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The relative abilities of an avirulent Salmonella typhimurium strain with wild-type lipopolysaccharide (LPS) character, SL5319, and a nearly isogenic LPS-deficient mutant, SL5325, to colonize the large intestines of streptomycin-treated CD-1 mice in vivo and to penetrate colonic mucus in vitro were studied. Previously it had been shown that, when fed simultaneously to streptomycin-treated mice (approximately 1010 CFU each), the S. typhimurium strain with wild-type LPS colonized at 108 CFU/g of feces indefinitely, whereas the LPS-deficient mutant dropped within 3 days to a level of only 104 CFU/g of feces. In the present investigation, when SL5325 was allowed to colonize for 8 days before feeding mice SL5319 or when it was fed to mice simultaneously with an Escherichia coli strain of human fecal origin (1010 CFU each), both strains colonized indefinitely at 107 CFU/g of feces. Moreover, when the wild-type and LPS-deficient mutant strains were fed to mice simultaneously in low numbers (approximately 105 CFU each) the strains survived equally well in the large intestines for 8 days, after which the LPS-deficient mutant was eliminated (less than 102 CFU/g of feces), whereas the wild-type colonized at a level of 107 CFU/g of feces. In addition although both strains were able to adhere to mucus and epithelial cell preparations in vitro, the wild-type strain was shown to have greater motility and chemotactic activity on CD-1 mouse colonic mucus in vitro and to more rapidly penetrate and form a stable association with immobilized colonic mucosal components in vitro. Based on these data, we suggest that the ability of an S. typhimurium strain to colonize the streptomycin-treated mouse large intestine may, in part, depend on its ability to penetrate deeply into the mucus layer on the intestinal wall and subsequently, through growth, colonize the mucosa.