Nitrous oxide production in sputum from cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection

Chronic lung infection by Pseudomonas aeruginosa is the major severe complication in cystic fibrosis (CF) patients, where P. aeruginosa persists and grows in biofilms in the endobronchial mucus under hypoxic conditions. Numerous polymorphonuclear leukocytes (PMNs) surround the biofilms and create local anoxia by consuming the majority of O2 for production of reactive oxygen species (ROS). We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N2O), an intermediate in the denitrification pathway. We measured N2O and O2 with electrochemical microsensors in 8 freshly expectorated sputum samples from 7 CF patients with chronic P. aeruginosa infection. The concentrations of NO 3- and NO2- in sputum were estimated by the Griess reagent. We found a maximum median concentration of 41.8 μM N2O (range 1.4-157.9 μM N2O). The concentration of N2O in the sputum was higher below the oxygenated layers. In 4 samples the N2O concentration increased during the initial 6 h of measurements before decreasing for approximately 6 h. Concomitantly, the concentration of NO3- decreased in sputum during 24 hours of incubation. We demonstrate for the first time production of N2O in clinical material from infected human airways indicating pathogenic metabolism based on denitrification. Therefore, P. aeruginosa may acquire energy for growth by denitrification in anoxic endobronchial mucus in CF patients. Such ability for anaerobic growth may be a hitherto ignored key aspect of chronic P. aeruginosa infections that can inform new strategies for treatment and prevention. © 2014 Kolpen et al

The paranasal sinuses are focus for colonisation and chronic biofilm lung infection in CF patients

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Physiological levels of nitrate support anoxic growth by denitrification of Pseudomonas aeruginosa at growth rates reported in cystic fibrosis lungs and sputum

© 2014 Line, Alhede, Kolpen, Kuhl, Ciofu, Bjarnsholt, Moser, Toyofuku, Nomura, H0i'by and Jensen. Chronic Pseudomonas aeruginosa lung infection is the most severe complication in patients with cystic fibrosis (CF). The infection is characterised by the formation of biofilm surrounded by numerous polymorphonuclear leukocytes (PMNs) and strong O2 depletion in the endobronchial mucus. We have reported that O2 is mainly consumed by the activated PMNs, while O2 consumption by aerobic respiration is diminutive and nitrous oxide (N2O) is produced in infected CF sputum. This suggests that the reported growth rates ofP. aeruginosa in lungs and sputum may result from anaerobic respiration using denitrification. The growth rate of P. aeruginosa achieved by denitrification at physiological levels (~400 μM) of nitrate (NO3-) is however, not known. Therefore, we have measured growth rates of anoxic cultures of PAO1 and clinical isolates (n = 12) in LB media supplemented with NO3- and found a significant increase of growth when supplementing PAO1 and clinical isolates with > 150 μM NO3- and 100 μM NO3-, respectively. An essential contribution to growth by denitrification was demonstrated by the inability to establish a significantly increased growth rate by a denitrification deficient ΔnirS-N mutant at <1 mM of NO3-. Activation of denitrification could be achieved by supplementation with as little as 62.5 μM of NO3- according to the significant production of N2O by the nitrous oxide reductase deficient ΔnosZ mutant. Studies of the promoter activity, gene transcripts and enzyme activity of the four N-oxide reductases in PAO1 (Nar, Nir, Nor, Nos) further verified the engagement of denitrification, showing a transient increase in activation and expression and rapid consumption of NO3- followed by a transient increase of NO2-. Growth rates obtained by denitrification in this study were comparable to our reported growth rates in the majority of P. aeruginosa cells in CF lungs and sputum. Thus, we have demonstrated that denitrification is required for P. aeruginosa growth in infected endobronchial CF mucus

A systematic approach to the interrogation and sharing of standardised biofilm signatures

Publicado em "6th International Conference on Practical Applications of Computational Biology & Bioinformatics", ISBN 978-3-642-28838-8The study of microorganism consortia, also known as biofilms, is associated to a number of applications in biotechnology, ecotechnology and clinical domains. A public repository on existing biofilm studies would aid in the design of new studies as well as promote collaborative and incremental work. However, bioinformatics approaches are hampered by the limited access to existing data. Scientific publications summarise the studies whilst results are kept in researchers’ private ad hoc files. Since the collection and ability to compare existing data is imperative to move forward in biofilm analysis, the present work has addressed the development of a systematic computer-amenable approach to biofilm data organisation and standardisation. A set of in-house studies involving pathogens and employing different state-of-the-art devices and methods of analysis was used to validate the approach. The approach is now supporting the activities of BiofOmics, a public repository on biofilm signatures (http://biofomics.org).The authors thank, among others, Rosario Oliveira, Maria Joao Vieira, Idalina Machado, Nuno Cerca, Mariana Henriques, Pilar Teixeira, Douglas Monteiro, Melissa Negri, Susana Lopes, Carina Almeida and Helder Lopes, for submitting their data. The financial support from IBB-CEB, Fundacao para a Ciencia e Tecnologia (FCT) and European Community fund FEDER (Program COMPETE), project PTDC/SAU-ESA/646091/2006/FCOMP-01-0124-FEDER-007480, are also gratefully acknowledged

