The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth of Erwinia amylovora and acts as a seed germination-arrest factor

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) shares biological activities with 4-formylaminooxyvinylglycine, a related molecule produced by Pseudomonas fluorescens WH6. We found that culture filtrates of a P.aeruginosa strain overproducing AMB weakly interfered with seed germination of the grassy weed Poa annua and strongly inhibited growth of Erwinia amylovora, the causal agent of the devastating orchard crop disease known as fire blight. AMB was active against a 4-formylaminooxyvinylglycine-resistant isolate of E.amylovora, suggesting that the molecular targets of the two oxyvinylglycines in Erwinia do not, or not entirely, overlap. The AMB biosynthesis and transport genes were shown to be organized in two separate transcriptional units, ambA and ambBCDE, which were successfully expressed from IPTG-inducible tac promoters in the heterologous host P.fluorescens CHA0. Engineered AMB production enabled this model biocontrol strain to become inhibitory against E.amylovora and to weakly interfere with the germination of several graminaceous seeds. We conclude that AMB production requires no additional genes besides ambABCDE and we speculate that their expression in marketed fire blight biocontrol strains could potentially contribute to disease control.

Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa.

The high-affinity siderophore salicylate is an intermediate in the biosynthetic pathway of pyochelin, another siderophore and chelator of transition metal ions, in Pseudomonas aeruginosa. The 2.5-kb region upstream of the salicylate biosynthetic genes pchBA was sequenced and found to contain two additional, contiguous genes, pchD and pchC, having the same orientation. The deduced amino acid sequence of the 60-kDa PchD protein was similar to those of the EntE protein (2,3-dihydroxybenzoate-AMP ligase) of Escherichia coli and other adenylate-forming enzymes, suggesting that salicylate might be adenylated at the carboxyl group by PchD. The 28-kDa PchC protein showed similarities to thioesterases of prokaryotic and eukaryotic origin and might participate in the release of the product(s) formed from activated salicylate. One potential product, dihydroaeruginoate (Dha), was identified in culture supernatants of iron-limited P. aeruginosa cells. The antifungal antibiotic Dha is thought to arise from the reaction of salicylate with cysteine, followed by cyclization of cysteine. Inactivation of the chromosomal pchD gene by insertion of the transcription and translation stop element omega Sm/Sp abolished the production of Dha and pyochelin, implying that PchD-mediated activation of salicylate may be a common first step in the synthesis of both metabolites. Furthermore, the pchD::omega Sm/Sp mutation had a strong polar effect on the expression of the pchBA genes, i.e., on salicylate synthesis, indicating that the pchDCBA genes constitute a transcriptional unit. A full-length pchDCBA transcript of ca. 4.4 kb could be detected in iron-deprived, growing cells of P. aeruginosa. Transcription of pchD started at tandemly arranged promoters, which overlapped with two Fur boxes (binding sites for the ferric uptake regulator) and the promoter of the divergently transcribed pchR gene encoding an activator of pyochelin biosynthesis. This promoter arrangement allows tight iron-mediated repression of the pchDCBA operon.

Methionine-321 in the C-terminal alpha-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity.

Pseudomonas aeruginosa has a pair of distinct ornithine carbamoyltransferases. The anabolic ornithine carbamoyltransferase encoded by the argF gene catalyzes the formation of citrulline from ornithine and carbamoylphosphate. The catabolic ornithine carbamoyltransferase encoded by the arcB gene promotes the reverse reaction in vivo; although this enzyme can be assayed in vitro for citrulline synthesis, its unidirectionality in vivo is determined by its high concentration at half maximum velocity for carbamoylphosphate ([S]0.5) and high cooperativity toward this substrate. We have isolated mutant forms of catabolic ornithine carbamoyltransferase catalyzing the anabolic reaction in vivo. The corresponding arcB mutant alleles on a multicopy plasmid specifically suppressed an argF mutation of P. aeruginosa. Two new mutant enzymes were obtained. When methionine 321 was replaced by isoleucine, the mutant enzyme showed loss of homotropic cooperativity at physiological carbamoylphosphate concentrations. Substitution of glutamate 105 by lysine resulted in a partial loss of the sigmoidal response to increasing carbamoylphosphate concentrations. However, both mutant enzymes were still sensitive to the allosteric activator AMP and to the inhibitor spermidine. These results indicate that at least two residues of catabolic ornithine carbamoyltransferase are critically involved in positive carbamoylphosphate cooperativity: glutamate 105 (previously known to be important) and methionine 321. Mutational changes in either amino acid will affect the geometry of helix H2, which contains several residues required for carbamoylphosphate binding.

