Audrey Perry

The effect of ex vivo lung perfusion on microbial load in human donor lungs

BACKGROUND Ex vivo lung perfusion (EVLP) has emerged as a technique to potentially recondition unusable donor lungs for transplantation. Beneficial effects of EVLP on physiologic function have been reported, but little is known about the effect of normothermic perfusion on the infectious burden of the donor lung. In this study, we investigated the effect of EVLP on the microbial load of human donor lungs. METHODS Lungs from 18 human donors considered unusable for transplantation underwent EVLP with a perfusate containing high-dose, empirical, broad-spectrum anti-microbial agents. Quantitative cultures of bacteria and fungi were performed on bronchoalveolar lavage fluid from the donor lung before and after 3 to 6 hours of perfusion. The identification of any organisms and changes in number of colony forming units before and after EVLP were assessed and anti-microbial susceptibilities identified. RESULTS Thirteen out of 18 lungs had positive cultures, with bacterial loads significantly decreasing after EVLP. Yeast loads increased when no anti-fungal treatment was given, but were reduced when prophylactic anti-fungal treatment was added to the circuit. Six lungs were ultimately transplanted into patients, all of whom survived to hospital discharge. There was 1 death at 11 months. CONCLUSIONS Our study shows that EVLP with high-dose, empirical anti-microbial agents in the perfusate is associated with an effective reduction in the microbial burden of the donor lung, a benefit that has not previously been demonstrated.

Polymicrobial airway bacterial communities in adult bronchiectasis patients.

BackgroundChronic airway infection contributes to the underlying pathogenesis of non-cystic fibrosis bronchiectasis (NCFBr). In contrast to other chronic airway infections, associated with COPD and CF bronchiectasis, where polymicrobial communities have been implicated in lung damage due to the vicious circle of recurrent bacterial infections and inflammation, there is sparse information on the composition of bacterial communities in NCFBr. Seventy consecutive patients were recruited from an outpatient adult NCFBr clinic. Bacterial communities in sputum samples were analysed by culture and pyrosequencing approaches. Bacterial sequences were analysed using partial least square discrimination analyses to investigate trends in community composition and identify those taxa that contribute most to community variation.ResultsThe lower airway in NCFBr is dominated by three bacterial taxa Pasteurellaceae, Streptococcaceae and Pseudomonadaceae. Moreover, the bacterial community is much more diverse than indicated by culture and contains significant numbers of other genera including anaerobic Prevotellaceae, Veillonellaceae and Actinomycetaceae. We found particular taxa are correlated with different clinical states, 27 taxa were associated with acute exacerbations, whereas 11 taxa correlated with stable clinical states. We were unable to demonstrate a significant effect of antibiotic therapy, gender, or lung function on the diversity of the bacterial community. However, presence of clinically significant culturable taxa; particularly Pseudomonas aeruginosa and Haemophilus influenzae correlated with a significant change in the diversity of the bacterial community in the lung.ConclusionsWe have demonstrated that acute exacerbations, the frequency of exacerbation and episodes of clinical stability are correlated, in some patients, with a significantly different bacterial community structure, that are associated with a presence of particular taxa in the NCFBr lung. Moreover, there appears to be an inverse relationship between the abundance of P. aeruginosa and that of of H. influenzae within the NCFBr lung bacterial community. This interaction requires further exploration.

Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty-two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterized the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, LPS pattern, biofilm formation, urease activity, and antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined, agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (P = 0.037). Transmissible CF strains generally lacked O-antigen, produced less pyocyanin and had low virulence in G. mellonella. Furthermore, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, this full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.

A novel culture medium for isolation of rapidly-growing mycobacteria from the sputum of patients with cystic fibrosis

BACKGROUND Isolation of mycobacteria from the sputum of patients with cystic fibrosis (CF) is challenging due to the overgrowth of cultures by other bacteria and fungi. In this setting, Burkholderia cepacia selective agar (BCSA) has been recommended as a convenient and effective culture medium for the isolation of rapidly-growing, non-tuberculous mycobacteria (NTM). A novel selective culture medium (RGM medium) was evaluated for the isolation of rapidly-growing NTM from the sputum of children and adults with CF. METHODS A total of 118 isolates of rapidly-growing mycobacteria and 98 other bacteria and fungi were inoculated onto RGM medium. These were assessed for growth at 30°C over a seven day period. A total of 502 consecutive sputum samples were collected from 210 patients with CF. Each sample was homogenized and cultured onto RGM medium and also onto BCSA. Cultures were incubated for 10days at 30°C. RESULTS Of 118 isolates of mycobacteria all but one grew well on RGM medium, whereas 94% of other bacteria and fungi were inhibited. A total of 55 sputum samples (from 33 distinct patients) yielded NTM using a combination of both RGM and BCSA (prevalence: 15.7%). NTM were recovered from 54 sputum samples using RGM medium compared with only 17 samples using BCSA (sensitivity 98% vs. 31%; P≤0.0001). A total of 419 isolates of non-mycobacteria were recovered from sputum samples on BCSA compared with 46 on RGM medium. CONCLUSIONS RGM medium offers a simple and effective culture method for the isolation of rapidly-growing mycobacteria from sputum samples from patients with CF without decontamination of samples. RGM medium allows for the systematic screening of all sputum samples routinely referred for culture from patients with CF.

