RESUMO
Soil ecosystems harbor diverse microorganisms and yet remain only partially characterized as neither single-cell sequencing nor whole-community sequencing offers a complete picture of these complex communities. Thus, the genetic and metabolic potential of this "uncultivated majority" remains underexplored. To address these challenges, we applied a pooled-cell-sorting-based mini-metagenomics approach and compared the results to bulk metagenomics. Informatic binning of these data produced 200 mini-metagenome assembled genomes (sorted-MAGs) and 29 bulk metagenome assembled genomes (MAGs). The sorted and bulk MAGs increased the known phylogenetic diversity of soil taxa by 7.2% with respect to the Joint Genome Institute IMG/M database and showed clade-specific sequence recruitment patterns across diverse terrestrial soil metagenomes. Additionally, sorted-MAGs expanded the rare biosphere not captured through MAGs from bulk sequences, exemplified through phylogenetic and functional analyses of members of the phylum Bacteroidetes Analysis of 67 Bacteroidetes sorted-MAGs showed conserved patterns of carbon metabolism across four clades. These results indicate that mini-metagenomics enables genome-resolved investigation of predicted metabolism and demonstrates the utility of combining metagenomics methods to tap into the diversity of heterogeneous microbial assemblages.IMPORTANCE Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism.
RESUMO
Life-threatening tension pneumothorax in neonates on extracorporeal membrane oxygenation (ECMO) has been associated with an increase in arterial oxygen tension and a decrease in peripheral perfusion, followed by a decrease in ECMO flow with progressive hemodynamic deterioration. To investigate this triad, chest tubes were placed bilaterally in 9 dogs to allow injection of air to produce tension pneumothorax. Six dogs were subsequently placed on standard venoarterial ECMO before the reinduction of tension pneumothorax. Measured values included arterial pulse pressure, inferior vena cava pressure, systemic arterial blood gases, peripheral arterial oxygen saturation, mixed venous oxygen saturation, and left heart cardiac output. Oxygen delivery was calculated from directly measured values. Each of the 6 dogs on ECMO demonstrated the triad of increased arterial oxygen tension (92 +/- 7 to 325 +/- 20 mm Hg; p less than 0.05), decreased peripheral perfusion (as evidenced by a decrease in pulse pressure from 55 +/- 4 to 31 +/- 5 mm Hg; p less than 0.05), and decreased mixed venous oxygen saturation (71% +/- 3% to 22% +/- 2% saturation; p less than 0.05) followed by a lower ECMO flow with progressive hemodynamic deterioration (oxygen delivery decreased from 285 +/- 11 to 111 +/- 12 mL/min; p less than 0.05). Aspiration of the intrathoracic air allowed return to baseline ECMO flow and hemodynamic stability in all dogs. The triad of increased arterial oxygen tension and decreased peripheral perfusion (as evidenced by a lower arterial pulse pressure and lower mixed venous oxygen saturation) followed by decreased ECMO flow with progressive hemodynamic deterioration consistently appears when tension pneumothorax occurs on ECMO.
