L. McIlreavey

Community dynamics and the lower airway microbiota in stable chronic obstructive pulmonary disease, smokers and healthy non-smokers

Rationale The role bacteria play in the progression of COPD has increasingly been highlighted in recent years. However, the microbial community complexity in the lower airways of patients with COPD is poorly characterised. Objectives To compare the lower airway microbiota in patients with COPD, smokers and non-smokers. Methods Bronchial wash samples from adults with COPD (n=18), smokers with no airways disease (n=8) and healthy individuals (n=11) were analysed by extended-culture and culture-independent Illumina MiSeq sequencing. We determined aerobic and anaerobic microbiota load and evaluated differences in bacteria associated with the three cohorts. Culture-independent analysis was used to determine differences in microbiota between comparison groups including taxonomic richness, diversity, relative abundance, ‘core’ microbiota and co-occurrence. Measurement and main results Extended-culture showed no difference in total load of aerobic and anaerobic bacteria between the three cohorts. Culture-independent analysis revealed that the prevalence of members of Pseudomonas spp. was greater in the lower airways of patients with COPD; however, the majority of the sequence reads for this taxa were attributed to three patients. Furthermore, members of Bacteroidetes, such as Prevotella spp., were observed to be greater in the ‘healthy’ comparison groups. Community diversity (α and β) was significantly less in COPD compared with healthy groups. Co-occurrence of bacterial taxa and the observation of a putative ‘core’ community within the lower airways were also observed. Conclusions Microbial community composition in the lower airways of patients with COPD is significantly different to that found in smokers and non-smokers, indicating that a component of the disease is associated with changes in microbiological status.

Antibiotic resistance in Prevotella species isolated from patients with cystic fibrosis

OBJECTIVES To compare the antimicrobial susceptibility of Prevotella spp. isolated from cystic fibrosis (CF) and non-CF patients and analyse the impact of antibiotic prescribing in the preceding year on resistance amongst CF isolates. METHODS The susceptibility of 80 CF Prevotella isolates to 12 antibiotics was compared with that of 50 Prevotella isolates from invasive infections in people who did not have CF and 27 Prevotella isolates from healthy controls. RESULTS All isolates were susceptible to chloramphenicol, meropenem and piperacillin/tazobactam, with only four isolates resistant to metronidazole. However, resistance to amoxicillin, ceftazidime and tetracycline was apparent in all groups. Significant differences in clindamycin resistance (UK CF, 56%; UK invasive, 10%) and co-amoxiclav non-susceptibility (UK CF, 32%; UK invasive, 12%) were observed between UK CF and UK invasive isolates. The likelihood of non-susceptibility to clindamycin and co-amoxiclav in UK CF isolates was 5.5-fold and 2.5-fold higher relative to that in UK invasive isolates, respectively. Azithromycin MICs were also significantly higher for CF isolates (P < 0.001), which was associated with current prescription of azithromycin. More than 50% of clinical isolates tested in this study were β-lactamase positive. CONCLUSIONS This study profiles antibiotic susceptibility in Prevotella spp. in CF and demonstrates that meropenem, piperacillin/tazobactam, chloramphenicol and metronidazole are likely to be the most effective antibiotics if treatment is indicated.

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.

Mechanisms of reduced susceptibility and genotypic prediction of antibiotic resistance in Prevotella isolated from cystic fibrosis (CF) and non-CF patients

