A. Reid

Reduced bacterial colony count of anaerobic bacteria is associated with a worsening in lung clearance index and inflammation in cystic fibrosis.

Anaerobic bacteria have been identified in abundance in the airways of cystic fibrosis (CF) subjects. The impact their presence and abundance has on lung function and inflammation is unclear. The aim of this study was to investigate the relationship between the colony count of aerobic and anaerobic bacteria, lung clearance index (LCI), spirometry and C-Reactive Protein (CRP) in patients with CF. Sputum and blood were collected from CF patients at a single cross-sectional visit when clinically stable. Community composition and bacterial colony counts were analysed using extended aerobic and anaerobic culture. Patients completed spirometry and a multiple breath washout (MBW) test to obtain LCI. An inverse correlation between colony count of aerobic bacteria (n = 41, r = -0.35; p = 0.02), anaerobic bacteria (n = 41, r = -0.44, p = 0.004) and LCI was observed. There was an inverse correlation between colony count of anaerobic bacteria and CRP (n = 25, r = -0.44, p = 0.03) only. The results of this study demonstrate that a lower colony count of aerobic and anaerobic bacteria correlated with a worse LCI. A lower colony count of anaerobic bacteria also correlated with higher CRP levels. These results indicate that lower abundance of aerobic and anaerobic bacteria may reflect microbiota disruption and disease progression in the CF lung.

Pandoraea apista isolated from a patient with cystic fibrosis: problems associated with laboratory identification.

