Biofilm formation by Pseudomonas aeruginosa: role of the C4-HSL cell-to-cell signal and inhibition by azithromycin

Objectives: In Pseudomonas aeruginosa, biofilm formation is controlled by a cell-to-cell signalling circuit relying on the secretion of 3-oxo-C12-HSL and C4-HSL. Previous studies suggested that C4-HSL plays no significant role in biofilm formation. However the wild-type PAO1 strain PAO-BI, used as a control in these studies is itself impaired in the production of C4-HSL. We wondered therefore whether the role of C4-HSL in biofilm formation might have been underestimated, and whether azithromycin inhibits biofilm formation by interfering with cell-to-cell signalling. Methods: We used isogenic mutants of wild-type PAO1 strains PAO-BI and PT5 in a static biofilm model. Biofilm formation was quantified using Crystal Violet staining and exopolysaccharide measurements. Results: Wild-type strain PAO-BI, as a result of its reduced C4-HSL secretion, produced 40% less biofilm compared with the wild-type PAO1 strain PT5. Using isogenic mutants of strain PT5 we have shown that whereas a lasI mutant (deficient in 3-oxo-C12-HSL) produced similar amounts of biofilm to the wild-type, a rhlI mutant (deficient in C4-HSL) produced 70% less biofilm. In the latter strain, biofilm formation could be restored by addition of exogenous C4-HSL. Azithromycin, known to reduce the production of both 3-oxo-C12-HSL and C4-HSL, inhibited biofilm formation of wild-type PT5 by 45%. This inhibition could be reversed by the addition of both cell-to-cell signals. Conclusions: Our results indicate that C4-HSL also plays a significant role in biofilm formation. Furthermore, we demonstrate the potential of using cell-to-cell signalling blocking agents such as azithromycin to interfere with biofilm formatio

Analysis of antibiotic resistance gene expression in Pseudomonas aeruginosa by quantitative real-time-PCR

In Pseudomonas aeruginosa many of the clinically relevant resistance mechanisms result from changes in gene expression as exemplified by the Mex drug efflux pumps, the AmpC β-lactamase and the carbapenem-specific porin OprD. We used quantitative real-time-PCR to analyze the expression of these genes in susceptible and antibiotic-resistant laboratory and clinical strains. In nalB mutants, which overexpress OprM, we observed a four- to eightfold increase in the expression of mexA, mexB, and oprM genes. MexX and mexY genes were induced eight to 12 times in the presence of 2 mg L−1 tetracycline. The mexC/oprJ and mexE/oprN gene expression levels were increased 30- to 250-fold and 100- to 760-fold in nfxB and nfxC mutants, respectively. We further found that in defined laboratory strains expression levels of ampC and oprD genes paralleled β-lactamase activity and OprD protein levels, respectively. Our data support the use of quantitative real-time-PCR chain reaction for the analysis of the antimicrobial resistance gene expression in P. aeruginos

Autoinducer production and quorum-sensing dependent phenotypes of Pseudomonas aeruginosa vary according to isolation site during colonization of intubated patients

