FIG. 3.

(A) In vitro stability of replicative and integrative bivalent rBCG strains containing the pBlaF*-nef-gag operon. One colony of rBCG strains transformed either with plasmid pJLN29 or integration-proficient vectors pOIP4 and pOIP33 was inoculated into liquid medium without antibiotic and grown for 100 days. At different time points, aliquots of each culture were sampled and serially diluted before plating onto selective and nonselective medium. Ratios of kanamycin-resistant (rBCG) versus kanamycin-sensitive (total BCG) colonies were calculated for each of the three strains over cell divisions. Generations equal the number of days of culture, since the generation time for BCG is approximately 24 h. The most representative of two experiments is shown. (B) In vivo stability of replicative and integrative rBCG(pBlaF*-nef-gag) strains. On day 0 three groups of 25 BALB/c mice were inoculated i.v. with 5 × 10⁶ CFU of rBCG(pJLN29), rBCG::pOIP4, or rBCG::pOIP33. At different times after initial inoculation, CFU recovered from spleen, liver, or lungs from five individual mice per group were numbered onto medium supplemented (rBCG colonies) or not supplemented (total BCG colonies) with kanamycin. Graph represents mean ratios of rBCG colonies/total BCG colonies ± standard deviations for the three mice groups.