Cointegrate/hybrid plasmids combine the genetic elements of two or more plasmids and generally carry abundant antimicrobial resistance determinants. Hence, the spread of cointegrate plasmids will accelerate the transmission of AMR genes. To evaluate the transmission risk caused by cointegrate plasmids, we investigated the structural diversity, fitness cost, and stability of a cointegrate plasmid in Klebsiella pneumoniae YZ6 and Escherichia coli EC600. The cointegrate plasmid pSL131_IncA/C_IncX3 was from a clinical Salmonella Lomita strain. After transferring the plasmid into E. coli EC600 by conjugation, we observed plasmids with different structures, including a full-length original plasmid and two truncated versions. By contrast, DNA fragment deletion and bla_CTX-M-14 gene insertion in the plasmid were detected in a transconjugant derived from K. pneumoniae YZ6. These results suggest that the structure of the plasmid was unstable during conjugation. Furthermore, both the full-length plasmid in EC600 and the structurally reorganized plasmid in YZ6 imposed a fitness cost on the bacterial host and enhanced biofilm formation ability. Serial passaging in antibiotic-free medium resulted in a rapid decline of the plasmid in YZ6. However, the stability of the structurally reorganized plasmid in YZ6 was improved via serial passaging in antibiotic-containing medium. SNP calling revealed that mutations of the outer membrane porin may play an essential role in this process. These findings indicate that structural versatility could contribute to the dissemination of cointegrate plasmids. Although the plasmid incurred a fitness cost in other Enterobacteriaceae species, positive selection could alleviate the adverse effects.

The emergence and spread of antibiotic resistance genes (ARGs) have imposed a serious threat on global public health. Horizontal gene transfer (HGT) via plasmids is mainly responsible for the spread of ARGs, and conjugation plays an important role in HGT. The conjugation process is very active in vivo and its effect on the spreading of ARGs may be underestimated. In this review, factors affecting conjugation in vivo, especially in the intestinal environment, are summarized. In addition, the potential mechanisms affecting conjugation in vivo are summarized from the perspectives of bacterial colonization and the conjugation process.

Plasmids incur a fitness cost that has the potential to restrict the dissemination of resistance in bacterial pathogens. However, bacteria can overcome this disadvantage by compensatory evolution to maintain their resistance. Compensatory evolution can occur via both chromosomes and plasmids, but there are a few reviews regarding this topic, and most of them focus on plasmids. In this review, we provide a comprehensive overview of the currently reported mechanisms underlying compensatory evolution on chromosomes and plasmids, elucidate key targets regulating plasmid fitness cost, and discuss future challenges in this field. We found that compensatory evolution on chromosomes primarily arises from mutations in transcriptional regulatory factors, whereas compensatory evolution of plasmids predominantly involves three pathways: plasmid copy number regulation, conjugation transfer efficiency, and expression of antimicrobial resistance (AMR) genes. Furthermore, the importance of reasonable selection of research subjects and effective integration of diverse advanced research methods is also emphasized in our future study on compensatory mechanisms. Overall, this review establishes a theoretical framework that aims to provide innovative ideas for minimizing the emergence and spread of AMR genes.

The large-scale epidemic of the tet(X4) gene in the livestock and poultry industry is threatening public health; however, there is still a lack of comparative studies on tet(X4)-bearing plasmids in chicken and pig Escherichia coli. To evaluate the prevalence trend of tet(X4)-bearing plasmids and the factors influencing their persistence in the livestock and poultry industry, we examined the fitness cost, stability under tetracyclines pressure, and conjugation frequencies at various temperatures of six tet(X4)-bearing plasmids in four representative pig E. coli isolates and chicken E. coli isolates. Compared with pig E. coli, the plasmid in chicken E. coli showed lower fitness cost, and stronger ability to promote bacterial biofilm formation and motility. Meanwhile, the presence of tetracycline may favor the stability of tet(X4)-bearing plasmids, which was more common in chicken E. coli. Furthermore, the optimal temperature for IncX1 tet(X4)-bearing plasmid conjugation was 42 °C, and its conjugation frequency in chicken E. coli was higher than that in pig E. coli, whereas the optimal temperature for IncFII tet(X4)-bearing plasmid conjugation was 37 °C and it performed better in pig E. coli, suggesting the predominant plasmid types circulating in chicken E. coli and pig E. coli may be distinct. Collectively, although tet(X4) currently appears to be more prevalent in pig E. coli, this is probably independent of the fitness cost caused by tet(X4)-plasmids. To curb the future spread of the tet(X4) gene, reduced tetracyclines usage and tailored interventions should be applied in different breeding industries.