Characterisation of key genotypic and phenotypic traits of clinical cystic fibrosis Staphylococcus aureus isolates.

Introduction. One third of people with CF in the UK are co-infected by both Staphylococcus aureus and Pseudomonas aeruginosa. Chronic bacterial infection in CF contributes to the gradual destruction of lung tissue, and eventually respiratory failure in this group.Gap Statement. The contribution of S. aureus to cystic fibrosis (CF) lung decline in the presence or absence of P. aeruginosa is unclear. Defining the molecular and phenotypic characteristics of a range of S. aureus clinical isolates will help further understand its pathogenic capabilities.Aim. Our objective was to use molecular and phenotypic tools to characterise twenty-five clinical S. aureus isolates collected from mono- and coinfection with P. aeruginosa from people with CF at the Royal Victoria Infirmary, Newcastle upon Tyne.Methodology. Genomic DNA was extracted and sequenced. Multilocus sequence typing was used to construct phylogeny from the seven housekeeping genes. A pangenome was calculated using Roary, and cluster of Orthologous groups were assigned using eggNOG-mapper which were used to determine differences within core, accessory, and unique genomes. Characterisation of sequence type, clonal complex, agr and spa types was carried out using PubMLST, eBURST, AgrVATE and spaTyper, respectively. Antibiotic resistance was determined using Kirby-Bauer disc diffusion tests. Phenotypic testing of haemolysis was carried out using ovine red blood cell agar plates and mucoid phenotypes visualised using Congo red agar.Results. Clinical strains clustered closely based on agr type, sequence type and clonal complex. COG analysis revealed statistically significant enrichment of COG families between core, accessory and unique pangenome groups. The unique genome was significantly enriched for replication, recombination and repair, and defence mechanisms. The presence of known virulence genes and toxins were high within this group, and unique genes were identified in 11 strains. Strains which were isolated from the same patient all surpassed average nucleotide identity thresholds, however, differed in phenotypic traits. Antimicrobial resistance to macrolides was significantly higher in the coinfection group.Conclusion. There is huge variation in genetic and phenotypic capabilities of S. aureus strains. Further studies on how these may differ in relation to other species in the CF lung may give insight into inter-species interactions.

Evaluation of Online Distant Synchronous Interprofessional Simulations.

INTRODUCTION: Formative interprofessional education is an accreditation standard for health professional student populations. This study examined the perception of midwifery students and obstetrics and gynecology (OB-GYN) residents participating in distance synchronous interprofessional simulation. METHODS: Students participated in an interprofessional simulation in an interactive video conferencing environment. Participants were midwifery students and OB-GYN residents from unaffiliated, geographically distant educational programs. Students' feedback was collected with a survey after the simulation session. RESULTS: Eighty-six percent of midwifery students strongly agreed they felt better prepared for team-based care in future practice after the simulation, whereas 59% of OB-GYN students strongly agreed. Seventy-seven percent of midwifery students strongly agreed they were more clear on the scope of practice of the other profession after the simulation, whereas 53% of OB-GYN students strongly agreed. Eighty-seven percent of midwifery students and 74% of OB-GYN residents strongly agreed the distance synchronous simulation was a positive learning experience. DISCUSSION: This study demonstrated that midwifery students and OB-GYN residents valued the experience of distance synchronous interprofessional education. Most learners reported feeling better prepared for team-based care and gained a better understanding of each other's scope of practice. Distance synchronous simulations can increase midwifery students' and OB-GYN residents' access to interprofessional education.

Improved Self-Assessed Collaboration Through Interprofessional Education: Midwifery Students and Obstetrics and Gynecology Residents Learning Together.

