Plasmid Stability Test

Plasmid stability was determined as previously reported with minor modifications (Gao et al., 2020). Briefly, cultures were incubated at 25, 30, 37, or 42°C in a shaking water bath (200 rpm) and were diluted 1,000-fold in antibiotic-free LB broth. After 24 h, cultures were serially diluted and plated on antibiotic-free LB agar and LB agar containing meropenem (0.5 μg ml⁻¹ L). The retention rate of the bla_NDM-1 gene was calculated by dividing the number of colonies that grew on meropenem-containing LB agar by the number of colonies on antibiotic-free LB agar. In addition, 10 colonies were randomly selected and subjected to PCR validation of the bla_NDM-1 gene. [It is unlikely that the chromosomal integration of NDM gene in plasmid stability test occurred. In our previous published work for IncX3 plasmid (Liu et al., 2020), we have proved that “PCR tests revealed that the blaNDM-1 gene was maintained by transconjugants and wild-type isolates for 1,000 generations. In addition, the location of the blaNDM-1 gene did not change in the decedents, as shown by Southern blotting.” The 24 h passage in this plasmid stability test could not lead to NDM-1 chromosomal integration].