Effect of the efflux inhibitors 1-(1-naphthylmethyl)-piperazine and phenyl-arginine-beta-naphthylamide on antimicrobial susceptibility and virulence factor production in Vibrio cholerae.

OBJECTIVES: The aim of the study was to test the hypothesis that the efflux pump inhibitors (EPIs) 1-(1-naphthylmethyl)-piperazine (NMP) and phenyl-arginine-beta-naphthylamide (PAbetaN) can inhibit the Vibrio cholerae resistance-nodulation-division (RND) family efflux systems, and thereby render V. cholerae susceptible to antimicrobial agents and inhibit the production of the virulence factors cholera toxin (CT) and the toxin coregulated pilus (TCP).
METHODS: The susceptibility of V. cholerae to antimicrobial compounds was determined in the presence or absence of NMP and PAbetaN. Transcriptional reporters were used to assess the effects of NMP and PAbetaN on the expression of the genes encoding the virulence factor regulators TcpP and ToxT, whereas CT and TCP production were determined by ELISA using GM1 ganglioside-coated microtitre plates and TcpA Western immunoblotting, respectively.
RESULTS: NMP and PAbetaN potentiated antimicrobial compounds that were substrates for the V. cholerae RND efflux systems. PAbetaN exhibited complete inhibition of the RND efflux systems for Triton X-100 and deoxycholate, but partial inhibition of the efflux systems for cholate and erythromycin. NMP exhibited partial inhibition for all compounds tested except for SDS. The presence of NMP reduced the MIC of SDS to a level that was lower than that observed in an RND efflux-deficient strain, whereas the SDS MIC was unaffected by the presence of PAbetaN. Neither EPI potentiated polymyxin B, penicillin, ampicillin or chloramphenicol. Both NMP and PAbetaN inhibited the production of CT and the TCP and appeared to have additional virulence gene repressing activity independent of RND efflux inhibition.
CONCLUSIONS: RND efflux inhibitors represent potential novel therapeutics for the treatment of cholera.