Assessment of panobacumab as adjunctive immunotherapy for the treatment of nosocomial Pseudomonas aeruginosa pneumonia.

The fully human anti-lipopolysaccharide (LPS) immunoglobulin M (IgM) monoclonal antibody panobacumab was developed as an adjunctive immunotherapy for the treatment of O11 serotype Pseudomonas aeruginosa infections. We evaluated the potential clinical efficacy of panobacumab in the treatment of nosocomial pneumonia. We performed a post-hoc analysis of a multicenter phase IIa trial (NCT00851435) designed to prospectively evaluate the safety and pharmacokinetics of panobacumab. Patients treated with panobacumab (n = 17), including 13 patients receiving the full treatment (three doses of 1.2 mg/kg), were compared to 14 patients who did not receive the antibody. Overall, the 17 patients receiving panobacumab were more ill. They were an average of 72 years old [interquartile range (IQR): 64-79] versus an average of 50 years old (IQR: 30-73) (p���= 0.024) and had Acute Physiology and Chronic Health Evaluation II (APACHE II) scores of 17 (IQR: 16-22) versus 15 (IQR: 10-19) (p���= 0.043). Adjunctive immunotherapy resulted in an improved clinical outcome in the group receiving the full three-course panobacumab treatment, with a resolution rate of 85 % (11/13) versus 64 % (9/14) (p���= 0.048). The Kaplan-Meier survival curve showed a statistically significantly shorter time to clinical resolution in this group of patients (8.0 [IQR: 7.0-11.5] versus 18.5 [IQR: 8-30] days in those who did not receive the antibody; p���= 0.004). Panobacumab adjunctive immunotherapy may improve clinical outcome in a shorter time if patients receive the full treatment (three doses). These preliminary results suggest that passive immunotherapy targeting LPS may be a complementary strategy for the treatment of nosocomial O11 P. aeruginosa pneumonia.

Molecular epidemiology of Pseudomonas aeruginosa in intensive care units over a 10-year period (1998-2007).

Pseudomonas aeruginosa is one of the leading nosocomial pathogens in intensive care units (ICUs). The source of this microorganism can be either endogenous or exogenous. The proportion of cases as a result of transmission is still debated, and its elucidation is important for implementing appropriate control measures. To understand the relative importance of exogenous vs. endogenous sources of P. aeruginosa, molecular typing was performed on all available P. aeruginosa isolated from ICU clinical and environmental specimens in 1998, 2000, 2003, 2004 and 2007. Patient samples were classified according to their P. aeruginosa genotypes into three categories: (A) identical to isolate from faucet; (B) identical to at least one other patient sample and not found in faucet; and (C) unique genotype. Cases in categories A and B were considered as possibly exogenous, and cases in category C as possibly endogenous. A mean of 34 cases per 1000 admissions per year were found to be colonized or infected by P. aeruginosa. Higher levels of faucet contamination were correlated with a higher number of cases in category A. The number of cases in category B varied from 1.9 to 20 cases per 1000 admissions. This number exceeded 10/1000 admissions on three occasions and was correlated with an outbreak on one occasion. The number of cases considered as endogenous (category C) was stable and independent of the number of cases in categories A and B. The present study shows that repeated molecular typing can help identify variations in the epidemiology of P. aeruginosa in ICU patients and guide infection control measures.

Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa.

The biosynthetic genes pchDCBA and pchEF, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the pchR regulatory gene on the chromosome of Pseudomonas aeruginosa. The 4.6-kb region located downstream of the pchEF genes was found to contain three additional, contiguous genes, pchG, pchH, and pchI, probably forming a pchEFGHI operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the Yersinia pestis and Y. enterocolitica proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in pchG abolished pyochelin formation, whereas mutations in pchH and pchI did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a P. aeruginosa mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed pchDCBA and pchEFG genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host Escherichia coli expressing pchDCBA and pchEFG together with the E. coli entD gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in P. aeruginosa.

