Cross-resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps: exposure of a susceptible mutant strain to triclosan selects nfxB mutants overexpressing MexCD-OprJ

Abstract

Triclosan is an antiseptic frequently added to items as diverse as soaps, lotions, toothpaste, and many commonly used household fabrics and plastics. Although wild-type Pseudomonas aeruginosa expresses the triclosan target enoyl-acyl carrier protein reductase, it is triclosan resistant due to expression of the MexAB-OprM efflux system. Exposure of a susceptible Delta(mexAB-oprM) strain to triclosan selected multidrug-resistant bacteria at high frequencies. These bacteria hyperexpressed the MexCD-OprJ efflux system due to mutations in its regulatory gene, nfxB. The MICs of several drugs for these mutants were increased up to 500-fold, including the MIC of ciprofloxacin, which was increased 94-fold. Whereas the MexEF-OprN efflux system also participated in triclosan efflux, this antimicrobial was not a substrate for MexXY-OprM.

FIG. 1

Western blots of P. aeruginosa cell lysates and mutations causing triclosan resistance. (A) Standardized amounts of whole-cell lysates were separated on a 0.1% SDS–10% PAGE gel and electroblotted on nitrocellulose membranes, and the membranes were probed with monoclonal antibodies against OprJ and OprN. The strains analyzed were PAO1 OprM⁺; KG3056 OprJ⁺; PAO7H OprN⁺; PAO200, an OprM null PAO1 mutant (Δ[mexAB-oprM]); and PAO200-2, PAO200-3, and PAO200-4, spontaneous triclosan-resistant nfxB derivatives of PAO200. (B) Mutations leading to triclosan resistance. The nfxB genes from PAO200 and its three triclosan-resistant derivatives, PAO200-2, PAO200-3, and PAO200-4, were amplified by PCR from genomic DNA templates and sequenced. The nfxB sequence from each strain shown is the consensus obtained from six separate sequencing reactions; it was determined in duplicate from two separate clones, as well as in duplicate by directly sequencing the PCR products. Only portions of the nfxB sequence are shown, and codons are numbered as previously described (24). Arrows, changes from the PAO200 sequence. Amino acid residues constituting the putative helix-turn-helix DNA binding domain of NfxB are bracketed.