Christian van Delden

Swarming of Pseudomonas aeruginosa Is Dependent on Cell-to-Cell Signaling and Requires Flagella and Pili

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.

Azithromycin Inhibits Quorum Sensing in Pseudomonas aeruginosa

ABSTRACT We report that 2 μg 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 geneslasR 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.

Overexpression of the MexEF-OprN Multidrug Efflux System Affects Cell-to-Cell Signaling in Pseudomonas 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 Virulence Analyzed in a Dictyostelium discoideum Host System

Pseudomonas aeruginosa is an important opportunistic pathogen that produces a variety of cell-associated and secreted virulence factors. P. aeruginosa infections are difficult to treat effectively because of the rapid emergence of antibiotic-resistant strains. In this study, we analyzed whether the amoeba Dictyostelium discoideum can be used as a simple model system to analyze the virulence of P. aeruginosa strains. The virulent wild-type strain PAO1 was shown to inhibit growth of D. discoideum. Isogenic mutants deficient in the las quorum-sensing system were almost as inhibitory as the wild type, while rhl quorum-sensing mutants permitted growth of Dictyostelium cells. Therefore, in this model system, factors controlled by the rhl quorum-sensing system were found to play a central role. Among these, rhamnolipids secreted by the wild-type strain PAO1 could induce fast lysis of D. discoideum cells. By using this simple model system, we predicted that certain antibiotic-resistant mutants of P. aeruginosa should show reduced virulence. This result was confirmed in a rat model of acute pneumonia. Thus, D. discoideum could be used as a simple nonmammalian host system to assess pathogenicity of P. aeruginosa.

Population structure of Pseudomonas aeruginosa

The metabolically versatile Gram-negative bacterium Pseudomonas aeruginosa inhabits terrestrial, aquatic, animal-, human-, and plant-host-associated environments and is an important causative agent of nosocomial infections, particularly in intensive-care units. The population genetics of P. aeruginosa was investigated by an approach that is generally applicable to the rapid, robust, and informative genotyping of bacteria. DNA, amplified from the bacterial colony by circles of multiplex primer extension, is hybridized onto a microarray to yield an electronically portable binary multimarker genotype that represents the core genome by single nucleotide polymorphisms and the accessory genome by markers of genomic islets and islands. The 240 typed P. aeruginosa strains of diverse habitats and geographic origin segregated into two large nonoverlapping clusters and 45 isolated clonal complexes with few or no partners. The majority of strains belonged to few dominant clones widespread in disease and environmental habitats. The most frequent genotype was represented by the sequenced strain PA14. Core and accessory genome were found to be nonrandomly assembled in P. aeruginosa. Individual clones preferred a specific repertoire of accessory segments. Even the most promiscuous genomic island, pKLC102, had integrated preferentially into a subset of clones. Moreover, some physically distant loci of the core genome, including oriC, showed nonrandom associations of genotypes, whereas other segments in between were freely recombining. Thus, the P. aeruginosa genome is made up of clone-typical segments in core and accessory genome and of blocks in the core with unrestricted gene flow in the population.

Effectiveness of Combination Antimicrobial Therapy for Pseudomonas aeruginosa Bacteremia

