Classification and molecular characteristics of (X)-carrying plasmids in species.

The rapid dissemination of plasmid-mediated (X) genes in species has compromised the clinical effectiveness of tigecycline, one of the last-resort antibiotics. However, the classification strategy and homology group of (X)-positive spp. plasmids remain largely unknown. In this study, we classified them by genome-based replicon typing, followed by analyses of structural characteristics, transferability and effect. A total of 34 plasmids distributed in at least nine species were collected, including three (X3)-positive plasmids and one (X6)-positive plasmid from our genome sequencing results. Among them, there were 28 plasmids carrying Rep_3 superfamily replicase genes and classified into six homology groups, consisting of GR31 (82.1%), GR26 (3.6%), GR41 (3.6%), GR59 (3.6%), and novel groups GR60 (3.6%) and GR61 (3.6%). Our (X3)-positive plasmids pYH16040-1, pYH16056-1, and pYH12068-1 belonged to the dominant GR31 group, whereas the (X6)-positive plasmid pYH12068-2 was unclassified. Structurally, all (X)-positive GR31 plasmids shared similar plasmid replication (), stability ( and ) and accessory modules [(X) and ], and 97.6% of plasmid-mediated (X) genes in species were adjacent to IS. Conjugation and susceptibility testing revealed pYH16040-1, pYH16056-1, and pYH12068-2, carrying plasmid transfer modules, were able to mediate the mobilization of multiple antibiotic resistance. Under the treatment of tigecycline, the mortality rate of infected by pYH16040-1-mediated (X3)-positive spp. isolate significantly increased when compared with its plasmid-cured strain ( < 0.0001). The spread of such plasmids is of great clinical concern, more effects are needed and will facilitate the future analysis of (X)-positive spp. plasmids.