Sputum containing zinc enhances carbapenem resistance, biofilm formation and virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa chronic lung infections are the leading cause of mortality in cystic fibrosis patients, a serious problem which is notably due to the numerous P. aeruginosa virulence factors, to its ability to form biofilms and to resist the effects of most antibiotics. Production of virulence factors and biofilm formation by P. aeruginosa is highly coordinated through complex regulatory systems. We recently found that CzcRS, the zinc and cadmium-specific two-component system is not only involved in metal resistance, but also in virulence and carbapenem antibiotic resistance in P. aeruginosa. Interestingly, zinc has been shown to be enriched in the lung secretions of cystic fibrosis patients. In this study, we investigated whether zinc might favor P. aeruginosa pathogenicity using an artificial sputum medium to mimic the cystic fibrosis lung environment. Our results show that zinc supplementation triggers a dual P. aeruginosa response: (i) it exacerbates pathogenicity by a CzcRS two-component system-dependent mechanism and (ii) it stimulates biofilm formation by a CzcRS-independent mechanism. Furthermore, P. aeruginosa cells embedded in these biofilms exhibited increased resistance to carbapenems. We identified a novel Zn-sensitive regulatory circuit controlling the expression of the OprD porin and modifying the carbapenem resistance profile. Altogether our data demonstrated that zinc levels in the sputum of cystic fibrosis patients might aggravate P. aeruginosa infection. Targeting zinc levels in sputum would be a valuable strategy to curb the increasing burden of P. aeruginosa infections in cystic fibrosis patients.

Comparison of double-locus sequence typing (DLST) and multilocus sequence typing (MLST) for the investigation of Pseudomonas aeruginosa populations.

Reliable molecular typing methods are necessary to investigate the epidemiology of bacterial pathogens. Reference methods such as multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) are costly and time consuming. Here, we compared our newly developed double-locus sequence typing (DLST) method for Pseudomonas aeruginosa to MLST and PFGE on a collection of 281 isolates. DLST was as discriminatory as MLST and was able to recognize "high-risk" epidemic clones. Both methods were highly congruent. Not surprisingly, a higher discriminatory power was observed with PFGE. In conclusion, being a simple method (single-strand sequencing of only 2 loci), DLST is valuable as a first-line typing tool for epidemiological investigations of P. aeruginosa. Coupled to a more discriminant method like PFGE or whole genome sequencing, it might represent an efficient typing strategy to investigate or prevent outbreaks.

Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis.

Bacterial-fungal interactions have important physiologic and medical ramifications, but the mechanisms of these interactions are poorly understood. The gut is host to trillions of microorganisms, and bacterial-fungal interactions are likely to be important. Using a neutropenic mouse model of microbial gastrointestinal colonization and dissemination, we show that the fungus Candida albicans inhibits the virulence of the bacterium Pseudomonas aeruginosa by inhibiting P. aeruginosa pyochelin and pyoverdine gene expression, which plays a critical role in iron acquisition and virulence. Accordingly, deletion of both P. aeruginosa pyochelin and pyoverdine genes attenuates P. aeruginosa virulence. Heat-killed C. albicans has no effect on P. aeruginosa, whereas C. albicans secreted proteins directly suppress P. aeruginosa pyoverdine and pyochelin expression and inhibit P. aeruginosa virulence in mice. Interestingly, suppression or deletion of pyochelin and pyoverdine genes has no effect on P. aeruginosa's ability to colonize the GI tract but does decrease P. aeruginosa's cytotoxic effect on cultured colonocytes. Finally, oral iron supplementation restores P. aeruginosa virulence in P. aeruginosa and C. albicans colonized mice. Together, our findings provide insight into how a bacterial-fungal interaction can modulate bacterial virulence in the intestine. Previously described bacterial-fungal antagonistic interactions have focused on growth inhibition or colonization inhibition/modulation, yet here we describe a novel observation of fungal-inhibition of bacterial effectors critical for virulence but not important for colonization. These findings validate the use of a mammalian model system to explore the complexities of polymicrobial, polykingdom infections in order to identify new therapeutic targets for preventing microbial disease.