INTRODUCTION: Research suggests that interprofessional education, bringing learners together to learn about, with, and from each other, improves health professions education and can improve health outcomes. Little research has measured outcomes of interprofessional education between midwifery students and obstetrics and gynecology residents. The purpose of this study was to examine self-assessed interprofessional and collaborative competencies among midwifery students and obstetrics and gynecology residents. METHODS: Baseline self-assessed interprofessional and collaborative competencies were compared with follow-up measurements to evaluate learners' experiences over an 11-month study period. Participants were midwifery students and obstetrics and gynecology residents who experienced interprofessional learning activities. The Interprofessional Education Collaborative Competency Self-Assessment Survey (IPEC Survey) and Interprofessional Collaborative Competency Attainment Survey (ICCAS) were used. RESULTS: Of 256 learners at 4 demonstration sites, 223 (87%) completed the baseline, and 121 of 237 eligible learners (51%) completed the follow-up surveys. The IPEC Survey total score (t = 2.31, P = .02) and interaction subscale (t = 2.85, P = .005) and ICCAS score (t = 4.04, P = .001) increased for midwifery students but not obstetrics and gynecology residents on the IPEC Survey (t = 0.32, P = .75) and ICCAS (t = -0.05, P = .96) measures. Midwifery students (87%) and residents (57%) reported improved overall ability to collaborate. Learners responding to 3 open-ended questions valued team-based experiences, including learning how to communicate with each other; appreciated learning each other's education and scope of practice; and recommended skills development including uncommon clinical events, case discussions, and direct clinical care. DISCUSSION: This study advanced knowledge about interprofessional education between midwifery students and obstetrics and gynecology residents. Midwifery students improved in self-assessed interprofessional and collaborative competencies. Most learners reported better interprofessional collaboration skills and were positive about future interprofessional learning. This evaluation approach is available for other programs implementing or extending interprofessional education.

Exposure to bile and gastric juice can impact the aerodigestive microbiome in people with cystic fibrosis.

Studies of microbiota reveal inter-relationships between the microbiomes of the gut and lungs. This relationship may influence the progression of lung disease, particularly in patients with cystic fibrosis (CF), who often experience extraoesophageal reflux (EOR). Despite identifying this relationship, it is not well characterised. Our hypothesis is that the gastric and lung microbiomes in CF are related, with the potential for aerodigestive pathophysiology. We evaluated gastric and sputum bacterial communities by culture and 16S rRNA gene sequencing in 13 CF patients. Impacts of varying levels of bile acids, pepsin and pH on patient isolates of Pseudomonas aeruginosa (Pa) were evaluated. Clonally related strains of Pa and NTM were identified in gastric and sputum samples from patients with symptoms of EOR. Bacterial diversity was more pronounced in sputa compared to gastric juice. Gastric and lung bile and pepsin levels were associated with Pa biofilm formation. Analysis of the aerodigestive microbiomes of CF patients with negative sputa indicates that the gut can be a reservoir of Pa and NTM. This combined with the CF patient's symptoms of reflux and potential aspiration, highlights the possibility of communication between microorganisms of the gut and the lungs. This phenomenon merits further research.

Evaluation of a Novel Chromogenic Medium for the Detection of Pseudomonas aeruginosa in Respiratory Samples from Patients with Cystic Fibrosis.

Pseudomonas aeruginosa is a dominant cause of respiratory infection in individuals with cystic fibrosis (CF), leading to significant morbidity and mortality. Detection of P. aeruginosa is conducted by culture of respiratory samples but this process may occasionally be compromised due to overgrowth by other bacteria and fungi. We aimed to evaluate a novel chromogenic medium, Pseudomonas aeruginosa chromogenic agar (PACA), for culture of P. aeruginosa from respiratory samples, from patients with CF. A total of 198 respiratory samples were cultured onto PACA and three other media: CHROMID® P. aeruginosa, CHROMagar™ Pseudomonas and MacConkey agar. P. aeruginosa was recovered from 66 samples (33%), using a combination of all media. After 72 h incubation, the sensitivity of the four chromogenic media was as follows: 91% for PACA and CHROMagar™ Pseudomonas, 85% for CHROMID® P. aeruginosa and 83% for MacConkey agar. For the three chromogenic media, the positive predictive value after 72 h was as follows: 95% for PACA, 56% for CHROMagar™ Pseudomonas and 86% for CHROMID® P. aeruginosa. PACA proved to be a highly effective culture medium for the isolation and specific detection of P. aeruginosa from respiratory samples.

Evaluation of Three Culture Media for Isolation of Burkholderia cepacia Complex from Respiratory Samples of Patients with Cystic Fibrosis.

Burkholderia cepacia complex (BCC) is a significant pathogen causing respiratory disease in individuals with cystic fibrosis (CF). Diagnosis is typically achieved by isolation of BCC on selective culture media following culture of sputum or other respiratory samples. The aim of this study was to compare the efficacy of three commercially available selective media for the isolation of BCC. The three media comprised Burkholderia cepacia selective agar (BCSA; bioMérieux), BD Cepacia medium (BD: Becton-Dickinson) and MAST Cepacia medium (MAST laboratories). Each medium was challenged with 270 respiratory samples from individuals with CF as well as an international collection of BCC (n = 26) and 14 other isolates of Burkholderia species at a range of inocula. The international collection was also used to artificially "spike" 26 respiratory samples. From a total of 34 respiratory samples containing BCC, 97% were recovered on BD and 94% were detected on MAST and BCSA. All three media were effective for isolation of BCC. BCSA was much more selective than the other two media (p < 0.0001) meaning that fewer isolates required processing to exclude the presence of BCC.

