Functional Identification and Evolutionary Analysis of Two Novel Plasmids Mediating Quinolone Resistance in Proteus vulgaris

Plasmid-mediated quinolone resistance (PMQR) remains one of the main mechanisms of bacterial quinolone resistance and plays an important role in the transmission of antibiotic resistance genes (ARGs). In this study, two novel plasmids, p3M-2A and p3M-2B, which mediate quinolone resistance in Proteus vulgaris strain 3M (P3M) were identified. Of these, only p3M-2B appeared to be a qnrD-carrying plasmid. Both p3M-2A and p3M-2B could be transferred into Escherichia coli, and the latter caused a twofold change in ciprofloxacin resistance, according to the measured minimum inhibitory concentration (MIC). Plasmid curing/complementation and qRT-PCR results showed that p3M-2A can directly regulate the expression of qnrD in p3M-2B under treatment with ciprofloxacin, in which process, ORF1 was found to play an important role. Sequence alignments and phylogenetic analysis revealed the evolutionary relationships of all reported qnrD-carrying plasmids and showed that ORF1–4 in p3M-2B is the most conserved backbone for the normal function of qnrD-carrying plasmids. The identified direct repeats (DR) suggested that, from an evolutionary perspective, p3M-2B may have originated from the 2683-bp qnrD-carrying plasmid and may increase the possibility of plasmid recombination and then of qnrD transfer. To the best of our knowledge, this is the first identification of a novel qnrD-carrying plasmid isolated from a P. vulgaris strain of shrimp origin and a plasmid that plays a regulatory role in qnrD expression. This study also sheds new light on plasmid evolution and on the mechanism of horizontal transfer of ARGs encoded by plasmids.