Quantification of biofilm by crystal violet staining

All mycobacterial strains were cultured in Sauton media, started at OD600nm 0.03, in 24-well (M. tuberculosis) or 48-well non-treated tissue culture plates (BCG strains), and were incubated at 37 °C, 5% CO₂. Each strain was inoculated into 6 different wells, and experiments were repeated three times for statistical analysis. After 10 days (BCG strains) and 14 days (BCG strains and M. tuberculosis strains) of incubation, liquid media was removed and the whole surface pellicle and biofilm attached to the wells (these samples are referred to as “biofilms”) was maintained. Plates were baked at 30 °C for 24 h and 1 ml of 100% methanol was added to each well and incubated at room temperature for 15 min. Then, methanol was removed, and plates were dried at 37 °C for 15 min. Crystal violet (CV) was added to each well and incubated at 37 °C for 5 min. CV was removed and each well was washed four times with deionized water. Plates were dried at 37 °C for 15 min. Dye was extracted with 30% acetic acid for 15 min at 37 °C. Then the extract from each well was diluted 1:10 (M. tuberculosis), 1:20 (BCG, 10 days cultures), or 1:40 (BCG, 14 days cultures) in 30% acetic acid and read for OD550nm.

Data distribution for qPCR, CFU, and biofilm quantification were analyzed using the Anderson–Darling and Shapiro–Wilk tests, and found to follow a normal distribution in all instances. Growth (as OD600nm readings) of rBCG strains was compared with that of parental BCG harboring the empty vector pMV361 (BCG WT::pMV361) using Two-Way ANOVA followed by Dunnett’s multiple comparison test. Growth (as doubling time) was compared by One-Way ANOVA (logarithmic phase cultures) or Brown–Forsythe and Welch ANOVA (stationary phase cultures) followed by Dunnett’s multiple comparison tests. Bacterial replication (CFU/mL) was compared by 2-Way ANOVA followed by Tukey’s multiple comparison test. For quantification of biofilms, statistical significance was determined using One Way ANOVA followed by Dunnett’s test for multiple comparison. For RT-qPCR analyses, Brown-Forsythe and Welch ANOVA followed by Dunnett’s multiple comparison test was used for comparing biofilm samples; multiple t tests followed by Holm–Sidak multiple comparison test was used for comparing planktonic samples. GraphPad Prism 8 for MacOS was used for performing statistical analyses. Assays were conducted in three independent times, and the number of replicates per experiment is indicated in each figure legend.