Overexpression of RamA, Which Regulates Production of the Multidrug Resistance Efflux Pump AcrAB-TolC, Increases Mutation Rate and Influences Drug Resistance Phenotype.

In Enterobacteriales, the AcrAB-TolC efflux pump exports substrates, including antimicrobials, from the cell. Overexpression of AcrAB-TolC can occur after exposure to fluoroquinolones, leading to multidrug resistance. The expression of AcrAB-TolC in Salmonella is primarily regulated by the transcriptional activator RamA. However, other transcriptional activators, such as MarA, SoxRS, and Rob, can influence AcrAB-TolC expression. This study determined whether the overproduction or absence of RamA influences the mutation rate or the phenotype of mutants selected in Salmonella enterica serovar Typhimurium SL1344 after ciprofloxacin exposure. The absence of RamA (SL1344 ramA::aph) resulted in mutation frequencies/rates similar to those of wild-type Salmonella Typhimurium SL1344. However, the overproduction of RamA (SL1344 ramR::aph) and, consequently, AcrB resulted in a significantly higher mutation frequency and rate than for wild-type Salmonella Typhimurium SL1344. Whole-genome sequencing revealed that in addition to selecting gyrA mutants resistant to quinolones, SL1344 and SL1344 ramA::aph also produced multidrug-resistant (MDR) mutants, associated with mutations in soxR Conversely, mutations in SL1344 ramR::aph occurred in gyrA only. Although transcriptional regulators such as SoxRS are believed to play a minor role in AcrAB-TolC regulation under antibiotic selective pressure, we show that soxR mutants can be selected after exposure to ciprofloxacin, including when RamA is absent. This demonstrates that under selective pressure, Salmonella can respond to increased efflux pump expression by mutating other AcrAB-TolC regulatory genes, allowing for the evolution of MDR. Understanding how Salmonella responds to antibiotic pressure in the absence/overproduction of RamA is important if targeting transcriptional regulators to alter efflux is to be considered an avenue for future drug discovery.

Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump.

Efflux is an important mechanism in Gram-negative bacteria conferring multidrug resistance. Inhibition of efflux is an encouraging strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their mode of action is poorly understood. Exposure of Salmonella enterica serovar Typhimurium and Escherichia coli to chlorpromazine selected for mutations within genes encoding RamR and MarR, regulators of the multidrug tripartite efflux pump AcrAB-TolC. Further experiments with S. Typhimurium containing AcrB D408A (a nonfunctional efflux pump) and chlorpromazine or amitriptyline resulted in the reversion of the mutant acrB allele to the wild type. Together, this suggests these drugs are AcrB efflux substrates. Subsequent docking studies with AcrB from S. Typhimurium and E. coli, followed by molecular dynamics simulations and free energy calculations showed that chlorpromazine and amitriptyline bind at the hydrophobic trap, a preferred binding site for substrates and inhibitors within the distal binding pocket of AcrB. Based on these simulations, we suggest that chlorpromazine and amitriptyline inhibit AcrB-mediated efflux by interfering with substrate binding. Our findings provide evidence that these drugs are substrates and inhibitors of AcrB, yielding molecular details of their mechanism of action and informing drug discovery of new efflux inhibitors.IMPORTANCE Efflux pumps of the resistance nodulation-cell division (RND) superfamily are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; however, several issues have thus far hindered all efforts at designing new efflux inhibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibitors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds.

Do phenothiazines possess antimicrobial and efflux inhibitory properties?

Antibiotic resistance is a global health concern; the rise of drug-resistant bacterial infections is compromising the medical advances that resulted from the introduction of antibiotics at the beginning of the 20th century. Considering that the presence of mutations within individuals in a bacterial population may allow a subsection to survive and propagate in response to selective pressure, as long as antibiotics are used in the treatment of bacterial infections, development of resistance is an inevitable evolutionary outcome. This, combined with the lack of novel antibiotics being released to the clinical market, means the need to develop alternative strategies to treat these resistant infections is critical. We discuss how the use of antibiotic adjuvants can minimise the appearance and impact of resistance. To this effect, several phenothiazine-derived drugs have been shown to potentiate the activities of antibiotics used to treat infections caused by Gram-positive and Gram-negative bacteria. Outside of their role as antipsychotic medications, we review the evidence to suggest that phenothiazines possess inherent antibacterial and efflux inhibitory properties enabling them to potentially combat drug resistance. We also discuss that understanding their mode of action is essential to facilitate the design of new phenothiazine derivatives or novel agents for use as antibiotic adjuvants.