Clinical insights from metagenomic analysis of sputum samples from patients with cystic fibrosis.
ABSTRACT: As DNA sequencing becomes faster and cheaper, genomics-based approaches are being explored for their use in personalized diagnoses and treatments. Here, we provide a proof of principle for disease monitoring using personal metagenomic sequencing and traditional clinical microbiology by focusing on three adults with cystic fibrosis (CF). The CF lung is a dynamic environment that hosts a complex ecosystem composed of bacteria, viruses, and fungi that can vary in space and time. Not surprisingly, the microbiome data from the induced sputum samples we collected revealed a significant amount of species diversity not seen in routine clinical laboratory cultures. The relative abundances of several species changed as clinical treatment was altered, enabling the identification of the climax and attack communities that were proposed in an earlier work. All patient microbiomes encoded a diversity of mechanisms to resist antibiotics, consistent with the characteristics of multidrug-resistant microbial communities that are commonly observed in CF patients. The metabolic potentials of these communities differed by the health status and recovery route of each patient. Thus, this pilot study provides an example of how metagenomic data might be used with clinical assessments for the development of treatments tailored to individual patients.
Project description:Current therapy for cystic fibrosis (CF) focuses on minimizing the microbial community and the host's immune response through the aggressive use of airway clearance techniques, broad-spectrum antibiotics, and treatments that break down the pervasive endobronchial biofilm. Antibiotic selection is typically based on the susceptibility of individual microbial strains to specific antibiotics in vitro. Often this approach cannot accurately predict medical outcomes because of factors both technical and biological. Recent culture-independent assessments of the airway microbial and viral communities demonstrated that the CF airway infection is considerably more complex and dynamic than previously appreciated. Understanding the ecological and evolutionary pressures that shape these communities is critically important for the optimal use of current therapies (in both the choice of therapy and timing of administration) and the development of newer strategies. The climax-attack model (CAM) presented here, grounded in basic ecological principles, postulates the existence of two major functional communities. The attack community consists of transient viral and microbial populations that induce strong innate immune responses. The resultant intense immune response creates microenvironments that facilitate the establishment of a climax community that is slower-growing and inherently resistant to antibiotic therapy. Newer methodologies, including sequence-based metagenomic analysis, can track not only the taxonomic composition but also the metabolic capabilities of these changing viral and microbial communities over time. Collecting this information for CF airways will enable the mathematical modeling of microbial community dynamics during disease progression. The resultant understanding of airway communities and their effects on lung physiology will facilitate the optimization of CF therapies.
Project description:The human respiratory tract is constantly exposed to a wide variety of viruses, microbes and inorganic particulates from environmental air, water and food. Physical characteristics of inhaled particles and airway mucosal immunity determine which viruses and microbes will persist in the airways. Here we present the first metagenomic study of DNA viral communities in the airways of diseased and non-diseased individuals. We obtained sequences from sputum DNA viral communities in 5 individuals with cystic fibrosis (CF) and 5 individuals without the disease. Overall, diversity of viruses in the airways was low, with an average richness of 175 distinct viral genotypes. The majority of viral diversity was uncharacterized. CF phage communities were highly similar to each other, whereas Non-CF individuals had more distinct phage communities, which may reflect organisms in inhaled air. CF eukaryotic viral communities were dominated by a few viruses, including human herpesviruses and retroviruses. Functional metagenomics showed that all Non-CF viromes were similar, and that CF viromes were enriched in aromatic amino acid metabolism. The CF metagenomes occupied two different metabolic states, probably reflecting different disease states. There was one outlying CF virome which was characterized by an over-representation of Guanosine-5'-triphosphate,3'-diphosphate pyrophosphatase, an enzyme involved in the bacterial stringent response. Unique environments like the CF airway can drive functional adaptations, leading to shifts in metabolic profiles. These results have important clinical implications for CF, indicating that therapeutic measures may be more effective if used to change the respiratory environment, as opposed to shifting the taxonomic composition of resident microbiota.
