Culture enriched molecular profiling of the cystic fibrosis airway microbiome.

The microbiome of the respiratory tract, including the nasopharyngeal and oropharyngeal microbiota, is a dynamic community of microorganisms that is highly diverse. The cystic fibrosis (CF) airway microbiome refers to the polymicrobial communities present in the lower airways of CF patients. It is comprised of chronic opportunistic pathogens (such as Pseudomonas aeruginosa) and a variety of organisms derived mostly from the normal microbiota of the upper respiratory tract. The complexity of these communities has been inferred primarily from culture independent molecular profiling. As with most microbial communities it is generally assumed that most of the organisms present are not readily cultured. Our culture collection generated using more extensive cultivation approaches, reveals a more complex microbial community than that obtained by conventional CF culture methods. To directly evaluate the cultivability of the airway microbiome, we examined six samples in depth using culture-enriched molecular profiling which combines culture-based methods with the molecular profiling methods of terminal restriction fragment length polymorphisms and 16S rRNA gene sequencing. We demonstrate that combining culture-dependent and culture-independent approaches enhances the sensitivity of either approach alone. Our techniques were able to cultivate 43 of the 48 families detected by deep sequencing; the five families recovered solely by culture-independent approaches were all present at very low abundance (<0.002% total reads). 46% of the molecular signatures detected by culture from the six patients were only identified in an anaerobic environment, suggesting that a large proportion of the cultured airway community is composed of obligate anaerobes. Most significantly, using 20 growth conditions per specimen, half of which included anaerobic cultivation and extended incubation times we demonstrate that the majority of bacteria present can be cultured.

Pseudomonas aeruginosa cystic fibrosis isolates from individual patients demonstrate a range of levels of lethality in two Drosophila melanogaster infection models.

Recently, two Drosophila melanogaster models of infection, fly feeding and fly nicking, have been developed that allow a determination of pathogenic potential of Pseudomonas aeruginosa isolates. In this study, control strains, isolates from burn wounds, and isolates from the sputa of cystic fibrosis (CF) patients were used to compare the two infection models to determine whether any of the isolates might be better adapted to either of the models. In addition, our goal was to determine the variability of isolates from individual CF patients. Three of four control strains (PAO1, PAK, and PA14) caused significant mortality in the flies in both models of infection. The remaining control strain, PA103, was lethal to flies in the nicking model but lacked significant lethality in the feeding model. The burn wound isolates had a high level of lethality in both models. Interestingly, the CF isolates had the largest diversity of lethality in both models of infection. The range of pathogenic potentials of the CF isolates occurred across a cohort of patients, both at the patient level and down to the level of individual sputum samples. The majority of all isolates had similar levels of lethality in both fly infection models. However, two CF isolates were significantly more lethal in the nicking model, and three CF isolates were significantly more lethal in the feeding model. In conclusion, the two Drosophila infection models were useful for the analysis of the diversity of pathogenic potentials of P. aeruginosa isolates.