We describe swarming in Pseudomonas aeruginosa as a third mode of surface translocation in addition to the previously described swimming and twitching motilities. Swarming in P. aeruginosa is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids. Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming. Cells from the edge of the swarm were about twice as long as cells from the swarm center. In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy. While a fliC mutant of P. aeruginosa displayed slightly diminished swarming, a pilR and a pilA mutant, both deficient in type IV pili, were unable to swarm. Furthermore, cells with mutations in the las cell-to-cell signaling system showed diminished swarming behavior, while rhl mutants were completely unable to swarm. Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of P. aeruginosa in this type of surface motility.Pseudomonas aeruginosa is a gram-negative bacterium living in soil and aqueous environments, where it survives due to its extraordinary metabolic abilities. P. aeruginosa is also a typical opportunistic pathogen which colonizes the lungs of cystic fibrosis patients and causes severe infections in immunocompromised hosts. Due to its notorious elevated intrinsic resistance to antimicrobial agents and its ability to attach to and to form biofilms on medical devices (9), P. aeruginosa is difficult to eradicate in the hospital environment.P. aeruginosa has a single polar flagellum which enables the cell to swim in aqueous environments and in low-agar (Ͻ0.4%) medium. The flagellum and the chemotaxis system, consisting of chemoreceptors (11, 49) and a signal relay system similar to that of Escherichia coli (25,31), allow the bacterium to respond to attractants and repellents. In addition, P. aeruginosa is able to propagate at surface interfaces by twitching motility, which is mediated by type IV pili (5,12,53). Twitching motility is believed to result from the extension and retraction of the pilus filament, which propels the cells across a surface. Pilus synthesis and assembly require at least 40 genes which are located in several unlinked regions on the chromosome (22). The nature of the environmental signal that triggers the expression of pili is not known. Pili are important for attachment to epithelial cells (8, 17) and contribute to the virulence of P. aeruginosa in animal models (19,50,51). Furthermore, twitching motility and, hence, type IV pili are required for the formation of biofilms on abiotic surfaces (38).Besides swimming and twitching, several gram-negative bacteria are able to propagate on semisolid surfaces (i.e., 0.4 to 1.0% agar) in a coordinated manner by swarming ...

Antibiotic-resistant mutants of Pseudomonas aeruginosa were generated using chloramphenicol and ciprofloxacin as selective agents. These mutants displayed a multidrug phenotype and overexpressed an outer membrane protein of 50 kDa, which was shown by Western blot analysis to correspond to OprN. A cosmid clone harbouring the oprN gene was isolated by partial complementation of a mutant deficient in OprM, the outer membrane component of the mexAB-oprM efflux operon. Antibiotic-accumulation studies indicated that OprN was part of an energy-dependent antibiotic-efflux system. Sequencing of a 6180bp fragment from the complementing cosmid revealed the presence of three open reading frames (ORFs), which exhibited amino acid similarity to the components of the mexAB-oprM and mexCD-oprJ efflux operons of P. aeruginosa. The ORFs were designated MexE, MexF and OprN. Mutation of the mexE gene eliminated the multidrug-resistance phenotype in an OprN-overexpressing strain, but did not affect the susceptibility profile of the wild-type strain. Expression of the mexEF-oprN operon was shown to be positively regulated by a protein encoded on a 1.5 kb DNA fragment located upstream of mexE and belonging to the LysR family of transcriptional activators. The presence of a plasmid containing this DNA fragment was sufficient to confer a multidrug phenotype onto the wild-type strain but not onto the mexE mutant. Evidence is provided to show that the mexEF-oprN operon may be involved in the excretion of intermediates for the biosynthesis of pyocyanin, a typical secondary metabolite of P. aeruginosa.

Intrinsic and acquired antibiotic resistance of the nosocomial pathogen Pseudomonas aeruginosa is mediated mainly by the expression of several efflux pumps of broad substrate specificity. Here we report that nfxC type mutants, overexpressing the MexEF-OprN efflux system, produce lower levels of extracellular virulence factors than the susceptible wild type. These include pyocyanin, elastase, and rhamnolipids, three factors controlled by the las and rhl quorum-sensing systems of P. aeruginosa. In agreement with these observations are the decreased transcription of the elastase gene lasB and the rhamnosyltransferase genes rhlAB measured in nfxC type mutants. Expression of the lasR and rhlR regulator genes was not affected in the nfxC type mutant. In contrast, transcription of the C4-homoserine lactone (C4-HSL) autoinducer synthase gene rhlI was reduced by 50% in the nfxC type mutant relative to that in the wild type. This correlates with a similar decrease in C4-HSL levels detected in supernatants of the nfxC type mutant. Transcription of an rhlAB-lacZ fusion could be partially restored by the addition of synthetic C4-HSL and Pseudomonas quinolone signal (PQS). It is proposed that the MexEF-OprN efflux pump affects intracellular PQS levels.Pseudomonas aeruginosa is an opportunistic pathogen which may cause pneumonia and bacteremia in immunocompromised hosts and is responsible for chronic destructive lung disease in patients suffering from cystic fibrosis. The pathogenicity of P. aeruginosa is attributable to an arsenal of virulence factors, some of which are cell associated (pili, nonpilus adhesins, lipopolysaccharide, and alginate) while others are secreted (proteases, rhamnolipids, exotoxin A, exoenzyme S, and pyocyanin). The production of many of these extracellular virulence factors is controlled by two cell-to-cell signaling systems, called las and rhl, which are both composed of a transcriptional regulator (LasR and RhlR, respectively) and an autoinducer synthase (LasI and RhlI, respectively). LasI and RhlI catalyze the last step in the synthesis of the cell-to-cell signaling molecules 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL) and C4-HSL, respectively; each of these molecules binds to, and activates, its corresponding transcriptional regulator. The systems are connected via a hierarchical cascade (19) and allow coordinated production of extracellular virulence factors, which occurs only when the bacterial cell density has reached a threshold (quorum). Recently, a novel signaling molecule, called PQS, for Pseudomonas quinolone signal (39), has been identified. Furthermore, the published genome sequence of PAO1 (53) has revealed a new modulator of cellto-cell signaling, termed QscR (4). This protein is homologous to both LasR and RhlR and seems to prevent premature transcription of quorum-sensing regulated genes.Besides its pathogenic capabilities, P. aeruginosa is well known for its intrinsic resistance to a wide range of antimicrobial agents and its ability to develop multidrug resistance following antibi...

