Emergence and Characterization of Tigecycline Resistance Gene tet(X4) in ST609 Escherichia coli Isolates from Wastewater in Turkey

Abstract

Emergence of pathogens harboring tigecycline resistance genes incurs great concerns. Wastewater is recognized as the important reservoir of antimicrobial resistance genes. Here we characterized the phenotypes and genotypes of bacteria carrying tet(X4) from wastewater in Turkey for the first time. Four tet(X4)-positive Escherichia coli isolates were identified and characterized by PCR, Sanger sequencing, antimicrobial susceptibility testing, conjugation assays, Illumina sequencing, nanopore sequencing and bioinformatic analysis. Four tet(X4)-harboring isolates were multidrug-resistant (MDR) bacteria and the tet(X4) gene was nontransferable in four isolates. Genetic analysis revealed that tet(X4) genes in four isolates were located on plasmids co-harboring two replicons IncFIA(HI1) and IncFIB(K). However, none of the four plasmids carried genes associated with horizontal transfer of plasmids. The coexistence of bla_SHV-12-bearing IncX3-type plasmid and tet(X4)-harboring plasmid was also found in one isolate. These findings indicate that continuous surveillance of the tet(X4)-bearing isolates in different environments worldwide should be strengthened. IMPORTANCE The emergence of tigecycline resistance genes in humans and animals in China seriously threatens the clinical utility of tigecycline, but the molecular epidemiology of tigecycline-resistant bacteria in other countries remained largely unknown. Therefore, it is necessary to learn the prevalence and molecular characteristics of bacteria carrying tigecycline resistance genes, particularly the mobilizable tet(X4), in other countries. In the study, we first described the presence and molecular characteristics of the tet(X4)-positive E. coli isolates from wastewater in Turkey. Four tet(X4)-bearing isolates belonged to ST609, an E. coli clone commonly found from humans, animals and the environment. These findings highlight the importance of monitoring the tet(X4) gene in different settings globally.

Keywords: Escherichia coli; ST609; blaSHV-12; tet(X4); wastewater.

FIG 1

The evolutionary relatedness of the ST609 E. coli isolates and comparison of resistance plasmid structures. (a) Phylogenetic tree of twenty-eight ST609 E. coli isolates including four tet(X4)-positive E. coli isolates in this study and twenty-four isolates from the NCBI SRA database. The distribution of replicons was displayed in the figure. The presence or absence of replicons is colored in light purple or light gray, respectively. Phylogenetic tree was visualized by iTOL. (b) Circular comparison of tet(X4)-bearing plasmids (pTKEC21-17-tetX-91kb and pTKEC21-59-tetX-86kb) with similar ones in the NCBI database. The outmost circle indicates the plasmid pTKEC21-17-tetX-91kb with genes annotated. (c) Comparison of sequences of tet(X4)-bearing plasmids pTKEC21-17-tetX-91kb and pTKEC21-59-tetX-86kb with draft genome sequences of TKEC21-15 and TKEC21-42. (d) Circular comparison of bla_SHV-12-bearing pTKEC21-17-46kb with similar IncX3 plasmids in the NCBI database. The outmost circle indicates the plasmid pTKEC21-17-46kb with genes annotated. Circular comparisons between plasmids were performed using the BRIG. Comparisons between plasmids and draft genome sequences were performed using the website server (https://server.gview.ca/).