Identification of networks counteracting a successful combination treatment of MDR Pseudomonas aeruginosa with β-lactams and ciprofloxacin

Multidrug resistance (MDR) of Pseudomonas aeruginosa (Pa) poses a growing challenge for the treatment of medical infections. Previous studies have demonstrated that combining different classes of drugs successfully reduces mortality among patients with severe MDR-Pa infection. Thus, a combination of fluorochinolones/β-lactams or aminoglycosides/β-lactams in treatment regimens can markedly enhance the susceptibility as opposed to monotherapy alone and might be a therapeutical option in some cases. Nevertheless, certain percentages of strains remain resistant.

Using the bloodstream isolate Pa ID40 which is resistant to most β-lactam antibiotics and ciprofloxacin, we aimed at elucidating secondary resistance genes that are additionally involved in promoting ciprofloxacin resistance on the one hand and resistance to a ceftazidime (CAZ)/ciprofloxacin (CIP) combination therapy on the other hand to identify potential targets.

For this purpose, a transposon directed insertion sequencing approach was used for screening. To further validate candidates derived from this screen, checkerboard assays were performed to determine minimal inhibitory concentrations (MIC) of unmarked deletion mutants.

Deletion of genes in DNA repair (recA, recG, xseA), efflux pumps (mexEFoprN), cell envelope components (mlaA/vacJ, oprF), as well as amgK, encoding an enzyme involved in peptidoglycan recycling and LPS biosynthesis, reduced MIC values to ciprofloxacin 2- to 4-fold but did not break resistance of Pa ID40. A marked impact on MIC values regarding CIP/CAZ combination treatment resulted from single deletion of amgK, recG or mexEFoprN, but the MIC values still remained above the breakpoints for both antibiotics. Double deletions addressing different biological processes, such as deletion of amgK/mexEFoprN or amgk/recG, but not of mexEFoprN/recG reduced MIC values below breakpoints for both antibiotics upon CIP/CAZ treatment.

Thus, a complex network involving efflux pumps, DNA repair mechanisms and the so far in this context unrecognized N-acetylmuramate/N-acetylglucosamine kinase AmgK counteracts effective treatment of MDR-Pa strains with a CIP/CAZ combination.