WS03.6: Estimation of total bacterial load in explanted cystic fibrosis (CF) lungs via qPCR

Objectives: DNA-based analyses of CF airway samples have challenged ideas about the etiology of CF infections. Two notable findings are: (1) In early CF lung disease, the microbial DNA in samples typically derives from diverse collections of microbes not considered conventional CF pathogens. (2) In established disease (when conventional CF pathogens dominate) non-conventional pathogens are still identified, although in lesser quantities. These findings are complicated because airway samples transit through the oropharynx where non-conventional pathogens are highly abundant. In addition, collection devices (e.g. bronchoscopes) and analysis reagents harbor microbial DNA. Methods: We collected 190 bronchoalveolar lavage (BAL) samples from 22 children with CF after endotracheal tube intubation to attempt to bypass oropharyngeal organisms. We included controls to identify contamination from reagents, bronchoscopes, and the oropharynx. BAL was sequentially performed at 4–5 sites in each subject. Results: BAL samples varied widely in the abundance of bacterial DNA (measured by qPCR). At sites with high levels of bacterial DNA, 16S rRNA gene sequencing showed that conventional CF pathogens dominated, and a relatively small fraction of microbial DNA mapped to non-conventional organisms. In contrast, sites with a low abundance of bacterial DNA contained DNA from a diverse collection of non-conventional organisms. Notably, the non-conventional organisms detected in most BALs were dissimilar from those found in the oropharynx, suggesting that intubation effectively bypassed upper airway contaminants. However, the non-conventional organisms in most BALs were highly similar to those found in control washes from bronchoscopes performed before the procedure. Conclusion: Most of the non-conventional organisms in BALs were likely contaminants. The findings highlight the need for careful sampling and controls when DNA-based methods are used. WS03.6 Estimation of total bacterial load in explanted cystic fibrosis (CF) lungs via qPCR Objectives: Regional variation in microbial load may contribute to variation in structural damage and disease progression in the CF lung. This study aimed to estimate total bacterial load in both tissue and sputum samples from distinct anatomical regions of explanted CF lungs using 16S rRNA quantitative PCR (qPCR). Methods: Explanted lungs of CF patients were collected at transplantation, air inflated, frozen and cut into slices 2 cm thick. Cores (diameter = 1.4 cm) were removed from each slice and any sputum plugs within the tissue were excised. Following DNA extraction, the number of 16S copies/ml was quantified via probe-based absolute quantification using the LightCycler qPCR platform. Results: Sixty-one tissue (n = 13 patients) and 11 sputum (n = 5 patients) samples were analysed. The number of 16S copies/ml was significantly higher (p < 0.0001, Mann-Whitney U test) in sputum compared to tissue. Furthermore, in 9 matched sputum and tissue samples (collected from n = 4 patients), the number of 16S copies/ml was also significantly higher (p < 0.05, Wilcoxon signed-rank test) in sputum compared to the surrounding tissue. Nine patients had n ≥ 3 cores analysed; in 8 of these patients, significant differences (p < 0.05, Kruskal-Wallis H test, Dunn's test post-hoc) in 16S copies/mL were apparent between different tissue cores.