Construction of genetically defined double aro mutants of Salmonella typhi

The construction of genetically defined, double aro mutant strains CVD906 and CVD908, which were derived from Salmonella typhi strain ISP1820 (a recent isolate of S. typhi from Chile) and from laboratory strain Ty2, respectively, is described. Strains CVD906 and CVD908 differ from previously described aro mutants of S. typhi as their aro deletion mutations do not extend beyond the limits of the mutated aro genes, and no antibiotic-resistance genes, plasmid sequences or S. typhimurium DNA sequences remain in the mutant strains. In minimal medium the aro mutants of S. typhi are unable to replicate whereas the wild type parent strains grow well in minimal medium. Using intraperitoneal inoculation of mice with S. typhi strains suspended in hog gastric mucin as a virulence assay, it is shown that the single aro mutants and the double aro mutants of Ty2 and ISP1820 are attenuated in mice. Trans complementation of the aro mutants with the aroC gene or aroD gene, or both, results in strains that are phenotypically identical to that of the wild type parents indicating that no measurable additional changes other than loss of the aro gene function occurred during strain construction.