S. McGrath

Use of culture and molecular analysis to determine the effect of antibiotic treatment on microbial community diversity and abundance during exacerbation in patients with cystic fibrosis

Background Anaerobic bacteria are increasingly regarded as important in cystic fibrosis (CF) pulmonary infection. The aim of this study was to determine the effect of antibiotic treatment on aerobic and anaerobic microbial community diversity and abundance during exacerbations in patients with CF. Methods Sputum was collected at the start and completion of antibiotic treatment of exacerbations and when clinically stable. Bacteria were quantified and identified following culture, and community composition was also examined using culture-independent methods. Results Pseudomonas aeruginosa or Burkholderia cepacia complex were detected by culture in 24/26 samples at the start of treatment, 22/26 samples at completion of treatment and 11/13 stable samples. Anaerobic bacteria were detected in all start of treatment and stable samples and in 23/26 completion of treatment samples. Molecular analysis showed greater bacterial diversity within sputum samples than was detected by culture; there was reasonably good agreement between the methods for the presence or absence of aerobic bacteria such as P aeruginosa (κ=0.74) and B cepacia complex (κ=0.92), but agreement was poorer for anaerobes. Both methods showed that the composition of the bacterial community varied between patients but remained relatively stable in most individuals despite treatment. Bacterial abundance decreased transiently following treatment, with this effect more evident for aerobes (median decrease in total viable count 2.3×107 cfu/g, p=0.005) than for anaerobes (median decrease in total viable count 3×106 cfu/g, p=0.046). Conclusion Antibiotic treatment targeted against aerobes had a minimal effect on abundance of anaerobes and community composition, with both culture and molecular detection methods required for comprehensive characterisation of the microbial community in the CF lung. Further studies are required to determine the clinical significance of and optimal treatment for these newly identified bacteria.

Dual functional ionic liquids as plasticisers and antimicrobial agents for medical polymers

Contamination of medical devices with bacteria such as Meticillin resistant Staphylococcus aureus (MRSA) is of great clinical concern. Poly(vinyl chloride) is widely used in the production of medical devices, such as catheters. The flexibility of catheter tubing is derived from the addition of plasticisers. Here, we report the design of two dual functional ionic liquids, 1-ethylpyridinium docusate and tributyl(2-hydroxyethyl)phosphonium docusate, which uniquely provide a plasticising effect, and exhibit antimicrobial and antibiofilm-forming activity to a range of antibiotic resistant bacteria. The plasticisation of poly(vinyl chloride) was tailored as a function of ionic liquid concentration. The effective antimicrobial behaviour of both ionic liquids originates from the chemical structure of the anion or cation and is not limited to the length of the alkyl chain on the anion/cation. The design approach adopted will be useful in developing ionic liquids as multi-functional additives for polymers.

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.

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.

Detection of anaerobic bacteria in bronchoalveolar lavage fluid from paediatric CF patients

One hundred and twenty-four cystic fibrosis patients (lung transplant recipients excluded) delivered 998 respiratory tract samples during a 12 month period, and Haemophilus influenzae was isolated from 245 samples from 79 patients. H. influenzae was cultured at least once from 27 of 27 (100%) patients below 6 years of age, from 34 of 57 (60%) patients aged 6−17 years, and from 10 of 40 (25%) adult patients ( 18 y). From 25 patients, H. influenzae was recovered from half of their samples, and these patients accounted for 55% of all isolates. Cultivation of H. influenzae elicited treatment with oral amoxicillin (or amoxicillin-clavulanic acid; in rare instances ciprofloxacin), also in the absence of symptoms. Severe exacerbations were treated with intravenous cefuroxime or cetftriaxone. Fifteen percent of the isolates produced b-lactamase, another 12% exhibited decreased susceptibility to ampicillin (low-BLNAR (beta-lactamase negative ampicillin-resistant) H. influenzae), as evaluated by reduced susceptibility to oral cephalosporins by a disc-diffusion assay. These rates were similar to the susceptibility levels observed with H. influenzae cultured from non-CF patients. Whether the repeated isolation of H. influenzae from the same patient is attributable to a chronic colonization with a single strain is investigated by pulsed-field gel electrophoresis.

