Employing Fluorescence-Assisted Two-Step Allelic Exchange to Investigate MexVW in P. aeruginosa

Authors

• A Ramkumar
• PP Patil
• AD Chiang
• AG Moller
• S Ghosh
• JP Dekker

Abstract

Pseudomonas aeruginosa is an important cause of serious infections in individuals with compromised immunity. A remarkable feature of this pathogen is its ability to generate resistance to most classes of clinical antibiotics, mediated in part by efflux pumps. A recent study in our lab revealed that mutations resulting in overexpression of the RND family efflux pump MexVW unexpectedly conferred resistance to the broad-spectrum antibiotics ceftazidime-avibactam (CZA) and ceftolozane-tazobactam (C/T) in P. aeruginosa lab strain MPAO1. However, the degree to which MexVW contributes to CZA and C/T resistance in clinical P. aeruginosa isolates is unclear. In this work, we assessed the role of MexVW in clinical resistance by generating in-frame deletions of MexVW in clinical P. aeruginosa isolates using a two-step allelic exchange approach. This approach was modified to employ GFP-based fluorescence for merodiploid selection in multidrug resistant isolates. CZA and C/T MICs were measured by E-test, and the impact of MexVW deletion was determined by direct comparison of MICs between parent and isogenic MexVW-deleted derivatives. Additionally, mexW and mexB transcription was quantified by digital PCR. We show that in a subset of CZA-resistant clinical isolates, there is an attributable 2- to 6-fold decrease in MICs for CZA with MexVW deletion. In some cases, this resulted in the restoration of CZA susceptibility, suggesting that MexVW may represent an attractive pharmacologic target for antibiotic design. Ongoing work aims to diversify the clinical isolate test set and incorporate additional antibiotics to characterize the contributions of MexVW to clinical resistance more broadly.

Scientific Focus Area: Microbiology and Infectious Diseases

This page was last updated on Tuesday, August 6, 2024