Deciphering the Structural Diversity and Classification of the Mobile Tigecycline Resistance Gene <i>tet</i> (X)-Bearing Plasmidome among Bacteria

Abstract

The emergence of novel plasmid-mediated resistance genes constitutes a great public concern. Recently, mobile <i>tet</i>(X) variants were reported in diverse pathogens from different sources. However, the diversity of <i>tet</i>(X)-bearing plasmids remains largely unknown. In this study, the phenotypes and genotypes of all the <i>tet</i>(X)-positive tigecycline-resistant strains isolated from a slaughterhouse in China were characterized by antimicrobial susceptibility testing, conjugation, pulsed-field gel electrophoresis with S1 nuclease (S1-PFGE), and PCR. The diversity and polymorphism of <i>tet</i>(X)-harboring strains and plasmidomes were investigated by whole-genome sequencing (WGS) and single-plasmid-molecule analysis. Seventy-four <i>tet</i>(X4)-harboring <i>Escherichia coli</i> strains and one <i>tet</i>(X6)-bearing <i>Providencia rettgeri</i> strain were identified. The <i>tet</i>(X4)-bearing elements in 27 strains could be transferred to the recipient strain via plasmids. All <i>tet</i>(X4)-bearing plasmids isolated in this study and 15 <i>tet</i>(X4)-bearing plasmids reported online were analyzed. <i>tet</i>(X4)-bearing plasmids ranged from 9 to 294 kb and were categorized as ColE2-like, IncQ, IncX1, IncA/C2, IncFII, IncFIB, and hybrid plasmids with different replicons. The core <i>tet</i>(X4)-bearing genetic contexts were divided into four major groups: IS<i>CR2</i>-<i>tet</i>(X4)-<i>abh</i>, △IS<i>CR2</i>-<i>abh</i>-<i>tet</i>(X4)-IS<i>CR2</i>, IS<i>CR2-abh</i>-<i>tet</i>(X4)-IS<i>CR2</i>-<i>virD2</i>-<i>floR</i>, and <i>abh</i>-<i>tet</i>(X4)-IS<i>CR2</i>-<i>yheS</i>-<i>cat</i>-<i>zitR</i>-IS<i>CR2</i>-<i>virD2</i>-<i>floR</i> Tandem repeats of <i>tet</i>(X4) were universally mediated by IS<i>CR2</i> Different <i>tet</i>(X)-bearing strains existed in the same microbiota. Reorganization of <i>tet</i>(X4)-bearing multidrug resistance plasmids was found to be mediated by IS<i>26</i> and other homologous regions. Finally, single-plasmid-molecule analysis captured the heterogenous state of <i>tet</i>(X4)-bearing plasmids. These findings significantly expand our knowledge of the <i>tet</i>(X)-bearing plasmidome among microbiotas, which establishes a baseline for investigating the structure and diversity of human, animal, and environmental tigecycline resistomes. Characterization of <i>tet</i>(X) genes among different microbiotas should be performed systematically to understand the evolution and ecology.<b>IMPORTANCE</b> Tigecycline is an expanded-spectrum tetracycline used as a last-resort antimicrobial for treating infections caused by superbugs such as carbapenemase-producing or colistin-resistant pathogens. Emergence of the plasmid-mediated mobile tigecycline resistance gene <i>tet</i>(X4) created a great public health concern. However, the diversity of <i>tet</i>(X4)-bearing plasmids and bacteria remains largely uninvestigated. To cover this knowledge gap, we comprehensively identified and characterized the <i>tet</i>(X)-bearing plasmidome in different sources using advanced sequencing technologies for the first time. The huge diversity of <i>tet</i>(X4)-bearing mobile elements demonstrates the high level of transmissibility of the <i>tet</i>(X4) gene among bacteria. It is crucial to enhance stringent surveillance of <i>tet</i>(X) genes in animal and human pathogens globally.