A 17-year-old male patient with cystic fibrosis (CF), with CF mutation homozygous ∆F508, was admitted to the Royal Belfast Hospital for Sick Children in August 2001 for a course of intravenous antibiotics to treat a chronic pulmonary infection. He had an 11-year history of pulmonary infection with both a mucoid and a non-mucoid Pseudomonas aeruginosa, which had remained relatively sensitive to several antibiotics used routinely in the treatment of P. aeruginosa CF chest infections, including gentamicin, tobramycin, aztreonam, ceftazidime, ciprofloxacin, piperacillin/tazobactam, imipenim and meropenem. During that time he was not reported to have been colonised/infected with any other bacterial organism in his respiratory tract. On admission, an unidentified Gram-negative rod was isolated in addition to his chronic strains of mucoid and nonmucoid P. aeruginosa. This new organism was isolated from freshly expectorated sputum as the sole organism type on Burkholderia cepacia selective agar (BCSA; MAST Diagnostics Ltd, Merseyside, UK) following incubation at 37 ̊C for 48 h. As this was a new isolate growing on BCSA, it was sent for molecular identification and characterisation. Subsequently, this organism was isolated from the patient’s sputum on five occasions over a two-month period, and presently remains a part of the established resident bacterial flora. Semi-quantitative determination of organism numbers demonstrated that it was consistently present at the ++ level, approximating to 10 10 colony forming units(cfu)/g sputum. Clinically, since the first isolation of this organism from the patient’s sputum, there has been deterioration in his clinical status, including increased cough, chest tightness, wheeze, shortness of breath, production of purulent sputum, fatigue and weight loss of 1.8 kg over this two-month period. In addition, his forced vital capacity (FVC) fell from 89% to 77% predicted. Drug therapy during this period included a twoweek course of intravenous (iv) antibiotics (ceftazidime and tobramycin), as well as an oral course of ciprofloxacin and nebulised colomycin. He also received a course of oral steroids and itraconazole during this period, as there was some evidence of allergic bronchopulmonary aspergillosis (ABPA). Microbiologically, this organism was poorly identified (48% identification) phenotypically by the API 20NE scheme as Alcaligenes faecalis, with the profile 0000457. The organism was resistant in vitro by standard NCCLS disc diffusion assay to gentamicin, temocillin, ceftazidime, azlocillin, meropenem, aztreonam and colistin, and was sensitive to tobramycin, piperacillin/tazobactam, imipenem and ciprofloxacin. Subsequently, in order to aid identification, the organism was examined using molecular techniques. All DNA isolation procedures were carried out in a Class II biological safety cabinet in a room geographically separate from that used to set up reaction mixes and also from the ‘post PCR’ room, in order to minimise false-positive results and in accordance with good molecular diagnostic practice (GMDP), as detailed in the guidelines of Millar et al. DNA was extracted from a single colony using the Roche High Purity PCR Template kit (Roche Diagnostics Ltd, UK), following the manufacturer’s instructions. All reaction mixes were set up in a PCR hood in a room separate from that used to extract DNA, and from the amplification and post-PCR room, in order to minimise contamination. Reaction mixes (50 μL) were set up as follows: 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.5 mmol/L MgCl2, 200 μmol/L (each) dATP, dCTP, dGTP and dTTP, 1.25 units Thermus aquaticus (Taq) DNA polymerase (Amplitaq; Perkin Elmer), 0.2 μmol/L (each) of the 16S rRNA primers P11P (forward) 5’ GAG GAA GGT GGG GAT GAC GT -3’ and P13P (reverse) 5’ AGG CCC GGG AAC GTA TTC AC -3’, as previously described, and 4 μL DNA template. Following a hot start, the reaction mixtures were subjected to the following thermal cycling parameters in a Perkin Elmer 2400 thermocycler: 96 ̊C for 3 min, followed by 40 cycles of 96 ̊C for 1 min, 55 ̊C for 1 min, 72 ̊C for 1 min, and a final extension at 72 ̊C for 10 min. During each run, molecular-grade water was included randomly as a negative control and appropriate DNA template from Staphylococcus aureus was included as a positive control. Following amplification, samples (15 μL) were removed from each reaction mixture and examined by electrophoresis (80 V, 45 min) in gels composed of 2% (w/v) agarose (Gibco, UK) in TAE buffer (40 mmol/L Tris, 20 mmol/L acetic acid, 1 mmol/L EDTA [pH 8.3]), stained with ethidium bromide (5 μg/100 mL). Gels were visualised under ultraviolet illumination, using a gel image analysis system (UVP Products, England), and all images archived as digital (*.bmp) graphic files. Subsequently, amplicons were purified (particularly to remove dNTPs, polymerases, salts and primers) using a QIAquick PCR purification kit (Qiagen Ltd., UK) and eluted in Tris-HCl (10 mmol/L [pH 8.5]) prior to sequencing. Cy-5’labelled primer (P11P) was prepared and used for sequencing in the forward direction with the ALF Express II (Amersham-Pharmacia Ltd., Bucks, UK) employing the Thermo Sequenase fluorescent-labelled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, UK; cat no: RPN 2438). Thermal cycling parameters were: 96 ̊C for 1 min, followed by 25 cycles of 96 ̊C for 10 sec, 50 ̊C for 5 sec, 60 ̊C for 5 sec, followed by a 4 ̊C hold. Sequences obtained were compared with those stored in the GenBank data system, using BLAST alignment software

Efficacy and long‐term outcomes of palivizumab prophylaxis to prevent respiratory syncytial virus infection in infants with cystic fibrosis in Northern Ireland

RSV causes considerable morbidity and mortality in children. In cystic fibrosis (CF) viral infections are associated with worsening respiratory symptoms and bacterial colonization. Palivizumab is effective in reducing RSV hospitalization in high risk patient groups. Evidence regarding its effectiveness and safety in CF is inconclusive. CF screening in N. Ireland enabled timely palivizumab prophylaxis, becoming routine in 2002.

