Discovery of natural phages in Eastern EUCOM region to target MDR ESKAPE pathogens for wound infection countermeasure development
The challenges of developing novel antimicrobial compounds in the era of crisis-level AMR has re-energized efforts to bring bacteriophage therapy into clinical armaments (11, 12). A global consortium research effort led by U.S. Army and Navy to develop phage therapeutic approaches is investigating "naturally" harvested phages to design fixed, modularized, or personalized phage cocktails. The constituent phages evaluated for inclusion in the various cocktail formulations are down-selected to enrich for those targeting specific MDROs. Participating OCONUS DoD laboratories then purify the phage suspensions collected from their unique respective geographic regions and send them to WRAIR-Main for more comprehensive characterization and evaluation (13-16).
Notably, USAMRD-G owns a collection of diverse, well-characterized high-risk global and novel MDR ESKAPE pathogens collected via its long-term GEIS-funded AMR surveillance activities (17). The majority of this collection is presented by carbapenem-resistant MDR K. pneumoniae, P. aeruginosa and A. baumannii, including latter two pathogens displaying extensive drug-resistant (XDR; defined as non-susceptibility to all but two or fewer antimicrobial classes) phenotypes. Under the continuous surveillance of two active AMR studies, we also have observed increasing antibiotic resistance over time. Last year (2021), for example, we observed for the first time potentially pan-drug resistant (PDR; defined as non-susceptibility to all drugs in all antimicrobial classes) clinical isolates of A. baumannii.
We plan to expand the catchment area of our AMR protocols to monitor resistance profiles of ESKAPE pathogens from other countries across Eastern Europe. Given our location in Tbilisi (the birthplace of modern phage research), active collaboration with the Eliava Institute (the world's foremost phage research and treatment center), access to environmental samples (through our widespread surveillance activities for vectors and vector-borne pathogens), and AMR surveillance across Eastern EUCOM (planned for Latvia, Romania, Bulgaria, Poland, and potentially Ukraine), rapidly growing sequencing capabilities, and close relationship with MRSN and BT, USAMRD-G is uniquely positioned to conduct its own advanced studies of the phage host-range, phage resistance, and acquisition/loss of antibiotic resistance among isolates over time with added phage selective pressure. For our ESKAPE isolates exhibiting phage resistant phenotypes, we will also study genomic elements and mutations that confer resistance, whether those mechanisms can be inactivated, and/or whether the resistance phenotypes can be overcome by other phage isolates.
We propose to fill the gaps of phage research activities and expand upon the ongoing Army-Navy phage therapeutic research activities by leveraging the strategic geographical position of USAMRD-G. If funded, we will harvest MDRO-targeting natural phages from environmental sources within the EUCOM AOR and evaluate them to select therapeutic options and thus, support DoD's efforts to identify potential MCM candidates for MDR infection treatment. We will also investigate relationships between phage and host bacteria to identify possible genetic elements contributing to phage resistance.
Discovery and propagation of the new natural phages from the Eastern EUCOM region will enrich the current phage library and support development of advanced phage products (global, population-focused, or individual) with global applicability. We hypothesize that we will discover novel natural bacteriophages that target unique MDR ESKAPE pathogens circulating in our surveillance areas.
Specific Aims, Objectives, and Milestones:
The main objective of the study is to collect and characterize new EUCOM region-specific phages targeting MDROs. The present research will be focused on A. baumannii, K. pneumoniae and P. aeruginosa, as they are most abundant MDR pathogens observed within our local strain collection. We will down-select for phages with desired characteristics for further consideration as cocktail constituents. Meeting this objective addresses the current gap in the broad phage library and contributes to the advancement of phage as a viable countermeasure against MDROs. To reach the goal we will complete the following study aims.
Aim 1: Identify phage-resistant MDROs from Eastern EUCOM
1.1 From the USAMRD-G's extant collection of phages against K. pneumoniae and P. aeruginosa from the PRMRP study, we will select 15-25 phages with the broadest lytic spectrum against each target MDRO species. Based on our prior phage hunts and screening under the scope of PRMRP program, the phages chosen for further analysis will have lytic activity against 60% of K. pneumoniae or P. aeruginosa strains.
1.2 From the diverse collection of clinical Georgian MDR ESKAPE isolates, we will choose strains of A. baumannii, K. pneumoniae and P. aeruginosa that were not tested previously against available phages. Our MDRO collection, which has been sequenced and characterized previously by MRSN to obtain AMR genotypes and phylogenetic relationships, can provide strains of K. pneumoniae, P. aeruginosa (n=30-50 strains of each species) and A. baumannii (n=10-20) for determining their susceptibility to available phages.
1.3 We will examine the lytic profile of selected phages using the panel of selected Georgian K. pneumoniae or P. aeruginosa strains as well as isolates obtained from Balkan and Baltic countries to: a) select phage preparations with broadest lytic activity and b) identify Eastern EUCOM MDRO strains that are non-susceptible to the most efficient, i.e., possessing the widest host range, phages in the collection. The non-susceptible MDROs will be used as hosts for subsequent phage hunts and enrichments, and will be investigated for genetic elements that confer phage resistance.
Aim 2: Collect new natural phages against these clinical MDROs circulating in Eastern EUCOM
2.1 - 2.3 Representative sets of A. baumannii and K. pneumoniae and P. aeruginosa exhibiting phage resistance under Aim 1 will be used to harvest natural phages from the environmental sources collected in different geographical areas (corresponding to subtasks in this aim) in Central and Eastern Europe.
Aim 3: Evaluate phage host response range using our collection of regional MDROs as well as select MDRO ESKAPE strains from MRSN
3.1 - 3.3 We will capabilities characterize the lytic profile of new phages using the collections of corresponding MDROs obtained from various countries within the EUCOM region and select subset of phages with the therapeutic potential.
Aim 4: Characterize selected phages using genomic analysis
4.1 - 4.3 We will collect, sequence, and characterize phages selected for lytic activity against phage-resistant pathogen strains of A. baumannii, K. pneumoniae and P. aeruginosa using MinION and/or Illumina-based sequencing technologies. We will analyze and annotate phage genomes and deposit the annotated genomes on NCBI.
Aim 5: Identify bacterial genetic elements contributing to the development of the phage-resistance
5.1 - 5.3 We will identify bacterial genetic elements contributing to the development of the phage-resistance of MDR strains of K. pneumoniae, P. aeruginosa and A. baumannii from Eastern Europe (mainly in Georgia, the Balkans, and the Baltic countries). Sequencing will be performed using Illumina MiSeq platform. We will analyze bacterial genetic elements contributing to the phage-resistance using Geneious and BLAST bioinformatics programs.
bacteriophage; phage; multi-drug resistance; MDRO; anti-microbial resistance; AMR; bacteria
bacteriophage; phage; multi-drug resistance; MDRO; anit-microbial resistance; AMR; bacteria