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas aeruginosa </it>frequently colonizes and is responsible for severe ventilator-associated pneumonia in intubated patients. A quorum-sensing (QS) circuit, depending on the production of the two QS-signaling molecules (autoinducers, AIs) 3-oxo-C₁₂-HSL and C₄-HSL, regulates the production by <it>P. aeruginosa </it>of several virulence factors and is required for biofilm formation. Therefore QS-inhibition has been suggested as a new target for preventive and/or therapeutic strategies. However the precise role of QS during colonization and subsequent infections of intubated patients remains unclear.</p> <p>Results</p> <p>We wondered whether QS is active during colonization of intubated patients, and whether <it>P. aeruginosa </it>isolates growing inside the biofilm covering the intubation devices and those resident in the lungs of colonized patients differ in their QS-dependent phenotypes. We collected the intubation devices of eight patients colonized by <it>P. aeruginosa</it>. We detected 3-oxo-C₁₂-HSL on eight, and C₄-HSL on six of these devices. In three of these patients we also obtained <it>P. aeruginosa </it>isolates from tracheal aspirates at the time of extubation (n = 18), as well as isolates from the intubation devices (n = 25). We genotyped these isolates, quantified their AIs production, and determined three QS-dependent phenotypes (adherence capacity, biofilm and elastase production). The production of 3-oxo-C₁₂-HSL was consistently increased for isolates from the intubation devices, whereas the production of C₄-HSL was significantly higher for isolates from tracheal aspirates. Isolates from tracheal aspirates produced significantly higher amounts of elastase but less biofilm, and had a marginally reduced adhesion capacity than isolates from the intubation devices. Levels of 3-oxo-C₁₂-HSL and elastase production correlated statistically for tracheal intubation isolates, whereas levels of 3-oxo-C₁₂-HSL production and adhesion ability, as well as biofilm production, correlated weakly amongst intubation device isolates.</p> <p>Conclusion</p> <p>Our findings demonstrate that autoinducers are produced during the colonization of intubated patients by <it>P. aeruginosa</it>. The microenvironment, in which <it>P. aeruginosa </it>grows, may select for bacteria with different capacities to produce autoinducers and certain QS-dependent phenotypes. QS-inhibition might therefore affect differently isolates growing inside the biofilm covering intubation devices and those resident in the lungs.</p

First case of Cryptococcus gattii multilobar pneumonia in Switzerland and associated challenges

n/

Microbial Communities of Conducting and Respiratory Zones of Lung-Transplanted Patients.

Background: Lung transplantation (LT) is a recognized treatment for end-stage pulmonary disease. Bacteria from the recipient nasopharynx seed the new lungs leading to infections and allograft damage. Understanding the characteristics and topological variations of the microbiota may be important to apprehend the pathophysiology of allograft dysfunction. Objectives: To examine the characteristics and relationship of bacterial compositions between conducting and respiratory zones of the allograft. Methods: We performed 16S rRNA gene sequencing on bronchial aspirates (BAs) and bronchoalveolar lavages (BALs) collected in pairs in 19 patients at several time-points post-LT. Results: The respiratory zone was characterized independently of the time post-LT by a higher bacterial richness than the conducting zone (p = 0.041). The phyla Firmicutes and Proteobacteria dominated both sampling zones, with an inverse correlation between these two phyla (Spearman r = -0.830). Samples of the same pair, as well as pairs from the same individual clustered together (Pseudo-F = 3.8652, p &lt; 0.01). Microbiota of BA and BAL were more closely related in samples from the same patient than each sample type across different patients, with variation in community structure being mainly inter-individual (p &lt; 0.01). Both number of antibiotics administered (p &lt; 0.01) and time interval post-LT (p &lt; 0.01) contributed to the variation in global microbiota structure. Longitudinal analysis of BA-BAL pairs of two patients showed dynamic wave like fluctuations of the microbiota. Conclusions: Our results show that post-transplant respiratory zones harbor higher bacterial richness, but overall similar bacterial profiles as compared to conductive zones. They further support an individual microbial signature following LT

Azithromycin to prevent Pseudomonas aeruginosa ventilator-associated pneumonia by inhibition of quorum sensing: a randomized controlled trial

Purpose: Anti-virulence strategies have not been evaluated for the prevention of bacterial infections. Prolonged colonization of intubated patients with Pseudomonas aeruginosa isolates producing high-levels of the quorum sensing (QS)-regulated virulence factor rhamnolipids has been associated with ventilator-associated pneumonia (VAP). In this pathogen, azithromycin reduces QS-regulated virulence. We aimed to assess whether azithromycin could prevent VAP in patients colonized by rhamnolipids producing isolates. Methods: In a randomized, double-blind, multicenter trial, intubated colonized patients received either 300mg/day azithromycin or placebo. Primary endpoint was the occurrence of P. aeruginosa VAP. We further identified those patients persistently colonized by isolates producing high-levels of rhamnolipids and therefore at the highest risk to develop VAP linked to this QS-dependent virulence factor. Results: Ninety-two patients were enrolled; 43 azithromycin-treated and 42 placebo patients were eligible for the per-protocol analysis. In the per-protocol population, the occurrence of P. aeruginosa VAP was reduced in the azithromycin group but without reaching statistical significance (4.7 vs. 14.3% VAP, p=0.156). QS-dependent virulence of colonizing isolates was similarly low in both study groups, and only five patients in each arm were persistently colonized by high-level rhamnolipids producing isolates. In this high-risk subgroup, the incidence of VAP was reduced fivefold in azithromycin versus placebo patients (1/5 vs. 5/5 VAP, p=0.048). Conclusions: There was a trend towards reduced incidence of VAP in colonized azithromycin-treated patients. In addition, azithromycin significantly prevented VAP in those patients at high risk of rhamnolipid-dependent VAP, suggesting that virulence inhibition is a promising anti-microbial strateg

