Efficacy of an eradication protocol in case of Pseudomonas aeruginosa primo-infections in children with cystic fibrosis

Introduction: Patients with cystic fibrosis (CF) are more susceptible to pathogens like P. aeruginosa (PA). PA primo-­‐infections require particular attention, as with failure in eradication, there is 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 the best is. Methods: Retrospective single centre study with data analysis from June 1st 2007 to June 1st 2011 of patients who had primo-­‐infections 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 were included in our analysis. 7 girls (38.9%) and 11 boys (61.1%) followed the eradication protocol. Boys had 12 primo-­‐infections and girls had 8. Among these 20 primo-­‐infections, 16 (80%) had an all-­‐overall success in eradication and 4 (20%) a failure. No significant statistical difference for age between these groups (t-­‐test = 0.07, p = 0.94), neither for FEV1% (t-­‐test = 0.96, p = 0.41) nor 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 is 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.

Mechanisms of antagonism of Pseudomonas fluorescens EPS62e against Erwinia amylovora, the causal agent of fire blight

Pseudomonas fluorescens EPS62e was selected during a screening procedure for its high efficacy in controlling infections by Erwinia amylovora, the causal agent of fire blight disease, on different plant materials. In field trials carried out in pear trees during bloom, EPS62e colonized flowers until the carrying capacity, providing a moderate efficacy of fire-blight control. The putative mechanisms of EPS62e antagonism against E. amylovora were studied. EPS62e did not produce antimicrobial compounds described in P. fluorescens species and only developed antagonism in King’s B medium, where it produced siderophores. Interaction experiments in culture plate wells including a membrane filter, which physically separated the cultures, confirmed that inhibition of E. amylovora requires cell-to-cell contact. The spectrum of nutrient assimilation indicated that EPS62e used significantly more or different carbon sources than the pathogen. The maximum growth rate and affinity for nutrients in immature fruit extract were higher in EPS62e than in E. amylovora, but the cell yield was similar. The fitness of EPS62e and E. amylovora was studied upon inoculation in immature pear fruit wounds and hypanthia of intact flowers under controlled-environment conditions. When inoculated separately, EPS62e grew faster in flowers, whereas E. amylovora grew faster in fruit wounds because of its rapid spread to adjacent tissues. However, in preventive inoculations of EPS62e, subsequent growth of EPS101 was significantly inhibited. It is concluded that cell-to-cell interference as well as differences in growth potential and the spectrum and efficiency of nutrient use are mechanisms of antagonism of EPS62e against E. amylovora

Compact portable biosensor for arsenic detection in aqueous samples with Escherichia coli bioreporter cells.

We present a compact portable biosensor to measure arsenic As(III) concentrations in water using Escherichia coli bioreporter cells. Escherichia coli expresses green fluorescent protein in a linearly dependent manner as a function of the arsenic concentration (between 0 and 100 μg/L). The device accommodates a small polydimethylsiloxane microfluidic chip that holds the agarose-encapsulated bacteria, and a complete optical illumination/collection/detection system for automated quantitative fluorescence measurements. The device is capable of sampling water autonomously, controlling the whole measurement, storing and transmitting data over GSM networks. We demonstrate highly reproducible measurements of arsenic in drinking water at 10 and 50 μg/L within 100 and 80 min, respectively.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr.

The Pseudomonas aeruginosa gene anr, which encodes a structural and functional analog of the anaerobic regulator Fnr in Escherichia coli, was mapped to the SpeI fragment R, which is at about 59 min on the genomic map of P. aeruginosa PAO1. Wild-type P. aeruginosa PAO1 grew under anaerobic conditions with nitrate, nitrite, and nitrous oxide as alternative electron acceptors. An anr deletion mutant, PAO6261, was constructed. It was unable to grow with these alternative electron acceptors; however, its ability to denitrify was restored upon the introduction of the wild-type anr gene. In addition, the activities of two enzymes in the denitrification pathway, nitrite reductase and nitric oxide reductase, were not detectable under oxygen-limiting conditions in strain PAO6261 but were restored when complemented with the anr+ gene. These results indicate that the anr gene product plays a key role in anaerobically activating the entire denitrification pathway.

