Emerging Opportunity and Destiny of - and (X4)-Coharboring Plasmids in Escherichia coli.

The emergence of the plasmid-mediated colistin resistance gene and the plasmid-mediated tigecycline resistance gene (X4) represents a significant threat to public health. Although and (X4) have been reported to coexist in the same isolate, there are no reports on the emergence of plasmids coharboring and (X4). In this study, we aimed to investigate the opportunities for the emergence of and (X4)-coharboring plasmids and their destiny in Escherichia coli. Two plasmids carrying both and (X4) were constructed through conjugation assays and confirmed by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Nanopore long-read sequencing. Seven evolved plasmids carrying and (X4) from one of the two plasmids were acquired after continuous evolutionary processes. The fitness effects of and (X4)-coharboring plasmids were studied by stability experiments, competition experiments, and growth curve measurements. A plasmid carrying and (X4) and conferring no fitness cost to its host strain E. coli C600 emerged after evolution during serial passages of bacteria. We proved that it can be anticipated that and (X4) could appear in a single plasmid, and the possibility of occurrence in field strains should be monitored constantly. The originally formed cointegrate plasmids coharboring and (X4) could evolve into a plasmid with lower fitness costs. This will undoubtedly accelerate the transmission of and (X4) globally. The findings highlighted the great possibility of novel hybrid plasmids positive for and (X4), and the risk is worthy of increasing attention and public concern globally. Tigecycline and colistin are used as last-resort therapies to treat infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, the emergence of the plasmid-mediated tigecycline resistance gene (X4) and the plasmid-mediated colistin resistance gene represents a significant threat to human health. A plasmid coharboring and (X4) has not emerged so far, but the potential risk should not be ignored. Plasmids coharboring such vital resistance genes will greatly accelerate the progression of pan-drug resistance among pathogens globally. Therefore, evaluation of the emerging opportunity for the and (X4)-coharboring plasmids and their destiny in E. coli is of great significance. We provide important insight into the contributions of , IS, a truncated IS (ΔIS), and ISR during the generation of cointegrate plasmids carrying and (X4) and highlight the importance of antimicrobials in the evolution and diversity of and (X4)-coharboring plasmids. We show that monitoring of the occurrence of -carrying MDR plasmids and (X4)-bearing MDR plasmids in the same strain should be strengthened to avoid the formation of and (X4)-coharboring plasmids.