A Quorum Sensing Regulated Small Volatile Molecule Reduces Acute Virulence and Promotes Chronic Infection Phenotypes

A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections

Pseudomonas aeruginosa Overrides the Virulence Inducing Effect of Opioids When It Senses an Abundance of Phosphate

The gut during critical illness represents a complex ecology dominated by the presence of healthcare associated pathogens, nutrient scarce conditions, and compensatory host stress signals. We have previously identified key environmental cues, opioids and phosphate depletion that independently activate the virulence of Pseudomonas aeruginosa. Opioids induce quinolone signal production (PQS), whereas phosphate depletion leads to a triangulated response between MvfR-PQS, pyoverdin, and phosphosensory/phosphoregulatory systems (PstS-PhoB). Yet how P. aeruginosa manages its response to opioids during nutrient scarce conditions when growth is limited and a quorum is unlikely to be achieved is important in the context of pathogenesis in gut during stress. To mimic this environment, we created nutrient poor conditions and exposed P. aeruginosa PAO1 to the specific k-opioid receptor agonist U-50,488. Bacterial cells exposed to the k-opioid expressed a striking increase in virulence- and multi-drug resistance-related genes that correlated to a lethal phenotype in C. elegans killing assays. Under these conditions, HHQ, a precursor of PQS, rather than PQS itself, became the main inducer for pqsABCDE operon expression. P. aeruginosa virulence expression in response to k-opioids required PqsE since ΔPqsE was attenuated in its ability to activate virulence- and efflux pumps-related genes. Extracellular inorganic phosphate completely changed the transcriptional response of PAO1 to the k- opioid preventing pqsABCDE expression, the activation of multiple virulence- and efflux pumps-related genes, and the ability of P. aeruginosa to kill C. elegans. These results indicate that when P. aeruginosa senses resource abundance in the form of phosphate, it overrides its response to compensatory host signals such as opioids to express a virulent and lethal phenotype. These studies confirm a central role for phosphate in P. aeruginosa virulence that might be exploited to design novel anti- virulence strategies

Electrocautery causes more ischemic peritoneal tissue damage than ultrasonic dissection

Contains fulltext : 96869.pdf (publisher's version ) (Open Access)BACKGROUND: Minimizing peritoneal tissue injury during abdominal surgery has the benefit of reducing postoperative inflammatory response, pain, and adhesion formation. Ultrasonic dissection seems to reduce tissue damage. This study aimed to compare electrocautery and ultrasonic dissection in terms of peritoneal tissue ischemia measured by microdialysis. METHODS: In this study, 18 Wistar rats underwent a median laparotomy and had a peritoneal microdialysis catheter implanted in the left lateral sidewall. The animals were randomly assigned to receive two standard peritoneal incisions parallel to the catheter by either ultrasonic dissection or electrocautery. After the operation, samples of microdialysis dialysate were taken every 2 h until 72 h postoperatively for measurements of pyruvate, lactate, glucose, and glycerol, and ratios were calculated. RESULTS: The mean lactate-pyruvate ratio (LPR), lactate-glucose ratio (LGR), and glycerol concentration were significantly higher in the electrocautery group than in the ultrasonic dissection group until respectively 34, 48, and 48 h after surgery. The mean areas under the curve (AUC) of LPR, LGR, and glycerol concentration also were higher in the electrocautery group than in the ultrasonic dissection group (4,387 vs. 1,639, P=0.011; 59 vs. 21, P=0.008; 7,438 vs. 4,169, P=0.008, respectively). CONCLUSION: Electrosurgery causes more ischemic peritoneal tissue damage than ultrasonic dissection.01 juni 201

The Pseudomonas Quinolone Signal (PQS) Balances Life and Death in Pseudomonas aeruginosa Populations

When environmental conditions deteriorate and become inhospitable, generic survival strategies for populations of bacteria may be to enter a dormant state that slows down metabolism, to develop a general tolerance to hostile parameters that characterize the habitat, and to impose a regime to eliminate damaged members. Here, we provide evidence that the pseudomonas quinolone signal (PQS) mediates induction of all of these phenotypes. For individual cells, PQS, an interbacterial signaling molecule of Pseudomonas aeruginosa, has both deleterious and beneficial activities: on the one hand, it acts as a pro-oxidant and sensitizes the bacteria towards oxidative and other stresses and, on the other, it efficiently induces a protective anti-oxidative stress response. We propose that this dual function fragments populations into less and more stress tolerant members which respond differentially to developing stresses in deteriorating habitats. This suggests that a little poison may be generically beneficial to populations, in promoting survival of the fittest, and in contributing to bacterial multi-cellular behavior. It further identifies PQS as an essential mediator of the shaping of the population structure of Pseudomonas and of its response to and survival in hostile environmental conditions

