Low-Level Tetracycline Resistance Gene tet(O) _3 in Campylobacter jejuni

Abstract

Campylobacter (C.) spp. are the most important foodborne, bacterial, and zoonotic pathogens worldwide. Resistance monitoring of foodborne bacterial pathogens is an important tool to control antimicrobial resistance as a part of the "One Health" approach. The detection and functionality of new resistance genes are of paramount importance in applying more effective screening methods based on whole genome sequencing (WGS). Most tetracycline-resistant C. spp. isolates harbor tet(O), a gene that encodes a ribosomal protection protein. Here we describe tet(O)_3, which has been identified in two food isolates of C. jejuni and is very similar to the tet(O) gene in Streptococcus pneumoniae, having a truncated promoter sequence. This gene confers resistance to tetracycline below 1 mg/L, which is the epidemiological cut-off value. We have analyzed the entire genome of these two isolates, together with a C. jejuni isolate found to have high-level resistance to tetracycline. In contrast to the highly resistant isolate, the promoter of tet(O)_3 is highly responsive to tetracycline, as observed by reverse transcription polymerase chain reaction (RT-PCR). In addition, the two isolates possess a CRISPR repeat, fluoroquinolone resistance due to the gyrA point mutation C257T, a β-lactamase resistance gene bla_OXA-184, a multidrug efflux pump CmeABC and its repressor CmeR, but no plasmid. Low-level antibiotic resistant C. jejuni might therefore have an advantage for surviving in non-host environments.

Keywords: epidemiological cutoff; foodborne pathogen; multidrug efflux pump; tetracycline resistance; β-lactamase.

Figure 1

A genome analysis of the region around tet(O)_1 and tet(O)_3 of (a) GC119, (b) FC77; (c) FC88. In FC77 and FC88 the tet(O)_3 is located between two truncated virulence genes, feoA and feoB. Both isolates possess another copy of the non-truncated genes in their genome.

Figure 2

RT-PCR on C. jejuni FC77 (green), FC88 (red), and GC119 (blue) grown in MH broth plus 5% lyophilized horse blood with and without the addition of 0.6 mg/L tetracycline (both bolt and dashed line) harvested at mid logarithmic growth condition. As a control, a 16S RNA RT-PCR was included with the same sample extracts.

Figure 3

The promoter analysis of tet(O)_1 in GC119 and Campylobacter 81–176 and tet(O)_3 of FC77 and FC88. The “ATG” start codon of the protein is boxed in red, the ribosomal binding site in blue, the purple box indicates the −10 region, and the proposed −35 region is boxed in green; * shows sequence homology.