Project description:Although the cystic fibrosis (CF) lung microbiota has been characterized in several studies, little is still known about the temporal changes occurring at the whole microbiome level using untargeted metagenomic analysis. The aim of this study was to investigate the taxonomic and functional temporal dynamics of the lower airway microbiome in a cohort of CF patients. Multiple sputum samples were collected over 15 months from 22 patients with advanced lung disease regularly attending three Italian CF Centers, given a total of 79 samples. DNA extracted from samples was subjected to shotgun metagenomic sequencing allowing both strain-level taxonomic profiling and assessment of the functional metagenomic repertoire. High inter-patient taxonomic heterogeneity was found with short-term compositional changes across clinical status. Each patient exhibited distinct sputum microbial communities at the taxonomic level, and strain-specific colonization of both traditional and atypical CF pathogens. A large core set of genes, including antibiotic resistance genes, were shared across patients despite observed differences in clinical status, and consistently detected in the lung microbiome of all subjects independently from known antibiotic exposure. In conclusion, an overall stability in the microbiome-associated genes was found despite taxonomic fluctuations of the communities.
Project description:Lung infections play a critical role in cystic fibrosis (CF) pathogenesis. CF respiratory tract is now considered to be a polymicrobial niche and advances in high-throughput sequencing allowed to analyze its microbiota and mycobiota. However, no NGS studies until now have characterized both communities during CF pulmonary exacerbation (CFPE). Thirty-three sputa isolated from patients with and without CFPE were used for metagenomic high-throughput sequencing targeting 16S and ITS2 regions of bacterial and fungal rRNA. We built inter-kingdom network and adapted Phy-Lasso method to highlight correlations in compositional data. The decline in respiratory function was associated with a decrease in bacterial diversity. The inter-kingdom network revealed three main clusters organized around Aspergillus, Candida, and Scedosporium genera. Using Phy-Lasso method, we identified Aspergillus and Malassezia as relevantly associated with CFPE, and Scedosporium plus Pseudomonas with a decline in lung function. We corroborated in vitro the cross-domain interactions between Aspergillus and Streptococcus predicted by the correlation network. For the first time, we included documented mycobiome data into a version of the ecological Climax/Attack model that opens new lines of thoughts about the physiopathology of CF lung disease and future perspectives to improve its therapeutic management.
Project description:BACKGROUND: Saturated brines are extreme environments of low diversity. Salinibacter ruber is the only bacterium that inhabits this environment in significant numbers. In order to establish the extent of genetic diversity in natural populations of this microbe, the genomic sequence of reference strain DSM 13855 was compared to metagenomic fragments recovered from climax saltern crystallizers and obtained with 454 sequencing technology. This kind of analysis reveals the presence of metagenomic islands, i.e. highly variable regions among the different lineages in the population. RESULTS: Three regions of the sequenced isolate were scarcely represented in the metagenome thus appearing to vary among co-occurring S. ruber cells. These metagenomic islands showed evidence of extensive genomic corruption with atypically low GC content, low coding density, high numbers of pseudogenes and short hypothetical proteins. A detailed analysis of island gene content showed that the genes in metagenomic island 1 code for cell surface polysaccharides. The strain-specific genes of metagenomic island 2 were found to be involved in biosynthesis of cell wall polysaccharide components. Finally, metagenomic island 3 was rich in DNA related enzymes. CONCLUSION: The genomic organisation of S. ruber variable genomic regions showed a number of convergences with genomic islands of marine microbes studied, being largely involved in variable cell surface traits. This variation at the level of cell envelopes in an environment devoid of grazing pressure probably reflects a global strategy of bacteria to escape phage predation.
Project description:Cystic fibrosis (CF) is a life-threatening genetic disorder, characterized by chronic microbial lung infections due to abnormally viscous mucus secretions within airways. The clinical management of CF typically involves regular respiratory-tract cultures in order to identify pathogens and to guide treatment. However, culture-based methods can miss atypical or slow-growing microbes. Furthermore, the isolated microbes are often not classified at the strain level due to limited taxonomic resolution.Here, we show that untargeted metagenomic sequencing of sputum DNA can provide valuable information beyond the possibilities of culture-based diagnosis. We sequenced the sputum of six CF patients and eleven control samples (including healthy subjects and chronic obstructive pulmonary disease patients) without prior depletion of human DNA or cell size selection, thus obtaining the most unbiased and comprehensive characterization of CF respiratory tract microbes to date. We present detailed descriptions of the CF and healthy lung microbiome, reconstruct near complete pathogen genomes, and confirm that the CF lungs consistently exhibit reduced microbial diversity. Crucially, the obtained genomic sequences enabled a detailed identification of the exact pathogen strain types, when analyzed in conjunction with existing multi-locus sequence typing databases. We also detected putative pathogenicity islands and indicators of antibiotic resistance, in good agreement with independent clinical tests.Unbiased sputum metagenomics provides an in-depth profile of the lung pathogen microbiome, which is complementary to and more detailed than standard culture-based reporting. Furthermore, functional and taxonomic features of the dominant pathogens, including antibiotics resistances, can be deduced-supporting accurate and non-invasive clinical diagnosis.
