Coexistence of tet(X4), mcr-1, and blaNDM-5 in ST6775 Escherichia coli Isolates of Animal Origin in China

Abstract

Emergence of pathogens harboring multiple resistance genes incurs great concerns. Cooccurrence of mobile resistance genes conferring resistance to tigecycline, colistin, and carbapenems in Escherichia coli has not been investigated. This study aimed to characterize three E. coli isolates coharboring tet(X4), mcr-1, and bla_NDM-5. Isolates coharboring tet(X4), mcr-1, and bla_NDM-5 were identified and characterized by PCR, Sanger sequencing, antimicrobial susceptibility testing, conjugation assays, Illumina sequencing, nanopore sequencing, and bioinformatic analysis. Three E. coli isolates carrying tet(X4), mcr-1, and bla_NDM-5 were identified from pigeons in China. They were resistant to almost all antimicrobials except enrofloxacin. tet(X4) and bla_NDM-5 could be conjugated into E. coli C600, but mcr-1 was nontransferable in three isolates. Three isolates belonged to sequence type 6775 (ST6775), and clonal dissemination of isolates carrying tet(X4), mcr-1, and bla_NDM-5 existed in the pigeon farm. Genetic analysis revealed that mcr-1 mediated by the Tn6330 was located on the chromosome, tet(X4) was located on the IncFII plasmid, and bla_NDM-5 was located on the IncX3 plasmid. We first characterized the E. coli isolates carrying tet(X4), mcr-1, and bla_NDM-5 simultaneously. Relevant measures should be taken to decrease the prevalence of pathogens carrying tet(X4), mcr-1, and bla_NDM-5. IMPORTANCE Tigecycline and colistin are regarded as vital antimicrobials to treat multidrug-resistant (MDR) bacterial infections, such as that caused by carbapenemase-producing Enterobacteriaceae (CPE). Cooccurrence of mobile resistance genes conferring resistance to last-resort antimicrobials in E. coli remains unknown. Here, we characterized E. coli strains coharboring tet(X4), mcr-1, and bla_NDM-5 phenotypically and genetically. Resistance genes tet(X4), mcr-1, and bla_NDM-5 were located on transposons or plasmids that were mobile genetic elements related to the capture, accumulation, and dissemination of such important resistance genes. The emergence of E. coli isolates carrying tet(X4), mcr-1, and bla_NDM-5 highlights the importance of monitoring the coexistence of novel mobile resistance genes in different settings with a One Health approach. Risk of transmission of such MDR pathogens from animals to humans should be evaluated comprehensively.

Keywords: Escherichia coli; ST6775; blaNDM-5; coexistence; mcr-1; tet(X4).

FIG 1

Comprehensive information of the assembled draft genomes from 10 ST6775 E. coli isolates. (a) The number of SNP differences among 10 ST6775 E. coli isolates, including three isolates in the study and seven isolates from the NCBI SRA database. (b) The distribution of plasmid replicons in 10 ST6775 E. coli isolates. The blue rectangles indicate the presence of plasmid replicons. (c) The distribution of antimicrobial resistance genes in 10 ST6775 E. coli isolates. The red rectangles indicate the presence of antimicrobial resistance genes. (d) The distribution of insert sequences in 10 ST6775 E. coli isolates. The green rectangles indicate the presence of insert sequences. Legend labels indicates the similarity (%).

FIG 2

Genetic environment of mcr-1 in the chromosome. Linear alignment of the selected chromosomal segment (23,152 bp) containing Tn6330 with other homologous sequences available in the NCBI database.

FIG 3

Genetic context of tet(X4) in the IncFII plasmid. (a) Circular comparison between the tet(X)-bearing IncFII plasmid pPT62-tetX-108kb in the study and other homologous plasmids pAB12-1-tetX4 and p47EC available in the NCBI database. The outermost circles indicate the plasmid pPT62-tetX-108kb with genes annotated. (b) Linear alignment of tet(X4)-bearing structure on plasmid pPT62-tetX-108kb with tet(X4)-carrying structure on plasmid pAB12-1-tetX4 available in the NCBI database. Green arrows indicate the directions of the primers used to screen the formation of the circular intermediate, and names of primers are marked next to the arrows. (c) The circular intermediate mediated by IS26. Green arrows indicate the directions of the primers. (d) A copy of IS26 left after the circular intermediate fell off. Green arrows indicate the directions of the primers.

FIG 4

Genetic environment of bla_NDM-5 in the IncX3 plasmid. Circular comparison of three bla_NDM-5-bearing plasmids. The bla_NDM-5-harboring IncX3 plasmid pPT62-NDM-47kb in this study was compared with two plasmids pEc-MW07_NDM and pNDM5_020031 in the NCBI database. The outermost circles indicate the plasmid pPT62-NDM-47kb with genes annotated.