Genetic inactivation of acrAB or inhibition of efflux induces expression of ramA

Abstract

Objectives: The transcriptional activator RamA regulates production of the multidrug resistance efflux AcrAB-TolC system in several Enterobacteriaceae. This study investigated factors that lead to increased expression of ramA.

Methods: In order to monitor changes in ramA expression, the promoter region of ramA was fused to a gfp gene encoding an unstable green fluorescence protein (GFP) on the reporter plasmid, pMW82. The ramA reporter plasmid was transformed into Salmonella Typhimurium SL1344 and a ΔacrB mutant. The response of the reporter to subinhibitory concentrations of antibiotics, dyes, biocides, psychotropic agents and efflux inhibitors was measured during growth over a 5 h time period.

Results: Our data revealed that the expression of ramA was increased in a ΔacrB mutant and also in the presence of the efflux inhibitors phenylalanine-arginine-β-naphthylamide, carbonyl cyanide m-chlorophenylhydrazone and 1-(1-naphthylmethyl)-piperazine. The phenothiazines chlorpromazine and thioridazine also increased ramA expression, triggering the greatest increase in GFP expression. However, inducers of Escherichia coli marA and soxS and 12 of 17 tested antibiotic substrates of AcrAB-TolC did not induce ramA expression.

Conclusions: This study shows that expression of ramA is not induced by most substrates of the AcrAB-TolC efflux system, but is increased by mutational inactivation of acrB or when efflux is inhibited.

Keywords: antibiotic resistance; efflux inhibitors; phenothiazines.

Figure 1.

Induction of ramA expression by deletion of acrB or addition of chlorpromazine. Fold change in fluorescence in SL1344 ΔacrB and SL1344 in the presence of 100 mg/L chlorpromazine relative to SL1344. Fold change was calculated from the maximum fluorescence value achieved for SL1344 after 5 h of exposure relative to SL1344 ΔacrB and SL1344 in the presence of 100 mg/L chlorpromazine at the same point. An asterisk indicates a statistically significant difference (P < 0.05). CPZ, chlorpromazine.

Figure 2.

Flow cytometry of strain L1232 in the presence and absence of chlorpromazine. (a) Fold change in geometric mean of fluorescence of strain L1232. An asterisk indicates a statistically significant difference (P < 0.05) between values for L1232 and L1232 in the presence of 50 mg/L chlorpromazine after 2 h of exposure. (b) A representative histogram showing the gfp fluorescence of L1232 (unfilled) and L1232 in the presence of chlorpromazine (filled). CPZ, chlorpromazine.

Figure 3.

Exposure of strain L1232 to efflux pump inhibitors, psychoactive drugs and substrates of AcrAB–TolC. Fold change in fluorescence after 5 h of exposure after the addition of: (a) CCCP, PAβN, NMP, BM-38 and the control value at the same timepoint; (b) chlorpromazine (CPZ), trifluoperazine (TRIF), orphenadrine (ORP) amitriptyline (AMI), thioridazine (THIO), sertraline (SERT), haloperidol (HAL) and the control value at the same timepoint; and (c) tetracycline (TET), minocycline (MIN), chloramphenicol (CHL), norfloxacin (NOR), ciprofloxacin (CIP), nalidixic acid (NAL), levofloxacin (LVX), trimethoprim (TMP), erythromycin (ERY), cloxacillin (CL), nafcillin (NAF), carbenicillin (CAR), cefamandole (FAM), rifampicin (RIF), doxorubicin (DXR), linezolid (LZD), novobiocin (NOV), SDS and the control value at the same timepoint. Concentrations of each compound are stated in the Results section and Table S1. An asterisk indicates a statistically significant difference (P < 0.05) between values for L1232 and L1232 in the presence of compound.