FIG. 3.

Plasmid maintenance in vitro (A) and in vivo (B). To determine in vitro stability, strains were cultured with (GG55-Chl) and without (GG55-no Chl) chloramphenicol (L. monocytogenes LLO-E7) or with and without d-alanine [Lmdd(pTV3)]. The cultures were diluted 1:1,000 daily into fresh LB. The CFU of the cultures were determined daily on BHI (BHI) and on BHI with chloramphenicol (BHI-Chl) for L. monocytogenes LLO-E7 or on BHI with d-alanine (BHI-Ala) for Lmdd(pTV3). All liquid medium and plates contained an additional 50 μg of streptomycin per ml, to which Listeria monocytogenes strain 10403S is naturally resistant. To determine in vivo plasmid maintenance, L. monocytogenes was injected intraperitoneally at a dose of 1/10 the LD₅₀ in C57BL/6 mice. Spleens were harvested at different time points postinjection and homogenized in phosphate-buffered saline. CFU counts were prepared on BHI plates with and without d-alanine for Lmdd(pTV3), on BHI plates with and without chloramphenicol for L. monocytogenes LLO-E7, and on BHI plates only for wild-type 10403S.