Metagenomics and metatranscriptomics: Windows on CF-associated viral and microbial communities

Abstract

Background Samples collected from CF patient airways often contain large amounts of host-derived nucleic acids that interfere with recovery and purification of microbial and viral nucleic acids. This study describes metagenomic and metatranscriptomic methods that address these issues. Methods Microbial and viral metagenomes, and microbial metatranscriptomes, were successfully prepared from sputum samples from five adult CF patients. Results Contaminating host DNA was dramatically reduced in the metagenomes. Each CF patient presented a unique microbiome; in some Pseudomonas aeruginosa was replaced by other opportunistic bacteria. Even though the taxonomic composition of the microbiomes are very different, the metabolic potentials encoded by the community are very similar. The viral communities were dominated by phages that infect major CF pathogens. The metatranscriptomes reveal differential expression of encoded metabolic potential with changing health status. Conclusions Microbial and viral metagenomics combined with microbial transcriptomics characterize the dynamic polymicrobial communities found in CF airways, revealing both the taxa present and their current metabolic activities. These approaches can facilitate the development of individualized treatment plans and novel therapeutic approaches.

A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation

“…The cystic fibrosis (CF) lung contains a complex community of interacting microbes and viruses (Willner et al, 2009;Sibley and Surette, 2011;Yang et al, 2011;Delhaes et al, 2012;Lim et al, 2012;Rabin and Surette, 2012;Zhao et al, 2012;. These communities shape microenvironments within the lung with their own biological, chemical and physical characteristics.…”

Biofilm development and enhanced stress resistance of a model, mixed-species community biofilm

“…For example, while P. aeruginosa dominates biofilms found in the lungs of cystic fibrosis patients, other species such as Abiotrophia adiacens, Bacteroides gracilis, Prevotella salivae, Rothia mucilaginosa and Staphylococcus hominis are also present (Rogers et al, 2004;Lim et al, 2013b). Interactions between different species in the mixed-biofilm communities are believed to affect their development, structures and functions (James et al, 1995;Harrison, 2007;Moons et al, 2009).…”

Clinical Insights from Metagenomic Analysis of Sputum Samples from Patients with Cystic Fibrosis

“…Microbial samples (i.e., the microbiome) were treated with ␤-mercaptoethanol to dissociate the mucus. The cells were repeatedly washed with sterilized deionized water to lyse the human cells and then treated with DNase to remove extracellular DNA (e.g., human DNA and that associated with biofilms [5]). Microbial DNA was extracted using the NucleoSpin tissue kit with the Gram-positive variation, which includes a lysozyme treatment.…”

“…Despite these significant advances, newer technologies have yet to be used in routine clinical microbiology practice. Treating polymicrobial infections will also require a personalized approach, because the taxonomic identities and functional characteristics of microbial communities are often patient specific (5). Here, we moved toward this goal by using metagenomics to monitor complex pulmonary infections in patients with cystic fibrosis (CF).…”

“…Thus, information on community function is typically only obtainable with metagenomic sequencing, although some functions can be predicted based on taxonomy alone (e.g., methanogenesis). Neither approach requires culturing bacteria, and when applied to CF patients, both methods have revealed the microbial diversity and community complexity in airways to be unexpectedly high (5,9). Furthermore, these sequence-based technologies have demonstrated that bacterial diversity decreases during treatment with broad-spectrum antibiotics and as the disease advances over longer periods of time (10,11).…”