Cycle Ergometer Tests in Children With Cystic Fibrosis: Reliability and Feasibility

The aim of this study was to assess the reliability and feasibility of cycle ergometer tests in young children with cystic fibrosis (CF). Children with CF aged 6–11 years and with stable lung disease performed two cycle ergometry tests (intermittent sprint and continuous incremental) on two occasions 1 week apart. Reliability was assessed using repeated‐measures ANOVA. Bias was considered to be significant at P < 0.05 level and a coefficient of variation (CV) below 10% was considered acceptable. Feasibility and acceptability data were also collected. Sixteen children with CF completed the study: (9M:7F), 8.7(1.8) years, FEV1%predicted: 88.1(17.4). Power measurements recorded during the intermittent sprint test demonstrated significant bias over days (P < 0.05) and CVs were between 10% and 15%. Peak work capacity recorded during the continuous incremental test was reliable (bias P < 0.05, CV < 10%), as was heart rate and SpO2 recorded during both tests (bias P < 0.05, CV < 10%). No problems were experienced in administering the tests and all children completed both tests on two separate occasions. There was a mixed response to questions on acceptability of tests. This is the first study to provide information on the reliability of performance measures recorded during an intermittent sprint protocol (peak power) and a continuous incremental cycle ergometry (peak work capacity) in children with CF. Pediatr Pulmonol. 2012; 47:1226–1234.

Paediatric hospital in the home (HITH) for cystic fibrosis exacerbations: equivalent outcomes with equivalent physiotherapy & nursing care

The high resolution, experimental 3D structures of complete ABC exporters, published since 2006, have been used to generate models of the 3D structure of the CFTR protein in different conformations. These models are useful to understand the molecular basis of the CFTR function, as well as to evaluate the impact of mutations on the CFTR 3D structure and function. The Sav1866 structure in an outward-facing conformation was first used to model the open form of the CFTR channel (1), whereas a MsbA structure in an inward-facing conformation (called “closed apo”) was afterwards considered to construct a plausible 3D model of the closed form of the channel (2). Despite the large reorganization of the membrane-spanning domains and movements of the nucleotide-binding domains, the coupling interfaces linking these domains are relatively well conserved, suggesting that these act as pivots around which the CFTR channel dynamics occur. We have further considered our previous models of the CFTR channel, based on the Sav1866 and MsbA 3D structures, as well as new ones constructed on the basis of the recent P-gp 3D structure, in order to highlight new structural features, which may account for specific functional features. The models especially support the hypothesis that CFTR may consist of a “broken” ABC transporter, having an “atrophied” gate at the cytoplasmic side (3). According to our models, this gate would be located at the level of the bundle formed by the four intracellular loops (ICLs). Moreover, a careful analysis of the 3D structure models revealed several potential ligand binding sites at the interface between the domains (NBDs heterodimer interface, but also MSDs:NBDs), suggesting that these could be privileged targets for therapeutic strategies. Supported by Vaincre La Mucoviscidose. 1. Mornon J-P, Lehn P, Callebaut I (2008) Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane spanning domains and coupling interfaces. Cell Mol Life Sci 65:2594–2612. 2. Mornon J-P, Lehn P, Callebaut I (2009) Molecular models of the open and closed states of the whole human CFTR protein. Cell Mol Life Sci 66: 3469-3486. 3. Gadsby DC (2009) Ion channels versus ion pumps: the principal difference, in principle. Nat Rev Mol Cell Biol 10:344–352.

Relative resistance index (RRI) – a scoring system for antibiotic resistance in Pseudomonas aeruginosa

Abstract Background: There is a need to measure antibiotic resistance of Pseudomonas aeruginosa (PA) in cystic fibrosis (CF), either qualitatively or quantitatively, to inform patient management. The aim of this study was to develop a simple method by which resistance can be quantified by calculating a relative resistance index (RRI), and to assess correlation of RRIs with clinical variables. Methods: In our model, RRIs were calculated based on resistance to aztreonam, ceftazidime, ciprofloxacin, colistin, meropenem, tazocin, temicillin and tobramycin. Eighty-five adults with CF and chronic PA colonisation were identified. For each, all PA cultures were allocated a score of 0 for susceptible, 0.5 for intermediate resistance or 1 for resistance for each antibiotic listed above, and the RRI calculated by dividing the sum of these by the number of antibiotics, giving a maximum score of 1. The mean RRIs for all cultures were correlated with key clinical variables monitored in CF patients (including age, FEV1, IV antibiotic days and BMI). Results: RRIs for non-mucoid PA exhibited moderate positive correlation with total number of IV days (r = 0.405; p < 0.001) and moderate negative correlation with FEV1 % predicted (r = −0.437; p < 0.001). RRIs were not significantly correlated with duration of colonisation, typing (clonal vs other strain) or BMI. Median RRIs were significantly higher for females (0.26, IQR 0.13–0.54) than males (0.18, IQR 0.07–0.37) for non-mucoid PA only (p = 0.03). Conclusions: RRI is an easily calculated measure that correlates with other clinical variables in CF patients and enables quantitative monitoring of resistance.