A repeated GGA motif is critical for the activity and stability of the riboregulator RsmY of Pseudomonas fluorescens.

The riboregulator RsmY of Pseudomonas fluorescens strain CHA0 is an example of small regulatory RNAs belonging to the global Rsm/Csr regulatory systems controlling diverse cellular processes such as glycogen accumulation, motility, or formation of extracellular products in various bacteria. By binding multiple molecules of the small regulatory protein RsmA, RsmY relieves the negative effect of RsmA on the translation of several target genes involved in the biocontrol properties of strain CHA0. RsmY and functionally related riboregulators have repeated GGA motifs predicted to be exposed in single-stranded regions, notably in the loops of hairpins. The secondary structure of RsmY was corroborated by in vivo cleavage with lead acetate. RsmY mutants lacking three or five (out of six) of the GGA motifs showed reduced ability to derepress the expression of target genes in vivo and failed to bind the RsmA protein efficiently in vitro. The absence of GGA motifs in RsmY mutants resulted in reduced abundance of these transcripts and in a shorter half-life (< or = 6 min as compared with 27 min for wild type RsmY). These results suggest that both the interaction of RsmY with RsmA and the stability of RsmY strongly depend on the GGA repeats and that the ability of RsmY to interact with small regulatory proteins such as RsmA may protect this RNA from degradation.

A Th17- and Th2-skewed Cytokine Profile in Cystic Fibrosis Lungs Represents a Potential Risk Factor for Pseudomonas aeruginosa Infection.

Rationale: Cystic fibrosis (CF) is characterized by progressive pulmonary inflammation that is infection-triggered. Pseudomonas aeruginosa represents a risk factor for deterioration of lung function and reduced life expectancy. Objectives: To assess T-cell cytokine/chemokine production in clinically stable children with CF and evaluate the association between T-cell subtypes and susceptibility for infection with P. aeruginosa. Methods: T-cell cytokine/chemokine profiles were measured in bronchoalveolar lavage fluid (BALF) from children with CF (n = 57; 6.1 ± 5.9 yr) and non-CF control subjects (n = 18; 5.9 ± 4.3 yr). Memory responses to Aspergillus fumigatus and P. aeruginosa were monitored. High-resolution computed tomography-based Helbich score was assessed. In a prospective observational trial the association between BALF cytokine/chemokine profiles and subsequent infection with P. aeruginosa was studied. Measurements and Main Results: Th1- (INF-γ), Th2- (IL-5, IL-13), Th17- (IL-17A), and Th17-related cytokines (IL-1β, IL-6) were significantly up-regulated in airways of patients with CF. IL-17A, IL-13, and IL-5 were significantly higher in BALF of symptomatic as compared with clinically asymptomatic patients with CF. IL-17A and IL-5 correlated with the percentage of neutrophils in BALF (r = 0.41, P < 0.05 and r = 0.46, P < 0.05, respectively). Th17- (IL-17A, IL-6, IL-1β, IL-8) and Th2-associated cytokines and chemokines (IL-5, IL-13, TARC/CCL17), but not IFN-γ levels, significantly correlated with high-resolution computed tomography changes (Helbich score; P < 0.05). P. aeruginosa- and A. fumigatus-specific T cells from patients with CF displayed significantly higher IL-5 and IL-17A mRNA expression. IL-17A and TARC/CCL17 were significantly augmented in patients that developed P. aeruginosa infection within 24 months. Conclusions: We propose a role for Th17 and Th2 T cells in chronic inflammation in lungs of patients with CF. High concentrations of these cytokines/chemokines in CF airways precede infection with P. aeruginosa.

Use of green fluorescent protein-based reporters to monitor balanced production of antifungal compounds in the biocontrol agent Pseudomonas fluorescens CHA0.

