Sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis

Abstract

Antibiotic resistance arises through mechanisms such as selection of naturally occurring resistant mutants and horizontal gene transfer. Recently, oxidative stress has been implicated as one of the mechanisms whereby bactericidal antibiotics kill bacteria. Here, we show that sublethal levels of bactericidal antibiotics induce mutagenesis, resulting in heterogeneous increases in the minimum inhibitory concentration for a range of antibiotics, irrespective of the drug target. This increase in mutagenesis correlates with an increase in ROS and is prevented by the ROS scavenger thiourea and by anaerobic conditions, indicating that sublethal concentrations of antibiotics induce mutagenesis by stimulating the production of ROS. We demonstrate that these effects can lead to mutant strains that are sensitive to the applied antibiotic but resistant to other antibiotics. This work establishes a radical-based molecular mechanism whereby sublethal levels of antibiotics can lead to multidrug resistance, which has important implications for the widespread use and misuse of antibiotics.

Figure 1. Low levels of bactericidal antibiotics increase mutation rate due to reactive oxygen species formation

(A) Fold change in mutation rate (mean +/− 95% CI) relative to an untreated control (no drug) for wildtype E. coli (MG1655) following an overnight treatment with 1µg/ml ampicillin, 1µg/ml kanamycin, 3µg/ml kanamycin, 15ng/ml norfloxacin, 50ng/ml norfloxacin, or 1mM hydrogen peroxide (H₂O₂). (B) Correlation between oxidative stress levels (HPF fluorescence) and fold change in mutation rate for wildtype E. coli for the treatments described in A. (C–D) Fold change in mutation rate (mean +/− 95% CI) relative to an untreated control (no drug) for wildtype E. coli following an overnight treatment with 100mM thiourea and no drug, 1µg/ml ampicillin, 1µg/ml kanamycin, 3µg/ml kanamycin, 15ng/ml norfloxacin, 50ng/ml norfloxacin, or 1mM hydrogen peroxide (H₂O₂) under (C) aerobic growth conditions with 100mM thiourea or (D) anaerobic growth conditions.

Figure 2. Low levels of bactericidal antibiotics can lead to broad-spectrum increases in MIC due to ROS-mediated mutagenesis

(A–B) Fold change in MIC relative to an aerobic no-drug control for ampicillin, norfloxacin, kanamycin, tetracycline and chloramphenicol, following 5 days of growth in the presence 1µg/ml ampicillin under (A) aerobic or (B) anaerobic growth conditions. See also Figure S1.

Figure 3. Ampicillin treatment of E. coli results in heterogeneous increases in MIC for ampicillin and norfloxacin

(A–B) Shown are the distributions of (A) ampicillin or (B) norfloxacin MICs for 44 ampicillin-treated isolates. The maximum growth inhibitory concentration tested for norfloxacin was 1000ng/ml, and the MICs for these isolates may be ≥ 1000ng/ml.

Figure 4. Ampicillin treatment leads to the formation of norfloxacin-resistant isolates with mutations in gyrA, gyrB or the acrAB promoter (P_acrAB) and kanamycin-resistant isolates with mutations in rpsL or arcA

(A–B) Isolates with point mutation resulting in (A) a D82G or D87Y substitution in GyrA, or (B) a S464F substitution in GyrB. (C) T-to-A DNA base pair mutation in the AcrR/EnvR binding site of the -35 region of P_acrAB. P_acrAB is partially annotated to show the -10 and -35 regions (bold), the transcription start site (capitalized A) and the AcrR/EnvR binding site (underlined). (D) Isolates with insertion between basepair 92 and 93 (K58) and between basepair 78 and 79 (K62) resulting in truncation of RpsL. (E) Isolate with a single basepair insertion between basepair 211 and 212 resulting in a truncated ArcA protein missing the majority of the helix-turn-helix (HTH) DNA binding domain.