lactoferrin and Pseudomonas-Infections

The aerobic Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen responsible for life-threatening acute and chronic infections in humans. As part of chronic infection P. aeruginosa forms biofilms, which shield the encased bacteria from host immune clearance and provide an impermeable and protective barrier against currently available antimicrobial agents. P. aeruginosa has an absolute requirement for iron for infection success. By influencing cell-cell communication (quorum sensing) and virulence factor expression, iron is a powerful regulator of P. aeruginosa behaviour. Consequently, the imposed perturbation of iron acquisition systems has been proposed as a novel therapeutic approach to the treatment of P. aeruginosa biofilm infection. In this review, we explore the influence of iron availability on P. aeruginosa infection in the lungs of the people with the autosomal recessive condition cystic fibrosis as an archetypal model of chronic P. aeruginosa biofilm infection. Novel therapeutics aimed at disrupting P. aeruginosa are discussed, with an emphasis placed on identifying the barriers that need to be overcome in order to translate these promising in vitro agents into effective therapies in human pulmonary infections.

Topics: Anti-Infective Agents; Biofilms; Chelating Agents; Cystic Fibrosis; Gene Expression Regulation, Bacterial; Homeostasis; Humans; Iron; Lactoferrin; Lung; Pseudomonas aeruginosa; Pseudomonas Infections; Quorum Sensing; Respiratory Tract Infections; Thiocyanates

Topics: Anti-Bacterial Agents; Anti-Infective Agents; Cells, Cultured; Dose-Response Relationship, Drug; Drug Synergism; Durapatite; Humans; Lactoferrin; Nanoparticles; Pseudomonas fluorescens; Pseudomonas Infections; Quercetin; U937 Cells

Lactoferrin (Lf), an iron-chelating glycoprotein of innate immunity, produced by exocrine glands and neutrophils in infection/inflammation sites, is one of the most abundant defence molecules in airway secretions. Lf, a pleiotropic molecule, exhibits antibacterial and anti-inflammatory functions. These properties may play a relevant role in airway infections characterized by exaggerated inflammatory response, as in Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) subjects. To verify the Lf role in Pseudomonas aeruginosa lung infection, we evaluated the efficacy of aerosolized bovine Lf (bLf) in mouse models of P. aeruginosa acute and chronic lung infections. C57BL/6NCrl mice were challenged with 10

Topics: Administration, Inhalation; Aerosols; Animals; Anti-Infective Agents; Anti-Inflammatory Agents; Bronchoalveolar Lavage Fluid; Cattle; Cytokines; Disease Models, Animal; Inflammation; Inflammation Mediators; Lactoferrin; Lung Diseases; Male; Mice; Mice, Inbred C57BL; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections

Lactoferrin, a multifunctional protein with antimicrobial activity, is a component of the innate immune system. It may possibly prevent clinical isolates of Pseudomonas aeruginosa from developing biofilm, but this hypothesis is yet to be widely accepted. We evaluated the in vitro effects of lactoferrin on biofilm formation by various clinical isolates of P. aeruginosa using a modified method of the microtiter plate biofilm assay. Lactoferrin significantly inhibited biofilm formation in these isolates. The effect was the most marked at 2 mg/ml, which suggested that an optimal concentration of lactoferrin might exist. Lactoferrin inhibited biofilm formation in eight of nine clinical isolates after 1 day of incubation; however, the inhibitory effects were maintained until 7 days of incubation in only two of those eight strains. Suppression of biofilm formation may be caused by a mechanism that is independent of the bactericidal effects of lactoferrin because the number of viable bacteria was not influenced by lactoferrin under the experimental conditions. Supplementation of lactoferrin to preformed biofilm demonstrated a reduction in biofilm, which suggests that lactoferrin may have a destructive effect on biofilm. Pretreatment with ferric chloride partially restored biofilm formation, suggesting an iron-chelating action may be involved in the inhibitory mechanism of lactoferrin. These results suggest that lactoferrin provides inhibitory effects on biofilm formation in many clinical isolates of P. aeruginosa and that it may also have destructive effects on preformed biofilm, but further research using multiple clinical strains should be undertaken to clarify if those effects are universal.