Activity of hypothiocyanite and lactoferrin (ALX-009) against respiratory cystic fibrosis pathogens in sputum

Objectives To determine the antimicrobial activity of ALX-009, a combination of bovine lactoferrin and hypothiocyanite, in sputum against Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc), key pathogens causing infection in the lungs of cystic fibrosis (CF) patients. Methods The antimicrobial activity of ALX-009 against clinical respiratory P. aeruginosa isolates was determined by time-kill assay. Sputum from CF patients was treated with ALX-009, either alone or in combination with tobramycin, and the effect on P. aeruginosa, Bcc and total sputum density was determined. Results Time-kill assay indicated that ALX-009 was bactericidal at 24 h against 4/4 P. aeruginosa isolates under aerobic conditions, and against 3/4 isolates under anaerobic conditions. ALX-009 was also bactericidal against P. aeruginosa in sputum samples at 6 h (n = 22/24 samples) and 24 h (n = 14/24 samples), and demonstrated significantly greater activity than tobramycin at both timepoints. Activity against Bcc in sputum samples (n = 9) was also demonstrated, but the magnitude of change in Bcc density was less than for P. aeruginosa. To determine the effect of treating sputum with two doses of ALX-009, similar to current regimens for inhaled antibiotics, aliquots of a further 10 sputum samples positive for P. aeruginosa were treated with one (t = 0 h) or two doses (t = 0 h, t = 12 h) of ALX-009; treatment with two doses resulted in bactericidal activity in 7/10 samples at 34 h compared with only 3/10 samples when treatment was with one dose. Conclusions ALX-009 demonstrates promise as a novel antimicrobial that could be used to decrease P. aeruginosa density in the lungs of people with CF.

Evidence of persistence of Prevotella spp. in the cystic fibrosis lung

Purpose. Prevotella spp. represent a diverse genus of bacteria, frequently identified by both culture and molecular methods in the lungs of patients with chronic respiratory infection. However, their role in the pathogenesis of chronic lung infection is unclear; therefore, a more complete understanding of their molecular epidemiology is required. Methodology. Pulsed Field Gel Electrophoresis (PFGE) and Random Amplified Polymorphic DNA (RAPD) assays were developed and used to determine the degree of similarity between sequential isolates (n=42) from cystic fibrosis (CF) patients during periods of clinical stability and exacerbation. Results. A wide diversity of PFGE and RAPD banding patterns were observed, demonstrating considerable within‐genus heterogeneity. In 8/12 (66.7%) cases, where the same species was identified at sequential time points, pre‐ and post‐antibiotic treatment of an exacerbation, PFGE/RAPD profiles were highly similar or identical. Congruence was observed between PFGE and RAPD (adjusted Rand coefficient, 0.200; adjusted Wallace RAPD‐>PFGE 0.459, PFGE‐>RAPD 0.128). Furthermore, some isolates could not be adequately assigned a species name on the basis of 16S rRNA analysis: these isolates had identical PFGE/RAPD profiles to Prevotella histicola. Conclusion. The similarity in PFGE and RAPD banding patterns observed in sequential CF Prevotella isolates may be indicative of the persistence of this genus in the CF lung. Further work is required to determine the clinical significance of this finding, and to more accurately distinguish differences in pathogenicity between species.

