THE POTENTIAL APPLICATION AND RISKS ASSOCIATED WITH THE USE OF PREDATORY BACTERIA AS A BIOCONTROL AGENT AGAINST WOUND INFECTIONS

Background: Disease-causing microorganisms that have become resistant to drug therapy are an increasing cause of burn, wound, blast, and bone infections, with many traditional antimicrobial agents becoming ineffective. Resistance can be considered as a natural response to the selective pressure of a drug and can develop in free-floating bacteria as well as in surface-attached bacteria or biofilms. One of the major difficulties in controlling surface-attached bacteria is their enhanced resistance to antimicrobial agents, i.e., biofilm bacteria can be up to 1,000 times more resistant to antimicrobial agents than their planktonic counterparts. Thus, the huge doses of antimicrobials that are required to rid wounds and medical devices of biofilms are impractical. The problem of multidrug-resistant (MDR) bacterial infections in the Wounded Warrior drove researchers to examine other potential anti-bacterial strategies. Among these alternative therapies is the use of biological control agents such as medical maggots, phage, biodebridement, and predatory bacteria.Hypothesis: Our central hypothesis is that predatory prokaryotes are able to serve as a novel topical therapeutic agent in controlling non-treatable, MDR, wound-related bacterial infections. In a previous study, supported by USAMRMC via a War Supplemental CDMRP grant, we confirmed that predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are able to prey on a wide range of pathogens including MDR bacteria isolated from Wounded Warrior and Gram-negative bacteria that form biofilms. Although our initial work highlighted the in vitro efficacy of predatory bacteria to control medically relevant MDR-bacteria, many questions still remain unclear regarding the impact and possible risks associated with using predatory bacteria as a live therapeutic.Aims: The aim of this proposal is to address key questions regarding the safety and efficacy of predatory bacteria in ex vivo and in vivo systems and will be divided in to three tasks: (1) Investigating predator prey/host bacteria interactions and resistance. (2) Determining the effect of predatory bacteria on eukaryotic cells. (3) Measuring the efficacy of predation and toxicity in animal models.Study Design: (1) Investigating predator prey interactions and resistance. Under this task, we will conduct a series of experiments aimed at examining the possible emergence of genetically stable resistance in host bacteria following predation and the ability of the predator to breach its host specificity and attack previously resistant bacteria. We will also attempt to enrich and obtain predatory bacteria lab-variants more adaptive to prey at 37 degrees Celsius, and on a given host, as these variants might be more suitable for specific medical applications. (2) Determining the effect of predatory bacteria on eukaryotic cells. Under this task, we will address questions regarding the impact of predatory bacteria on non-microbial cells and whether predatory bacteria have an adverse or toxic effect on eukaryotic cells. We will also assess the predators' ability to attach, attack, and invade mammalian cells and/or provoke the induction of a pro-inflammatory response. (3) Efficacy and toxicity in animal models. Under this task, we will address the toxicity of and the immune response to predator bacteria in established animal models of wound infection. First, we will inoculate infected and uninfected wounds in a mouse puncture model with a range of predator doses. Then, the immune response to predator inoculation will be evaluated using histology, RT-PCR, and ELISA-based assays. Separately, known markers of sepsis will be examined to address a possible concern that excess predator could be harmful to the host. We will compare our results to a porcine wound model since this model is more representative of human skin as pigs heal similarly to humans via re-epithelization.Clinical Impact and Military Relevance: With the emergence of new MDR microorganisms becoming more prevalent in combat-related injuries, the need for new anti-infection and anti-biofilm topical countermeasures is essential. We believe that our research will provide caregivers with a safe and effective biological-based product to treat wound infection, allowing faster healing and minimizing morbidity and mortality, as well as limiting the chances of spreading new pathogens in hospitals to which injured military personnel are transported. The unique ability of predatory bacteria to attack MDR organisms and biofilms will provide military and civilian caregivers the means to treat these extremely persistent infection that no longer respond to 'conventional' therapeutics before they become life-threatening.