Lung function and disease severity in cystic fibrosis patients heterozygous for p.Arg117His

Expression of p.Arg117His cystic fibrosis (CF) transmembrane conductance regulator is influenced by a polythymidine (poly-T) tract and a thymidine–guanine (TG) repeat on intron 9, which vary in length and affect exon 10 skipping. We compared clinical characteristics and the rate of progression of lung disease of CF patients carrying the p.Arg117His mutation with different intron 9 varying sequences (poly-T) and mutation classes in trans. Data were collected from patients in Northern Ireland, UK, including diagnostic features, sweat chloride, nutritional status, sputum microbiology, CF-related complications and lung function. Poly-T and TG repeats were determined by PCR. Forced expiratory volume in 1 s (FEV1) decline was determined from linear regression of FEV1 measurements of patients over time. We identified 62 patients with p.Arg117His, 55 with a class I/II mutation in trans and six with p.Arg117His/p.Gly551Asp. 42 patients had 5T and 13 had 7T. All patients had 12 TG repeats. Patients with p.Arg117His-5T had greater lung function decline, sweat chloride concentrations, pancreatic insufficiency and prevalence of Pseudomonas aeruginosa infection compared with patients with p.Arg117His-7T. Lung function decline and disease severity in p.Arg117His is determined by the poly-T tract length and identity of the mutation in trans. Patients with p.Arg117His-5T and a second class I/II mutation have a severity similar to p.Phe508del homozygous patients, although lung function decline is delayed to an older age. There may be linkage disequilibrium between p.Arg117His and 12 TG repeats. p.Arg117His CFTR with 5T repeats is associated with accelerated lung function decline compared with p.Arg117His-7T http://ow.ly/yAdS308q3dn

Molecular conservation within LES9F and PS21 liverpool epidemic strain (LES) markers in wild-type clinical pseudomonas aeruginosa isolated from the sputum of adult patients with cystic fibrosis.

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Comparison of in vitro susceptibilities to levofloxacin and ciprofloxacin with Pseudomonas aeruginosa and Stenotrophomonas maltophilia isolated from cystic fibrosis patients in Northern Ireland

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Muddy puddles - the microbiology of puddles located outside tertiary university teaching hospitals

In the British Isles, the frequency of rain results in the formation of puddles on footpaths and roads in/around hospitals. No data are available demonstrating the microbiological composition of such puddles and therefore a study was undertaken to examine the microbiology of puddles in the grounds of two tertiary university‐teaching hospitals (18 sites) and compared with control puddles from non‐hospital rural environments (eight sites), estimating (i) total viable count; (ii) identification of organisms in puddles; (iii) enumeration of Escherichia coli: (iv) detection of Extended Spectrum β‐Lactamase producing organisms and (v) direct antimicrobial susceptibility testing. A mean count of 2·3 × 103 CFU per ml and 1·0 × 109 CFU per ml was obtained for hospital and non‐hospital puddles respectively. Isolates (n = 77; 54 hospital and 23 non‐hospital) were isolated comprising of 23 species among 17 genera (hospital sites), where the majority (10/16; 62·5%) of genera identified were Gram‐negative approximately, a fifth (20·6%) were shared by hospital and non‐hospital rural samples. Escherichia coli was detected in half of the hospital puddles and under‐half (37·5%) of the rural puddles extended spectrum β‐lactamase organisms were not detected in any samples examined. Rainwater puddles from the hospital and non‐hospital environments contain a diverse range of bacteria, which are capable of causing infections.