Persistence of plasmid and (X4) in an isolate coharboring and after acquiring an IncFII (X4)-positive plasmid.

The prevalence of plasmid-mediated tigecycline resistance gene (X4) is presenting an increasing trend. Once (X4)-bearing plasmids are captured by multidrug-resistant bacteria, such as and -coharboring bacteria, it will promote bacteria to develop an ultra-broad resistance spectrum, limiting clinical treatment options. However, little is known about the destiny of such bacteria or how they will evolve in the future. Herein, we constructed a multidrug-resistant bacteria coharboring (X4), , and by introducing a (X4)-bearing plasmid into a and positive strain. Subsequently, the stability of (X4) and the plasmid was measured after being evolved under tigecycline or antibiotic-free circumstance. Interestingly, we observed both (X4)-bearing plasmids in tigecycline treated strains and non-tigecycline treated strains were stable, which might be jointly affected by the increased conjugation frequency and the structural alterations of the (X4)-positive plasmid. However, the stability of (X4) gene showed different scenarios in the two types of evolved strains. The (X4) gene in non-tigecycline treated strains was stable whereas the (X4) gene was discarded rapidly in tigecycline treated strains. Accordingly, we found the expression levels of (X4) gene in tigecycline-treated strains were several times higher than in non-tigecycline treated strains and ancestral strains, which might in turn impose a stronger burden on the host bacteria. SNPs analysis revealed that a myriad of mutations occurred in genes involving in conjugation transfer, and the missense mutation of gene in chromosome of tigecycline treated strains might account for the completely different stability of (X4)-bearing plasmid and (X4) gene. Collectively, these findings shed a light on the possibility of the emergence of multidrug resistant bacteria due to the transmission of (X4)-bearing plasmid, and highlighted that the antibiotic residues may be critical to the development of such bacteria.