AIMS: To develop reporter constructs based on stable and unstable variants of the green fluorescent protein (GFP) for monitoring balanced production of antifungal compounds that are crucial for the capacity of the root-colonizing Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogenic fungi. METHODS AND RESULTS: Pseudomonas fluorescens CHA0 produces the three antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT) and pyrrolnitrin (PRN). The gfp[mut3] and gfp[AAV] reporter genes were fused to the promoter regions of the DAPG, PLT and PRN biosynthetic genes. The reporter fusions were then used to follow the kinetics of expression of the three antifungal metabolites in a microplate assay. DAPG and PLT were found to display an inverse relationship in which each metabolite activates its own biosynthesis while repressing the synthesis of the other metabolite. PRN appears not to be involved in this balance. However, the microbial and plant phenolic metabolite salicylate was found to interfere with the expression of both DAPG and PLT. CONCLUSIONS: The results obtained provide evidence that P. fluorescens CHA0 may keep the antifungal compounds DAPG and PLT at a fine-tuned balance that can be affected by certain microbial and plant phenolics. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, the present study is the first to use stable and unstable GFP variants to study antibiotic gene expression in a biocontrol pseudomonad. The developed reporter fusions will be a highly valuable tool to study in situ expression of this bacterial biocontrol trait on plant roots, i.e. at the site of pathogen suppression.

Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1.

Virulence in the opportunistic human pathogen Pseudomonas aeruginosa is controlled by cell density via diffusible signalling molecules ('autoinducers') of the N-acylhomoserine lactone (AHL) type. Two Bacillus sp. isolates (A23 and A24) with AHL-degrading activity were identified among a large collection of rhizosphere bacteria. From isolate A24 a gene was cloned which was similar to the aiiA gene, encoding an AHL lactonase in another Bacillus strain. Expression of the aiiA homologue from isolate A24 in P. aeruginosa PAO1 reduced the amount of the quorum sensing signal N-oxododecanoyl-L-homoserine lactone and completely prevented the accumulation of the second AHL signal, N-butyryl-L-homoserine lactone. This strongly reduced AHL content correlated with a markedly decreased expression and production of several virulence factors and cytotoxic compounds such as elastase, rhamnolipids, hydrogen cyanide and pyocyanin, and strongly reduced swarming. However, no effect was observed on flagellar swimming or on twitching motility, and aiiA expression did not affect bacterial adhesion to a polyvinylchloride surface. In conclusion, introduction of an AHL degradation gene into P. aeruginosa could block cell-cell communication and exoproduct formation, but failed to interfere with surface colonization.

Domain shuffling in a sensor protein contributed to the evolution of insect pathogenicity in plant-beneficial Pseudomonas protegens.

Pseudomonas protegens is a biocontrol rhizobacterium with a plant-beneficial and an insect pathogenic lifestyle, but it is not understood how the organism switches between the two states. Here, we focus on understanding the function and possible evolution of a molecular sensor that enables P. protegens to detect the insect environment and produce a potent insecticidal toxin specifically during insect infection but not on roots. By using quantitative single cell microscopy and mutant analysis, we provide evidence that the sensor histidine kinase FitF is a key regulator of insecticidal toxin production. Our experimental data and bioinformatic analyses indicate that FitF shares a sensing domain with DctB, a histidine kinase regulating carbon uptake in Proteobacteria. This suggested that FitF has acquired its specificity through domain shuffling from a common ancestor. We constructed a chimeric DctB-FitF protein and showed that it is indeed functional in regulating toxin expression in P. protegens. The shuffling event and subsequent adaptive modifications of the recruited sensor domain were critical for the microorganism to express its potent insect toxin in the observed host-specific manner. Inhibition of the FitF sensor during root colonization could explain the mechanism by which P. protegens differentiates between the plant and insect host. Our study establishes FitF of P. protegens as a prime model for molecular evolution of sensor proteins and bacterial pathogenicity.

