There is an increasing appreciation of the polymicrobial nature of many bacterial infections such as those associated with cystic fibrosis (CF) and of the potentially important role for interspecies interactions in influencing both bacterial virulence and response to therapy. Patients with CF are often co-infected with Pseudomonas aeruginosa and other pathogens including Burkholderia cenocepacia and Stenotrophomonas maltophilia. These latter bacteria produce signal molecules of the diffusible signal factor (DSF) family, which are cis-2-unsaturated fatty acids. We have previously shown by in vitro studies that DSF from S. maltophilia leads to altered biofilm formation and increased resistance to antibiotics by P. aeruginosa; these responses of P. aeruginosa require the sensor kinase PA1396. Here we show that DSF signals are present in sputum taken from patients with CF. Presence of these DSF signals was correlated with patient colonization by S. maltophilia and/or B. cenocepacia. Analysis of 50 clinical isolates of P. aeruginosa showed that each responded to the presence of synthetic DSF by increased antibiotic resistance and these strains demonstrated little sequence variation in the PA1396 gene. In animal experiments using CF transmembrane conductance regulator knockout mice, the presence of DSF promoted P. aeruginosa persistence. Furthermore, antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells was enhanced in the presence of DSF. Taken together, these data provide substantial evidence that interspecies DSF-mediated bacterial interactions occur in the CF lung and may influence the efficacy of antibiotic treatment, particularly for chronic infections involving persistence of bacteria.

Chronic polymicrobial infections of the lung are the foremost cause of morbidity and mortality in cystic fibrosis (CF) patients. The composition of the microbial flora of the airway alters considerably during infection, particularly during patient exacerbation. An understanding of which organisms are growing, their environment and their behaviour in the airway is of importance for designing antibiotic treatment regimes and for patient prognosis. To this end, we have analysed sputum samples taken from separate cohorts of CF and non-CF subjects for metabolites and in parallel, and we have examined both isolated DNA and RNA for the presence of 16S rRNA genes and transcripts by high-throughput sequencing of amplicon or cDNA libraries. This analysis revealed that although the population size of all dominant orders of bacteria as measured by DNA- and RNA- based methods are similar, greater discrepancies are seen with less prevalent organisms, some of which we associated with CF for the first time. Additionally, we identified a strong relationship between the abundance of specific anaerobes and fluctuations in several metabolites including lactate and putrescine during patient exacerbation. This study has hence identified organisms whose occurrence within the CF microbiome has been hitherto unreported and has revealed potential metabolic biomarkers for exacerbation.

Background and objective: Standard nodal staging of lung cancer consists of positron emission tomography/ computed tomography (PET/CT), followed by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) if PET/CT shows mediastinal lymphadenopathy. Sensitivity of EBUS-TBNA in patients with N0/N1 disease by PET/CT is unclear and largely based on retrospective studies. We assessed the sensitivity of EBUS-TBNA in this setting. Methods: We enrolled patients with proven or suspected lung cancer staged as N0/N1 by PET/CT and without metastatic disease (M0), who underwent staging EBUS-TBNA. Primary outcome was sensitivity of EBUS-TBNA compared with a composite reference standard of surgical stage or EBUS-TBNA stage if EBUS demonstrated N2/N3 disease. Results: Seventy-five patients were included in the analysis. Mean tumour size was 3.52 cm (AE1.63). Fifteen of 75 patients (20%) had N2 disease. EBUS-TBNA identified six while nine were only identified at surgery. Sensitivity of EBUS-TBNA for N2 disease was 40% (95% CI: 16.3-67.7%). Conclusion: A significant proportion of patients with N0/N1 disease by PET/CT had N2 disease (20%) and EBUS-TBNA identified a substantial fraction of these patients, thus improving diagnostic accuracy compared with PET/CT alone. Sensitivity of EBUS-TBNA however appears lower compared with historical data from patients with larger volume mediastinal disease. Therefore, strategies to improve EBUS-TBNA accuracy in this population should be further explored