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One of the hallmarks of Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients is very-high-celldensity (HCD) replication in the lung, allowing this bacterium to induce virulence controlled by the quorumsensing systems. However, the nutrient sources sustaining HCD replication in this chronic infection are largely unknown. Here, we performed microarray studies of P. aeruginosa directly isolated from the lungs of CF patients to demonstrate its metabolic capability and virulence in vivo. In vivo microarray data, confirmed by real-time reverse transcription-PCR, indicated that the P. aeruginosa population expressed several genes for virulence, drug resistance, and utilization of multiple nutrient sources (lung surfactant lipids and amino acids) contributing to HCD replication. The most abundant lung surfactant lipid molecule, phosphatidylcholine (PC), induces key genes of P. aeruginosa pertinent to PC degradation in vitro as well as in vivo within the lungs of CF patients. The results support recent research indicating that P. aeruginosa exists in the lungs of CF patients as a diverse population with full virulence potential. The data also indicate that there is deregulation of several pathways, suggesting that there is in vivo evolution by deregulation of a large portion of the transcriptome during chronic infection in CF patients. To our knowledge, this is the first in vivo transcriptome analysis of P. aeruginosa in a natural infection in CF patients, and the results indicate several important aspects of P. aeruginosa pathogenesis, drug resistance, nutrient utilization, and general metabolism within the lungs of CF patients.Pseudomonas aeruginosa is the major cause of morbidity and mortality in lung diseases, including cystic fibrosis (CF) (6, 11, 32) and nosocomial pneumonia (3,40). Over 93% of CF patients between 18 and 24 years old have been reported to have P. aeruginosa infections (11). In addition, nosocomial pneumonia is the second most common of all nosocomial infections, and P. aeruginosa was the most frequently isolated microbe involved from 1992 to 1997 (37). The pathogenesis of this organism has been intensively studied with respect to virulence and virulence expression (5,26,31,36,39), biofilm production (10, 43), and quorum sensing (15,27,28,30). Several virulence factors that P. aeruginosa expresses (e.g., exotoxin A, exoenzyme S, cytotoxin, proteases, lipases, phospholipases, alginate, and hydrogen cyanide) all contribute to severe lung damage. High-cell-density (HCD) replication is necessary for many of these events to occur, and Ͼ10 9 bacteria/ml of sputum have been found in the lungs of CF patients (43, 46). The ability of P. aeruginosa to obtain nutrients in the lung for HCD replication and maintenance is the quintessential factor leading to quorum-sensing-induced virulence expression, which is a hallmark of chronic lung infections in CF patients. However, the nutrient requirements of P. aeruginosa in vivo are unknown. A recent in vitro study by Palmer et al. (25), in which P. aerugi...

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 (...

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