Combined exposure to non-antibiotic pharmaceutics and antibiotics in the gut synergistically promote the development of multi-drug-resistance in Escherichia coli

Abstract

The gut microbiota represents an important reservoir of antibiotic-resistant bacteria (ARB), which poses a significant threat to public health. However, little is known about the emergence of ARB in the gut after the combined exposure to antibiotics and non-antibiotic pharmaceutics. Here, Escherichia coli , a common opportunistic pathogen in the gut microbiota, was exposed to the antidepressant duloxetine (2.5 µg/L–25 mg/L) and/or chloramphenicol (6 µg/L–4 mg/L). The resistant strains were isolated to determine the minimum inhibition concentration (MIC) of 29 antibiotics. Then, genome-wide DNA sequencing, global transcriptomic sequencing, and real-time quantitative polymerase chain reaction were performed to quantify the synergy between duloxetine and chloramphenicol. Combined exposure synergistically increased the mutation frequency of chloramphenicol resistance by 2.45–9.01 fold compared with the independent exposure. A combination index reaching 187.7 indicated strong duloxetine and chloramphenicol synergy. The resultant mutants presented heritable enhanced resistance to 12 antibiotics and became ARB to eight antibiotics. Furthermore, combined exposure significantly increased the transcriptomic expression of acrA, acrB , and marA in E. coli , and generated a more robust oxidative stress response. Together with the occurrence of DNA mutations in marR in the mutants, stronger triggers to the AcrAB-TolC transport system and the MlaFEDB ABC transporter via reactive oxygen species (ROS)-induced mutagenesis, verified by gene knockout, contributed to the synergistic enhancement of antibiotic resistance in the combined exposure group. Regardless of whether their formation was induced by duloxetine, chloramphenicol, or their combination, the E. coli mutants showed 1.1–1.7-fold increases in the expression levels of acrA, acrB, acrZ, mdtE , and mdtF . This pattern indicated that the mutants shared the same resistance mechanisms against chloramphenicol, involving the improved efflux pumps AcrAB-TolC and mdtEF . Our findings demonstrated that antibiotics and non-antibiotic pharmaceutics synergistically accelerate the evolution of ARB and may enhance their spread.

Simulated Gastric Acid Promotes the Horizontal Transfer of Multidrug Resistance Genes across Bacteria in the Gastrointestinal Tract at Elevated pH Levels

“…Previous studies have revealed that nonantibiotic pharmaceuticals, including fluoxetine and duloxetine, could induce multidrug resistance in E. coli through mutagenesis ( 15 , 16 ). To assess the effects of drug consumption on the horizontal transfer of ARGs in SGF, we used four antidepressants (fluoxetine, amitriptyline, duloxetine, and sertraline) and assessed the conjugative transfer of ARGs between E. coli HB101 and Salmonella Aberdeen in SGF at pH 4.5 ( Fig.…”

“… E. coli HB101 containing the RP4 plasmid (60,099 bp) and exhibiting Amp, Kan, and Tet resistance was chosen as the donor. E. coli K-12 exhibiting Chl resistance, obtained from our previous mutation study ( 16 ), and Salmonella Aberdeen exhibiting Str resistance, purchased from the American Type Culture Collection (ATCC 50312), were chosen as the recipients. E. coli HB101 was cultured in Luria-Bertani (LB) medium (Oxoid, UK) containing 100 mg/L Amp, 60 mg/L Kan, and 40 mg/L Tet.…”

Anti-infective Efficacy of Duloxetine against Catheter-Associated Urinary Tract Infections Caused by Gram-Positive Bacteria

“…aeruginosa, E. coli, Klebsiella pneumoniae, and Clostridium difficile. , These studies have promoted the importance of drug repurposing for various new applications.…”

TCA and SSRI Antidepressants Exert Selection Pressure for Efflux-Dependent Antibiotic Resistance Mechanisms in Escherichia coli

“…We also showed that spontaneously resistant mutants to chloramphenicol were simultaneously resistant to antidepressants due to selection for AcrAB-TolC activity. Recently, the selective serotonin and norepinephrine reuptake inhibitor, duloxetine, and chloramphenicol were reported to impose a selection pressure for marR mutations in E. coli ( 83 ). Thus, there is cross-resistance between antidepressants and antibiotics that may be more wide-spread and suggests that treatment by one class of drug could influence the efficacy of treatment by the other class of drug due to the changes in microbial diversity and associated resistance mechanisms.…”