An intrinsically disordered antimicrobial peptide dendrimer from stereorandomized virtual screening.

Membrane-disruptive amphiphilic antimicrobial peptides behave as intrinsically disordered proteins by being unordered in water and becoming α-helical in contact with biological membranes. We recently discovered that synthesizing the α-helical antimicrobial peptide dendrimer L-T25 ((KL)8(KKL)4(KLL)2 KKLL) using racemic amino acids to form stereorandomized sr-T25, an analytically pure mixture of all possible diastereoisomers of L-T25, preserved antibacterial activity but abolished hemolysis and cytotoxicity, pointing to an intrinsically disordered antibacterial conformation and an α-helical cytotoxic conformation. In this study, to identify non-toxic intrinsically disordered homochiral antimicrobial peptide dendrimers (AMPDs), we surveyed sixty-three sr-analogs of sr-T25 selected by virtual screening. One of the analogs, sr-X18 ((KL)8(KLK)4(KLL)2 KLLL), lost antibacterial activity as L-enantiomer and became hemolytic due to α-helical folding. By contrast, the L- and D-enantiomers of sr-X22 ((KL)8(KL)4(KKLL)2 KLKK) were equally antibacterial, non-hemolytic, and non-toxic, implying an intrinsically disordered bioactive conformation. Screening stereorandomized libraries may be generally useful to identify or optimize intrinsically disordered bioactive peptides

Remission after treatment of osteoarticular infections due to Pseudomonas aeruginosa versus Staphylococcus aureus: a case-controlled study

Purpose: Osteoarticular infections due to methicillin-susceptible Staphylococcus aureus (MSSA) or its methicillin-resistant variant (MRSA) are feared due to treatment failures. According to clinical experience, Pseudomonas aeruginosa may reveal less long-term remission than S. aureus. Methods: A case-controlled study comparing outcomes of osteoarticular infections due to P. aeruginosa vs S. aureus was performed at Geneva University Hospitals. Results: A total of 111 S. aureus (including 37 MRSA) and 20 P. aeruginosa osteoarticular infections were analysed in 131 patients: arthroplasties (n = 38), fracture fixation devices (n = 56), native joint arthritis (n = 7) and osteomyelitis without implant (n = 30). The median active follow-up time was 4years. The patients underwent a median number of two surgical interventions for P. aeruginosa infections compared to two for S. aureus (two for MRSA), while the median duration of antibiotic treatment was 87days for P. aeruginosa and 46days for S. aureus infections (58days for MRSA) (all p > 0.05). Overall, Pseudomonas-infected patients tended towards a lower remission rate than those infected with S. aureus (12/20 vs 88/111; p = 0.06). This was similar when P. aeruginosa was compared with MRSA alone (12/20 vs 30/37; p = 0.08). In multivariate logistic regression analyses adjusting for case mix, odds ratios (OR) for remission were as follows: P. aeruginosa vs S. aureus [OR 0.4, 95% confidence interval (CI) 0.1-1.2], number of surgical interventions (OR 0.6, 95% CI 0.5-1.0) and duration of antibiotic treatment (OR 1.0, 95% CI 1.0-1.0). Conclusions: Despite a similar number of surgical interventions and longer antibiotic treatment, osteoarticular infections due to P. aeruginosa tended towards a lower remission rate than infections due to S. aureus in general or MRSA in particula