Efficacy of an eradication schema in case of Pseudomonas aeruginosa primo-infection in children with cystic fibrosis

Introduction: Patients with Cystic fibrosis (CF) are more susceptible to pathogens like P. aeruginosa (PA). PA primo-infection requires particular attention as failure in eradication is associated with accelerated lung deterioration. The main aim of this study is to assess the rate of PA eradication according to our particular protocol with inhaled tobramycin and oral ciprofloxacin, as there is no consensus in the literature on what eradication protocol is optimal. Methods: Retrospective single centre study with data analysis from June 1st 2007 to June 1st 2011 of patients with PA primo-infection exclusively treated by 3 x 28 days of inhaled tobramycin and oral ciprofloxacin for the first and last 21 days. Success in eradication is defined by ≥ 3 negative bacteriologies for 6 months after the beginning of the protocol. If ≥ 1 bacteriology is positive, we consider the eradication as a failure. Results: Out of 41 patients, 18 followed the eradication protocol and were included in our analysis (7 girls (38.9%) and 11 boys (61.1%)). Boys had 12 primo-infections and girls had 8. Among these 20 primo-infections, 16 (80%) had an overall success in eradication and 4 (20%) a failure. There was no significant statistical differences in age between these groups (t-test = 0.07, p = 0.94), nor for FEV1% (t-test = 0.96, p = 0.41) or BMI (t-test = 1.35, p = 0.27). Rate of success was 100% for girls and 66.6% for boys. Conclusion: Our protocol succeeded in an overall eradication rate of 80%, without statistical significant impact on FEV1 % and BMI values. However, there was a sex difference with eradication rates in girls (100%) and boys (66.6%). A sex difference has not yet been reported in the literature. This should be evaluated in further studies.

Cinética e caracterização de ramnolip��deos produzidos por Pseudomonas aeruginosa MSIC02 utilizando glicerol como fonte de carbono

Glycerol, a co-product of biodiesel production, was used as a carbon source for the kinetics studies and production of biosurfactants by P. aeruginosa MSIC02. The highest fermentative parameters (Y PX = 3.04 g g-1; Y PS = 0.189 g g-1, P B = 31.94 mg L-1 h-1 and P X = 10.5 mg L-1 h-1) were obtained at concentrations of 0.4% (w/v) NaNO3 and 2% (w/v) glycerol. The rhamnolipid exhibited 80% of emulsification on kerosene, surface tension of 32.5 mN m-1, CMC = 28.2 mg L-1, C20 (concentration of surfactant in the bulk phase that produces a reduction of 20 dyn/cm in the surface tension of the solvent) = 0.99 mg L-1, Γm (surface concentration excess) = 2.4 x 10-26 mol Å-2 and S (surface area) = 70.4 Ų molecule-1 with solutions containing 10% NaCl. A mathematical model based on logistic equation was considered to representing the process. Model parameters were estimated by non-linear regression method. This approach was able to give a good description of the process.

Typing of Pseudomonas aeruginosa from hospitalized patients: a comparison of susceptibility and biochemical profiles with genotype

Typing techniques are essential for understanding hospital epidemiology, permitting the elucidation of the source of infection and routes of bacterial transmission. Although DNA-based techniques are the "gold standard" for the epidemiological study of Pseudomonas aeruginosa, antibiotic profiles and biochemical results are used because they are easy to perform and to interpret and relatively inexpensive. Antibiotypes (susceptibility profiles) and biotypes (biochemical profiles) were compared to genotypes established by DNA restriction enzyme analysis in 81 clinical isolates of P. aeruginosa from three hospitals in Porto Alegre, Brazil. The epidemiological relationship among patients was also evaluated. Susceptibility and restriction profiles were discrepant in more than 50% of the cases, and many antibiotypes were observed among isolates from the same genotype. Furthermore, susceptibility profiles did not allow the distinction of isolates from unrelated genotypes. Since a large number of isolates (63%) yielded the same biochemical results, only 10 biotypes were detected, showing that this typing method has a low discriminatory power. On the other hand, DNA restriction enzyme typing allowed us to establish 71 distinct types. Epidemiological data about the relation among P. aeruginosa isolates were not conclusive. The results of the present study indicate that the only method that can establish a clonal relation is DNA restriction enzyme typing, whereas the other methods may cause misleading interpretations and are inadequate to guide proper infection control measures.