Decontamination Strategies Used for AFB Culture Significantly Reduce the Viability of Mycobacterium abscessus Complex in Sputum Samples from Patients with Cystic Fibrosis.

Nontuberculous mycobacteria are important respiratory pathogens in patients with cystic fibrosis (CF). For diagnosis, international guidelines recommend culture of sputum that has been decontaminated via chemical treatment. Fifty-six sputum samples from 32 patients known to be previously colonized or infected with NTM were subdivided, and the aliquots were subjected to six different decontamination strategies, followed by quantitative culture for NTM. Thirty sputum samples contained Mycobacterium abscessus complex (MABSC) and 11 contained Mycobacterium avium complex (MAC). Decontamination strategies included treatment with N-acetyl L-cysteine with 2% sodium hydroxide (NALC-NaOH), 4% NaOH, 1% chlorhexidine, 0.5 N sulfuric acid, 5% oxalic acid, double decontamination with NALC-NaOH, followed by 5% oxalic acid, and saline (0.85%) as a control. The samples were also cultured directly with no treatment. Treatment with NALC-NaOH resulted in an average reduction in colony count of 87% for MABSC when compared with direct culture. NaOH at 4% caused a 98.3% average reduction in colony count. All treatments that included NaOH resulted in colony counts that were statistically lower than those obtained from direct culture or the saline-treated control (p < 0.05). Standard treatments using sulfuric or oxalic acids were less deleterious, but still resulted in an average reduction in colony count of at least 30%. The viability of MAC was much less affected by most decontamination treatments. In conclusion, the viability of MABSC was severely compromised by standard decontamination regimens. This supports recent evidence showing that optimal recovery of MABSC is achieved by culture on an appropriate selective agar without decontamination of sputum samples.

Automated antimicrobial susceptibility testing of slow-growing Pseudomonas aeruginosa strains in the presence of tetrazolium salt WST-1.

Slow growing, mucoid isolates of Pseudomonas aeruginosa require adaptation of the protocol used for automated antimicrobial susceptibility testing (AST). In the present study we used a water soluble tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) in combination with menadione for possibly improving AST of slow growing and biofilm-forming P. aeruginosa isolates from cystic fibrosis (CF) patients. WST-1 and menadione addition ensures sensitive detection of microbial growth increase in the presence of antibiotics that may remain undetected with the automated VITEK® 2 method. We observed that 32.8% of P. aeruginosa isolates from CF and bronchiectasis patients produced an elevated absorbance signal intensity thereby increasing the sensitivity while maintaining the accuracy of VITEK 2. Our study merits future investigation with other slow growing pathogenic bacterial species.

Phenotypic and Genomic Variability of Serial Peri-Lung Transplantation Pseudomonas aeruginosa Isolates From Cystic Fibrosis Patients.

Cystic fibrosis (CF) represents one of the major genetic and chronic lung diseases affecting Caucasians of European descent. Patients with CF suffer from recurring infections that lead to further damage of the lungs. Pulmonary infection due to Pseudomonas aeruginosa is most prevalent, further increasing CF-related mortality. The present study describes the phenotypic and genotypic variations among 36 P. aeruginosa isolates obtained serially from a non-CF and five CF patients before, during and after lung transplantation (LTx). The classical and genomic investigation of these isolates revealed a common mucoid phenotype and only subtle differences in the genomes. Isolates originating from an individual patient shared ≥98.7% average nucleotide identity (ANI). However, when considering isolates from different patients, substantial variations in terms of sequence type (ST), virulence factors and antimicrobial resistance (AMR) genes were observed. Whole genome multi-locus sequence typing (MLST) confirmed the presence of unique STs per patient regardless of the time from LTx. It was supported by the monophyletic clustering found in the genome-wide phylogeny. The antibiogram shows that ≥91.6% of the isolates were susceptible to amikacin, colistin and tobramycin. For other antibiotics from the panel, isolates frequently showed resistance. Alternatively, a comparative analysis of the 36 P. aeruginosa isolates with 672 strains isolated from diverse ecologies demonstrated clustering of the CF isolates according to the LTx patients from whom they were isolated. We observed that despite LTx and associated measures, all patients remained persistently colonized with similar isolates. The present study shows how whole genome sequencing (WGS) along with phenotypic analysis can help us understand the evolution of P. aeruginosa over time especially its antibiotic resistance.

