PROJECT SUMMARY/ABSTRACT Medical device-associated microbial infection arises from pathogenic bacterial adhesion and subsequent biofilm formation on devices. It is known that bacterial intracellular nucleotide second messenger signaling plays an important role in biofilm development. Interference with these nucleotides signaling could provide a novel approach to address the problem of pathogenic biofilm formation on biomaterial surfaces. This application proposes to synthesize small molecule derivates of 4-arylazo-3,5-diamino-1H-pyrazole (named as SP02, SP03, and SP04) and tether them to polyurethane (PU) and polydimethylsiloxane (PDMS) biomaterial surfaces so that the small molecules can interfere with nucleotide signaling and interrupt biofilm formation in a way that will both reduce the formation of biofilms and increase the antibiotic efficacy for treating biofilms that do develop. Through testing 4 clinically relevant pathogenic biofilm bacteria on modified surfaces in vitro and in vivo, a new approach for creation of improved antimicrobial biomaterials for implantable medical devices such as catheters will be developed. The central hypothesis is that “biomaterial surfaces tethered with small molecule derivates of 4-arylazo-3,5-diamino-1H-pyrazole can inhibit and interrupt biofilm formation and growth by interfering with intracellular nucleotide signaling. This leads to disruption of biofilm formation and increases efficacy of antibiotics, therefore making microbial infection treatable using standard antibiotic therapy.” To test this hypothesis, four specific aims are proposed. Aims 1 and 2 will focus on in vitro assessment of the effectiveness of three small molecules and these small molecules bonded PU and PDMS biomaterial surfaces for inhibiting biofilm formation and increasing antibiotic efficacies, as well as addressing biocompatibility. The nucleotide levels and RNAseq will be quantified to determine the effects of small molecules on bacterial intracellular nucleotide signaling. Through these experiments, the molecule tethering approach that leads to the greatest inhibition of biofilm formation will be identified for in vivo studies. Aim 3 will test the antibacterial properties and tissue response to small molecule modified biomaterials using a 7-day subcutaneous infection rat model to validate the findings of small molecule functionalized polymers identified from Aims 1 and 2. To accelerate the application of new approach in medical devices, commercial pediatric central venous catheters will be modified with small molecules. Biofilm formation and antibiotic efficacies will be tested using a total implantable venous access port (TIVAP) in vitro and in vivo in Aim 4. The success of experiments described will allow progression to in vivo studies of other medical devices and large animal studies for preclinical trials, as well as providing important basic science information on nucleotide messenger signaling in biofilm formation and cont...