Successful spread ofmcr-1-bearing IncX4 plasmids is associated with variant in replication protein of IncX4 plasmids

IncX4 plasmids are one of the most epidemiologically successful vehicles for mcr-1 spread. Here we found that the IncX4 plasmids carried two different replication proteins encoded by genes pir-1 and pir-2 , respectively, but mcr-1 was only carried by IncX4 plasmid encoding pir-1 . The copy number of pir-2 encoding plasmids (3.15±0.9 copies) are higher than that of pir-1 encoding plasmids (0.85±0.5 copies). When mcr-1 was cloned into IncX4 plasmid encoding pir-2 , the higher copy number of these plasmids resulted in increased expression of mcr-1 and a greater fitness burden on their host cells. However, these plasmids exhibited a lower rate of invasion into the bacterial population compared to mcr-1 positive plasmids encoding the pir-1 gene. These findings collectively explain the absence of mcr-1 in all IncX4 plasmids encoding pir-2 . Our results further confirmed that low-copy numbers are important for the spread of mcr-1 plasmid from the perspective of natural evolution.