This SBIR Phase I project will develop a hand-held, point-of-care, sensing strip and instrument for immediate identification of which bacterial species are causing a urinary tract infection (UTI) in a companion animal. This technology will allow veterinarians to judiciously prescribe antibiotics for an animal during the initial examination. In the United States alone, there are nearly 1.5 million veterinary visits annually for UTIs in dogs and cats. The average time to initial UTI test results is 48 hours, during which time the animal is generally consuming antibiotics. Less than half of urine cultures show any bacterial growth, indicating that the majority of antibiotic prescriptions were unnecessary. Further, it is important to identify which bacterial species are causing the UTI, so that the proper antibiotic can be administered. Prudent use of antibiotics will control the spread of infection and the emergence of antibiotic resistance, thus resulting in healthier animals and reducing the need for new antibiotic drug development. This project will help translate the initial research and discoveries that were funded previously by the National Science Foundation. The underlying principle for this technology is that each bacterial species produces and excretes its own unique set of chemicals that are used to coordinate intercellular activities, analogous to pheromones produced by animals. It is possible to identify what bacterial cells are present in a sample by detecting these chemicals. There are much greater quantities of the chemicals in the sample than bacterial cells, making this approach more sensitive and easier to implement than other technologies. The proposed research will result in the creation of a label-free, aptamer-based, electrochemical sensing platform for the detection and identification of the four most common Gram-negative bacterial pathogens causing UTIs in companion animals, within two minutes of sample collection. The novelty of the approach is to measure unique quorum sensing molecules (QSMs) that are secreted by the bacteria. These QSMs are produced by all bacterial cells continuously at a rate of approximately 10,000 molecules per hour, thereby improving the limit of detection by four orders of magnitude. An array of electrodes, modified with novel aptamers and functionalized with electro-active molecules, will be used to detect the QSMs, acting as biomarkers, produced by each bacterial species. Combining optimized aptamer molecules with electrochemical sensing and a proprietary signal processing algorithm allows for unprecedented limits of detection, sensitivity, and specificity. The technology will work directly in complex fluids without any reagent addition or separation steps.