Molecular epidemiological analysis suggests cross-infection with Pseudomonas aeruginosa is rare in non-cystic fibrosis bronchiectasis

To the Editor: In both cystic fibrosis (CF) and non-CF bronchiectasis (NCFBr) chronic Pseudomonas aeruginosa infection is adversely prognostic [1, 2]. In CF, epidemic infections with specific clones of P. aeruginosa are associated with further adverse outcomes [3, 4]. This cross-infection risk has led to segregation of patients [5]. There are few data on P. aeruginosa cross-infection in NCFBr. As a result, segregation in NCFBr has not been addressed in guidelines [6]. Our aim was to undertake a cross-infection study in NCFBr. This was undertaken in an adult bronchiectasis service in the north-east of England (UK) that is separated from the regional CF unit (sited 2 miles (3 km) away). The service was initiated in 2007 with a weekly specialist clinic without a Pseudomonas -specific clinic. When NCFBr patients are hospitalised, there is a preference for cubicle-based (single-patient room) management, but when cubicles are unavailable, patients are managed in six-bed bays. All patients had computed tomographic confirmation and had predominantly idiopathic or post-infectious bronchiectasis with CF excluded following current guidelines [6]. The study had ethical permission and Caldicott approval (Newcastle and North Tyneside National Research Ethics Service Committee). 56 isolates were selected for analysis. Six were chosen from CF patients as laboratory controls. 50 were NCFBr isolates collected between 2008 and 2011 from …

Microbiological profiles of sputum and gastric juice aspirates in Cystic Fibrosis patients.

Gastro-Oesophageal Reflux (GOR) is a key problem in Cystic Fibrosis (CF), but the relationship between lung and gastric microbiomes is not well understood. We hypothesised that CF gastric and lung microbiomes are related. Gastric and sputum cultures were obtained from fifteen CF patients receiving percutaneous endoscopic gastrostomy feeding. Non-CF gastric juice data was obtained through endoscopy from 14 patients without lung disease. Bacterial and fungal isolates were identified by culture. Molecular bacterial profiling used next generation sequencing (NGS) of the 16S rRNA gene. Cultures grew bacteria and/or fungi in all CF gastric juice and sputa and in 9/14 non-CF gastric juices. Pseudomonas aeruginosa(Pa) was present in CF sputum in 11 patients, 4 had identical Pa strains in the stomach. NGS data from non-CF gastric juice samples were significantly more diverse compared to CF samples. NGS showed CF gastric juice had markedly lower abundance of normal gut bacteria; Bacteroides and Faecalibacterium, but increased Pseudomonas compared with non-CF. Multivariate partial least squares discriminant analysis demonstrated similar bacterial profiles of CF sputum and gastric juice samples, which were distinct from non-CF gastric juice. We provide novel evidence suggesting the existence of an aerodigestive microbiome in CF, which may have clinical relevance.

Evaluation of Various Culture Media for Detection of Rapidly-Growing Mycobacteria from Patients with Cystic Fibrosis.

ABSTRACT Isolation of nontuberculous mycobacteria (NTM) from the sputum of patients with cystic fibrosis (CF) is challenging due to overgrowth by rapidly growing species that colonize the lungs of patients with CF. Extended incubation on Burkholderia cepacia selective agar (BCSA) has been recommended as an expedient culture method for the isolation of rapidly growing NTM in this setting. The aim of this study was to assess five selective media designed for the isolation of Burkholderia cepacia complex, along with two media designed for the isolation of mycobacteria (rapidly growing mycobacteria [RGM] medium and Middlebrook 7H11 agar), for their abilities to isolate NTM. All seven media were challenged with 147 isolates of rapidly growing mycobacteria and 185 isolates belonging to other species. RGM medium was then compared with the most selective brand of BCSA for the isolation of NTM from 224 sputum samples from patients with CF. Different agars designed for the isolation of B. cepacia complex varied considerably in their inhibition of other bacteria and fungi. RGM medium supported the growth of all isolates of mycobacteria and was more selective than any other medium. NTM were recovered from 17 of 224 sputum samples using RGM medium, compared with only 7 samples using the most selective brand of BCSA (P = 0.023). RGM medium offers a superior option, compared to other selective agars, for the isolation of rapidly growing mycobacteria from the sputum of patients with CF. Furthermore, the convenience of using RGM medium enables routine screening for rapidly growing NTM in all submitted sputum samples from patients with CF.

