Strand-biased circularizing integrative elements spread tmexCD-toprJ gene clusters encoding RND-type multidrug efflux pumps by repeated transpositions

Antimicrobial resistance genes (ARGs) are associated with mobile genetic elements (MGEs) that conscript useful genes into the human-microbe and microbe-microbe battlefields. Thus, under intense selective pressure, ARGs have been constantly adapting and evolving, spreading among microbes. tmexCD-toprJ gene clusters, which encode resistance-nodulation-cell division (RND)-type efflux pumps, confer multidrug-resistance to clinically important antimicrobials, including tigecycline. Noteworthily, these gene clusters have emerged in gram-negative bacteria in humans, animals, and the environment worldwide by MGE-mediated transfer. Here we show a hidden MGE, strand-biased circularizing integrative element (SE), that is recently recognized to mediate transpositions of ARGs, associated with the spread of tmexCD-toprJ gene clusters. We identified multidrug-resistant isolates of Aeromonas species in a water environment in Vietnam that harbored multiple copies of tmexCD-toprJ in their chromosomes that were associated with SEs. In particular, Aeromonas hydrophila NUITM-VA1 was found to harbor two copies of a novel variant of tmexC3.3D3.3-topJ1 within cognate SEs, whereas Aeromonas caviae NUITM-VA2 harbored four copies of a novel variant of tmexC2D2.3-topJ2 within cognate SEs. Based on the nature of SE to incorporate a neighboring sequence into the circular form and reinsert it into target sites during transposition, we identified the order of intragenomic movements of tmexCD-toprJ gene clusters. Altogether, our findings suggest that most known subgroups of tmexCD-toprJ and their subvariants underwent transpositions among bacterial chromosomes and plasmids via SEs. Hence, a tmexCD-toprJ gene cluster ancestor may have been initially mobilized via SE, subsequently spreading among bacteria and evolving in new hosts.