Stable Listeria monocytogenes live vaccine candidate strains with graded attenuation on the mouse model

Metabolic drift (antibiotics resistance) mutations were used to construct stable two (and three) marker vaccine candidate strains of the predominant Listeria monocytogenes serotypes 1/2a and 4b by stepwise selection. Derived from wild-type strains, spontaneous chromosomal streptomycin-resistant clones with their i.p. LD50 elevated from less than or equal to 10(5.0) c.f.u. to approximately 10(6.1) c.f.u. were used in the second step to isolate the rifampicin-resistant mutants with i.p. LD50 values ranging from 10(6.6) to 10(7.4). On i.p. immunization with fully tolerated doses (less than or equal to 1% LD50), these potential vaccine strains were found to protect not less than 95% of the mice against a lethal (approximately 100 LD50) challenge with the homologous wild-type strain. Further elevation of the i.p. LD50 to greater than 10(8.3) c.f.u. by means of a third attenuating fosfomycin-resistance marker resulted in overattenuation and reduced protective capacity.