WGS-Based Prediction and Analysis of Antimicrobial Resistance in Campylobacter jejuni Isolates From Israel

Abstract

Rapid developments in the field of whole genome sequencing (WGS) make in silico antimicrobial resistance (AMR) a target within reach. Campylobacter jejuni is a leading cause of foodborne infections in Israel with increasing rates of resistance. We applied WGS analysis to study the prevalence and genetic basis of AMR in 263 C. jejuni human and veterinary representative isolates retrieved from a national collection during 2003-2012. We evaluated the prediction of phenotypic AMR from genomic data. Genomes were screened by the NCBI AMRFinderPlus and the BioNumerics tools for acquired AMR genes and point mutations. The results were compared to phenotypic resistance determined by broth microdilution. The most prevalent resistant determinants were the multi-drug efflux transporter gene cmeABC (100%), the tetracycline resistance tet(O) gene (82.1%), the quinolone resistance gyrA T861 point mutation (75.7%), and the aadE streptomycin resistance gene. A variety of 12 known β lactam resistance genes (bla_OXA variants) were detected in 241 (92%) isolates, the most prevalent being bla_OXA-193, bla_OXA-461, and bla_OXA-580 (56, 16, and 7%, respectively). Other aminoglycoside resistance genes and the macrolide resistance point mutation were rare (<1%). The overall correlation rate between WGS-based genotypic prediction and phenotypic resistance was 98.8%, sensitivity, specificity, positive, and negative predictive values being 98.0, 99.3, 99.1, and 98.5%, respectively. wgMLST-based phylogeny indicated a high level of clonality and clustering among the studied isolates. Closely related isolates that were part of a genetic cluster (single linkage distance ≤ 15 alleles) based on wgMLST phylogeny mostly shared a homogenous AMR determinant profile. This was observed in 18 of 20 (90.0%) clusters within clonal complex-21, suggesting clonal expansion of resistant isolates. Strong association to lineage was noted for the aadE gene and the various bla_OXA genes. High resistance rates to tetracycline and quinolones and a low resistance rate to macrolides were detected among the Israeli C. jejuni isolates. While a high genotypic-phenotypic correlation was found, some resistance phenotypes could not be predicted by the presence of AMR determinants, and particularly not the level of resistance. WGS-based prediction of antimicrobial resistance in C. jejuni requires further optimization in order to integrate this approach in the routine workflow of public health laboratories for foodborne surveillance.

Keywords: Campylobacter jejuni; antimicrobial resistance; antimicrobial-susceptibility testing; bioinformatics; whole-genome sequencing.

Figure 1

wgMLST-based phylogeny of 263 C. jejuni isolates. The minimum spanning tree is based on wgMLST analyses of 239 clinical and 24 veterinary isolates. Isolates are represented by circles connected by branches proportional to the allelic distance. The distribution of clonal complexes among the studied population is denoted by color. Partitioned nodes represent closely clustered isolates (≤ 15 allelic distance threshold).

Figure 2

Correlation between quinolone MIC and the presence of point mutations in the GyrA protein among 219 C. jejuni isolates. The different mutations are denoted by color. Resistance breakpoints: ciprofloxacin (>0.5 μg/ml), nalidixic acid (>16 μg/ml).

Figure 3

AMR genetic determinants among a wgMLST phylogeny of Clonal Complex 21. A wgMLST-based minimum spanning tree of 104 CC-21 isolates is shown. Each node represents a strain. Partitioned nodes represent closely clustered isolates (≤ 15 allelic distance threshold). Sequence types are denoted in black. The number of allelic differences is shown on the branches connecting the nodes. The AMR gene profile detected by AMRFinderPlus is denoted by color.

Figure 4

wgMLST-based phylogeny of 263 C. jejuni isolates. The distribution of the tet(O) gene associated with tetracycline resistance is shown in color.

Figure 5

wgMLST-based phylogeny of 263 C. jejuni isolates. The distribution of the gyrA T86I point mutation associated with quinolone resistance is shown in color.

Figure 6

wgMLST-based phylogeny of 263 C. jejuni isolates. The distribution of the aadE (ant(6)-Ia) gene associated with streptomycin resistance is shown in color.

Figure 7

The distribution of bla_OXA gene variants across an MLST-based phylogeny of 263 C. jejuni isolates. Each node in the minimum spanning tree represents a sequence type (ST), and the number of allelic differences is denoted on the branches connecting the nodes. The presence of β-lactamase (bla_OXA) gene variants is denoted by color. The G → T promoter transversion associated with high-level ampicillin resistance in bla_OXA−193 is shown in black.

Figure 8

Trends in the prevalence of the gyrA T86I point mutation among 239 human C. jejuni isolates collected over a decade.

Figure 9

Trends in the prevalence of the tet(O) gene among 239 human C. jejuni isolates collected over a decade.