Topics: Anti-Bacterial Agents; Biofilms; Cell Proliferation; Chlorides; Ferric Compounds; Humans; Lactoferrin; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Pseudomonas Infections

To determine the feasibility of formulating and aerosolizing powders containing bacteriophages KS4-M and ΦKZ for lung delivery and treatment of pulmonary Burkholderia cepacia complex and Pseudomonas aeruginosa infections.. Endotoxin-removed bacteriophages KS4-M and ΦKZ were lyophilized in lactose/lactoferrin 60 : 40 w/w matrix and deagglomerated in a mixer mill (without beads) to formulate respirable powders. The powders were then aerosolized using an Aerolizer(®) capsule inhaler. Mass median aerodynamic diameter (MMAD) of this inhalable aerosol was determined using Andersen cascade impactor at 60 l min(-1). Measured MMAD for both types of powders was 3·4 μm, and geometric standard deviation was 1·9-2·0. Viability of bacteriophages delivered distal to an idealized mouth-throat replica was determined from bioassays of samples collected on filters placed after the idealized replica. As a percentage of inhaler load, amount of powder delivered distal to the mouth-throat replica, which is a measure of lung delivery, was 33·7 ± 0·3% for KS4-M and 32·7 ± 0·9% for ΦKZ. Titres collected downstream of the mouth throat were (3·4 ± 2·5) × 10(6) PFU for KS4-M with an Aerolizer capsule load of (9·8 ± 4·8) × 10(6) and (1·9 ± 0·6) × 10(7) for ΦKZ with an Aerolizer capsule load of (6·5 ± 1·9) × 10(7).. Bacteriophages KS4-M and ΦKZ can be lyophilized without significant loss of viability in a lactose/lactoferrin 60 : 40 w/w matrix. The resulting powders can be aerosolized to deliver viable bacteriophages to the lungs..   Development of lactoferrin-based bacteriophage aerosol powders solidifies the ground for future research on developing novel formulations as an alternative to inhaled antibiotic therapy in patients with cystic fibrosis.

Topics: Aerosols; Bacteriophages; Burkholderia cepacia complex; Burkholderia Infections; Cystic Fibrosis; Dry Powder Inhalers; Freeze Drying; Humans; Lactoferrin; Lung; Nebulizers and Vaporizers; Pseudomonas aeruginosa; Pseudomonas Infections

Pseudomonas aeruginosa is naturally resistant to many antibiotics, and infections caused by this organism are a serious threat, especially to hospitalized patients. The intrinsic low permeability of P. aeruginosa to antibiotics results from the coordinated action of several mechanisms, such as the presence of restrictive porins and the expression of multidrug efflux pump systems. Our goal was to develop antimicrobial peptides with an improved bacterial membrane-permeabilizing ability, so that they enhance the antibacterial activity of antibiotics. We carried out a structure activity relationship analysis to investigate the parameters that govern the permeabilizing activity of short (8- to 12-amino-acid) lactoferricin-derived peptides. We used a new class of constitutional and sequence-dependent descriptors called PEDES (peptide descriptors from sequence) that allowed us to predict (Spearman's ρ = 0.74; P < 0.001) the permeabilizing activity of a new peptide generation. To study if peptide-mediated permeabilization could neutralize antibiotic resistance mechanisms, the most potent peptides were combined with antibiotics, and the antimicrobial activities of the combinations were determined on P. aeruginosa strains whose mechanisms of resistance to those antibiotics had been previously characterized. A subinhibitory concentration of compound P2-15 or P2-27 sensitized P. aeruginosa to most classes of antibiotics tested and counteracted several mechanisms of antibiotic resistance, including loss of the OprD porin and overexpression of several multidrug efflux pump systems. Using a mouse model of lethal infection, we demonstrated that whereas P2-15 and erythromycin were unable to protect mice when administered separately, concomitant administration of the compounds afforded long-lasting protection to one-third of the animals.

Topics: Animals; Anti-Bacterial Agents; Drug Synergism; Female; Lactoferrin; Mice; Microbial Sensitivity Tests; Peptides; Pseudomonas aeruginosa; Pseudomonas Infections

