Antidepressant fluoxetine induces multiple antibiotics resistance in Escherichia coli via ROS-mediated mutagenesis

Antibiotic resistance poses a great threat to global public health. Overuse of antibiotics is generally considered as the major factor contributing to it. However, little is known about whether non-antibiotic drugs could play potential roles in the emergence of antibiotic resistance.We aimed to investigate whether antidepressant fluoxetine induces multiple antibiotic resistances and reveal underlying mechanisms.Escherichia coli K12 was exposed to different concentrations of fluoxetine (0, 0.5, 5, 50 and 100 mg/L) and the resistant strains were isolated by plating on antibiotic containing plates. Resistant strains were randomly selected to determine the increase of minimum inhibition concentration (MIC) of multiple antibiotics. Genome-wide DNA sequencing was performed on cells cultured in lysogeny broth (LB) without any fluoxetine or antibiotics exposure. RNA sequencing and proteomic profiling of isolated mutants grown in LB with 100 mg/L fluoxetine were analyzed to reveal the underlying mechanisms.Exposure of Escherichia coli to fluoxetine at 5-100 mg/L after repeated subculture in LB for 30 days promoted its mutation frequency resulting in increased resistance against the antibiotics chloramphenicol, amoxicillin and tetracycline. This increase was up to 5.0 × 107 fold in a dose-time pattern. Isolated mutants with resistance to one of these antibiotics also exhibited multiple resistances against fluoroquinolone, aminoglycoside, β-lactams, tetracycline and chloramphenicol. According to global transcriptional and proteomic analyses, the AcrAB-TolC pump together with the YadG/YadH transporter, a Tsx channel and the MdtEF-TolC pump have been triggered to export the antibiotics to the exterior of the cell. Whole-genome DNA analysis of the mutants further revealed that ROS-mediated mutagenesis (e.g., deletion, insertion, and substitution) of DNA-binding transcriptional regulators (e.g., marR, rob, sdiA, cytR and crp) to up-regulate the expression of efflux pumps, may further enhance the antibiotic efflux.Our findings for the first time demonstrated that the exposure to antidepressant fluoxetine induces multiple antibiotic resistance in E. coli via the ROS-mediated mutagenesis.