Genus Cronobacter is a member of the family Enterobacteriaceae consisting of several opportunistic species. The primary focus of this study was to utilize an in vitro co-culture model of the blood brain barrier to investigate a bovine fecal strain of C. sakazakii to investigate pathogenicity. The strain was found to have the same effect on the barrier’s integrity as the positive Escherichia coli control. Additionally, C. sakazakii strain BAA-894 was found to have the same effect as the negative E. coli control. This study also focused on the development of a site-specific mutagenesis procedure for C. sakazakii. A procedure using linear transformation was able to replace the putative virulence gene zpx (zinc-containing metalloprotease) in C. sakazakii. A future virulence study would involve using this mutagenesis procedure to induce a mutation in genes of C. sakazakii speculated to play a role in BBB translocation followed by challenge in the BBB model.