Disbalancing Envelope Stress Responses as a Strategy for Sensitization of Escherichia coli to Antimicrobial Agents

Esther Recacha,Ana García-Duque,Álvaro Pascual,Fernando Docobo-Pérez,Valeria Fox,Sara Díaz-Díaz,José Manuel Rodríguez-Martínez

doi:10.3389/fmicb.2021.653479

Abstract

Disbalancing envelope stress responses was investigated as a strategy for sensitization of Escherichia coli to antimicrobial agents. Seventeen isogenic strains were selected from the KEIO collection with deletions in genes corresponding to the σE, Cpx, Rcs, Bae, and Psp responses. Antimicrobial activity against 20 drugs with different targets was evaluated by disk diffusion and gradient strip tests. Growth curves and time-kill curves were also determined for selected mutant-antimicrobial combinations. An increase in susceptibility to ampicillin, ceftazidime, cefepime, aztreonam, ertapenem, and fosfomycin was detected. Growth curves for Psp response mutants showed a decrease in optical density (OD) using sub-MIC concentrations of ceftazidime and aztreonam (ΔpspA and ΔpspB mutants), cefepime (ΔpspB and ΔpspC mutants) and ertapenem (ΔpspB mutant). Time-kill curves were also performed using 1xMIC concentrations of these antimicrobials. For ceftazidime, 2.9 log10 (ΔpspA mutant) and 0.9 log10 (ΔpspB mutant) decreases were observed at 24 and 8 h, respectively. For aztreonam, a decrease of 3.1 log10 (ΔpspA mutant) and 4 log1010 (ΔpspB mutant) was shown after 4–6 h. For cefepime, 4.2 log10 (ΔpspB mutant) and 2.6 log10 (ΔpspC mutant) decreases were observed at 8 and 4 h, respectively. For ertapenem, a decrease of up to 6 log10 (ΔpspB mutant) was observed at 24 h. A deficient Psp envelope stress response increased E. coli susceptibility to beta-lactam agents such as cefepime, ceftazidime, aztreonam and ertapenem. Its role in repairing extensive inner membrane disruptions makes this pathway essential to bacterial survival, so that disbalancing the Psp response could be an appropriate target for sensitization strategies.

Highlights

Since antimicrobial resistance is increasing worldwide, new targets (Dickey et al, 2017; Recacha et al, 2017; Cattoir and Felden, 2019) need to be sought, either to find new antimicrobial families or to increase the susceptibility of bacterial populations (Laxminarayan et al, 2013)
After 24 h, no growth was observed in the presence of aztreonam at 0.5xMIC (p < 0.01, compared to wild-type, optical density (OD) value 0.08) (Figures 1D–F), a decrease in OD was observed in the presence of ceftazidime at 0.25xMIC (OD value 0.33) (p < 0.0001, compared to wild-type BW25113, OD value 0.5) and at 0.5xMIC (OD value 0.16) (p < 0.05, relative to wild-type, OD value 0.3) (Figures 1M–O), and in cefepime at 0.5xMIC (OD value 0.11) (p < 0.05, relative to wild-type, OD value 0.21) (Figures 1D–F)
The pspA and pspC mutants showed no growth at 0.5xMIC of aztreonam (OD value 0.10) (p < 0.01, compared to wild-type, OD value 0.27) (Figures 1A–C) until 12 h and no growth was observed at 0.5xMIC of cefepime (OD value 0.08) (p < 0.0001, relative to wild-type, OD value 0.30) (Figures 1J–L) at 24 h, respectively

Summary

Introduction

Since antimicrobial resistance is increasing worldwide, new targets (Dickey et al, 2017; Recacha et al, 2017; Cattoir and Felden, 2019) need to be sought, either to find new antimicrobial families or to increase the susceptibility of bacterial populations (Laxminarayan et al, 2013). The Psp response is activated by changes linked to the aberrant localization of OM secretin complexes and other conditions that disrupt the IM, including the dissipation of the proton motive force These signals are transduced through changes in interactions between a set of Psp proteins that lead to the liberation of the PspF transcription factor from the inhibitor PspA and the upregulated production of a limited set of adaptive factors capable of fostering endurance and survival (Flores-Kim and Darwin, 2014, 2016). This pathway is activated by antimicrobial compounds made by plants, animals, and microbes, as well as metals, and can stimulate resistance to broad classes of these substances, primarily, it appears, through the regulation of the multidrug RND efflux pumps AcrD and MdtABC, together with the common OM component TolC (Baranova and Nikaido, 2002; Raffa and Raivio, 2002; Cordeiro et al, 2014; Lin et al, 2014)

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