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 MexABOprM efflux system. Exposure of a susceptible ⌬(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.Pseudomonas aeruginosa is a clinically significant pathogen, particularly in immunocompromised hosts (36). Infections caused by this bacterium are difficult to treat due to its many intrinsic and acquired antibiotic resistances. Intrinsic resistance is mostly attributable to the expression of several multidrug resistance (MDR) efflux systems. The P. aeruginosa genome (35) contains structural genes for at least 12 resistance nodulation type efflux systems, of which only 4, i.e., MexABOprM (27), MexCD-OprJ (26), MexEF-OprN (13), and MexXY (1, 21, 38), have been characterized. Exposure to selected substrates can select for their upregulated or constitutive expression (13,14,26,38).2-Hydroxyphenylethers are a class of compounds that exhibit broad-spectrum antimicrobial activity. Triclosan is the most potent and widely used member of this class (2, 5) and is used in hand soaps, lotions, toothpastes, and oral rinses, as well as in fabrics and plastics. It was long thought to act as a nonspecific "biocide" (29), but recent biochemical and genetic studies have shown that triclosan acts on a defined bacterial target in the fatty acid biosynthetic pathway, enoyl-acyl carrier protein (ACP) reductase (FabI) (7,9,10,12,18,20) or its homolog InhA in mycobacteria (18). Some bacteria possess triclosan-resistant enoyl-ACP reductase homologs (FabK), and to date P. aeruginosa is unique among gram-negative bacteria in that it possesses both triclosan-sensitive and -resistant enzymes (8). Alterations in FabI active-site residues confer resistance to triclosan (9,10,20). Of particular concern is that such amino acid changes selected by exposure to triclosan lead to cross-resistance with other antimicrobial agents (9), including clinically used front-line drugs, since some mutations leading to triclosan resistance in Mycobacterium smegmatis also caused resistance to isoniazid (18). Moreover, triclosan is a substrate of a multidrug efflux pump in clinical and laboratory Escherichia coli strains (19). We have recently shown that P. aeruginosa strain PAO1 is intrinsically resistant to triclosan by virtue of expression of the MexAB-OprM efflux pump (32), and the same is true for all strains of this species tested to date (...

The genomes of the two clonally derived Pseudomonas aeruginosa prototypic strains PAO1 and DSM-1707 differ by the presence of a 2.19-Mb inversion including oriC. Integration of two Flp recombinase target sites near the rrn operons containing the inversion endpoints in PAO1 led to Flp-catalyzed inversion of the intervening 1.59-Mb fragment, including oriC, at high frequencies (83%), favoring the chromosome configuration found in DSM-1707. The results indicate that the oriC-containing region of the P. aeruginosa chromosome can readily undergo and tolerate large inversions.Pseudomonas aeruginosa is an opportunistic pathogen that can be found in diverse habitats. It causes a variety of acute infections and is also responsible for chronic life-threatening lung infections of cystic fibrosis (CF) patients (3,9,18). CF isolates are characterized by certain phenotypes, including rough lipopolysaccharide structure, mucoid phenotype, and loss of motility (3,9,17). Comparative genome mapping of Pseudomonas aeruginosa PAO with P. aeruginosa C, which belongs to a major clone found in CF patient infections and aquatic habitats, also revealed variations at the genomic level (13). CF isolates contained large chromosomal inversions, and the exclusive detection of inversions in isolates from the lungs of patients with CF, which represent atypical habitats for this bacterium, was cited as supporting the theory that features of this particular ecological niche may select, cause, or tolerate the observed genomic changes (10). This is what might have occurred between Escherichia coli and several closely related Salmonella species, including Salmonella enterica serovar Typhimurium (6). Since large chromosomal inversions could be constructed and stably maintained under laboratory conditions in E. coli (6), Salmonella serovar Typhimurium (8), and Bacillus subtilis (1), bacteria evidently have the inherent ability to tolerate and even select for gross chromosomal rearrangements. During construction of a ⌬(mexAB-oprM) ⌬(mexCDoprJ) chromosomal double mutant in the PAO1 background by using a Flp recombinase-based method (5), we observed that the intervening 1.59-Mb region containing oriC (Fig. 1A) underwent inversions at high frequencies and decided to further investigate this phenomenon.Deletion of the mexCD-oprJ operon from a ⌬(mexAB-oprM) strain. Strain PAO200 [⌬(mexAB-oprM)::FRT] was previously described (14). The ⌬(mexCD-oprJ)::Gm r -FRT strains PAO236 and PAO237 were derived from PAO200 in several steps. First, the mexC-mexD-oprJ genes were deleted from pKMJ002 (2) by digestion with ClaI, followed by religation to form pPS1088.One of the delimiting ClaI sites is located 156 bp upstream of the mexC operon start at the ATG codon of nfxB, and the second ClaI site is located 209 bp downstream of the oprJ termination codon. Second, the deleted 6,138-bp DNA segment was replaced by the gentamycin resistance (Gm r )-Flp recombinase target (FRT) cassette from pPS856 (5), followed by return of the deletion into the PAO200 chromosome by a previou...

The contribution of efflux pumps to multidrug resistance in 12 Pseudomonas aeruginosa isolates from various animal sources was assessed. Western immunoblot analyses demonstrated that all twelve isolates expressed significant levels of the MexAB^OprM efflux system whereas two isolates simultaneously expressed the MexEF^OprN or MexXY systems, respectively. One strain contained a single mutation in mexR, a regulator of mexAB^oprM expression, that did not adversely affect the MexR amino acid sequence, and three isolates contained the same, single base change in the mexA^mexR intergenic region. The MexXY-expressing strain contained two base substitutions in its mexZ regulatory gene which did not alter the MexR sequence. ß

The contribution of efflux pumps to multidrug resistance in 12 Pseudomonas aeruginosa isolates from various animal sources was assessed. Western immunoblot analyses demonstrated that all twelve isolates expressed significant levels of the MexAB OprM efflux system whereas two isolates simultaneously expressed the MexEF OprN or MexXY systems, respectively. One strain contained a single mutation in mexR, a regulator of mexAB-oprM expression, that did not adversely affect the MexR amino acid sequence, and three isolates contained the same, single base change in the mexA-mexR intergenic region. The MexXY-expressing strain contained two base substitutions in its mexZ regulatory gene which did not alter the MexR sequence.