Constitutive soxR mutations contribute to multiple-antibiotic resistance in clinical Escherichia coli isolates

Abstract

The soxRS regulon of Escherichia coli and Salmonella enterica is induced by redox-cycling compounds or nitric oxide and provides resistance to superoxide-generating agents, macrophage-generated nitric oxide, antibiotics, and organic solvents. We have previously shown that constitutive expression of soxRS can contribute to quinolone resistance in clinically relevant S. enterica. In this work, we have carried out an analysis of the mechanism of constitutive soxS expression and its role in antibiotic resistance in E. coli clinical isolates. We show that constitutive soxS expression in three out of six strains was caused by single point mutations in the soxR gene. The mutant SoxR proteins contributed to the multiple-antibiotic resistance phenotypes of the clinical strains and were sufficient to confer multiple-antibiotic resistance in a fresh genetic background. In the other three clinical isolates, we observed, for the first time, that elevated soxS expression was not due to mutations in soxR. The mechanism of such increased soxS expression remains unclear. The same E. coli clinical isolates harbored polymorphic soxR and soxS DNA sequences, also seen for the first time.

FIG. 1.

Expression of soxS mRNA in clinical E. coli isolates. Overnight cultures were diluted 1/100 in fresh LB broth and grown with vigorous shaking (250 rpm) for 105 min at 30°C (A and C) or for 90 min at 37°C (B). At that point, the cultures were split, the appropriate samples were treated with 1.3 mM PQ, and shaking resumed at 250 rpm and 30°C or 37°C as appropriate for another 30 to 40 min. RNA was then harvested for Northern analysis. (A) Expression of soxS mRNA in E. coli strains GC4468 (soxR⁺), E3, E17, E19, and M1, with or without 1.3 mM paraquat treatment. (B) Basal expression of soxS mRNA in E. coli strains GC4468, JTG936 (soxR^c), I236, I237, I242, I243, I244, I246, I248, I251, I253, and I254.

FIG. 2.

Regulation of soxS::lacZ by mutant SoxR proteins. Plasmids pACYC177, pAK-WT, pAK-E3, pAK-E17, pAK-E19, pAK-M1, and pAK-I237 carrying no soxR, wild-type soxR, or mutant alleles of soxR were introduced into strain EH46 (ΔsoxR soxS::lacZ) (Table 1), and the transcriptional activity of the expressed SoxR proteins was assayed by measuring expression of β-galactosidase in the presence or absence of 250 μM PQ. The data shown are the means and standard errors of two determinations.

FIG. 3.

Antibiotic resistance conferred by mutant soxR genes. Plasmids pACYC177, pAK-WT, pAK-E3, pAK-E17, pAK-E19, pAK-M1, and pAK-I237 encoding no soxR, wild-type soxR, or a mutant allele of soxR were introduced into strain TN402 (ΔsoxR) (Table 2), and their contribution to the antibiotic resistance phenotype of the recipient strain was assayed on antibiotic gradient plates in the presence or absence of 50 μM PQ. (A) Chloramphenicol (CHL), 0 to 30 μg/ml; (B) nalidixic acid (NAL), 0 to 15 μg/ml; (C) ciprofloxacin (CIP), 0 to 0.125 μg/ml. The data shown are the means and standard errors of three determinations.

FIG. 4.

Multicopy suppression of antibiotic resistance with wild-type soxR. Clinical isolates E17, M1, and I242, as well as control laboratory strains GC4468 (soxR⁺), DJ901 (ΔsoxRS), JTG936 (soxR^c), and JTG1052 (soxR^c), were transformed with the IPTG-inducible plasmid pSXR (Table 1) or the vector alone. The antibiotic resistance of the control and pSXR-containing strains was assayed on antibiotic gradient plates in the presence of 5 μM IPTG. The maximum antibiotic concentrations per plate were as follows: for strain E17, chloramphenicol (CHL) at 1.5 mg/ml, ciprofloxacin (CIP) at 250 μg/ml, and tetracycline (TET) at 750 μg/ml; for strain M1, chloramphenicol at 40 μg/ml, ciprofloxacin at 5 μg/ml, and tetracycline at 19 μg/ml; for strain I242, chloramphenicol at 20 μg/ml, ciprofloxacin at 1 μg/ml, and tetracycline at 300 μg/ml; for strains GC4468, DJ901, JTG936, and JTG1052, chloramphenicol at 20 μg/ml, ciprofloxacin at 0.05 μg/ml, and tetracycline at 15 μg/ml. The data shown are the means and standard deviations of three determinations.