Human infections due to extraintestinal pathogenic E. coli (ExPEC) strains, including urinary tract infections (UTIs), newborn meningitis (NBM), abdominal sepsis, and septicemia, result in significant morbidity, mortality and are estimated to cost the US health care system billions of dollars annually. ExPEC strains also infect chickens causing economic losses for the poultry industry, a significant economic sector in the US and globally, due to costs of containment, mortality, and disposal of carcasses. Chicken products are suspected to be a source of ExPEC infections in humans, and the ExPEC strains are now considered a new food-born pathogen. In addition, the treatment of ExPEC infections often fails because of multidrug- resistance. The increase in numbers of immunocompromised populations, including the elderly, coupled with multidrug-resistance among ExPEC strains, will challenge the treatment of ExPEC infections and likely increase cost of treatment in the near future. The difficulty of developing an effective vaccine against ExPEC is related to their phylogenic diversity. A polyvalent vaccine although challenging, is needed. We have spent the last few years elucidating the virulence factors of ExPECs and Salmonella. We have constructed recombinant attenuated Salmonella vaccine vector systems for animals and humans that are capable of inducing cross-protective immunity to avian pathogenic E. coli (APEC) and other enteric pathogens. The results of recent studies of this system are very encouraging and suggest the success of using attenuated Salmonella to prevent bacterial infections;however more studies are needed to increase both the safety and efficacy of the treatment in humans. We propose to use our proven technologies to develop vaccines to prevent and reduce ExPEC infections in humans. Our objectives include: (i) complete construction of S. Typhimurium to express multiple antigens carried on an ExPEC plasmid to protect against multiple ExPEC infections in mice;(ii) construction and evaluation of a regulated delayed lysis in RASV using a toxin-antitoxin system as a new biological containment system consistent with an efficacious vaccination;and (iii) evaluation of our RASV with a regulated delayed lysis system to protect against urethral, meningitis, and sepsis infections in mice. PUBLIC HEALTH RELEVANCE: Our project proposes a unique strategy to engineer a safe, easy to use Salmonella-based treatment that will be effective in eradicating ExPEC infections that are responsible for significant loss of life and cost billions of dollars to the US health care system annually. Moreover, if successful, the delayed-attenuated lysis system proposed can be used in any live bacterial vaccine to increase their safety and efficacy in delivering antigens.