Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules. RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P. aeruginosa. An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P. aeruginosa. A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P. aeruginosa. Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation. Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA.

Susceptibility of clinical isolates of multiresistant Pseudomonas aeruginosa to a hospital disinfectant and molecular typing

The aim of this study was to evaluate the susceptibility of 35 resistant Pseudomonas aeruginosa clinical isolates to a quaternary ammonium hospital disinfectant. The methodology was the AOAC Use-Dilution Test, with disinfectant at its use-concentration. In addition, the chromosomal DNA profile of the isolates were determined by macro-restriction pulsed field gel electrophoresis (PFGE) method aiming to verify the relatedness among them and the behavior of isolates from the same group regarding the susceptibility to the disinfectant. Seventy one percent of the isolates were multiresistant to antibiotics and 43% showed a reduced susceptibility to the disinfectant. The PFGE methodology detected 18 major clonal groups. We found isolates with reduced susceptibility to the disinfectant and we think that these are worrying data that should be further investigated including different organisms and chemical agents in order to demonstrate that microorganisms can be destroyed by biocide as necessary. We also found strains of the same clonal groups showing different susceptibility to the disinfectant. This is an interesting observation considering that only few works are available about this subject. PFGE profile seems not to be a reliable marker for resistance to disinfectants.

Emergence of dhfrXVb and blaCARB-4 gene cassettes in class 1 integrons from clinical Pseudomonas aeruginosa isolated in Amazon region

Integrons play a role in horizontal acquisition and expression of genes, as well as gene reservoir, contributing for the resistance phenotype, particularly relevant to bacteria of clinical importance. We aimed to determine the composition and the organization of the class 1 integron variable region present in Pseudomonas aeruginosa clinical isolates from Brazil. Strains carrying class 1 integrons were resistant to the majority of antibiotics tested, except to imipenem and ceftazidime. Sequence analysis of the integron variable region revealed the presence of the blaCARB-4 gene into two distinct cassette arrays: aacA4-dhfrXVb-blaCARB-4 and aadB-aacA4-blaCARB-4 . dhfrXVb gene cassette, which is rare in Brazil and in P. aeruginosa species, was found in one isolate. PFGE analysis showed the spread of blaCARB-4 among P. aeruginosa clones. The occurrence of blaCARB-4 and dhfrXVb in Brazil may contribute for developing resistance to clinically important antibiotics, and shows a diversified scenarium of these elements occurring in Amazon clinical settings, where no study about integron dinamycs was performed to date.

PchC thioesterase optimizes nonribosomal biosynthesis of the peptide siderophore pyochelin in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the antibiotic dihydroaeruginoate (Dha) and the siderophore pyochelin are produced from salicylate and cysteine by a thiotemplate mechanism involving the peptide synthetases PchE and PchF. A thioesterase encoded by the pchC gene was found to be necessary for maximal production of both Dha and pyochelin, but it was not required for Dha release from PchE and could not replace the thioesterase function specified by the C-terminal domain of PchF. In vitro, 2-aminobutyrate, a cysteine analog, was adenylated by purified PchE and PchF proteins. In vivo, this analog strongly interfered with Dha and pyochelin formation in a pchC deletion mutant but affected production of these metabolites only slightly in the wild type. Exogenously supplied cysteine overcame the negative effect of a pchC mutation to a large extent, whereas addition of salicylate did not. These data are in agreement with a role for PchC as an editing enzyme that removes wrongly charged molecules from the peptidyl carrier protein domains of PchE and PchF.

Host and invader impact of transfer of the clc genomic island into Pseudomonas aeruginosa PAO1

Genomic islands, large potentially mobile regions of bacterial chromosomes, are a major contributor to bacteria evolution. Here, we investigated the fitness cost and phenotypic differences between the bacterium Pseudomonas aeruginosa PAO1 and a derivative carrying one integrated copy of the clc element, a 103-kb genomic island [and integrative and conjugative element (ICE)] originating in Pseudomonas sp. strain B13 and a close relative of genomic islands found in clinical and environmental isolates of P. aeruginosa. By using a combination of whole genome transcriptome profiling, phenotypic arrays, competition experiments, and biofilm formation studies, only few differences became apparent, such as reduced biofilm growth and fourfold stationary phase repression of genes involved in acetoin metabolism in PAO1 containing the clc element. In contrast, PAO1 carrying the clc element acquired the capacity to grow on 3-chlorobenzoate and 2-aminophenol as sole carbon and energy substrates. No fitness loss >1% was detectable in competition experiments between PAO1 and PAO1 carrying the clc element. The genes from the clc element were not silent in PAO1, and excision was observed, although transfer of clc from PAO1 to other recipient bacteria was reduced by two orders of magnitude. Our results indicate that newly acquired mobile DNA not necessarily invoke an important fitness cost on their host. Absence of immediate detriment to the host may have contributed to the wide distribution of genomic islands like clc in bacterial genomes

A cell biological view of the siderophore pyochelin iron uptake pathway in Pseudomonas aeruginosa.

