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ABSTRACT Conjugative plasmids play a vital role in bacterial evolution and promote the spread of antibiotic resistance. They usually cause fitness costs that diminish the growth rates of the host bacteria. Compensatory mutations are known as an effective evolutionary solution to reduce the fitness cost and improve plasmid persistence. However, whether the plasmid transmission by conjugation is sufficient to improve plasmid persistence is debated since it is an inherently costly process. Here, we experimentally evolved an unstable and costly mcr-1 plasmid pHNSHP24 under laboratory conditions and assessed the effects of plasmid cost and transmission on the plasmid maintenance by the plasmid population dynamics model and a plasmid invasion experiment designed to measure the plasmid’s ability to invade a plasmid-free bacterial population. The persistence of pHNSHP24 improved after 36 days evolution due to the plasmid-borne mutation A51G in the 5′UTR of gene traJ. This mutation largely increased the infectious transmission of the evolved plasmid, presumably by impairing the inhibitory effect of FinP on the expression of traJ. We showed that increased conjugation rate of the evolved plasmid could compensate for the plasmid loss. Furthermore, we determined that the evolved high transmissibility had little effect on the mcr-1-deficient ancestral plasmid, implying that high conjugation transfer is vital for maintaining the mcr-1-bearing plasmid. Altogether, our findings emphasized that, besides compensatory evolution that reduces fitness costs, the evolution of infectious transmission can improve the persistence of antibiotic-resistant plasmids, indicating that inhibition of the conjugation process could be useful to combat the spread of antibiotic-resistant plasmids. IMPORTANCE Conjugative plasmids play a key role in the spread of antibiotic resistance, and they are well-adapted to the host bacteria. However, the evolutionary adaptation of plasmid-bacteria associations is not well understood. In this study, we experimentally evolved an unstable colistin resistance (mcr-1) plasmid under laboratory conditions and found that increased conjugation rate was crucial for the persistence of this plasmid. Interestingly, the evolved conjugation was caused by a single-base mutation, which could rescue the unstable plasmid from extinction in bacterial populations. Our findings imply that inhibition of the conjugation process could be necessary for combating the persistence of antibiotic-resistance plasmids.

The objective of this study was to assess the impact of diverse plasmids bearing colistin resistance gene mcr-1 on host fitness. Forty-seven commensal E. coli isolates recovered from the pig farm where mcr-1 was first identified were screened for mcr-1. mcr-1-bearing plasmids were characterized by sequencing. The fitness impact of mcr-1-bearing plasmids was evaluated by in vitro competition assays. Twenty-seven (57.5%) E. coli isolates were positive for mcr-1. The mcr-1 genes were mainly located on plasmids belonging to IncI2 (n = 5), IncX4 (n = 11), IncHI2/ST3 (n = 8), IncFII (n = 2), and IncY (n = 2). InHI2 plasmids also carried other resistance genes (floR, blaCTX−M, and fosA3) and were only detected in isolates from nursery pigs. Sequences of the representative mcr-1–bearing plasmids were almost identical to those of the corresponding plasmid types reported previously. An increase in the fitness of IncI2- and IncX4-carrying strains was observed, while the presence of IncHI2, IncFII and IncY plasmids showed a fitness cost although an insignificant fitness increase was initially observed in IncFII or IncY plasmids-containing strains. Acquisition of IncI2-type plasmid was more beneficial for host E. coli DH5α than either IncHI2 or IncX4 plasmid, while transformants with IncHI2-type plasmid presented a competitive disadvantage against IncI2 or IncX4 plasmid containing strains. In conclusion, IncI2, IncX4, and IncHI2 were the major plasmid types driving the dissemination of mcr-1 in this farm. Increased fitness or co-selection by other antimicrobials might contribute to the further dissemination of the three epidemic mcr-1–positive plasmids (IncI2, IncX4, and IncHI2) in this farm and worldwide.