Effects of Stress, Reactive Oxygen Species, and the SOS Response on De Novo Acquisition of Antibiotic Resistance in Escherichia coli

Abstract

Strategies to prevent the development of antibiotic resistance in bacteria are needed to reduce the threat of infectious diseases to human health. The de novo acquisition of resistance due to mutations and/or phenotypic adaptation occurs rapidly as a result of interactions of gene expression and mutations (N. Handel, J. M. Schuurmans, Y. Feng, S. Brul, and B. H. Ter Kuile, Antimicrob Agents Chemother 58:4371-4379, 2014, http://dx.doi.org/10.1128/AAC.02892-14). In this study, the contribution of several individual genes to the de novo acquisition of antibiotic resistance in Escherichia coli was investigated using mutants with deletions of genes known to be involved in antibiotic resistance. The results indicate that recA, vital for the SOS response, plays a crucial role in the development of antibiotic resistance. Likewise, deletion of global transcriptional regulators, such as gadE or soxS, involved in pH homeostasis and superoxide removal, respectively, can slow the acquisition of resistance to a degree depending on the antibiotic. Deletion of the transcriptional regulator soxS, involved in superoxide removal, slowed the acquisition of resistance to enrofloxacin. Acquisition of resistance occurred at a lower rate in the presence of a second stress factor, such as a lowered pH or increased salt concentration, than in the presence of optimal growth conditions. The overall outcome suggests that a central cellular mechanism is crucial for the development of resistance and that genes involved in the regulation of transcription play an essential role. The actual cellular response, however, depends on the class of antibiotic in combination with environmental conditions.

FIG 1

Acquisition of resistance to amoxicillin in the E. coli MG1655 and BW25113 wild-type strains and ΔsoxS, ΔompF, and ΔgadE knockout strains in mineral medium. Cells were adapted by stepwise increasing the drug concentration by a factor of 2 when growth was comparable to that of the wild-type cells.

FIG 2

Acquisition of resistance to enrofloxacin in E. coli MG1655 and BW25113 wild-type cells and ΔsoxS, ΔompF, and ΔgadE cells in mineral medium.

FIG 3

Acquisition of resistance to amoxicillin in E. coli MG1655 and BW25113 wild-type cells and ΔrecA cells in complex LB medium.

FIG 4

Acquisition of resistance to enrofloxacin in E. coli MG1655 and BW25113 wild-type cells and ΔrecA cells in complex LB medium.

FIG 5

Acquisition of resistance to amoxicillin in the presence of pH 6 (A) or 2% NaCl (B) in the E. coli MG1655 and BW25113 wild-type strains. The lines without points represent the averages for the three controls (MG1655 twice and BW25113 once) at pH 6.9 without additional NaCl. Cells were adapted to pH 6 or 2% NaCl by daily passaging to fresh medium without any antibiotic for 7 days.

FIG 6

Acquisition of resistance to enrofloxacin in the presence of pH 6 (A) or 2% NaCl (B) in the E. coli MG1655 and BW25113 wild-type strains. The lines without points represent the average for the three controls at pH 6.9 without additional NaCl. Cells were adapted to pH 6 or 2% NaCl by daily passaging to fresh medium without any antibiotic for 7 days.

FIG 7

Change in expression levels of selected genes in enrofloxacin-resistant (Enro) and E. coli MG1655 wild-type (WT) cells cultured in mineral medium at pH 6 or with additional salt (2% NaCl) compared to that in the wild-type strain (pH 6.9, 0% additional NaCl). Expression levels were compared to those by the wild type in the absence or presence (exp) of 0.125 μg/ml enrofloxacin. Data are the means ± SDs for two biological replicates.