Efflux pump in genomic island GI-M202a mediates transfer of Polymyxin B resistance in Pandoraea pnomenusa M202

Abstract

Background Polymyxin B was thought to be last-line therapeutic options against multidrug-resistant Gram-negative bacteria, especially in COVID-19 co-infections or other serious infections. The risk of antimicrobial resistance and its spread to the environment should be brought to the forefront. Methods P. pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage. The genome of M202 was sequenced by PacBio RS II and Illumina HiSeq 4000 platforms. MFS transporter recombinant E. coli strain Mrc-3 was constructed by transferring encoding gene FKQ53_RS21695, a gene in Genomic islands (GIs) of M202, to E. coli 25DN. Mating experiments were performed to evaluate transfer of MFS transporter to Escherichia coli 25DN. And influences of efflux pump inhibitors on MICs were determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on its homologous model. Results MICs of multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, for polymyxin B is 96 mg/L. Genomic Island GI-M202a with major facilitator superfamily (MFS) transporter encoding gene and conjugative transfer proteins encoding genes of type IV secretion system was identified in strain M202. The mating experiment between M202 and Escherichia coli 25DN reflected the transferability of polymyxin B resistant GI-M202a. Efflux pump inhibitor and heterogeneous expression assays also suggested that MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible to polymyxin B resistance. Molecular docking revealed that polymyxin B fatty acyl group insert into hydrophobic region of transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B turns over around Tyr43 to left the peptide group to outside during efflux process, accompanies with conformation change of MFS transporter from inward to outward. Additionally, verapamil and CCCP demonstrated significant inhibition by competing binding sites. Conclusions These findings demonstrated that GI-M202a along with MFS transporter FKQ53_RS21695 in P. pnomenusa M202 could mediate the transmission of polymyxin B resistances.