Pyochelin (PCH) is a siderophore produced and secreted by Pseudomonas aeruginosa for iron capture. Using (55) Fe uptake and binding assays, we showed that PCH-Fe uptake in P. aeruginosa involves, in addition to the highly studied outer membrane transporter FptA, the inner membrane permease FptX, which recognizes PCH-(55) Fe with an affinity of 0.6 ± 0.2 nM and transports the ferri-siderophore complex from the periplasm into the cytoplasm: fptX deletion inhibited (55) Fe accumulation in the bacterial cytoplasm. Chromosomal replacement was used to generate P. aeruginosa strains producing fluorescent fusions with FptX, PchR (an AraC regulator), PchA (the first enzyme involved in the PCH biosynthesis) and PchE (a non-ribosomic peptide-synthetase involved in a further step). Fluorescence imaging and cellular fractionation showed a uniform repartition of FptX in the inner membrane. PchA and PchE were found in the cytoplasm, associated to the inner membrane all over the bacteria and also concentrated at the bacterial poles. PchE clustering at the bacterial poles was dependent on PchA expression, but on the opposite PchA clustering and membrane association was PchE-independent. PchA and PchE cellular organization suggests the existence of a siderosome for PCH biosynthesis as previously proposed for pyoverdine biosynthesis (another siderophore produced by P. aeruginosa).

Azithromycin inhibits expression of the GacA-dependent small RNAs RsmY and RsmZ in Pseudomonas aeruginosa.

Azithromycin at clinically relevant doses does not inhibit planktonic growth of the opportunistic pathogen Pseudomonas aeruginosa but causes markedly reduced formation of biofilms and quorum-sensing-regulated extracellular virulence factors. In the Gac/Rsm signal transduction pathway, which acts upstream of the quorum-sensing machinery in P. aeruginosa, the GacA-dependent untranslated small RNAs RsmY and RsmZ are key regulatory elements. As azithromycin treatment and mutational inactivation of gacA have strikingly similar phenotypic consequences, the effect of azithromycin on rsmY and rsmZ expression was investigated. In planktonically growing cells, the antibiotic strongly inhibited the expression of both small RNA genes but did not affect the expression of the housekeeping gene proC. The azithromycin treatment resulted in reduced expression of gacA and rsmA, which are known positive regulators of rsmY and rsmZ, and of the PA0588-PA0584 gene cluster, which was discovered as a novel positive regulatory element involved in rsmY and rsmZ expression. Deletion of this cluster resulted in diminished ability of P. aeruginosa to produce pyocyanin and to swarm. The results of this study indicate that azithromycin inhibits rsmY and rsmZ transcription indirectly by lowering the expression of positive regulators of these small RNA genes.

Negative control of quorum sensing by RpoN (sigma54) in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa PAO1, the expression of several virulence factors such as elastase, rhamnolipids, and hydrogen cyanide depends on quorum-sensing regulation, which involves the lasRI and rhlRI systems controlled by N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, respectively, as signal molecules. In rpoN mutants lacking the transcription factor sigma(54), the expression of the lasR and lasI genes was elevated at low cell densities, whereas expression of the rhlR and rhlI genes was markedly enhanced throughout growth by comparison with the wild type and the complemented mutant strains. As a consequence, the rpoN mutants had elevated levels of both signal molecules and overexpressed the biosynthetic genes for elastase, rhamnolipids, and hydrogen cyanide. The quorum-sensing regulatory protein QscR was not involved in the negative control exerted by RpoN. By contrast, in an rpoN mutant, the expression of the gacA global regulatory gene was significantly increased during the entire growth cycle, whereas another global regulatory gene, vfr, was downregulated at high cell densities. In conclusion, it appears that GacA levels play an important role, probably indirectly, in the RpoN-dependent modulation of the quorum-sensing machinery of P. aeruginosa.

Changing the epidemiology of carbapenem-resistant Pseudomonas aeruginosa in a Brazilian teaching hospital: the replacement of São Paulo metallo-β-lactamase-producing isolates

In Brazil, carbapenem-resistant Pseudomonas aeruginosa isolates are closely related to the São Paulo metallo-β-lactamase (SPM) Brazilian clone. In this study, imipenem-resistant isolates were divided in two sets, 2002/2003 and 2008/2009, analysed by pulsed field gel electrophoresis and tested for the Ambler class B metallo-β-lactamase (MBL) genes blaSPM-1, blaIMP and blaVIM. The results show a prevalence of one clone related to the SPM Brazilian clone in 2002/2003. In 2008/2009, P. aeruginosa isolates were mostly MBL negative, genetically diverse and unrelated to those that had been detected earlier. These findings suggest that the resistance to carbapenems by these recent P. aeruginosa isolates was not due to the spread of MBL-positive SPM-related clones, as often observed in Brazilian hospitals.

The infection of microvascular endothelial cells with ExoU-producing Pseudomonas aeruginosa triggers the release of von Willebrand factor and platelet adhesion

An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.