We report that 2 g of azithromycin/ml inhibits the quorum-sensing circuitry of Pseudomonas aeruginosa strain PAO1. Addition of synthetic autoinducers partially restored the expression of the trancriptional activator-encoding genes lasR and rhlR but not that of the autoinducer synthase-encoding gene lasI. We propose that azithromycin interferes with the synthesis of autoinducers, by an unknown mechanism, leading to a reduction of virulence factor production.

During a 6-month period, 21 pairs of Pseudomonas aeruginosa isolates susceptible (pretherapy) and resistant (posttherapy) to antipseudomonal ␤-lactam antibiotics were isolated from hospitalized patients. In vivo emergence of ␤-lactam resistance was associated with the overexpression of AmpC ␤-lactamase in 10 patients. In the other 11 patients, the posttherapy isolates produced only low, basal levels of ␤-lactamase and had increased levels of resistance to a variety of non-␤-lactam antibiotics (e.g., quinolones, tetracyclines, and trimethoprim) compared with the levels of ␤-lactamase production and resistance of their pretherapy counterparts. These data suggested the involvement of the MexA-MexB-OprM active efflux system in the multidrug resistance phenotype of the posttherapy strains. Immunoblotting of the outer membrane proteins of these 11 bacterial pairs with a specific polyclonal antibody raised against OprM demonstrated the overexpression of OprM in all the posttherapy isolates. To determine whether mutations in mexR, the regulator gene of the mexA-mexB-oprM efflux operon, could account for the overproduction of the efflux system, sequencing experiments were carried out with the 11 bacterial pairs. Eight posttherapy isolates were found to contain insertions or deletions that led to frameshifts in the coding sequences of mexR. Two resistant strains had point mutations in mexR that yielded single amino acid changes in the protein MexR, while another strain did not show any mutation in mexR or in the promoter region upstream of mexR. Introduction of a plasmid-encoded wild-type mexR gene into five posttherapy isolates partially restored the susceptibility of the bacteria to selected antibiotics. These results indicate that in the course of antimicrobial therapy multidrug-resistant active efflux mutants overexpressing the MexA-MexB-OprM system may emerge as a result of mutations in the mexR gene.Bacterial strains and culture conditions. Clinical strains of P. aeruginosa were recovered from urine (n ϭ 3), transtracheal aspirate (n ϭ 4), or stool (n ϭ 1) specimens or from surgical wounds (n ϭ 3) from patients hospitalized at the University Hospital of Besançon, Besançon, France, during a 6-month survey in 1996. The isolates were identified by conventional methods (2). The other P. aeruginosa strains used in the study were PAO1 (a wild-type strain obtained from B. W. Holloway), 4098 (a mutant of PAO1 producing basal, noninducible levels of AmpC ␤-lactamase) (15), 4098E (a single step-mutant of 4098 overexpressing the MexA-MexB-OprM efflux system) (16), 4098ET (an oprM::⍀-Hg transductant of 4098E) (22), PT75 (a mutant of 4098ET overproducing the MexE-MexF-OprN efflux system) (12), and ERYRT (a mutant of oprM::⍀-Hg PAO1 overexpressing the MexC-MexD-OprJ pump system) (22). Escherichia coli INF␣ was used as a host for DNA cloning and plasmid preparation. All bacterial strains were routinely cultured at 37°C on Mueller-Hinton agar medium (Becton Dickinson) or grown in brain heart infusion broth (Difco Laboratories), unle...

We investigated the regulation of the MexEF-OprN multidrug efflux system of Pseudomonas aeruginosa, which is overexpressed innfxC-type mutants and confers resistance to quinolones, chloramphenicol and trimethoprim. Sequencing of the DNA region upstream of the mexEF-oprN operon revealed the presence of an open reading frame (ORF) of 304 amino acids encoding a LysR-type transcriptional activator, termed MexT. By using T7-polymerase, a 34-kDa protein was expressed in Escherichia coli from a plasmid carrying the mexT gene. Expression of amexE::lacZ fusion was 10-fold higher in nfxC-type mutants than in the wild-type strain; however, transcription of mexT as well as the mexT DNA region was unchanged. Located adjacent to mexT but transcribed in opposite direction, the beginning of an ORF termedqrh (quinone oxidoreductase homologue) was identified. Expression of a qrh::lacZ fusion was also found to be activated by MexT. Further, we present evidence for coregulation at the transcriptional and the posttranscriptional level between the MexEF-OprN efflux system and the OprD porin responsible for cross-resistance of nfxC-type mutants to carbapenem antibiotics.

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