There is an urgent need to identify therapies that prevent SARS-CoV-2 infection and improve the outcome of COVID-19 patients. Although repurposed drugs with favorable safety profiles could have significant benefit, widely available prevention or treatment options for COVID-19 have yet to be identified. Efforts to identify approved drugs with in vitro activity against SARS-CoV-2 resulted in identification of antiviral sigma-1 receptor ligands, including antihistamines in the histamine-1 receptor binding class. We identified antihistamine candidates for repurposing by mining electronic health records of usage in population of more than 219,000 subjects tested for SARS-CoV-2. Usage of diphenhydramine, hydroxyzine and azelastine was associated with reduced incidence of SARS-CoV-2 positivity in subjects greater than age 61. We found diphenhydramine, hydroxyzine and azelastine to exhibit direct antiviral activity against SARS-CoV-2 in vitro. Although mechanisms by which specific antihistamines exert antiviral effects is not clear, hydroxyzine, and possibly azelastine, bind Angiotensin Converting Enzyme-2 (ACE2) and the sigma-1 receptor as off-targets. Clinical studies are needed to measure the effectiveness of diphenhydramine, hydroxyzine and azelastine for disease prevention, for early intervention, or as adjuvant therapy for severe COVID-19.

Bacterial cooperative associations and dynamics in the biofilm microenvironments are of special interest in recent years. Knowledge of localized gene-expression and corresponding bacterial behaviors within the biofilm architecture at a global scale has been limited, due to a lack of robust technology to study limited number of cells in stratified layers of biofilms. With our recent pioneering developments in single bacterial cell transcriptomic analysis technology, we generated herein an unprecedented spatial transcriptome map of the mature in vitro Pseudomonas aeruginosa biofilm model, revealing contemporaneous yet altered bacterial behaviors at different layers within the biofilm architecture (i.e., surface, middle, and interior of the biofilm). Many genes encoding unknown functions were highly expressed at the biofilm-solid interphase, exposing a critical gap in the knowledge of their activities that maybe unique to this interior niche. Several genes of unknown functions are critical for biofilm formation. The in vivo importance of these unknown proteins was validated in invertebrate (fruit fly) and vertebrate (mouse) models. We envisage the future value of this report to the community, in aiding the further pathophysiological understanding of P. aeruginosa biofilms. Our approach will open doors to the study of bacterial functional genomics of different species in numerous settings.

The fatty acid (FA) degradation pathway of Pseudomonas aeruginosa, an opportunistic pathogen, was recently shown to be involved in nutrient acquisition during BALB/c mouse lung infection model. The source of FA in the lung is believed to be phosphatidylcholine, the major component of lung surfactant. Previous research indicated that P. aeruginosa has more than two fatty acyl-CoA synthetase genes (fadD; PA3299 and PA3300), which are responsible for activation of FAs using ATP and coenzyme A. Through a bioinformatics approach, 11 candidate genes were identified by their homology to the Escherichia coli FadD in the present study. Four new homologues of fadD (PA1617, PA2893, PA3860, and PA3924) were functionally confirmed by their ability to complement the E. coli fadD mutant on FA-containing media. Growth phenotypes of 17 combinatorial fadD mutants on different FAs, as sole carbon sources, indicated that the four new fadD homologues are involved in FA degradation, bringing the total number of P. aeruginosa fadD genes to six. Of the four new homologues, fadD4 (PA1617) contributed the most to the degradation of different chain length FAs. Growth patterns of various fadD mutants on plant-based perfumery substances, citronellic and geranic acids, as sole carbon and energy sources indicated that fadD4 is also involved in the degradation of these plant-derived compounds. A decrease in fitness of the sextuple fadD mutant, relative to the ΔfadD1D2 mutant, was only observed during BALB/c mouse lung infection at 24 h.

Prokaryotic cell transcriptomics has been limited to mixed or sub-population dynamics and individual cells within heterogeneous populations, which has hampered further understanding of spatiotemporal and stage-specific processes of prokaryotic cells within complex environments. Here we develop a ‘TRANSITomic’ approach to profile transcriptomes of single Burkholderia pseudomallei cells as they transit through host cell infection at defined stages, yielding pathophysiological insights. We find that B. pseudomallei transits through host cells during infection in three observable stages: vacuole entry; cytoplasmic escape and replication; and membrane protrusion, promoting cell-to-cell spread. The B. pseudomallei ‘TRANSITome’ reveals dynamic gene-expression flux during transit in host cells and identifies genes that are required for pathogenesis. We find several hypothetical proteins and assign them to virulence mechanisms, including attachment, cytoskeletal modulation, and autophagy evasion. The B. pseudomallei ‘TRANSITome’ provides prokaryotic single-cell transcriptomics information enabling high-resolution understanding of host-pathogen interactions.