subclade.

FIGURE 3

Pairwise SNP analysis show that C. coli clade II isolates were less diverse than C. coli clade III isolates. Heatmap color code of pairwise number of SNPs is shown on the right. NCBI closed genomes of C. coli clade II and clade III are included for comparison.

FIGURE 4

Phylogenetic tree of the ceuE target gene for C. coli species differentiation. ceuE of C. coli clade I, for which typing methods have been optimized, is significantly different to clade II and clade II C. coli ceuE. The C. coli hybrid isolate in between C. jejuni and C. coli clade I was originally detected by ambiguous real-time PCR results which were caused by C. jejuni introgression into the C. coli genome (Golz et al., 2020). The scale bar represents the distance between the sequences determined by the number of SNPs. Dotted line is out of scale. *, subclade.

FIGURE 5

Phylogenetic tree of plasmid-located genes of wild bird isolates based on presence or absence. Strains are depicted on the left. Blue bars indicate presence of plasmid-located genes (Supplementary Table 4).