Periodic Reporting for period 3 - PHARMS (Bacteriophage inhibition of antibiotic-resistant pathogenic microbes and founding for novel therapeutic strategies) Reporting period: 2022-01-01 to 2023-06-30 Summary of the context and overall objectives of the project The emergence of antimicrobial resistance (AMR) is a major threat to global health, global economies, and humanity itself. Worldwide, the death toll is forecasted to exceed those caused by cancer, diabetes and cardiovascular diseases by 2050 in total - if no new therapies are developed. In the last two decades, only one new class of antibiotics has been brought to the market. This is partly due to the fact that “classical” targets in bacteria have been addressed extensively, now only leading to the rapid development of resistance. Thus, there is an enormous need to catch up on innovative therapeutic strategies that are beyond the beaten path of classic antibiotic development. Phage therapy, a promising complement to antibiotics, utilizes viruses of bacteria (bacteriophages) or phage-derived inhibitors as natural ways to fight AMR. The main obstacles in the clinical application of phage-based AMR therapy are the limited number of phage isolates and the unknown molecular mechanisms of phage-delivered bactericidal action. Building on the recent advances of my group in high-throughput, culture-independent but host-targeted methodologies, PHARMS aims to deploy a revolutionary approach: to screen for all possible phages of a resistant bacterial isolate, characterize multiple lines of their bactericidal functions, and use this information for the design of a whole battery of phage-based therapies that employ multifaceted modes of action. By elucidating universal and specific mechanisms of phage-delivered inhibition of AMR pathogens, PHARMS is positioned to provide the rational framework for the design of novel therapeutic strategies aimed at treating common and life-threatening infectious diseases. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We used the high throughput ‘Viral Tagging’ combined with metagenomic analyses to screen environmental and human-derived samples to isolate and sequence unculturable phages infecting three multi-resistant bacteria: multiple Helicobacter pylori strains, Acinetobacter baumannii, and Haemophilus influenzae. This has expanded our understanding of viral diversity infecting these pathogens. We identified over a hundred major novel viruses against H. pylori, H. influenza, and A. baumannii. Among those, no taxonomy was assigned to the majority of the viral contigs based on the previously isolated bacteriophages, which again indicates the giant cultivation bias. We observed high diversity among those newly identified novel phages as well and could identify their phylogenetic relationship.Next, we looked into the infection mechanisms of novel phages on A. Baumannii cells to showcase the novel infection strategies those new phages encode. We studied phage-host interaction in different physiological states of the bacteria, and using a multi-omics approach that integrates genomics, transcriptomics, proteomics, and metabolomics. We revealed significant changes in host metabolism co-occurred with a twentyfold increase in phage mRNAs involved in the regulatory, metabolic, and antibacterial activities. Yet, many phage genes upregulated were of unknown functions. In addition, we have identified over a hundred proteins and metabolites specific to phage-infected bacteria, which varied based on the host's physiology and time of infection. Moreover, the network analyses showed dynamic correlations between different omes through the infection period. The strongest correlations were observed for omes involved in fatty acid, nucleotide, and amino acid metabolisms, suggesting significant phage-specific reprogramming of the host's metabolic activities.In addition, to join the fight against health complications caused by the COVID-19 pandemic, we isolated, characterized, and formulated a combination of phages for treating acute pneumonia caused by co-infection with multiresistant bacteria. The top-notch cocktail that was developed following extensive kinetics analyses consists of highly efficient phages against the most common sources of co-infections in COVID-19 patients: multiresistant Klebsiella pneumonia, Pseudomonas aeruginosa, and Staphylococcus aureus. Our analyses showed high genetic diversity among the selected phages suggesting diverse infection strategies used by them for infecting the target bacteria; this further complicates the development of resistance in host bacteria and holds its potential for further therapeutic development.To date, we have identified over a hundred major novel viruses and discovered novel phage infection strategies. Multi-omics approaches were tailored to generate a list of novel phage genes which hold potential for further therapeutic development; in vitro and in vivo evaluation are ongoing. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The knowledge gained in PHARMS has the potential to change the current paradigms in AMR control, by elaborating the molecular mechanisms of phage inhibition of AMR strains that is currently a “black box” and beyond our classical view of phage activity. PHARMS will provide a systematic analysis of phage inhibition on AMR bacteria, and functional characterization of the resulting genes of interest. I expect these results to lead to the discovery of novel molecular mechanisms of phage bactericidal activity, and insights into the general mechanisms controlling the regulation of phage infection that may also lead to bactericidal activity to other AMR pathogenic bacteria, thus can be exploited as therapeutic products with superior bactericidal kinetics. Furthermore, given the extreme and almost unexplored diversity of phages, discoveries in PHARMS may lead to the development of novel tools for metabolic engineering and cell cycle control, which might become as important as recombineering or CRISPR/Cas already has.