The objectives of this study were to determine whether a synergistic effect could be obtained in vitro between bovine lactoferricin (B-LFcin) and antibiotics against Pseudomonas aeruginosa and Staphylococcus aureus isolates from ocular infections, and to evaluate the use of B-LFcin as an adjunct to the antibiotic treatment of corneal infection in vivo.. Chequerboard and time-kill assays were performed to investigate the combined effects of B-LFcin and conventional antibiotics, including ciprofloxacin, ceftazidime and gentamicin, against 17 strains of P. aeruginosa (8) and S. aureus (9) isolated from ocular infection and inflammation, and 1 reference strain of S. aureus. Corneas of C57BL/6 mice were topically challenged with a multidrug-resistant strain of P. aeruginosa. Nine hours post-challenge, mice were treated topically and hourly with either vehicle, B-LFcin, ciprofloxacin or ciprofloxacin containing B-LFcin for 8 h. Corneas were then clinically examined, and bacterial numbers and levels of myeloperoxidase (MPO) evaluated.. Synergy between B-LFcin and ciprofloxacin or ceftazidime was identified in most P. aeruginosa isolates, including multidrug-resistant strains, whereas no synergistic effect was seen between B-LFcin and gentamicin. Synergy was only observed with B-LFcin and ciprofloxacin against 2/10 S. aureus strains, and there was no synergy between B-LFcin and any of the other antibiotics tested. Combined B-LFcin and ciprofloxacin treatment significantly improved the clinical outcome, and reduced bacterial numbers and MPO in infected mouse corneas. B-LFcin alone was also able to reduce levels of MPO in infected corneas.. These findings indicate that B-LFcin may have advantages as an adjunct therapy with both antimicrobial and anti-inflammatory properties in the treatment of corneal infection.

Topics: Administration, Topical; Animals; Anti-Bacterial Agents; Cattle; Corneal Diseases; Drug Synergism; Lactoferrin; Mice; Mice, Inbred C57BL; Microbial Sensitivity Tests; Microbial Viability; Pseudomonas aeruginosa; Pseudomonas Infections; Staphylococcal Infections; Staphylococcus aureus; Treatment Outcome

The medical impact of bacterial biofilms has increased with the recognition of biofilms as a major contributor to chronic wounds such as diabetic foot ulcers, venous leg ulcers and pressure ulcers. Traditional methods of treatment have proven ineffective, therefore this article presents in vitro evidence to support the use of novel antimicrobials in the treatment of Pseudomonas aeruginosa biofilm. An in vitro biofilm model with a clinical isolate of P. aeruginosa was subjected to treatment with either lactoferrin or xylitol alone or in combination. Combined lactoferrin and xylitol treatment disrupted the structure of the P. aeruginosa biofilm and resulted in a >2log reduction in viability. In situ analysis indicated that while xylitol treatment appeared to disrupt the biofilm structure, lactoferrin treatment resulted in a greater than two-fold increase in the number of permeabilised bacterial cells. The findings presented here indicated that combined treatment with lactoferrin and xylitol significantly decreases the viability of established P. aeruginosa biofilms in vitro and that the antimicrobial mechanism of this treatment includes both biofilm structural disruption and permeablisation of bacterial membranes.

Topics: Anti-Bacterial Agents; Biofilms; Cell Membrane Permeability; Drug Synergism; Humans; Lactoferrin; Microbial Viability; Pseudomonas aeruginosa; Pseudomonas Infections; Wound Infection; Xylitol

Lactoferrin-induced cell depolarization and a delayed tobramycin-killing effect on Pseudomonas aeruginosa cells were correlated. This antibiotic tolerance effect (ATE) reflects the ability of a defense protein to modify the activity of an antibiotic as a result of its modulatory effect on bacterial physiology. P. aeruginosa isolates from cystic fibrosis patients showed higher ATE values (< or = 6-fold) than other clinical strains.

Topics: Anti-Bacterial Agents; Cystic Fibrosis; Drug Resistance, Bacterial; Humans; Lactoferrin; Pseudomonas aeruginosa; Pseudomonas Infections; Tobramycin

Macrolide antibiotics have clinical benefits in patients with diffuse panbronchiolitis and in patients with cystic fibrosis. Although many mechanisms have been proposed, the precise mechanisms are still uncertain. We examined the effects of erythromycin on bactericidal activity of airway surface liquid secreted by cultured human tracheal epithelial cells. Airway surface liquid was collected by washing the surface of human tracheal epithelial cells with a sodium solution (40 meq/l). Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa were incubated with airway surface liquid, and the number of surviving bacteria was examined. The number of bacteria in airway surface liquid from the cells cultured in medium alone was significantly lower than that in the sodium solution. Furthermore, the number of bacteria in airway surface liquid from the cells treated with erythromycin was significantly lower than that in airway surface liquid from the cells treated with solvent alone. The production of mRNA and protein of human beta-defensin-1 and human beta-defensin-2 was significantly increased by erythromycin. Bactericidal activity of airway surface liquid was observed at low concentrations (40 meq/l) of sodium but not at higher concentrations (> or =80 meq/l). Airway surface liquid did not contain significant amounts of antibiotics supplemented in the culture medium. Erythromycin at the levels in airway surface liquid and in culture medium did not inhibit bacterial growth. These results suggest that erythromycin may increase bactericidal activity of airway surface liquid in human airway epithelial cells through human beta-defensins production and reduce susceptibility of the airway to bacterial infection.

