The pathogen most frequently recovered from neonates with peri-natally acquired gram negative bacteremia and meningitis is E. coli K1; one of the principal causes of nosocomial gram negative infection in the neonate is Klebsiella penumoniae. We plan to study the feasibility of preventing infections due to both pathogens by blocking colonization of the oropharynx, thus intervening before bacteria penetrate mucosal barriers and invade the bloodstream. We have previously shown that adherence of E. coli K1 and Klebsiella to oral epithelial cells is specifically mediated by mannose-sensitive (MS) pili in both rat and human neonates, and that these pili play an important role in oropharyngeal colonization in a neonatal rat model. MS pili purified from E. coli K1 induce a brisk antibody response in the breast milk of pregnant rats, and neonatal rats suckled by immunized dams have a significantly reduced rate of bacteremia when challenged with the homologous E. coli strain. Based on these promising studies, we now propose to determine whether immunization of pregnant rats with purified E. coli K1 pili can protect their offspring from challenge with heterologous E. coli K1 and klebsiella strains. Serological studies will be performed to establish the antigenic relatedness of MS pili from multiple clinical strains so as to facilitate production of a vaccine with pili from as few strains as possible. Concurrently, we will perform histopathological studies in the rat model to determine the site and mechanism of mucosal invasion by E. coli K1. Ultimately, it will be necessary to show that human milk containing high titers of anti-pilus antibody -- whether naturally acquired or stimulated by pilus vaccine -- can prevent neonatal E. coli and klebsiella colonization and infection. As a first step, we will study the prevalence and titer of natural anti-pilus antibody in colostrum and breast milk. We will then determine whether intensive care babies who receive high-titer breast feedings are protected from nosocomial colonization with Klebsiella and other Enterobacteriaceae that express MS pili.