Metallo-β-lactamase-production in meropenem-susceptible Pseudomonas aeruginosa isolates: risk for silent spread

The aim of this study was to characterize two metallo-β-lactamases (MBLs)-producing Pseudomonas aeruginosa clinical isolates showing meropenem susceptibility. Antimicrobial susceptibility was assessed by automated testing and Clinical and Laboratory Standards Institute agar dilution method. MBL production was investigated by phenotypic tests. Molecular typing was determined by pulsed field gel electrophoresis (PFGE). MBL-encoding genes, as well as their genetic context, were identified by polymerase chain reaction (PCR) and sequencing. The location of blaIMP-16 was determined by plasmid electrophoresis, Southern blot and hybridization. Transcriptional levels of blaIMP-16, mexB, mexD, mexF, mexY, ampC and oprD were determined by semi-quantitative real time PCR. The P. aeruginosa isolates studied, Pa30 and Pa43, showed imipenem and meropenem susceptibility by automated testing. Agar dilution assays confirmed meropenem susceptibility whereas both isolates showed low level of imipenem resistance. Pa30 and Pa43 were phenotypically detected as MBL producers. PFGE revealed their clonal relatedness. blaIMP-16 was identified in both isolates, carried as a single cassette in a class 1 integron that was embedded in a plasmid of about 60-Kb. Pa30 and Pa43 overexpressed MexAB-OprM, MexCD-OprJ and MexXY-OprM efflux systems and showed basal transcriptional levels of ampC and oprD. MBL-producing P. aeruginosa that are not resistant to meropenem may represent a risk for therapeutic failure and act as silent reservoirs of MBL-encoding genes.

Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model.

The human pathogen Pseudomonas aeruginosa has been shown previously to use similar virulence factors when infecting mammalian hosts or Dictyostelium amoebae. Here we randomly mutagenized a clinical isolate of P. aeruginosa, and identified mutants with attenuated virulence towards Dictyostelium. These mutant strains also exhibited a strong decrease in virulence when infecting Drosophila and mice, confirming that P. aeruginosa makes use of similar virulence traits to confront these very different hosts. Further characterization of these bacterial mutants showed that TrpD is important for the induction of the quorum-sensing circuit, while PchH and PchI are involved in the induction of the type III secretion system. These results demonstrate the usefulness and the relevance of the Dictyostelium host model to identify and analyse new virulence genes in P. aeruginosa.

The global posttranscriptional regulator RsmA modulates production of virulence determinants and N-acylhomoserine lactones in Pseudomonas aeruginosa.

Posttranscriptional control is known to contribute to the regulation of secondary metabolism and virulence determinants in certain gram-negative bacteria. Here we report the isolation of a Pseudomonas aeruginosa gene which encodes a global translational regulatory protein, RsmA (regulator of secondary metabolites). Overexpression of rsmA resulted in a substantial reduction in the levels of extracellular products, including protease, elastase, and staphylolytic (LasA protease) activity as well as the PA-IL lectin, hydrogen cyanide (HCN), and the phenazine pigment pyocyanin. While inactivation of rsmA in P. aeruginosa had only minor effects on the extracellular enzymes and the PA-IL lectin, the production of HCN and pyocyanin was enhanced during the exponential phase. The influence of RsmA on N-acylhomoserine lactone-mediated quorum sensing was determined by assaying the levels of N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL) and N-butanoylhomoserine lactone (C4-HSL) produced by the rsmA mutant and the rsmA-overexpressing strain. RsmA exerted a negative effect on the synthesis of both 3-oxo-C12-HSL and C4-HSL, which was confirmed by using lasI and rhlI translational fusions. These data also highlighted the temporal expression control of the lasI gene, which was induced much earlier and to a higher level during the exponential growth phase in an rsmA mutant. To investigate whether RsmA modulates HCN production solely via quorum-sensing control, hcn translational fusions were employed to monitor the regulation of the cyanide biosynthesis genes (hcnABC). RsmA was shown to exert an additional negative effect on cyanogenesis posttranscriptionally by acting on a region surrounding the hcnA ribosome-binding site. This suggests that, in P. aeruginosa, RsmA functions as a pleiotropic posttranscriptional regulator of secondary metabolites directly and also indirectly by modulating the quorum-sensing circuitry.

Dual control of hydrogen cyanide biosynthesis by the global activator GacA in Pseudomonas aeruginosa PAO1.

The global response regulator GacA of Pseudomonas aeruginosa PAO1 positively controls the production of the quorum sensing signal molecule N-butanoyl-homoserine-lactone (C4-HSL) and hence the synthesis of several C4-HSL-dependent virulence factors, including hydrogen cyanide (HCN). This study presents evidence that GacA positively influences the transcription of the rhlI gene, specifying C4-HSL synthase, explaining the quorum sensing-dependent transcriptional control of the HCN biosynthetic genes (hcnABC). In addition, GacA was found to modulate hcn gene expression positively at a post-transcriptional level involving the hcnA ribosome-binding site. Thus, the activating effect of GacA on cyanogenesis results from both transcriptional and post-transcriptional mechanisms.