Phenotypic and genomic analysis reveals Riemerella anatipestifer as the potential reservoir of tet(X) variants.

Abstract

Tigecycline is regarded as one of the last-resort antimicrobials clinically. Emergence of plasmid-mediated tet(X) undermines such an important drug. However, the origins of tet(X) remain largely unexplored. Riemerella anatipestifer strains were characterized by PCR, antimicrobial susceptibility testing, WGS and bioinformatics analysis. Functional analysis of tet(X) was verified by cloning experiments. Genomic structures of chromosome- and plasmid-mediated tet(X) were analysed. Thirty-eight R. anatipestifer strains were collected and found to be positive for tet(X). These strains were resistant to multiple antimicrobials; 55.3% (21/38) of the strains were resistant to tigecycline and all of the strains demonstrated resistance to tetracycline. The complete genome sequences of 18 representative strains were obtained. WGS analysis of 38 genomes identified 13 tet(X) variants located on chromosomes, which increased MICs of tigecycline (16-256-fold) for Escherichia coli, although most of them could not confer high-level resistance to tigecycline in the original R. anatipestifer hosts. Genomic environment analysis indicated that the occurrence of multiple tet(X) variants is common and other resistance genes, such as catB, tet(Q), floR, blaOXA, ereD and ermF, could be located in the same chromosomal regions. Two types of tet(X)-bearing segments were identified, one of which was floR-ISCR2-tet(X). This indicates that tet(X) variants were not conserved in chromosomal structures, but in regions with potential transferability. Furthermore, an MDR plasmid carrying tet(X18) was found in R. anatipestifer 20190305E2-2, different from the chromosomal tet(X21). This study confirmed that tet(X) is highly prevalent in R. anatipestifer. The transfer risk of tet(X) across R. anatipestifer to other clinical pathogens warrants further investigations.