Ac Champion

Increased airway iron as a potential factor in the persistence of Pseudomonas aeruginosa infection in cystic fibrosis

Iron availability is critical to many bacteria and increased iron has been described in airway secretions in cystic fibrosis (CF). The main aim of the present study was to assess the relationship between iron in CF sputum and the quantitative bacterial burden. Iron, ferritin and total cell counts (TCC) were assessed in sputum samples obtained from 15 clinically stable CF patients chronically infected with Pseudomonas aeruginosa. Sputum samples were also obtained at the commencement of episodes of acute exacerbation in 10 subjects and analyses were repeated in six of these exacerbation cases after i.v. antibiotic treatment. The relationship between iron indices and the presence of P. aeruginosa, as well as total anaerobic bacterial load, was determined. Sputum was also obtained from 10 CF patients with no evidence of infection with P. aeruginosa and 11 normal healthy controls. Sputum iron, ferritin and TCC were significantly elevated in all CF patients, even in those not infected with P. aeruginosa, compared with healthy controls. There was a strong positive relationship between sputum iron and P. aeruginosa in clinically stable patients, but not in samples obtained during an acute exacerbation. There was no relationship between sputum iron and anaerobic bacterial load. Antibiotic treatment significantly reduced sputum TCC and anaerobic bacterial load, but not iron, ferritin or the presence of P. aeruginosa during an exacerbation. In conclusion, the present study suggests that increased airway iron may be important to Pseudomonas aeruginosa persistence in cystic fibrosis.

Poor clinical outcomes associated with a multi-drug resistant clonal strain of Pseudomonas aeruginosa in the Tasmanian cystic fibrosis population

Background and objective:  Clonal strains of Pseudomonas aeruginosa have been identified in large cystic fibrosis (CF) centres. Whether such strains are more virulent or whether cross‐infection between patients explains their widespread prevalence is unknown. This study described the epidemiology of P. aeruginosa infection in CF patients in Tasmania, Australia, an area with a high CF birth incidence. Patients in Tasmania are geographically dispersed and when this study was conducted (2003) there was no central CF clinic, with patients receiving treatment in regional hospitals.

Virulence gene distribution in clinical, nosocomial and environmental isolates of Pseudomonas aeruginosa.

The virulence factor genotypes of a large cohort of clinical, nosocomial environment and community environment isolates (184 in total) of Pseudomonas aeruginosa from Tasmania, Australia, were determined by PCR. The virulence factor genotype of the majority of isolates was highly conserved, with the exception of the virulence gene exoU, which demonstrated low prevalence (33 isolates; 18 %) in the population tested. Isolates collected from the environment of intensive therapy wards (intensive care unit and neurosurgical units) of the major tertiary referral hospital in Tasmania were found to be more likely (P<0.001 and P<0.05, respectively) to possess the virulence factor gene exoU than all other isolates. Adult cystic fibrosis isolates showed a decreased prevalence of the exoU gene (P<0.01) when compared to other clinical isolates (P<0.01), which may indicate decreased virulence. No specific virulence factor genotype was associated with the cystic fibrosis epidemic strains tested.

Bacterial cyanogenesis occurs in the cystic fibrosis lung

The cystic fibrosis (CF) lung environment is poorly defined, but data suggest that bacteria may encounter reduced oxygen tensions and possibly an anaerobic environment. Pseudomonas aeruginosa produces the potent toxin cyanide under strictly microaerobic conditions. Evidence of bacterial cyanogenesis in the CF lung was investigated in the present study by measuring sputum cyanide concentrations. Sputum cyanide was measured in seven stable CF patients, as well as before and after intravenous antibiotic therapy during a hospital admission in a further eight patients experiencing acute exacerbations. All patients were chronically infected with P. aeruginosa. Comparative sputum data were obtained from nine CF patients with no documented P. aeruginosa infection and 10 healthy, nonsmoking normal controls. High levels of cyanide were detected in all the P. aeruginosa-infected stable CF patients (median (range) 0.56 (0.37–2.81) μg·mL−1), and in seven out of eight acute sputum samples (0.73 (0–1.43) μg·mL−1). In contrast, cyanide was not detectable in sputum from eight out of nine CF patients without P. aeruginosa infection or in any of the normal controls. Intravenous antibiotic treatment significantly reduced sputum cyanide levels (median 0.73 to median 0.0 µg·mL−1). The cyanide detected indicates that the cystic fibrosis lung provides a predominantly microaerobic environment for Pseudomonas aeruginosa. Cyanide is likely to be a potentially important virulence factor in Pseudomonas aeruginosa-infected cystic fibrosis patients.

Epidemiology of Pseudomonas aeruginosa in a tertiary referral teaching hospital.

A genotypically indistinguishable strain of Pseudomonas aeruginosa (Australian epidemic strain III: AES III) has previously been found in a proportion of adults with cystic fibrosis (CF) in Tasmania, Australia. The aim of this study was to identify a source of these infections within the major tertiary referral hospital for the State of Tasmania, and to determine if this strain could be isolated from settings other than the CF lung. A total of 120 isolates of P. aeruginosa were collected from clinical and environmental sources within the hospital and from environmental locations in the hospital vicinity. These isolates were genotyped by random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR) and antimicrobial susceptibility testing was performed using the Clinical and Laboratory Standards Institute method. Confirmation of similar genotypes identified by RAPD-PCR was performed using pulsed-field gel electrophoresis with restriction enzyme SpeI. AES III was not recovered from any source other than the respiratory secretions of CF patients. P. aeruginosa in the non-CF settings was found to be panmictic, and no cross-infection or acquisition of hospital environment strains by patients was observed.

