RamA confers multidrug resistance in Salmonella enterica via increased expression of acrB, which is inhibited by chlorpromazine

Abstract

Salmonella enterica serovar Typhimurium SL1344, in which efflux pump genes (acrB, acrD, acrF, tolC) or regulatory genes thereof (marA, soxS, ramA) were inactivated, was grown in the presence of 240 antimicrobial and nonantimicrobial agents in the Biolog Phenotype MicroArray. Mutants lacking tolC, acrB, and ramA grew significantly worse than other mutants in the presence of 48 agents (some of which have not previously been identified as substrates of AcrAB-TolC) and particularly poorly in the presence of phenothiazines, which are human antipsychotics. MIC testing revealed that the phenothiazine chlorpromazine had antimicrobial activity and synergized with common antibiotics against different Salmonella serovars and SL1344. Chlorpromazine increased the intracellular accumulation of ethidium bromide, which was ablated in mutants lacking acrB, suggesting an interaction with AcrB. High-level but not low-level overexpression of ramA increased the expression of acrB; conferred resistance to chloramphenicol, tetracycline, nalidixic acid, and triclosan and organic solvent tolerance; and increased the amount of ethidium bromide accumulated. Chlorpromazine induced the modest overproduction of ramA but repressed acrB. These data suggest that phenothiazines are not efflux pump inhibitors but influence gene expression, including that of acrB, which confers the synergy with antimicrobials observed.

FIG. 1.

PM growth data showing clustering of the strains which grew better (enclosed by black lines) or more poorly (enclosed by dashed lines). The dotted line indicates the mean growth of these four strains (group A) compared with of SL1344 in the presence different compounds; ⋄, L561 (ΔacrF); ▪, L130 (marA::aph); ▴, L133 (ramA::aph); ×, L106 (acrD::aph).

FIG. 2.

Accumulation of ethidium bromide in the presence of chlorpromazine at 200 μg/ml (unfilled bars) and in the absence of chlorpromazine (gray bars). *, statistically significant increase in accumulation in the presence of chlorpromazine (P < 0.05). All units are arbitrary (arb.).

FIG. 3.

Efflux of ethidium bromide from Salmonella serovar Typhimurium SL1344 in the presence and absence of chlorpromazine (CPZ) at 200 μg/ml. ▴, SL1344; ▪, SL1344 plus chlorpromazine at 200 μg/ml. Fluorescence units are arbitrary (arb.).