OBJECTIVES To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cultured from patients with cystic fibrosis (CF), patients with invasive infection and healthy control subjects and to determine whether genotype can be used to predict phenotypic resistance. METHODS The susceptibility of 157 Prevotella isolates to seven antibiotics was compared, with detection of resistance genes (cfxA-type gene, ermF and tetQ), mutations within the CfxA-type β-lactamase and expression of efflux pumps. RESULTS Prevotella isolates positive for a cfxA-type gene had higher MICs of amoxicillin and ceftazidime compared with isolates negative for this gene (P < 0.001). A mutation within the CfxA-type β-lactamase (Y239D) was associated with ceftazidime resistance (P = 0.011). The UK CF isolates were 5.3-fold, 2.7-fold and 5.7-fold more likely to harbour ermF compared with the US CF, UK invasive and UK healthy control isolates, respectively. Higher concentrations of azithromycin (P < 0.001) and clindamycin (P < 0.001) were also required to inhibit the growth of the ermF-positive isolates compared with ermF-negative isolates. Furthermore, tetQ-positive Prevotella isolates had higher MICs of tetracycline (P = 0.001) and doxycycline (P < 0.001) compared with tetQ-negative isolates. Prevotella spp. were also shown, for the first time, to express resistance nodulation division (RND)-type efflux pumps. CONCLUSIONS This study has demonstrated that Prevotella isolated from various sources harbour a common pool of resistance genes and possess RND-type efflux pumps, which may contribute to tetracycline resistance. The findings indicate that antibiotic resistance is common in Prevotella spp., but the genotypic traits investigated do not reflect phenotypic antibiotic resistance in every instance.

Production of extended-spectrum β-lactamases and the potential indirect pathogenic role of Prevotella isolates from the cystic fibrosis respiratory microbiota

Extended-spectrum β-lactamase (ESBL) production and the prevalence of the β-lactamase-encoding gene blaTEM were determined in Prevotella isolates (n=50) cultured from the respiratory tract of adults and young people with cystic fibrosis (CF). Time-kill studies were used to investigate the concept of passive antibiotic resistance and to ascertain whether a β-lactamase-positive Prevotella isolate can protect a recognised CF pathogen from the action of ceftazidime in vitro. The results indicated that approximately three-quarters (38/50; 76%) of Prevotella isolates produced ESBLs. Isolates positive for ESBL production had higher minimum inhibitory concentrations (MICs) of β-lactam antibiotics compared with isolates negative for production of ESBLs (P<0.001). The blaTEM gene was detected more frequently in CF Prevotella isolates from paediatric patients compared with isolates from adults (P=0.002), with sequence analysis demonstrating that 21/22 (95%) partial blaTEM genes detected were identical to blaTEM-116. Furthermore, a β-lactamase-positive Prevotella isolate protected Pseudomonas aeruginosa from the antimicrobial effects of ceftazidime (P=0.03). Prevotella isolated from the CF respiratory microbiota produce ESBLs and may influence the pathogenesis of chronic lung infection via indirect methods, including shielding recognised pathogens from the action of ceftazidime.

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.

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.

95 Susceptibility of CF Prevotella isolates to neutrophil and complement mediated immune clearance

Objectives Prevotella spp. are the predominant anaerobes detected in CF sputum samples. To contribute to disease development bacteria, such as Prevotella, must first survive the hosts immune response. Therefore, this study aimed to determine the susceptibility of CF Prevotella isolates to neutrophil and complement mediated immune clearance. Methods Prevotella spp. (N = 8) isolated from CF patients (N = 8) were incubated with neutrophils from healthy controls (T = 20 mins) and with serum complement (T = 60 mins). Decreases in bacterial viability were calculated relative to controls. Prevotella and P. aeruginosa co-culture neutrophil killing assays were also performed. Table . Survival of isolates Species % Survival in 10% serum (N = 3), mean±SD %Survival in neutrophils (N = 3), mean±SD % Survival of P. aeruginosa, co-cultured with Prevotella, in neutrophils (N = 3), mean±SD P. denticola 100.5±6.4 * 66.13±4.16 44.74±10.56 P. salivae 42.64±4.24 114.3±15.04 * 43.05±2.28 P. nigrescens 70.9±6.99 67.8 48.19±3.17 P. veroralis 61.72±20.1 30.69±8.31 − P. melaninogenica 3.84±1.09 61.43±6.0 66.80±3.26 P. histicola 0 8.19±2.68 − P. pallens 113.3±9.43 * 23.33±5.77 − P. oulorum 101.6±5.4 * 46.03±8.56 − * Resistant. Results See the table. Prevotella isolates displayed resistance to serum killing (N = 3) and phagocytosis (N = 1). Four Prevotella isolates were more resistant to phagocytosis (>60% survival) than P. aeruginosa and S. aureus (∼42 and 54% survival, respectively). When co-cultured with P. melaninogenica, the resistance of P. aeruginosa to phagocytosis increased (P = 0.001). Conclusion Complement resistance & neutrophil evasion/inhibition strategies utilised by Prevotella spp. may enable persistence within the CF lung.

