ISOLATION OF MICROBIAL-DERIVED COMPOUNDS FOR MITIGATING DRUG-RESISTANT BACTERIA ASSOCIATED WITH BATTLEFIELD WOUND INFECTIONS

Background: Disease-causing microorganisms that have become resistant to drug therapy have emerged as a serious threat to military personnel and wounded soldiers. Multidrug-resistant (MDR) infections, which frequently develop after battlefield trauma, are particularly devastating in Wounded Warriors suffering from burns, blast wounds, and bone injuries. These infections cause severe negative outcome to the soldier and frequently lead to septicemia, wound dehiscence, osteomyelitis, limb losses, and possible death. With the emergence of new MDR bacteria, the need for discovering new antimicrobials has become increasingly urgent.Objective/Hypothesis: Throughout millions of years of evolution, bacteria have developed a vast array of mechanisms that allowed them to survive in their environment, including the secretion of antibiotics, bacteriocins, chelators, enzymes, biosurfactants and more. We hypothesize that bacteria growing as a biofilm produce natural compounds that help them to survive and compete in their environment. We also demonstrated that these compounds could be more readily isolated from biofilms. The goal of this proposal is to isolate novel antimicrobial and antibiofilm compounds from colony biofilms that are effective specifically against drug and and multidrug-resistant bacteria. We have recently developed a colony biofilm assay that enables us to detect antimicrobial agents that diffuse from the biofilm. We also established a new method to extract biofilm-matrix associated metabolites and measure their antimicrobial and antibiofilm properties.Specific Aims: The specific aims of this proposal are (1) assembling a diverse bacterial library and examining their antimicrobial attributes against MDR bacteria most prevalent in Wounded Worriers; (2) isolating crude microbial-derived compounds from biofilms and evaluating their anti-biofilm properties; and (3) preliminary chemical identification of the active compounds.Study Design: A diverse library of about 400 microorganisms will be assembled. A colony biofilm test will be used to identify antimicrobial compounds that are active against clinical relevant MDR pathogens including MDR strains of Acinetobacter spp, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and the yeast Candida albicans. The library assembled will be extremely diverse and include commercially available reference strains, clinical isolates, and freshly isolated environmental samples. Bacteria associated with combat-related infections will also be examined, as they might produce self-controlling metabolites. Using a new biofilm extraction protocol, high-density biofilms will be grown and biofilm-associated extracts will be collected directly from the established biofilms and examined for their antimicrobial and antibiofilm properties. Finally, the biofilm extracts that demonstrated desired biological activity will be further characterized.Impact: With MDR microorganisms becoming more prevalent in combat-related injuries and infections, as well as in many other medical settings, the need for new antimicrobials and antibiofilm drugs for controlling human pathogens is becoming essential. By re-tapping into the unlimited potential of bacteria to produce antimicrobial compounds and exploiting the biofilm's distinctive ability to accumulate and produce unique secondary metabolites, we aim to identify new antimicrobial countermeasures. The proposed experiments are expected to lead to the discovery of new antimicrobial and antibiofilm countermeasures that might be used in the near future in the fight against drug-resistant wound infections.Military Relevance: To specifically address the need to discover new antimicrobial and antibiofilm drugs that could be used to treat Wounded Warriors, we will place our attention on finding and isolating compounds that are active against MDR bacteria most prevalent in battlefield wounds. Among the strains we aim to examine will be drug and multidrug-resistant clinical samples isolated from Wounded Warriors. These samples will be kindly provided by our colleague at Walter Reed Army Institute of Research. Additional drug-resistant clinical strains will be available from our strain collection at the University of Medicine and Dentistry of New Jersey, as well as from colleagues from other medical institutions and commercially available strain deposits. We believe that this approach will allow us to identify clinically relevant drugs that could be used as a topical treatment to prevent and treat an infected wound or systemically to prevent or eradicate the spread of the microbial infection throughout the body.