Project description:For centuries ecologists have studied how the diversity and functional traits of plant and animal communities vary across biomes. In contrast, we have only just begun exploring similar questions for soil microbial communities despite soil microbes being the dominant engines of biogeochemical cycles and a major pool of living biomass in terrestrial ecosystems. We used metagenomic sequencing to compare the composition and functional attributes of 16 soil microbial communities collected from cold deserts, hot deserts, forests, grasslands, and tundra. Those communities found in plant-free cold desert soils typically had the lowest levels of functional diversity (diversity of protein-coding gene categories) and the lowest levels of phylogenetic and taxonomic diversity. Across all soils, functional beta diversity was strongly correlated with taxonomic and phylogenetic beta diversity; the desert microbial communities were clearly distinct from the nondesert communities regardless of the metric used. The desert communities had higher relative abundances of genes associated with osmoregulation and dormancy, but lower relative abundances of genes associated with nutrient cycling and the catabolism of plant-derived organic compounds. Antibiotic resistance genes were consistently threefold less abundant in the desert soils than in the nondesert soils, suggesting that abiotic conditions, not competitive interactions, are more important in shaping the desert microbial communities. As the most comprehensive survey of soil taxonomic, phylogenetic, and functional diversity to date, this study demonstrates that metagenomic approaches can be used to build a predictive understanding of how microbial diversity and function vary across terrestrial biomes.
Project description:Pseudomonas aeruginosa (Pa), normally a soil commensal, is an important opportunistic pathogen in Cystic Fibrosis (CF) and non-Cystic Fibrosis Bronchiectasis (nCFBR). Persistent infection correlates with accelerated decline in lung function and early mortality. The horizontal transfer of DNA by temperate bacteriophages can add gene function and selective advantages to their bacterial host within the constrained environment of the lower lung. In this study, we chemically induce temperate bacteriophages from clonal cultures of Pa and identify their mixed viral communities employing metagenomic approaches. We compared 92 temperate phage metagenomes stratified from these clinical backgrounds (47 CF and 45 nCFBR Pa isolates) using MG-RAST and GeneWise2. KEGG analysis shows the complexity of temperate phage accessory gene carriage increases with duration and severity of the disease. Furthermore, we identify the presence of Ig-like motifs within phage structural genes linked to bacterial adhesion and carbohydrate binding including Big_2, He_Pig, and Fn3. This study provides the first clinical support to the proposed bacteriophage adherence to mucus (BAM) model and the evolution of phages interacting at these mucosal surfaces over time.
Project description:We report the soil microbial diversity and functional aspects related to degradation of recalcitrant compounds, determined using a metagenomic approach, in a landfill lysimeter prepared with soil from Ghazipur landfill site, New Delhi, India. Metagenomic analysis revealed the presence and functional diversity of complex microbial communities responsible for waste degradation.
Project description:Kitchen sponges massively absorb and spread microorganisms, leading to contamination of kitchen appliances, surfaces, and food. Microwaving as an effective and widespread technique can rapidly reduce the microbial load of kitchen sponges. However, long-term effects of such treatments are largely unknown. Notably, it has been speculated that regularly applied domestic cleaning and disinfection may select for microbial communities with a higher pathogenic potential and/or malodorous properties. In this study, we distributed newly purchased polyurethane kitchen sponges to 20 participants, with the instruction to use them under normal household conditions for four weeks. Ten of the participants sanitized their sponges regularly by a standardized microwaving protocol, while the remaining ten sponges remained untreated. Metagenomic sequence data evaluation indicated that, in addition to bacteria, viruses, eukaryotes, and archaea were also part of the kitchen sponge microbiome. Comparisons of sanitized and untreated kitchen sponges indicated a trend towards a reduced structural microbial diversity while functional diversity increased. Microwave sanitization appeared to alter composition and metabolic properties of the microbial communities. Follow-up studies will have to show whether these changes are more positive or negative in terms of domestic hygiene, human health, and well-being.