124 Lack of antimicrobial activity of ivacaftor against clinical CF respiratory isolates

Objectives Ivacaftor contains a quinolone ring within its structure, similar to that of Ciprofloxacin. A previous study demonstrated that ivacaftor had some antimicrobial activity against laboratory and non-CF clinical isolates of S. aureus and S. pneumoniae . The aim of this study was to investigate the antimicrobial activity of ivacaftor against clinical respiratory CF isolates. Methods The MIC of ivacaftor against clinical isolates ( P. aeruginosa , S. aureus , Streptococcus sp.; n=5 from each genus) was determined using a radial diffusion assay. Bactericidal activity of ivacaftor against selected isolates growing planktonically and in biofilm was determined using a time-kill and a microtitre tray biofilm assay, respectively. Results were compared with ciprofloxacin. Results The radial diffusion assay indicated that ivacaftor had no bacteriostatic activity with an MIC of >50 mg/mL for all isolates tested. In time-kill assays bactericidal activity was observed for Streptococcus sp. only (Table 1). Ivacaftor had no effect on biofilm formation. Table 1Log change in CFU/mL (±SD) over 24 hours in time-kill assaysIvacaftor (32 mg/mL)Ciprofloxacin (5 mg/mL)ControlP. aeruginosa4.44±0.30−5.78±0.404.45±0.33S. aureus3.00±0.24−5.63±0.023.39±0.60Streptococcus sp.−3.17±2.26−4.50±1.353.75±0.51Achromobacter sp.2.83±0.27−1.63±3.843.01±0.17Stenotrophomonas sp.2.69±0.16−6.293.11±0.01 Conclusion Ivacaftor did not have a direct antimicrobial action against the clinical CF isolates tested, and did not slow the growth of isolates or inhibit biofilm formation. Work supported by a DEL NI studentship and a US-Ireland Project Partnership Grant.

100 Macrolide resistance is common among members of the CF airway microbiota

Objectives Chronic use of azithromycin has been associated with increased macrolide resistance in recognised CF pathogens. The aims of this study were to determine 1.macrolide resistance amongst other members of the CF airway microbiota 2.if resistance is associated with current patient prescription of azithromycin. Methods The susceptibility (MIC) of CF respiratory isolates (n = 26: Streptococcus, n=8; Rothia, n=5; Veillonella, n=5; Prevotella, n=4; Actinomyces, n=3; Fusobacterium , n=1) to azithromycin, clarithromycin and erythromycin was determined by Etest®. Isolates were separated into two groups depending on whether they were isolated from CF patients currently prescribed or not currently prescribed azithromycin and macrolide susceptibility was compared between the groups using the Mann–Whitney test. Results High-level macrolide resistance (>256 mg/mL) was common among the isolates tested: azithromycin, n=10/26 (38%); clarithromycin, n=6/26 (23%); erythromycin, n=11/26 (42%). Isolates from CF patients currently prescribed azithromycin (n = 9) had significantly higher MICs for the three macrolide antibiotics compared to isolates from patients not prescribed azithromycin (n = 17) [Table]. TableAzithromycin prescription versus susceptibilityMIC50 (mg/mL)PCurrent prescription of azithromycin (n = 9)No current prescription of azithromycin (n = 17)Azithromycin>25620.005Clarithromycin470.50.001Erythromycin>25640.01 Conclusion Resistance to three different macrolide antibiotics was common in bacteria present in the CF airway microbiota and was associated with current azithromycin prescription in CF patients. Work supported by a Wellcome Trust (FA) Vacation Scholarship.