ABSTRACT It remains controversial whether combination therapy, given empirically or as definitive treatment, for Pseudomonas aeruginosa bacteremia is associated with a better outcome than monotherapy. The aim of the present study was to compare the rates of survival among patients who received either combination therapy or monotherapy for P. aeruginosa bacteremia. We assembled a historical cohort of 115 episodes of P. aeruginosa bacteremia treated with empirical antipseudomonal therapy between 1988 and 1998. On the basis of susceptibility testing of the bacteremic P. aeruginosa isolate, we defined categories of empirical treatment, including adequate combination therapy, adequate monotherapy, and inadequate therapy, as well as corresponding categories of definitive therapy. Neither the adequacy of the empirical treatment nor the use of combination therapy predicted survival until receipt of the antibiogram. However, the risk of death from the date of receipt of the antibiogram to day 30 was higher for both adequate empirical monotherapy (adjusted hazard ratio [aHR], 3.7; 95% confidence interval [CI], 1.0 to 14.1) and inadequate empirical therapy (aHR, 5.0; 95% CI, 1.2 to 20.4) than for adequate empirical combination therapy. Compared to adequate definitive combination therapy, the risk of death at 30 days was also higher with inadequate definitive therapy (aHR, 2.6; 95% CI, 1.1 to 6.7) but not with adequate definitive monotherapy (aHR, 0.70; 95% CI, 0.30 to 1.7). In this retrospective analysis the use of adequate combination antimicrobial therapy as empirical treatment until receipt of the antibiogram was associated with a better rate of survival at 30 days than the use of monotherapy. However, adequate combination antimicrobial therapy given as definitive treatment for P. aeruginosa bacteremia did not improve the rate of survival compared to that from the provision of adequate definitive monotherapy.

Stringent response activates quorum sensing and modulates cell density-dependent gene expression in Pseudomonas aeruginosa

During nutrient starvation, Escherichia coli elicits a stringent response involving the ribosome-associated protein RelA. Activation of RelA results in a global change in the cellular metabolism including enhanced expression of the stationary-phase sigma factor RpoS. In the human pathogen Pseudomonas aeruginosa, a complex quorum-sensing circuitry, linked to RpoS expression, is required for cell density-dependent production of many secreted virulence factors, including LasB elastase. Quorum sensing relies on the activation of specific transcriptional regulators (LasR and RhlR) by their corresponding autoinducers (3-oxo-C(12)-homoserine lactone [HSL] and C(4)-HSL), which function as intercellular signals. We found that overexpression of relA activated the expression of rpoS in P. aeruginosa and led to premature, cell density-independent LasB elastase production. We therefore investigated the effects of the stringent response on quorum sensing. Both lasR and rhlR gene expression and autoinducer synthesis were prematurely activated during the stringent response induced by overexpression of relA. Premature expression of lasR and rhlR was also observed when relA was overexpressed in a PAO1 rpoS mutant. The stringent response induced by the amino acid analogue serine hydroxamate (SHX) also led to premature production of the 3-oxo-C(12)-HSL autoinducer. This response to SHX was absent in a PAO1 relA mutant. These findings suggest that the stringent response can activate the two quorum-sensing systems of P. aeruginosa independently of cell density.

CzcR-CzcS, a Two-component System Involved in Heavy Metal and Carbapenem Resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an environmental bacterium involved in mineralization of organic matter. It is also an opportunistic pathogen able to cause serious infections in immunocompromised hosts. As such, it is exposed to xenobiotics including solvents, heavy metals, and antimicrobials. We studied the response of P. aeruginosa upon exposure to heavy metals or antibiotics to investigate whether common regulatory mechanisms govern resistance to both types of compounds. We showed that sublethal zinc concentrations induced resistance to zinc, cadmium, and cobalt, while lethal zinc concentrations selected mutants constitutively resistant to these heavy metals. Both zinc-induced and stable zinc-resistant strains were also resistant to the carbapenem antibiotic imipenem. On the other hand, only 20% of clones selected on imipenem were also resistant to zinc. Heavy metal resistance in the mutants could be correlated by quantitative real time PCR with increased expression of the heavy metal efflux pump CzcCBA and its cognate two-component regulator genes czcR-czcS. Western blot analysis revealed reduced expression of the basic amino acid and carbapenem-specific OprD porin in all imipenem-resistant mutants. Sequencing of the czcR-czcS DNA region in eight independent zinc- and imipenem-resistant mutants revealed the presence of the same V194L mutation in the CzcS sensor protein. Overexpression in a susceptible wild type strain of the mutated CzsS protein, but not of the wild type form, resulted in decreased oprD and increased czcC expression. We further show that zinc is released from latex urinary catheters into urine in amounts sufficient to induce carbapenem resistance in P. aeruginosa, possibly compromising treatment of urinary tract infections by this class of antibiotics.

