Cephem potentiation by inactivation of non-essential genes involved in cell wall biogenesis of {beta}-lactamase-producing Escherichia coli [PublishAheadOfPrint]

Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for "helper" drugs reversing cephem resistance in Escherichia coli producing β-lactamases. A CMY-2-encoding plasmid was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hyper-susceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dapF and mrcB, restored susceptibility to cefoxitin and reduced the MICs of cefotaxime and ceftazidime, respectively, from the resistance to intermediate category according to clinical breakpoints. The same mutants harboring a CTX-M-1-encoding plasmid fell into the intermediate or susceptible category for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness cost in mrcB, whereas dapF had a 3-fold longer lag phase compared to the wild type, suggesting that drugs targeting DapF may display antimicrobial activity in addition to synergize with selected cephems. DapF appeared as a potential FOX helper drug target candidate since dapF inactivation resulted in synergistic potentiation of FOX in the genetic backgrounds tested. The study showed that individual inactivation of two non-essential genes involved in cell wall biogenesis potentiates cephem activity according to drug- and strain-specific patterns.

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