Characterization of bacteriophages infecting clinical isolates of Pseudomonas aeruginosa stored in a culture collection

Some clinical isolates of Pseudomonas aeruginosa stored in our culture collection did not grow or grew poorly and showed lysis on the culture plates when removed from the collection and inoculated on MacConkey agar. One hypothesis was that bacteriophages had infected and killed those clinical isolates. To check the best storage conditions to maintain viable P. aeruginosa for a longer time, clinical isolates were stored at various temperatures and were grown monthly. We investigated the presence of phage in 10 clinical isolates of P. aeruginosa stored in our culture collection. Four strains of P. aeruginosa were infected by phages that were characterized by electron microscopy and isolated to assess their ability to infect. The best condition to maintain the viability of the strains during storage was in water at room temperature. Three Siphoviridae and two Myoviridae phages were visualized and characterized by morphology. We confirmed the presence of bacteriophages infecting clinical isolates, and their ability to infect and lyse alternative hosts. Strain PAO1, however, did not show lysis to any phage. Mucoid and multidrug resistant strains of P. aeruginosa showed lysis to 50% of the phages tested.

Variação da microbiota natural e de pseudomonas aeruginosa em água mineral não carbonatada embalada em diferentes materiais durante o armazenamento a 30°c ± 1°c

Garrafas de PVC, polipropileno e vidro adequadamente limpas e sanificadas foram enxaguadas e enchidas com água mineral de uma fonte localizada no município de Lindóia-SP, e a seguir armazenadas a 30°C ± 1°C. Foram enumerados os microrganismos heterotróficos totais nos meios Ágar Padrão para Contagem e Ágar R2A imediatamente ap��s engarrafamento. A variação da contaminação foi seguida através de exames periódicos. A variação da população de Pseudomonas aeruginosa foi estudada inoculando garrafas contendo água mineral com uma suspensão de P. aeruginosa ATCC 10145 de maneira a se obter uma contaminação de aproximadamente 10² UFC/ml. A população da bactéria foi avaliada periodicamente durante o tempo de armazenamento usando o meio Ágar Pseudomonas P. Houve aumento da população dos microrganismos heterotróficos totais nos primeiros 30 dias de armazenamento para depois diminuir de maneira irregular e ficar aproximadamente constante até completar 6 meses de observação. As contagens de P. aeruginosa aumentaram sensivelmente nas 2 primeiras semanas de estocagem diminuindo ligeiramente a seguir até atingir níveis próximos ao inicial. Não foram constatadas diferenças entre os 3 tipos de materiais de embalagem comparados.

Effets des fluorures sur l'activité antimicrobienne des antibiotiques contre Staphylococcus aureus et Pseudomonas aeruginosa

L’émergence des souches bactériennes résistantes aux antibiotiques est un phénomène inquiétant, qui se répand à travers le monde. Staphylococcus aureus et Pseudomonas aeruginosa sont des bactéries pathogènes opportunistes multi résistantes qui peuvent causer plusieurs maladies. Cependant, ces bactéries deviennent difficiles à traiter avec des antibiotiques sans occasionner de toxicité. Alors pour trouver des solutions, c’est nécessaire de développer de nouvelles molécules afin de combattre les agents pathogènes résistants. Grâce à leur action pharmacologique, les fluorures exercent un certain effet antibactérien au niveau de l'émail des dents; donc, leur association aux antibiotiques pourrait bien a méliorer l’activité antimicrobienne. De ce fait, nous nous sommes proposés d’étudier les activités in vitro de la vancomycine (VAN), l’oxacilline (OXA), la ceftazidime (CFT) et la méropenème (MER) libre ou associée au fluorure de sodium (NaF) et fluorure de lithium (LiF) qui ont été évaluées sur des souches S.aureus et P.aeruginosa sensibles et résistantes, par la méthode de la microdilution en bouillon, déterminant leur concentration minimale inhibitrice (CMI), leur concentration minimale bactéricide (CMB), leur courbe cinétique (Time-Kill). Leur cytotoxicité sur les globules rouges humains, et leur stabilité à la temp��rature de 4°C et 22°C ont été étudiées. Les associations des antimicrobiens aux dérivés des fluorures ont montré une amélioration de l’effet des antibiotiques par la réduction des leurs concentrations et toxicité pour traiter correctement ces pathogènes résistants. Par conséquent, des antibiotiques associés aux dérivés de fluorure pourraient devenir une option de traitement contre des souches résistantes afin de diminuer la toxicité causée par de fortes doses des antibiotiques conventionnels.