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Pneumonia and other lower respiratory tract infections are the leading contributors to global mortality of any communicable disease [1]. During normal pulmonary homeostasis, competing microbial immigration and elimination produce a transient microbiome with distinct microbial states [2-4]. Disruption of underlying ecological forces, like aspiration rate and immune tone, are hypothesized to drive microbiome dysbiosis and pneumonia progression [5-7]. However, the precise microbiome transitions that accompany clinical outcomes in severe pneumonia are unknown. Here, we leverage our unique systematic and serial bronchoscopic sampling to combine quantitative PCR and culture for bacterial biomass with 16S rRNA gene amplicon, shotgun metagenomic, and transcriptomic sequencing in patients with suspected pneumonia to distill microbial signatures of clinical outcome. These data support the presence of four distinct microbiota states-oral-like, skin-like, Staphylococcus-predominant, and mixed-each differentially associated with pneumonia subtype and responses to pneumonia therapy. Infection-specific dysbiosis, quantified relative to non-pneumonia patients, associates with bacterial biomass and elevated oral-associated microbiota. Time series analysis suggests that microbiome shifts from baseline are greater with successful pneumonia therapy, following distinct trajectories dependent on the pneumonia subtype. In summary, our results highlight the dynamic nature of the lung microbiome as it progresses through community assemblages that parallel patient prognosis. Application of a microbial ecology framework to study lower respiratory tract infections enables contextualization of the microbiome composition and gene content within clinical phenotypes. Further unveiling the ecological dynamics of the lung microbial ecosystem provides critical insights for future work toward improving pneumonia therapy.

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The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains of P. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages.IMPORTANCEPseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.

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BACKGROUND: While indoor microbiomes impact our health and well-being, much remains unknown about taxonomic and functional transitions that occur in human-derived microbial communities once they are transferred away from human hosts. Toothbrushes are a model to investigate the potential response of oral-derived microbiota to conditions of the built environment. Here, we characterize metagenomes of toothbrushes from 34 subjects to define the toothbrush microbiome and resistome and possible influential factors. RESULTS: Toothbrush microbiomes often comprised a dominant subset of human oral taxa and less abundant or site-specific environmental strains. Although toothbrushes contained lower taxonomic diversity than oral-associated counterparts (determined by comparison with the Human Microbiome Project), they had relatively broader antimicrobial resistance gene (ARG) profiles. Toothbrush resistomes were enriched with a variety of ARGs, notably those conferring multidrug efflux and putative resistance to triclosan, which were primarily attributable to versatile environmental taxa. Toothbrush microbial communities and resistomes correlated with a variety of factors linked to personal health, dental hygiene, and bathroom features. CONCLUSIONS: Selective pressures in the built environment may shape the dynamic mixture of human (primarily oral-associated) and environmental microbiota that encounter each other on toothbrushes. Harboring a microbial diversity and resistome distinct from human-associated counterparts suggests toothbrushes could potentially serve as a reservoir that may enable the transfer of ARGs. Video abstract.

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