An efflux pump in genomic island GI-M202a mediates the transfer of polymyxin B resistance in Pandoraea pnomenusa M202

Abstract

Background: Polymyxin B is considered a last-line therapeutic option against multidrug-resistant gram-negative bacteria, especially in COVID-19 coinfections or other serious infections. However, the risk of antimicrobial resistance and its spread to the environment should be brought to the forefront.

Methods: Pandoraea pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage and then was sequenced by the PacBio RS II and Illumina HiSeq 4000 platforms. Mating experiments were performed to evaluate the transfer of the major facilitator superfamily (MFS) transporter in genomic islands (GIs) to Escherichia coli 25DN. The recombinant E. coli strain Mrc-3 harboring MFS transporter encoding gene FKQ53_RS21695 was also constructed. The influence of efflux pump inhibitors (EPIs) on MICs was determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on homology modeling.

Results: The MIC of polymyxin B for the multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, was 96 mg/L. GI-M202a, harboring an MFS transporter-encoding gene and conjugative transfer protein-encoding genes of the type IV secretion system, was identified in P. pnomenusa M202. The mating experiment between M202 and E. coli 25DN reflected the transferability of polymyxin B resistance via GI-M202a. EPI and heterogeneous expression assays also suggested that the MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible for polymyxin B resistance. Molecular docking revealed that the polymyxin B fatty acyl group inserts into the hydrophobic region of the transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B rotates around Tyr43 to externally display the peptide group during the efflux process, accompanied by an inward-to-outward conformational change in the MFS transporter. Additionally, verapamil and CCCP exhibited significant inhibition via competition for binding sites.

Conclusions: These findings demonstrated that GI-M202a along with the MFS transporter FKQ53_RS21695 in P. pnomenusa M202 could mediate the transmission of polymyxin B resistance.

Keywords: Genomic island; MFS transporter; Pandoraea pnomenusa; Polymyxin B resistance.

Fig. 1

Identification and genomic characteristics of M202. A Neighbor-joining tree generated on the basis of 16S rDNA gene sequences of M202. B PATRIC annotation of the genome of M202. C Subsystem analysis of the M202 genome

Fig. 2

Identification of GI-M202a in the M202 genome. Top image, GIs predicted by IslandViewer 4. Putative GIs predicted by the SIGI-HMM method (yellow squares) or IslandPath-DIMOB method (blue squares). The integrated results are indicated by red squares. Bottom line, gene arrangement in GI-M202a. Genes are denoted by arrows. MFS transporter, green arrows; DNA transfer-related genes, gray arrows; genes of unknown function, purple arrows; type IV secretion system, orange arrows; others, blue arrows

Fig. 3

A MICs of polymyxin B for different strains. M202-TC1: transconjugant of M202 and E. coli 25DN; Mrc-3: DH5α with heterogeneous expression of FKQ53_RS21695. B RT-qPCR analyses of FKQ53_RS21695 gene in P. pnomenusa M202, E. coli M202-TC1, and E. coli Mrc-3. **: p < 0.001

Fig. 4

Overall structure of the MFS transporter FKQ53_RS21695 of GI-M202a. A Cartoon representation of the FKQ53_RS21695 structure. The N-domain, C-domain, and α′ are represented in blue, orange, and green, respectively. Numbers indicate the TM helixes. B Active conformation of motif A and protonation/deprotonation-related residues. C The overall hydrophobic surface of FKQ53_RS21695. D The cavity-facing sides of the N and C domains have contrasting surface electrostatic potentials. The figure was generated using DS

Fig. 5

Polymyxin B efflux mediated by the MFS transporter FKQ53_RS21695. A PBC in the inward-open conformation. B PBC in outward-open conformation. PBC, polymyxin B binding cavity; PDR, protonation and deprotonation residues. C Interactions of PBC with polymyxin B in the inward-open conformation. D Interactions of PBC with polymyxin B in the outward-open conformation. Left: location of polymyxin B; right: interacting amino acid residues and chemical bonds

Fig. 6

Binding cavities for verapamil and CCCP in FKQ53_RS21695. A Locations of the polymyxin B- and verapamil-binding cavities. B Locations of the polymyxin B- and CCCP-binding cavities

Fig. 7

Proposed polymyxin B transport mechanism mediated by FKQ53_RS21695. Polymyxin B “turn around” transport mode