93 Production of virulence factors by Prevotella isolates belonging to the cystic fibrosis (CF) respiratory microbiota

Objectives The pathogenesis of Prevotella in CF lung infection is not clear. The aim of this study was to determine production of putative virulence factors by CF Prevotella isolates. Methods Prevotella isolates (CF, n=40; non-CF, n=50) were characterised phenotypically/genotypically for extended-spectrum β-lactamase (ESβL) production (Table). The presence of a capsule was determined for CF Prevotella isolates (n = 40) using both light and transmission electron microscopy. Following culture, the haemolytic capacity of cell-free supernatant from CF isolates (n = 40) was ascertained using a semi-quantitative assay. Proteolysis by Prevotella (n = 40) was investigated following growth on agar containing 2% w/v skimmed milk powder. Results ESβL production is summarised in the Table. Twenty-seven of 40 (68%) Prevotella isolates were encapsulated. All isolates (n = 40) exhibited an ability to degrade horse erythrocytes (Range, 2.07–41.95%; Mean, 23.45%) demonstrating the production of haemolysins. Protease activity was identified in 37/40 (93%) isolates. TableESBL productionPhenotypeMethodPositive, n (%)PCFNon-CFESβLCombined disc31/40 (78)27/50 (54)0.036blaTEMPCR & sequencing19/40 (48)7/50 (14)0.001 Conclusion Virulence factor production was common amongst Prevotella spp. CF Prevotella may potentially contribute to treatment failure of CF lung infection with b-lactams (ES3L production), evade the defence mechanism (capsule production) or contribute to host tissue damage (haemolysin/protease production). Funded by DEL NI studentships, HSC R&D, Public Health Agency, NI and the MRC through a US-Ireland Partnership Grant. Non-CF isolates provided by Dr Hall, Anaerobe Reference Unit, Wales.

ePS06.8 Cigarette smoke-induced changes in phenotype and virulence in Pseudomonas aeruginosa

Objectives The health risks of either direct or passive smoking in Cystic Fibrosis (CF) have been well established (Kopp et al., 2015). Chronic infection with Pseudomonas aeruginosa (PA) is associated with considerable morbidity and mortality in CF, but the direct effect of cigarette smoke (CS) on PA has not been established. This study aimed to determine the impact of CS on PA phenotype and virulence. Methods Cigarette smoke extract (CSE) was prepared as described previously (Comer et al, 2012). Respiratory PA isolates (n = 4) and type strain (n = 1; ATCC 27853) were grown aerobically at 37°C +/– CSE and total viable count cfu/ml (TVC) determined at a range of CSE concentrations. Pyocyanin production was quantified following chloroform extraction. Biofilm formation was examined using a microtitre tray assay with quantification by crystal violet staining (Stepanovic et al., 2000). Virulence of PA was determined in the Galleria mellonella infection model. Results Growth of PA isolates was not completely inhibited by any concentration of CSE used. Both pyocyanin production and biofilm formation were significantly greater when isolates were exposed to CSE (biofilm: p G. mellonella model when PA isolates were exposed to CSE, indicating increased virulence. Conclusion Exposure of PA to CSE in vitro increases PA virulence. Further clinical studies are required to more fully correlate these changes in phenotype with clinical outcomes.

94 Isolation and characterisation of bacteriophage infecting Prevotella spp. recovered from the cystic fibrosis (CF) airways

Objectives Prevotella spp. are the predominant anaerobes detected in CF sputum samples. Whether these organisms contribute to the pathogenicity of chronic lung disease is currently unclear. Bacterial pathogenicity is increasingly being associated with the carriage of temperate bacteriophage. As such, this study aimed to isolate and characterise bacteriophage infecting Prevotella spp. recovered from CF sputum samples. Methods Prevotella spp. (N = 6) were cultivated in basal anaerobic media, under anaerobic conditions, until late stationary/decline phase. Bacteria were pelleted by centrifugation and the supernatant passed through 0.22 µm filters before bacteriophage were concentrated using high speed centrifugation. Phage particles were stained with 2% uranyl acetate and viewed using a transmission electron microscope. Results Bacteriophage like particles with uniformity and icosahedral capsid structures that characterise many bacteriophage species were recovered from all Prevotella cultures. Conclusion CF Prevotella isolates harbour a variety of bacteriophage like particles. Preliminary investigations suggest that the production of these particles may occur in response to increases in bacterial population density. Ongoing genomic analysis may allow further characterisation of these bacteriophage like particles and provide insight into any potential effects they may have on bacterial virulence.