Interprofessional Education Between Midwifery Students and Obstetrics and Gynecology Residents: An American College of Nurse-Midwives and American College of Obstetricians and Gynecologists Collaboration.

Despite areas of excellence, US perinatal care outcomes lag behind most developed countries. In addition, a shortage and maldistribution of health care providers exists. The American College of Nurse-Midwives and the American College of Obstetricians and Gynecologists (ACOG) partnered to obtain funding to develop interprofessional education modules and other learning activities for midwifery students and obstetrics and gynecology residents in 4 demonstration sites. The multidisciplinary 2016 ACOG document Collaboration in Practice: Implementing Team-Based Care was adopted as a framework. Core competencies of values and ethics, roles and responsibilities, interprofessional communication, and teams and teamwork developed by the Interprofessional Education Collaborative were used to guide the work. Seven modules have been developed including guiding principles, patient-centered care, role clarification, collaborative practice, history and culture, care transition, and difficult conversations. Learners participate in laboratory and simulation activities and work together in clinical care settings. Stakeholder experiences as well as barriers to implementation are discussed. Learning materials and activity descriptions are open resourced and shared on a project website for use by programs interested in implementing an interprofessional curriculum. Ongoing formal evaluation including pilot testing of a program evaluation method is described.

In vitro effects of combined iron chelation, antibiotics and matrix disruption on clinical isolates of Pseudomonas aeruginosa.

OBJECTIVES: Pseudomonas aeruginosa is an important pathogen in chronic suppurative respiratory diseases, with adverse effects on severity, healthcare utilization and quality of life. Aerosolized combined biofilm disruption and iron chelators offer novel proof-of-concept for improving airway antimicrobial efficacy. Our aim was to assess the activity of desferrioxamine, Dornase alfa (DNase) and antibiotics on biofilm formation and against mature preformed biofilms of P. aeruginosa. METHODS: Fifty-six isolates of P. aeruginosa were screened for biofilm production and seven isolates with varying capacity to form biofilms were referred for further study. Three antibiotics (colistin, tobramycin and ciprofloxacin) as well as desferrioxamine and DNase were assessed for their ability to prevent biofilm formation using the crystal violet assay. The same method was used to assess their impact on mature biofilms. Each agent, as well as combinations of these agents, was also assessed for its effect on the metabolic activity and viability of preformed P. aeruginosa biofilm by the resazurin reduction assay and by performing viable counts. RESULTS: Antibiotics alone prevented the development of biofilms and partly reduced the viability of mature biofilms. Desferrioxamine and DNase did not reduce biofilm formation. For most isolates, desferrioxamine and DNase did not offer any clear advantage over the use of antibiotics alone with respect to reducing the viability of Pseudomonas biofilms. CONCLUSIONS: Colistin, tobramycin and ciprofloxacin prevented biofilm formation by P. aeruginosa and reduced the viability of mature biofilms. For most isolates, there was no clear advantage of combining these antimicrobials with desferrioxamine or DNase.

Temperate Bacteriophages from Chronic Pseudomonas aeruginosa Lung Infections Show Disease-Specific Changes in Host Range and Modulate Antimicrobial Susceptibility.