Molecular epidemiology of pseudomonas aeruginosae in intensive care units (ICUs) over a 10-year period (1998-2007)

Pseudomonas aeruginosa, une bactérie environnementale ubiquitaire, est un des pathogènes nosocomiaux les plus fréquents aux soins intensifs. La source de ce microorganisme peut être soit endogène, 2,6 à 24 % des patients hospitalisés étant colonisés au niveau digestif, soit exogène. La proportion des cas d'infections à P. aeruginosa d'origine exogène, donc secondaires à une transmission par manuportage ou par l'eau du réseau utilisée pour la toilette ou d'autres soins, reste débattue. Or une meilleure évaluation du taux d'infections exogènes est importante pour la mise en place de mesures de contrôle appropriées. Le but de cette étude était de déterminer sur une période de 10 ans les rôles respectifs des sources exogènes (robinets, autres patients) et endogène dans la colonisation et/ou l'infection par P.aeruginosa chez les patients des Soins Intensifs, ainsi que de documenter les variations épidémiologiques au cours du temps. L'étude a été menée dans les unités de Soins Intensifs du Centre Hospitalier Universitaire Vaudois (CHUV). Les patients colonisés et/ou infectés par P. aeruginosa entre 1998 et 2007ont été identifiés via la base de données du laboratoire de microbiologie. Ils ont été inclus dans l'étude s'ils étaient hospitalisés dans une des unités de Soins Intensifs, Durant cette période, des prélèvements pour recherche de P. aeruginosa ont été effectués sur des robinets des soins intensifs. Un typage moléculaire a été effectué sur toutes les souches cliniques et environnementales isolées en 1998, 2000, 2003, 2004 et 2007. Les patients inclus dans l'étude ont été répartis en quatre catégories (A-D) selon le résultat du typage moléculaire leur souche de P. aeruginosa. La catégorie A inclut les cas pour lesquels le génotype de P. aeruginosa est identique à un des génotypes retrouvé dans l'environnement. La catégorie B comprend les cas pour lesquels le génotype est identique à celui d'au moins un autre patient. La catégorie C comprend les cas avec un génotype unique et la catégorie D comprend les cas pour lesquels la souche était non disponible pour le typage. Les cas des catégories A et B sont considérés comme ayant une origine exogène. Au cours des années de l'étude, le nombre d'admissions aux soins intensifs est resté stable. En moyenne, 86 patients par année ont été identifiés colonisés ou infectés par P. aeruginosa aux Soins Intensifs. Durant la première année d'investigation, un grand nombre de patients colonisés par une souche de P. aeruginosa identique à une de celles retrouvées dans l'environnement a été mis en évidence. Par la suite, possiblement suite à l'augmentation de la température du réseau d'eau chaude, le nombre de cas dans la catégorie A a diminué. Dans la catégorie B, le nombre de cas varie de 1,9 à 20 cas/1000 admissions selon les années. Ce nombre est supérieur à 10 cas/1000 admissions en 1998, 2003 et 2007 et correspond à des situations épidémiques transitoires. Tout au long des 10 ans de l'étude, le nombre de cas dans la catégorie C (source endogène) est demeuré stable et indépendant des variations du nombre de cas dans les catégories A et B. En conclusion, la contribution relative des réservoirs endogène et exogène dans la colonisation et/ou l'infection des patients de soins Intensifs varie au cours du temps. Les facteurs principaux qui contribuent à de telles variations sont probablement le degré de contamination de l'environnement, la compliance des soignants aux mesures de contrôle des infections et la génétique du pathogène lui-même. Etant donné que ce germe est ubiquitaire dans l'environnement aqueux et colonise jusqu'à 15% des patients hospitalisés, la disparition de son réservoir endogène semble difficile. Cependant, cette étude démontre que son contrôle est possible dans l'environnement, notamment dans les robinets en augmentant la température de l'eau. De plus, si une souche multi-résistante est retrouvée de manière répétée dans l'environnement, des efforts doivent être mis en place pour éliminer cette souche. Des efforts doivent être également entrepris afin de limiter la transmission entre les patients, qui est une cause importante et récurrente de contamination exogène. - 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. aeru¬ginosa, 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 cat¬egories A and Β 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 Β varied from 1.9 to 20 cases per 1000 admissions. This num¬ber exceeded 10/1000 admissions on three occasions and was correlated with an outbreak on one occasion. The number of cases con¬sidered 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.

