Background: Metagenomic sequencing of respiratory microbial communities for pathogen identification in pneumonia may help overcome the limitations of culture-based methods. We examined the feasibility and clinical validity of rapid-turnaround metagenomics with Nanopore™ sequencing of clinical respiratory specimens. Methods: We conducted a case-control study of mechanically-ventilated patients with pneumonia (nine culturepositive and five culture-negative) and without pneumonia (eight controls). We collected endotracheal aspirates and applied a microbial DNA enrichment method prior to metagenomic sequencing with the Oxford Nanopore MinION device. For reference, we compared Nanopore results against clinical microbiologic cultures and bacterial 16S rRNA gene sequencing. Results: Human DNA depletion enabled in depth sequencing of microbial communities. In culture-positive cases, Nanopore revealed communities with high abundance of the bacterial or fungal species isolated by cultures. In four cases with resistant clinical isolates, Nanopore detected antibiotic resistance genes corresponding to the phenotypic resistance in antibiograms. In culture-negative pneumonia, Nanopore revealed probable bacterial pathogens in 1/5 cases and Candida colonization in 3/5 cases. In controls, Nanopore showed high abundance of oral bacteria in 5/8 subjects, and identified colonizing respiratory pathogens in other subjects. Nanopore and 16S sequencing showed excellent concordance for the most abundant bacterial taxa. Conclusions: We demonstrated technical feasibility and proof-of-concept clinical validity of Nanopore metagenomics for severe pneumonia diagnosis, with striking concordance with positive microbiologic cultures, and clinically actionable information obtained from sequencing in culture-negative samples. Prospective studies with real-time metagenomics are warranted to examine the impact on antimicrobial decision-making and clinical outcomes.

Insecticide and fungicide seed treatments are commonly used to control pests and pathogens in conventional maize and soybean culture, but little is known about their effects on the communities of non-target microbes that inhabit the rhizospheres or leaves of these crops. Because rhizosphere bacterial and fungal communities influence carbon and nutrient turnover, nutrient transformation, nutrient uptake and disease suppression, and because leaf endophyte fungal communities influence many aspects of stress tolerance in plants, any effect of a pesticide seed treatment on these microbial communities could have unintended and possibly adverse effects on seedling performance. We conducted a three-year field experiment in which maize (2013, 2015) and soybean (2014) were grown in rotation from seeds that were either coated or not coated with common pesticide treatments, which included contact and systemic fungicides and systemic insecticides. We sampled seedling rhizosphere soil (maize in 2013, soybean in 2014) and seedling leaves (soybean in 2014, maize in 2015) and characterized their microbial communities. For maize, the rhizosphere fungal and bacterial communities were significantly affected by the seed treatment, but leaf endophytic fungal communities were not. For soybean, the rhizosphere fungal community was significantly affected, as was the leaf endophytic fungal community, but not the rhizosphere bacterial community. These results show that pesticide seed treatments may affect rhizosphere soil microbial communities and endophytic leaf fungal communities more than one month after planting and, therefore, may have significant, unintended effects on non-target organisms. Additional research must determine the consequences of these effects and the nature of their context dependency.

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Background: Metagenomic sequencing of respiratory microbial communities for etiologic pathogen identification in pneumonia may help overcome the limitations of current culture-based methods. We examined the feasibility and clinical validity of rapid-turnaround metagenomics with Nanopore sequencing of respiratory samples for severe pneumonia diagnosis. Methods and Findings: We conducted a case-control study of mechanically-ventilated patients with pneumonia (nine culture-positive and five culture-negative) and without pneumonia (eight controls). We collected endotracheal aspirate samples (ETAs) and applied a microbial DNA enrichment method prior to performing metagenomic sequencing with the Oxford Nanopore MinION device. We compared Nanopore results against clinical microbiologic cultures and bacterial 16S rRNA gene sequencing. In nine culture-positive cases, Nanopore revealed communities with low alpha diversity and high abundance of the bacterial (n=8) or fungal (n=1) species isolated by clinical cultures. In four culture-positive cases with resistant organisms, Nanopore detected antibiotic resistance genes corresponding to the phenotypic resistance identified by clinical antibiograms. In culture-negative pneumonia, Nanopore revealed probable bacterial pathogens in 1/5 cases and airway colonization by Candida species in 3/5 cases. In controls, Nanopore showed high abundance of oral bacteria in 5/8 subjects, and identified colonizing respiratory pathogens in the three other subjects. Nanopore and 16S sequencing showed excellent concordance for the most abundant bacterial taxa. Conclusion: We demonstrated technical feasibility and proof-of-concept clinical validity of Nanopore metagenomics for severe pneumonia diagnosis, with striking concordance with positive microbiologic cultures and clinically actionable information offered from the sequencing profiles of culture-negative samples. Prospective studies with real-time metagenomics are warranted to examine the impact on antimicrobial decision-making and clinical outcomes.

BackgroundLung microbiota profiles in patients with early idiopathic pulmonary fibrosis (IPF) have been associated with disease progression; however, the topographic heterogeneity of lung microbiota and their roles in advanced IPF are unknown.MethodsWe performed a retrospective, case-control study of explanted lung tissue obtained at the time of lung transplantation or rapid autopsy from patients with IPF and other chronic lung diseases (connective tissue disease-associated interstitial lung disease (CTD-ILD), cystic fibrosis (CF), COPD and donor lungs unsuitable for transplant from Center for Organ Recovery and Education (CORE)). We sampled subpleural tissue and airway-based specimens (bronchial washings and airway tissue) and quantified bacterial load and profiled communities by amplification and sequencing of the 16S rRNA gene.FindingsExplants from 62 patients with IPF, 15 patients with CTD-ILD, 20 patients with CF, 20 patients with COPD and 20 CORE patients were included. Airway-based samples had higher bacterial load compared with distal parenchymal tissue. IPF basilar tissue had much lower bacterial load compared with CF and CORE lungs (p<0.001). No microbial community differences were found between parenchymal tissue samples from different IPF lobes. Dirichlet multinomial models revealed an IPF cluster (29%) with distinct composition, high bacterial load and low alpha diversity, exhibiting higher odds for acute exacerbation or death.InterpretationIPF explants had low biomass in the distal parenchyma of all three lobes with higher bacterial load in the airways. The discovery of a distinct subgroup of patients with IPF with higher bacterial load and worse clinical outcomes supports investigation of personalised medicine approaches for microbiome-targeted interventions.