Sputum neutrophils in cystic fibrosis patients display a reduced respiratory burst

BACKGROUND Few data exist on the functional activity of airway neutrophils in the milieu of the cystic fibrosis (CF) lung. We assessed reactive oxygen species (ROS) production by sputum neutrophils and the relationship to neutrophil viability. Identical assessments were made on peripheral blood neutrophils from CF patients. METHODS ROS production in sputum neutrophils was assessed in 31 CF patients at varying phases of clinical disease using flow cytometry. Twenty patients provided blood samples (including 16 who also provided a matched sputum sample). Neutrophil viability was determined using dual annexin V (apoptosis) and propidium iodide (necrosis) staining. Comparative peripheral blood data were obtained from 7 healthy controls. RESULTS ROS production was reduced in sputum compared to blood neutrophils and they demonstrated a higher level of necrosis. Subpopulations of neutrophils with different ROS production capacity were apparent in peripheral blood. Lung function was positively associated with both the proportion of blood neutrophils demonstrating increased ROS production and the proportion of apoptotic sputum neutrophils. CONCLUSIONS CF airway neutrophils display functional exhaustion. Healthier lungs in CF appear to be associated with subpopulations of blood neutrophils with increased oxidative burst capacity and evidence for increased neutrophil apoptosis within the airway.

Decreased virulence of cystic fibrosis Pseudomonas aeruginosa in Dictyostelium discoideum

The characteristics of clinical and environmental isolates of Pseudomonas aeruginosa from both hospital and community settings were analyzed in a eukaryotic virulence model employing the AX2 and X22 mutants of Dictyostelium discoideum. Thirty‐one strains, including two Australian epidemic strains, of P. aeruginosa were analyzed, five from environmental sources, six from clinical sources other than cystic fibrosis (CF) patients and nineteen from CF patients’ respiratory secretions. The majority of CF isolates almost uniquely supported the growth of D. discoideum. CF isolates of P. aeruginosa were found to be less virulent than isolates from other sources. Varying degrees of inhibition of the developmental cycle of D. discoideum when growing on CF isolates were also noted. This is the first description of P. aeruginosa isolates from clinical and environmental sources supporting the growth of D. discoideum.

Antimicrobial susceptibility testing of cystic fibrosis and non-cystic fibrosis clinical isolates of Pseudomonas aeruginosa: a comparison of three methods

Abstract Pseudomonas aeruginosa is an important pathogen in humans, particularly in the context of nosocomial infection and infections of the cystic fibrosis (CF) lung. In order to provide clinicians with information about the likely effectiveness of specific antimicrobial treatment for P. aeruginosa infections, clinical laboratories employ in vitro antimicrobial susceptibility testing. Two commonly employed methods are the CLSI disc-diffusion and Etest methods. The purpose of this study is to compare the accuracy of susceptibility results generated by these two methods against agar dilution as the reference method. Susceptible or nonsusceptible (resistant and intermediate) results of the Etest and CLSI disc-diffusion methods are compared with CLSI agar dilution results for a large cohort of clinical cystic fibrosis (n=71) and non-cystic fibrosis (n=83) isolates using CLSI interpretive criteria. An unacceptable number of major and very major errors were observed for various antimicrobials tested against both CF and non-CF isolates when using the Etest and CLSI disc-diffusion methods. The potential for error in standard laboratory antimicrobial susceptibility testing should be considered by clinicians when being guided by the results of such tests in the prescription of antimicrobial agents for P. aeruginosa infection.

Preliminary feasibility and modelling of a liquid matrix Dictyostelium discoideum virulence assay for Pseudomonas aeruginosa

Abstract Objectives: To develop and determine the feasibility of using a liquid matrix adaptation of the Dictyostelium discoideum bacterial virulence assay by testing on well-characterised clinical and environmental isolates of Pseudomonas aeruginosa. Materials and methods: Axenic AX2 D. discoideum were co-cultured with clinical and environmental isolates of P. aeruginosa in costar 24-well tissue culture plates for 24 h. A P. aeruginosa PAO1 positive control was tested in biological quintuplicate. Wells were then inspected using an inverted microscope and the degree of cytotoxic changes (sparse growth compared to control combined with rounding of cells and cytoplasmic shrinkage) on the D. discoideum cells was observed. A Klebsiella aerogenes negative control was included with each assay series. Results: Sixty-five clinical and 20 environmental P. aeruginosa isolates were tested in the model. Cystic fibrosis respiratory isolates were found to be significantly (P < 0.05) less cytotoxic than P. aeruginosa from other sources. Limitations attached to the funding of this paper did not allow validation against previously employed models or animal models. Discussion: A liquid matrix D. discoideum model for the analysis of P. aeruginosa virulence in a eukaryotic host is feasible, but further validation of the model is required before it may be employed in routine setting.

Urease production as a marker of virulence in Pseudomonas aeruginosa

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