Nutrient Availability as a Mechanism for Selection of Antibiotic Tolerant Pseudomonas aeruginosa within the CF Airway

Microbes are subjected to selective pressures during chronic infections of host tissues. Pseudomonas aeruginosa isolates with inactivating mutations in the transcriptional regulator LasR are frequently selected within the airways of people with cystic fibrosis (CF), and infection with these isolates has been associated with poorer lung function outcomes. The mechanisms underlying selection for lasR mutation are unknown but have been postulated to involve the abundance of specific nutrients within CF airway secretions. We characterized lasR mutant P. aeruginosa strains and isolates to identify conditions found in CF airways that select for growth of lasR mutants. Relative to wild-type P. aeruginosa, lasR mutants exhibited a dramatic metabolic shift, including decreased oxygen consumption and increased nitrate utilization, that is predicted to confer increased fitness within the nutrient conditions known to occur in CF airways. This metabolic shift exhibited by lasR mutants conferred resistance to two antibiotics used frequently in CF care, tobramycin and ciprofloxacin, even under oxygen-dependent growth conditions, yet selection for these mutants in vitro did not require preceding antibiotic exposure. The selection for loss of LasR function in vivo, and the associated adverse clinical impact, could be due to increased bacterial growth in the oxygen-poor and nitrate-rich CF airway, and from the resulting resistance to therapeutic antibiotics. The metabolic similarities among diverse chronic infection-adapted bacteria suggest a common mode of adaptation and antibiotic resistance during chronic infection that is primarily driven by bacterial metabolic shifts in response to nutrient availability within host tissues

A eukaryotic-type signalling system of Pseudomonas aeruginosa contributes to oxidative stress resistance, intracellular survival and virulence

<p>Abstract</p> <p>Background</p> <p>The genome of <it>Pseudomonas aeruginosa </it>contains at least three genes encoding eukaryotic-type Ser/Thr protein kinases, one of which, <it>ppkA</it>, has been implicated in <it>P. aeruginosa </it>virulence. Together with the adjacent <it>pppA </it>phosphatase gene, they belong to the type VI secretion system (H1-T6SS) locus, which is important for bacterial pathogenesis. To determine the biological function of this protein pair, we prepared a <it>pppA-ppkA </it>double mutant and characterised its phenotype and transcriptomic profiles.</p> <p>Results</p> <p>Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pyoverdine. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the plant model of infection. Whole-genome transcriptome analysis revealed that <it>pppA-ppkA </it>deletion affects the expression of oxidative stress-responsive genes, stationary phase σ-factor RpoS-regulated genes, and quorum-sensing regulons. The transcriptome of the <it>pppA-ppkA </it>mutant was also analysed under conditions of oxidative stress and showed an impaired response to the stress, manifested by a weaker induction of stress adaptation genes as well as the genes of the SOS regulon. In addition, expression of either RpoS-regulated genes or quorum-sensing-dependent genes was also affected. Complementation analysis confirmed that the transcription levels of the differentially expressed genes were specifically restored when the <it>pppA </it>and <it>ppkA </it>genes were expressed ectopically.</p> <p>Conclusions</p> <p>Our results suggest that in addition to its crucial role in controlling the activity of <it>P. aeruginosa </it>H1-T6SS at the post-translational level, the PppA-PpkA pair also affects the transcription of stress-responsive genes. Based on these data, it is likely that the reduced virulence of the mutant strain results from an impaired ability to survive in the host due to the limited response to stress conditions.</p

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10(-5) on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels.By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system

Potential therapeutic applications of microbial surface-activecompounds

Numerous investigations of microbial surface-active compounds or biosurfactants over the past two decades have led to the discovery of many interesting physicochemical and biological properties including antimicrobial, anti-biofilm and therapeutic among many other pharmaceutical and medical applications. Microbial control and inhibition strategies involving the use of antibiotics are becoming continually challenged due to the emergence of resistant strains mostly embedded within biofilm formations that are difficult to eradicate. Different aspects of antimicrobial and anti-biofilm control are becoming issues of increasing importance in clinical, hygiene, therapeutic and other applications. Biosurfactants research has resulted in increasing interest into their ability to inhibit microbial activity and disperse microbial biofilms in addition to being mostly nontoxic and stable at extremes conditions. Some biosurfactants are now in use in clinical, food and environmental fields, whilst others remain under investigation and development. The dispersal properties of biosurfactants have been shown to rival that of conventional inhibitory agents against bacterial, fungal and yeast biofilms as well as viral membrane structures. This presents them as potential candidates for future uses in new generations of antimicrobial agents or as adjuvants to other antibiotics and use as preservatives for microbial suppression and eradication strategies