A metagenomic approach to characterize temperate bacteriophage populations from Cystic Fibrosis and non-Cystic Fibrosis bronchiectasis patients

Pseudomonas aeruginosa (Pa), normally a soil commensal, is an important opportunistic pathogen in Cystic Fibrosis (CF) and non-Cystic Fibrosis Bronchiectasis (nCFBR). Persistent infection correlates with accelerated decline in lung function and early mortality. The horizontal transfer of DNA by temperate bacteriophages can add gene function and selective advantages to their bacterial host within the constrained environment of the lower lung. In this study, we chemically induce temperate bacteriophages from clonal cultures of Pa and identify their mixed viral communities employing metagenomic approaches. We compared 92 temperate phage metagenomes stratified from these clinical backgrounds (47 CF and 45 nCFBR Pa isolates) using MG-RAST and GeneWise2. KEGG analysis shows the complexity of temperate phage accessory gene carriage increases with duration and severity of the disease. Furthermore, we identify the presence of Ig-like motifs within phage structural genes linked to bacterial adhesion and carbohydrate binding including Big_2, He_Pig, and Fn3. This study provides the first clinical support to the proposed bacteriophage adherence to mucus (BAM) model and the evolution of phages interacting at these mucosal surfaces over time.

Anti-bacterial antibody and T cell responses in bronchiectasis are differentially associated with lung colonization and disease

BackgroundAs a way to determine markers of infection or disease informing disease management, and to reveal disease-associated immune mechanisms, this study sought to measure antibody and T cell responses against key lung pathogens and to relate these to patients’ microbial colonization status, exacerbation history and lung function, in Bronchiectasis (BR) and Chronic Obstructive Pulmonary Disease (COPD).MethodsOne hundred nineteen patients with stable BR, 58 with COPD and 28 healthy volunteers were recruited and spirometry was performed. Bacterial lysates were used to measure specific antibody responses by ELISA and T cells by ELIspot. Cytokine secretion by lysate-stimulated T cells was measured by multiplex cytokine assay whilst activation phenotype was measured by flow cytometry.ResultsTypical colonization profiles were observed in BR and COPD, dominated by P.aeruginosa, H.influenzae, S.pneumoniae and M.catarrhalis. Colonization frequency was greater in BR, showing association with increased antibody responses against P.aeruginosa compared to COPD and HV, and with sensitivity of 73% and specificity of 95%. Interferon-gamma T cell responses against P.aeruginosa and S.pneumoniae were reduced in BR and COPD, whilst reactive T cells in BR had similar markers of homing and senescence compared to healthy volunteers. Exacerbation frequency in BR was associated with increased antibodies against P. aeruginosa, M.catarrhalis and S.maltophilia. T cell responses against H.influenzae showed positive correlation with FEV1% (r = 0.201, p = 0.033) and negative correlation with Bronchiectasis Severity Index (r = − 0.287, p = 0.0035).ConclusionOur findings suggest a difference in antibody and T cell immunity in BR, with antibody being a marker of exposure and disease in BR for P.aeruginosa, M.catarrhalis and H.influenzae, and T cells a marker of reduced disease for H.influenzae.

Risk factors for lung disease progression in children with cystic fibrosis

We read with interest the recent paper by van Horck et al. [1], which studied 545 children followed for 5 years with longitudinal data from the Dutch Cystic Fibrosis registry. Data from 2009 to 2014 showed that proton pump inhibitor (PPI) use was associated with annual decline of % predicted forced expiratory volume in 1 s and future pulmonary exacerbation rates. In a discussion of potential mechanisms, the authors considered that bacteria are normally killed by acid conditions in the stomach but that gastric pH is raised following PPI use. It was therefore hypothesised that with extra-oesophageal reflux, surviving pathogens could reach the upper airway and be aspirated. We call for studies of widespread use of proton pump inhibitor therapy in people with cystic fibrosis and chronic lung disease; these should evaluate patient benefit and potential iatrogenic effects, including dysregulation of aerodigestive homeostasis http://ow.ly/ym6H30lxxZu