Occurrence of secondary attenuating mutations in avirulent Salmonella typhimurium vaccine strains

The attenuating ΔaroA554 mutation in Salmonella typhimurium strain SL3261 was complemented in vitro by selecting for aroA⁺ recombinant DNA clones. SL3261 containing cloned aroA⁺ genes did not require exogenous phenylalanine, tryptophan, tyrosine, p-aminobenzoic acid, or dihydroxybenzoicacid for growth in defined media. Cloned aroA⁺ genes did not restore wild-type virulence to SL3261, however, in a murine typhoid model. The J1aroA554 mutation was transduced into S. typhimurium strain SR-11, a mouse-virulent strain recently passaged in mice. The SR-11 ΔaroA554 mutant washighly attenuated for mice challenged parenterally. The same cloned aroA⁺ genes isolated in SL3261 restored the virulence of the SR-11 ΔaroA554 mutant to that of wild-type SR-11. These results suggest that while the ΔaroA554 allele remains effectivein reducing S. typhimurium virulence, laboratory passage of attenuated vaccine strains may lead to the accumulation of additional attenuating defects.