J. McCaughan

Clinical outcome after acquisition of Burkholderia cepacia in patients with cystic fibrosis.

BackgroundRespiratory disease is the major cause of morbidity and mortality in cystic fibrosis (CF). The significance ofBurkholderia cepacia (B. cepacia) in the pathogenesis of lung disease in CF is debated, but its exact role remains unclear.AimTo assess the impact of respiratory tract colonisation withB. cepacia in patients with CF by measuring changes in pulmonary function and body mass index (BMI).MethodsThree groups of patients were defined based on sputum culture isolates: Group 1 wereB. cepacia andPseudomonas aeruginosa (P. aeruginosa) positive patients; Group 2 wereP. aeruginosa positive; and Group 3 were colonised with neither organism. Forced expiratory volume (FEV1) and BMI were measured annually from 1987 to 1995 and the year of acquisition ofP. aeruginosa orB. cepacia was recorded.ResultsThe mean annual decrease in FEV1 was significantly different in all three groups: Group 1, −5.4 (5.1)%; Group 2, −3.9 (6.5)%; and Group 3, −1.6 (1.0)%, (p<0.05). The mean percentage decrease in FEV1 of a sub-group of Group 1 patients where theB. cepacia acquisition date was known was 6.1% per year versus 1.55% in Group 2 patients (p<0.05).ConclusionsAcquisition ofB. cepacia may be a cause of, rather than a marker for, a decrease in pulmonary function.

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

Comparison of antibiotic susceptibility patterns in Pseudomonas aeruginosa isolated from adult patients with cystic fibrosis (CF) with invasive Pseudomonas aeruginosa from non-CF patients

a Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Lisburn Road, Belfast, Northern Ireland, BT9 7AD, UK b Bloomfield Collegiate, Astoria Gardens, Belfast, Northern Ireland, BT5 6HW, UK c Department of Medical Microbiology, Royal Group of Hospitals, Grosvenor Road, Belfast, Northern Ireland, UK d Northern Ireland Regional Adult Cystic Fibrosis Unit, Belfast City Hospital, Lisburn Road, Belfast, Northern Ireland, BT9 7AB, UK e Centre for Infection & Immunity, Queens University, Lisburn Road, Belfast, Northern Ireland, BT9 7BL, UK f School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Northern Ireland, BT52 1SA, UK

MRSA eradication of newly acquired lower respiratory tract infection in cystic fibrosis

UK cystic fibrosis (CF) guidelines recommend eradication of methicillin-resistant Staphylococcus aureus (MRSA) when cultured from respiratory samples. As there is no clear consensus as to which eradication regimen is most effective, we determined the efficacy of eradication regimens used in our CF centre and long-term clinical outcome. All new MRSA positive sputum cultures (n=37) that occurred between 2000 and 2014 were reviewed. Eradication regimen characteristics and clinical, microbiological and long-term outcome data were collected. Rifampicin plus fusidic acid was the most frequently used regimen (24 (65%) out of 37 patients), with an overall success rate of 79% (19 out of 24 patients). Eradication failure was more likely in patients with an additional MRSA-positive peripheral screening swab (p=0.03) and was associated with worse survival (p=0.04). Our results demonstrate the feasibility and clinical benefits of MRSA eradication. As peripheral colonisation was associated with lower eradication success, strategies combining systemic and topical treatments should be considered to optimise outcomes in CF patients. In CF, eradication of MRSA from the respiratory tract is feasible and is associated with better clinical outcome http://ow.ly/YfbqD

Pathogen eradication” and “Emerging pathogens”: Difficult definitions in cystic fibrosis

Infection is a common complication of cystic fibrosis (CF) airway disease. Current treatment approaches include early intervention with the intent to eradicate pathogens in the hope of delaying the development of chronic infection and the chronic use of aerosolized antibiotics to suppress infection. ABSTRACT Infection is a common complication of cystic fibrosis (CF) airway disease. Current treatment approaches include early intervention with the intent to eradicate pathogens in the hope of delaying the development of chronic infection and the chronic use of aerosolized antibiotics to suppress infection. The use of molecules that help restore CFTR (cystic fibrosis transmembrane conductance regulator) function, modulate pulmonary inflammation, or improve pulmonary clearance may also influence the microbial communities in the airways. As the pipeline of these new entities continues to expand, it is important to define when key pathogens are eradicated from the lungs of CF patients and, equally important, when new pathogens might emerge as a result of these novel therapies.

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

Do veterinary antibiotics have efficacy against highly resistant Gram-negative pathogens from patients with cystic fibrosis?

Apramycin Cefquinome Cefapirin Cefoperazone Cefquinome Cefapirin Cefalonium Ceftiofur Cefovecin Chlortetracycline HCl Ciclosporin Cloxacillin Danofloxacin mesylate Dihydrostreptomycin Diethanolamine fusidate Enrofloxacin Florfenicol Framycetin sulphate Gamithromycin Kanamycin Lincomycin Marbofloxacin Novobiocin Orbifloxacin Penethamate hydrochloride Pirlimycin hydrochloride Pradofloxacin Spectinomycin Sulfadoxine Tilmicosin Tiamulin Tildipirosin Tulathromycin Tylosin Tylvalosin Ampicillin Amoxicillin Benzylpenicil Cefalexin Clindamycin Colistin Co-amoxiclav Doxycycline Erythromycin

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.

1 Grazioli B, Matera G, Laratta C et al. Giardia lamblia infection in patients with irritable bowel syndrome and dyspepsia: a prospective study. World J Gastroenterol 2006; 12: 1941–4. 2 Brown LM. Helicobacter pylori: epidemiology and routes of transmission. Epidemiol Rev 2000; 22: 283–97. 3 Thomas JE, Gibson GR, Darboe MK, Dale A, Weaver LT. Isolation of Helicobacter pylori from human faeces. Lancet 1992; 340: 1194–5. 4 Moreira ED Jr, Nassri VB, Santos RS et al. Association of Helicobacter pylori infection and giardiasis: results from a study of surrogate markers for fecal exposure among children. World J Gastroenterol 2005; 11: 2759–63. 5 Zeyrek D, Zeyrek F, Cakmak A, Cekin A. Association of Helicobacter pylori and giardiasis in children with recurrent abdominal pain. Turkiye Parazitol Derg 2008; 32 (1): 4–7. 6 Abou El-Hoda MM, Osman HM, Rasha MM, Douidar NL, Enany AY. Impact of Helicobacter pylori infection on the activities of urease and lipase enzymes in patients with giardiasis. J Egypt Public Health Assoc 2007; 82: 273–82.