Quorum sensing inhibition selects for virulence and cooperation in Pseudomonas aeruginosa

With the rising development of bacterial resistance the search for new medical treatments beyond conventional antimicrobials has become a key aim of public health research. Possible innovative strategies include the inhibition of bacterial virulence. However, consideration must be given to the evolutionary and environmental consequences of such new interventions. Virulence and cooperative social behaviour of the bacterium Pseudomonas aeruginosa rely on the quorum-sensing (QS) controlled production of extracellular products (public goods). Hence QS is an attractive target for anti-virulence interventions. During colonization, non-cooperating (and hence less virulent) P. aeruginosa QS-mutants, benefiting from public goods provided by wild type isolates, naturally increase in frequency providing a relative protection from invasive infection. We hypothesized that inhibition of QS-mediated gene expression removes this growth advantage and selection of less virulent QS-mutants, and maintains the predominance of more virulent QS-wild type bacteria. We addressed this possibility in a placebo-controlled trial investigating the anti-QS properties of azithromycin, a macrolide antibiotic devoid of bactericidal activity on P. aeruginosa, but interfering with QS, in intubated patients colonized by P. aeruginosa. In the absence of azithromycin, non-cooperating (and hence less virulent) lasR (QS)-mutants increased in frequency over time. Azithromycin significantly reduced QS-gene expression measured directly in tracheal aspirates. Concomitantly the advantage of lasR-mutants was lost and virulent wild-type isolates predominated during azithromycin treatment. We confirmed these results in vitro with fitness and invasion experiments. Azithromycin reduced growth rate of the wild-type, but not of the lasR-mutant. Furthermore, the lasR-mutant efficiently invaded wild-type populations in the absence, but not in the presence of azithromycin. These in vivo and in vitro results demonstrate that anti-virulence interventions based on QS-blockade diminish natural selection towards reduced virulence and therefore may increase the prevalence of more virulent genotypes in the Hospital environment. More generally, the impact of intervention on the evolution of virulence of pathogenic bacteria should be assessed. Trial Registration: ClinicalTrials.gov NCT00610623

Structure of Pseudomonas aeruginosa Populations Analyzed by Single Nucleotide Polymorphism and Pulsed-Field Gel Electrophoresis Genotyping

Pseudomonas aeruginosa has a wide ecological distribution that includes natural habitats and clinical settings. To analyze the population structure and distribution of P. aeruginosa, a collection of 111 isolates of diverse habitats and geographical origin, most of which contained a genome with a different SpeI macrorestriction profile, was typed by restriction fragment length polymorphism based on 14 single nucleotide polymorphisms (SNPs) located at seven conserved loci of the core genome (oriC, oprL, fliC, alkB2, citS, oprI, and ampC). The combination of these SNPs plus the type of fliC present (a or b) allowed the assignment of a genetic fingerprint to each strain, thus providing a simple tool for the discrimination of P. aeruginosa strains. Thirteen of the 91 identified SNP genotypes were found in two or more strains. In several cases, strains sharing their SNP genotype had different SpeI macrorestriction profiles. The highly virulent CHA strain shared its SNP genotype with other strains that had different SpeI genotypes and which had been isolated from nonclinical habitats. The reference strain PAO1 also shared its SNP genotype with other strains that had different SpeI genotypes. The P. aeruginosa chromosome contains a conserved core genome and variable amounts of accessory DNA segments (genomic islands and islets) that can be horizontally transferred among strains. The fact that some SNP genotypes were overrepresented in the P. aeruginosa population studied and that several strains sharing an SNP genotype had different SpeI macrorestriction profiles supports the idea that changes occur at a higher rate in the accessory DNA segments than in the conserved core genome.