Temperate bacteriophages are a common feature of Pseudomonas aeruginosa genomes, but their role in chronic lung infections is poorly understood. This study was designed to identify the diverse communities of mobile P. aeruginosa phages by employing novel metagenomic methods, to determine cross infectivity, and to demonstrate the influence of phage infection on antimicrobial susceptibility. Mixed temperate phage populations were chemically mobilized from individual P. aeruginosa, isolated from patients with cystic fibrosis (CF) or bronchiectasis (BR). The infectivity phenotype of each temperate phage lysate was evaluated by performing a cross-infection screen against all bacterial isolates and tested for associations with clinical variables. We utilized metagenomic sequencing data generated for each phage lysate and developed a novel bioinformatic approach allowing resolution of individual temperate phage genomes. Finally, we used a subset of the temperate phages to infect P. aeruginosa PAO1 and tested the resulting lysogens for their susceptibility to antibiotics. Here, we resolved 105 temperate phage genomes from 94 lysates that phylogenetically clustered into 8 groups. We observed disease-specific phage infectivity profiles and found that phages induced from bacteria isolated from more advanced disease infected broader ranges of P. aeruginosa isolates. Importantly, when infecting PAO1 in vitro with 20 different phages, 8 influenced antimicrobial susceptibility. This study shows that P. aeruginosa isolated from CF and BR patients harbors diverse communities of inducible phages, with hierarchical infectivity profiles that relate to the progression of the disease. Temperate phage infection altered the antimicrobial susceptibility of PAO1 at subinhibitory concentrations of antibiotics, suggesting they may be precursory to antimicrobial resistance.IMPORTANCE Pseudomonas aeruginosa is a key opportunistic respiratory pathogen in patients with cystic fibrosis and non-cystic fibrosis bronchiectasis. The genomes of these pathogens are enriched with mobile genetic elements including diverse temperate phages. While the temperate phages of the Liverpool epidemic strain have been shown to be active in the human lung and enhance fitness in a rat lung infection model, little is known about their mobilization more broadly across P. aeruginosa in chronic respiratory infection. Using a novel metagenomic approach, we identified eight groups of temperate phages that were mobilized from 94 clinical P. aeruginosa isolates. Temperate phages from P. aeruginosa isolated from more advanced disease showed high infectivity rates across a wide range of P. aeruginosa genotypes. Furthermore, we showed that multiple phages altered the susceptibility of PAO1 to antibiotics at subinhibitory concentrations.

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

BACKGROUND: As 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). METHODS: One 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. RESULTS: Typical 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). CONCLUSION: Our 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.

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

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 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.

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 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.

Non cystic fibrosis bronchiectasis: A longitudinal retrospective observational cohort study of Pseudomonas persistence and resistance.

BACKGROUND: The hallmark of non-cystic fibrosis bronchiectasis is recurrent bronchial infection, yet there are significant gaps in our understanding of pathogen persistence, resistance and exacerbation frequencies. Pseudomonas aeruginosa is a key pathogen thought to be a marker of disease severity and progression, yet little is known if the infection risk is seen in those with milder disease or if there is any potential for eradication. These data are important in determining risk stratification and follow up. METHODS AND PATIENT COHORT: A retrospective review of consecutive adult patients attending a specialist UK bronchiectasis clinic over a two-year recruitment period between July 2007 and June 2009 was performed. Analysis of our primary outcome, longitudinal microbiological status, was recorded based on routine clinical follow-up through to data capture point or date of death. Patients were stratified by lung function and infecting organism. RESULTS: 155 patients (mean (SD) age 62.2 (12.4) years; 60.1% female) were identified from clinic records with microbiological data for a median (IQR) follow up duration of 46 (35-62) months. Baseline mean FEV1% predicted was 60.6% (24.8) with mean exacerbation frequency of 4.42/year; 73.6% reported 3 or more exacerbations/year. Haemophilus influenzae was isolated in 90 (58.1%) patients and P. aeruginosa in 78 (50.3%) patients with persistent infection in 51 (56.7%) H. influenzae and 47 (60.3%) P. aeruginosa, respectively. Of the P. aeruginosa colonised patients, 16 (34%) became culture negative on follow-up with a mean of 5.2 negative sputum cultures/patient. P. aeruginosa was isolated from 5 out of 39 patients (12.8%) with minimal airflow limitation as compared to 18 out of 38 patients (47.4%) with severe airflow limitation. Although hospital admissions were significantly higher in the P. aeruginosa infected group (1.3 vs. 0.7 admissions per annum, p = 0.035), overall exacerbation rates were the same (4.6 vs. 4.3, p = 0.58). Independent predictors of P. aeruginosa colonisation were low FEV1% predicted (OR 2.46; 95% CI 1.27-4.77) and polymicrobial colonisation (OR 4.07; 95% CI 1.56-10.58). 17 (11%) patients were infected with multi-resistant strains; however, none were pan-resistant. CONCLUSIONS: P. aeruginosa is associated with greater persistent infection rates and more hospital admissions than H. influenzae. Exacerbation rates, however, were similar; therefore H. influenzae causes significant out-patient morbidity. P. aeruginosa infection occurs across all strata of lung function impairment but is infrequently multi-resistant in bronchiectasis. Careful microbiology follow up is required even in those with well-preserved lung function.