Phenotypes of Non-Attached Pseudomonas aeruginosa Aggregates Resemble Surface Attached Biofilm

For a chronic infection to be established, bacteria must be able to cope with hostile conditions such as low iron levels, oxidative stress, and clearance by the host defense, as well as antibiotic treatment. It is generally accepted that biofilm formation facilitates tolerance to these adverse conditions. However, microscopic investigations of samples isolated from sites of chronic infections seem to suggest that some bacteria do not need to be attached to surfaces in order to establish chronic infections. In this study we employed scanning electron microscopy, confocal laser scanning microscopy, RT-PCR as well as traditional culturing techniques to study the properties of Pseudomonas aeruginosa aggregates. We found that non-attached aggregates from stationary-phase cultures have comparable growth rates to surface attached biofilms. The growth rate estimations indicated that, independently of age, both aggregates and flow-cell biofilm had the same slow growth rate as a stationary phase shaking cultures. Internal structures of the aggregates matrix components and their capacity to survive otherwise lethal treatments with antibiotics (referred to as tolerance) and resistance to phagocytes were also found to be strikingly similar to flow-cell biofilms. Our data indicate that the tolerance of both biofilms and non-attached aggregates towards antibiotics is reversible by physical disruption. We provide evidence that the antibiotic tolerance is likely to be dependent on both the physiological states of the aggregates and particular matrix components. Bacterial surface-attachment and subsequent biofilm formation are considered hallmarks of the capacity of microbes to cause persistent infections. We have observed non-attached aggregates in the lungs of cystic fibrosis patients; otitis media; soft tissue fillers and non-healing wounds, and we propose that aggregated cells exhibit enhanced survival in the hostile host environment, compared with non-aggregated bacterial populations

The cystic fibrosis microbiome in an ecological perspective and its impact in antibiotic therapy

The recent focus on the cystic fibrosis (CF) complex microbiome has led to the recognition that the microbes can interact between them and with the host immune system, affecting the disease progression and treatment routes. Although the main focus remains on the interactions between traditional pathogens, growing evidence supports the contribution and the role of emergent species. Understanding the mechanisms and the biological effects involved in polymicrobial interactions may be the key to improve effective therapies and also to define new strategies for disease control. This review focuses on the interactions between microbe-microbe and host-microbe, from an ecological point of view, discussing their impact on CF disease progression. There are increasing indications that these interactions impact the success of antimicrobial therapy. Consequently, a new approach where therapy is personalized to patients by taking into account their individual CF microbiome is suggested.Portuguese Foundation for Science and Technology (FCT), the strategic funding of UID/BIO/04469/2013-CEB and UID/EQU/00511/2013-LEPABE units. This study was also supported by FCT and the European Community fund FEDER, through Program COMPETE, under the scope of the Projects “DNA mimics” PIC/IC/82815/2007, RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), “BioHealth—Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027 and NORTE-07-0124-FEDER-000025—RL2_ Environment and Health, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. The authors also acknowledge the grant of Susana P. Lopes (SFRH/BPD/95616/2013) and of the COST-Action TD1004: Theragnostics for imaging and therapy

Combined use of bacteriophage K and a novel bacteriophage to reduce Staphylococcus aureus biofilm formation

Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-resistant S. aureus (MRSA). Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host, releasing their progeny. We isolated a novel phage, DRA88, which has a broad host range among S. aureus bacteria. Morphologically, the phage belongs to the Myoviridae family and comprises a large double-stranded DNA (dsDNA) genome of 141,907 bp. DRA88 was mixed with phage K to produce a high-titer mixture that showed strong lytic activity against a wide range of S. aureus isolates, including representatives of the major international MRSA clones and coagulase-negative Staphylococcus. Its efficacy was assessed both in planktonic cultures and when treating established biofilms produced by three different biofilm-producing S. aureus isolates. A significant reduction of biofilm biomass over 48 h of treatment was recorded in all cases. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus biofilms