Topics: Anti-Bacterial Agents; beta-Defensins; Cell Survival; Cells, Cultured; Erythromycin; Gene Expression; Humans; Lactoferrin; Methicillin Resistance; Muramidase; Pseudomonas Infections; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium; Staphylococcal Infections; Trachea

The acquisition of Pseudomonas cepacia in patients with cystic fibrosis is associated with increasing deterioration in lung function and more frequent hospital admissions. Pseudomonas cepacia is usually resistant to several antibiotics in vitro, but the response of patients colonised with the organism has not been extensively studied in vivo.. A three month prospective study was performed to investigate the response of 14 Ps cepacia positive patients and 10 Ps cepacia negative patients to a two week course of intravenous antibiotics. All those who were Ps cepacia negative and six of the 14 Ps cepacia positive patients had Ps aeruginosa in their sputum which was sensitive to the prescribed therapy. The inflammatory markers C-reactive protein, white blood cell count, serum lactoferrin, neutrophil elastase/alpha 1-antitrypsin complex, and tumour necrosis factor alpha were measured at the start and end of each antibiotic course.. The median (range) % improvement in baseline FEV1 and FVC following treatment in the group as a whole was 15.2% (-23.5% to 156.3%) and 23.9% (-36.8% to 232.7%) respectively. There was no statistical difference in improvement in lung function, body weight, or inflammatory markers between individuals who were Ps cepacia positive and those who were Ps cepacia negative.. Patients who are Ps cepacia positive appear to respond as well to intravenous antibiotics as those who are Ps cepacia negative, despite having lower lung function and a bacterium in their sputum which is resistant in vitro to the antibiotics used.

Topics: Adult; Anti-Bacterial Agents; Biomarkers; Burkholderia cepacia; C-Reactive Protein; Cystic Fibrosis; Female; Humans; Lactoferrin; Leukocyte Count; Lung; Male; Prospective Studies; Pseudomonas Infections; Tumor Necrosis Factor-alpha

Bacterium- and neutrophil-derived proteases have been suggested to contribute to tissue injury at sites of Pseudomonas aeruginosa infection. Pseudomonas elastase cleavage of transferrin enhances in vitro iron removal from this protein by the P. aeruginosa siderophore pyoverdin. This cleavage also generates new iron chelates which, in contrast to iron bound to transferrin, are able to catalyze formation of the highly cytotoxic hydroxyl radical from neutrophil-derived superoxide and hydrogen peroxide via the Haber-Weiss reaction. In order to determine whether this cleavage occurs in vivo, a chemiluminescence immunoblot system was developed to detect the presence of proteolysis products of transferrin or the related iron-binding protein, lactoferrin. Using this immunoblot system, we detected transferrin and lactoferrin cleavage products in bronchoalveolar lavage (BAL) samples from 21 of 22 and 20 of 21 cystic fibrosis (CF) patients, respectively. Three of eleven and two of nine BAL samples from individuals with other forms of chronic inflammatory lung disease had transferrin and lactoferrin cleavage products, respectively. Each patient in whom such products were detected was also infected with P. aeruginosa. No such products were detected in normal individuals. In the CF patients, there was no clear correlation between the extent of transferrin or lactoferrin cleavage and BAL neutrophil or P. aeruginosa concentration or the disease status of the patient. In contrast, in the non-CF patients with chronic inflammatory lung disease, transferrin and lactoferrin cleavage products were detected only in those BAL samples which contained the greatest concentration of both neutrophils and P. aeruginosa. These data provide evidence that P. aeruginosa- and/or human-derived protease cleavage of transferrin and lactoferrin occurs in vivo in the airways of individuals with CF and other forms of chronic lung disease, suggesting that this process could contribute to P. aeruginosa-associated lung injury in these patients.

Topics: Adult; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Endopeptidases; Humans; Lactoferrin; Middle Aged; Neutrophils; Pneumonia; Pseudomonas Infections; Transferrin