OBJECTIVE: Develop a novel plasmid curing therapeutic capable of displacing antibiotic resistance and/or virulence causing plasmids from bacteria. Therapeutic interventions are sought that will be efficacious against a range of human pathogens of interest to the DoD. DESCRIPTION: The combined threat of the increasing prevalence of drug-resistant bacteria and a diminishing antibiotic pipeline places our warfighters at risk not only from health care associated and community acquired infections, but also from pandemics, emerging infectious pathogens and the intentional use of resistant pathogens for bioterrorism. One of the major routes by which bacterial pathogens become resistant to antibiotics and more virulent is through Horizontal Gene Transfer (HGT), which allows for genetic material transfer in the form of extrachromosomal plasmids from one cell to another. This phenomenon is capable of transferring resistance and/or virulence genes to normally antibiotic susceptible and avirulent bacteria. This creates a severe risk to front line antibiotic treatments, illustrated by the recent occurrence of isolates from methicillin-resistant Staphylococcus aureus (MRSA) that contain vancomycin resistance genes (in plasmid form) transferred from vancomycin-resistant enterococci (VRE). Likewise, G9241, a benign form of Bacillus cerus has acquired a B. anthracis virulence plasmid, demonstrating transfer of virulence plasmids by HGT. One way to reverse the resistance of emerging or engineered bacteria created by HGT may be to specifically target the plasmids being transferred between the cells, rather than using methods to directly kill the cells. This idea is known as Plasmid Curing. Proposals are sought that will develop novel plasmid curing therapeutics against plasmid encoded antibiotic resistant and highly virulent pathogens. Studies working with ESKAPE bacteria (Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, and bacteria that produce Extended Spectrum Beta Lactamase (ESBL) enzymes (Enterobacter and Escherichia coli)) are encouraged. The therapeutic should be clinically relevant and therefore shown to be non-toxic to humans and appropriate regulatory approval that would be needed in bringing forth such a therapeutic in the drug development pipeline should be considered. Developing such a safe intervention may help protect and provide appropriate treatment to our warfighters against the dangerous pathogens they encounter in theatre. PHASE I: Demonstrate via in vitro experiments that the proposed therapeutic is capable of removing any stable plasmid from a bacterial model (identified by proposer). Therapeutic approaches that are effective against both Gram (+) and Gram () will be prioritized. Metrics should demonstrate clearance and include clearance from two separate bacteria. If only partial clearance is achieved, state how this is still appropriate as therapeutic treatment. Propose an infectious in vivo animal model capable of assessing the health of the microbiome after treatment in addition to the efficacy of the treatment. Criteria also include providing details of the therapeutic; delivery method, proposed dosage, storage and stability, etc. Please note: Animal Subject Research (ASR) and Human Subject Research (HSR) are NOT expected or required for Phase I. PHASE II: Demonstrate the efficacy of the therapeutic to cure two plasmid containing pathogens of interest (identified by the proposer and relevant to the warfighter) that are either antibiotic resistant or virulent in an in vivo animal model. Demonstrate further the ability of the therapeutic to remove two or more plasmids from a pathogenic bacteria within the same animal model. Therapeutic approaches that are effective against both Gram (+) and Gram () will be prioritized. Appropriate toxicology studies of the therapeutic in an animal model to support an IND application should also be conducted. The overall health of the microbiome after use of the therapeutic in vivo should be described. Make sure to adhere to biosafety and ethical guidelines. PHASE III: Successful or promising approaches identified in Phase II would continue the development pathways for FDA approval and would support protecting the warfighter against such microbial threats. In addition, these therapeutics can be used as a medical countermeasure against any pathogen that may strike the general population. Phase III and IND approval would lead to appropriate clinical trials to gain FDA approval that may be funded through additional government and/or private funding sources. This SBIR Topic addresses the biomedical key technology area identified in the Defense Technology Area Plan from February 2003. Specifically drug resistant microbes are a significant current and future threat to US military personnel deployed overseas. Military personnel suffer significant life and limb threatening injuries and survive or resuscitated only to face months of hospitalization and multiple surgeries trying to combat extensively antibiotic resistant microbial pathogens. In the current military medical system we encounter microbes that are not responsive to any known antibiotics. In addition, naturally emergent or purposely engineered extensively antibiotic resistant microbes pose a significant threat to military operational activities. Most antibiotic resistance and many virulence genes are carried on portable and easily transferable circles of DNA called plasmids that live inside bacteria. Research and development under our topic will identify innovative ways of"curing"plasmids, that is, to directly attack the plasmids instead of the bacteria. Although high risk, if successful this approach could open a new way of countering biological threats.