Identification of Drug Resistance Determinants in a Clinical Isolate of Pseudomonas aeruginosa by High-Density Transposon Mutagenesis

Abstract

With the aim to identify potential new targets to restore antimicrobial susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa isolates, we generated a high-density transposon (Tn) insertion mutant library in an MDR P. aeruginosa bloodstream isolate (isolate ID40). The depletion of Tn insertion mutants upon exposure to cefepime or meropenem was measured in order to determine the common resistome for these clinically important antipseudomonal β-lactam antibiotics. The approach was validated by clean deletions of genes involved in peptidoglycan synthesis/recycling, such as the genes for the lytic transglycosylase MltG, the murein (Mur) endopeptidase MepM1, the MurNAc/GlcNAc kinase AmgK, and the uncharacterized protein YgfB, all of which were identified in our screen as playing a decisive role in survival after treatment with cefepime or meropenem. We found that the antibiotic resistance of P. aeruginosa can be overcome by targeting usually nonessential genes that turn essential in the presence of therapeutic concentrations of antibiotics. For all validated genes, we demonstrated that their deletion leads to the reduction of ampC expression, resulting in a significant decrease in β-lactamase activity, and consequently, these mutants partly or completely lost resistance against cephalosporins, carbapenems, and acylaminopenicillins. In summary, the determined resistome may comprise promising targets for the development of drugs that may be used to restore sensitivity to existing antibiotics, specifically in MDR strains of P. aeruginosa.

A tunable anthranilate-inducible gene expression system for Pseudomonas species

“…It requires-beside the promoter region-the two regulatory proteins RhaS and RhaR, which are encoded in divergent direction immediately upstream of the P rhaBAD promoter (Egan and Schleif 1993). The rhamnose-inducible system has been found to function better than the still often used E. coli P araB -based system in P. aeruginosa (Meisner and Goldberg 2016), and has since then been successfully used in P. aeruginosa (Sonnabend et al 2020). At first, we analyzed gfp expression in P. fluorescens strain A506 by fluorescence microscopy 1 h, 2 h, and 3 h after induction with inducer concentrations ranging from 10 to 0.001 mM (Fig.…”

Identification of Genes Required for Resistance to Peptidomimetic Antibiotics by Transposon Sequencing

“…While the exact function of AsmA is unclear, it has been speculated that AsmA plays a role in OM biogenesis by coordinating the assembly of OM proteins with the biosynthesis of LPS (Martorana et al, 2014). We also identified three genes potentially important for resistance to POLs and chimeras that encode proteins involved in the recycling of peptidoglycan (Sonnabend et al, 2020). The lytic transglycosylase SltB1 (PA14_12080) and the murein endopeptidase MepM1 (PA14_08540), which cleave peptidoglycan cross-connections, and the AmpG permease (PA14_57100), which imports peptidoglycan catabolites into the cytoplasm where they are degraded.…”

YgfB increases β-lactam resistance inPseudomonas aeruginosaby counteracting AlpA-mediatedampDh3expression

“…The MDR Pa strain ID40 is a bloodstream isolate carrying a loss of function mutation in dacB which inactivates Penicillin binding protein 4 (PBP4) 10, 11 . In a recent approach we identified various genes that contribute to resistance toward β-lactam antibiotics in ID40.…”

“…In a recent approach we identified various genes that contribute to resistance toward β-lactam antibiotics in ID40. Among them is the so far uncharacterized gene ygfB , whose expression significantly increases the levels of AmpC, β-lactamase activity and consequently, resistance to various classes of β-lactam antibiotics such as carbapenems, monobactams, and 3 rd and 4 th generation cephalosporins 10 . YgfB belongs to the UPF0149 family of proteins and orthologs are found in many γ-proteobacteria (Fig.…”

“…On the contrary, 1,6-anhydro-N-acetyl-MurNAc-peptides (anhMurNAc-peptides) originating from the turnover and recycling of the PG cell wall 9 can displace UDP-MurNAc-pentapeptide from AmpR. This allows AmpR to transactivate various promoters such as the ampC promoter.Interestingly, those mutations in ampD or dacB that cause derepression of ampC lead to a rise in the intracellular pool levels of anhMurNAc-peptides such as 1,6-MurNAc-L-alanyl-Dglutamyl-meso-diaminopimelic acid-D-alanyl-D-alanine, i.e., anhMurNAc-pentapeptide 7 .The MDR Pa strain ID40 is a bloodstream isolate carrying a loss of function mutation in dacB which inactivates Penicillin binding protein 4 (PBP4) 10,11 . In a recent approach we identified various genes that contribute to resistance toward β-lactam antibiotics in ID40.…”