Ferripyochelin uptake genes are involved in pyochelin-mediated signalling in Pseudomonas aeruginosa.

In response to iron starvation, Pseudomonas aeruginosa produces the siderophore pyochelin. When secreted to the extracellular environment, pyochelin chelates iron and transports it to the bacterial cytoplasm via its specific outer-membrane receptor FptA and the inner-membrane permease FptX. Exogenously added pyochelin also acts as a signal which induces the expression of the pyochelin biosynthesis and uptake genes by activating PchR, a cytoplasmic regulatory protein of the AraC/XylS family. The importance of ferripyochelin uptake genes in this regulation was evaluated. The fptA and fptX genes were shown to be part of the fptABCX ferripyochelin transport operon, which is conserved in Burkholderia sp. and Rhodospirillum rubrum. The fptB and fptC genes were found to be dispensable for utilization of pyochelin as an iron source, for signalling and for pyochelin production. By contrast, mutations in fptA and fptX not only interfered with pyochelin utilization, but also affected signalling and diminished siderophore production. It is concluded from this that pyochelin-mediated signalling operates to a large extent via the ferripyochelin transport system.

Inactivation of the regulatory gene algU or gacA can affect the ability of biocontrol Pseudomonas fluorescens CHA0 to persist as culturable cells in nonsterile soil.

Rifampin-resistant Pseudomonas fluorescens CHA0-Rif and mutants in which the regulatory gene algU (encoding sigma factor sigma(E)) or gacA (encoding a global regulator of secondary metabolism) was inactivated were compared for persistence in three nonsterile soils. Functional algU and (particularly) gacA were needed for CHA0-Rif to maintain cell culturability in soil.

Biotic Factors Affecting Expression of the 2,4-Diacetylphloroglucinol Biosynthesis Gene phlA in Pseudomonas fluorescens Biocontrol Strain CHA0 in the Rhizosphere.

ABSTRACT Production of the polyketide antimicrobial metabolite 2,4-diacetyl-phloroglucinol (DAPG) is a key factor in the biocontrol activity of Pseudomonas fluorescens CHA0. Strain CHA0 carrying a translational phlA'-'lacZ fusion was used to monitor expression of the phl biosynthetic genes in vitro and in the rhizosphere. Expression of the reporter gene accurately reflected actual production of DAPG in vitro and in planta as determined by direct extraction of the antimicrobial compound. In a gnotobiotic system containing a clay and sand-based artificial soil, reporter gene expression was significantly greater in the rhizospheres of two monocots (maize and wheat) compared with gene expression in the rhizospheres of two dicots (bean and cucumber). We observed this host genotype effect on bacterial gene expression also at the level of cultivars. Significant differences were found among six additional maize cultivars tested under gnotobiotic conditions. There was no difference between transgenic maize expressing the Bacillus thuringiensis insecticidal gene cry1Ab and the near-isogenic parent line. Plant age had a significant impact on gene expression. Using maize as a model, expression of the phlA'-'lacZ reporter gene peaked at 24 h after planting of pregerminated seedlings, and dropped to a fourth of that value within 48 h, remaining at that level throughout 22 days of plant growth. Root infection by Pythium ultimum stimulated bacterial gene expression on both cucumber and maize, and this was independent of differences in rhizosphere colonization on these host plants. To our knowledge, this is the first comprehensive evaluation of how biotic factors that commonly confront bacterial inoculants in agricultural systems (host genotype, host age, and pathogen infection) modulate the expression of key biocontrol genes for disease suppression.