Journal: Antimicrobial Agents and Chemotherapy

Article Title: Point Mutations within the Fatty Acid Synthase Type II Dehydratase Components HadA or HadC Contribute to Isoxyl Resistance in Mycobacterium tuberculosis

doi: 10.1128/AAC.01972-12

Figure Lengend Snippet: Mutations within HadA or HadC confer growth resistance during ISO treatment. Mid-log-phase cultures of wild-type (WT) M. tuberculosis and strains carrying either the C61G mutation in HadA (MTTR3) or the V85F mutation in HadC (MTTR18) were streaked onto Middlebrook 7H10 OADC plates supplemented with increasing concentrations of ISO (1, 2.5, and 5 μg/ml). Plates were incubated for 2 to 3 weeks at 37°C, after which growth was visualized.

Article Snippet: Mid-log-phase cultures of wild-type (WT) M. tuberculosis and strains carrying either the C61G mutation in HadA (MTTR3) or the V85F mutation in HadC (MTTR18) were streaked onto Middlebrook 7H10 OADC plates supplemented with increasing concentrations of ISO (1, 2.5, and 5 μg/ml).

Techniques: Mutagenesis, Incubation

Journal: Scientific Reports

Article Title: Mycobacterial DNA-binding protein 1 is critical for BCG survival in stressful environments and simultaneously regulates gene expression

doi: 10.1038/s41598-023-40941-9

Figure Lengend Snippet: Survival of BCG MDP1 cKD in macrophages and in vivo ( A ) Intracellular survival of MDP1-cKD BCG compared to VC, over 7 days. After confirmation of MDP1 expression in VC and suppression in MDP1-cKD BCG, THP-1 cells were infected with indicated strains at a multiplicity of infection (MOI) of 1:1 for 4 hrs. The cultures were subsequently washed twice with warm serum-free DMEM to remove uninfected BCG and finally cultured in DMEM supplemented with 200ng/ml ATc every 48 hrs. The cells were incubated at 37 °C in a humidified 5% CO 2 incubator. BCG CFU was determined at indicated timepoints. Data represent mean ± SE from three biological replicates. Statistical differences between VC ATc and MDP1-cKD BCG were assessed by unpaired Welch’s T test, *P < 0.05 **P < 0.01 ***P < 0.001; # p < 0.05 indicate differences between MDP1_#1-cKD and MDP1_#2-cKD. ( B ) In vivo survival of indicated strains over 28 days. Female C57BL/6J mice (n = 2–8) were intraperitoneally infected with 5 × 10 6 CFU of indicated BCG strains, suspended in 200 µl PBS. MDP1 expression was maintained by supplementing mice drinking water with 20 µg/ml Doxycycline (Doxy). BCG CFU was quantified from harvested mice organ homogenates plated on 7H10/OADC agar at indicated time points. Data represent mean ± SE from 2 to 8 mice per group. Statistical differences were analyzed using Mann-Whitney U test, *P < 0.05

Article Snippet: At indicated time points 100 µl of each culture was harvested, and 10-fold serial dilutions were made with Saline Tyloxapol Buffer (STB) to disrupt bacteria clots before plating 10 µl on Middlebrook 7H10/OADC agar supplemented with appropriate antibiotics.

Techniques: In Vivo, Expressing, Infection, Cell Culture, Incubation, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Mycobacterial DNA-binding protein 1 is critical for BCG survival in stressful environments and simultaneously regulates gene expression

doi: 10.1038/s41598-023-40941-9

Figure Lengend Snippet: Growth kinetics of BCG MDP1 cKD ( A ) Position of hupB target sequence start site shown by purple vertical lines for MDP1_ #1 and MDP1 #2 cKD BCG. Numbering indicates first nucleotide on the target sequence relative to annotated start coding sequence. Arrow indicates position of transcription start site. BCG growth kinetics depicted by ( B ) optical density (OD 600 nm) and ( C ) CFU over 21 days. Bacteria were cultured in 7H9/ADC medium supplemented with 200ng/ml ATc every 48 hours to induce sgRNA expression. At indicated time points culture aliquots were harvested to determine the OD and enumerate CFU after culturing on 7H10/OADC agar. Data represent mean ± SE from three biological replicates. Statistical differences between VC ATc and MDP1-cKD BCG were assessed by unpaired Welch’s T test, *P<0.05 **P<0.01 ***P<0.001; # p<0.05 indicate differences between MDP1_#1-cKD and MDP1_#2-cKD. ( D ) Representative western blot images cropped from different gels delineated with black border lines confirming the expression of MDP1: InhA serves as a loading control. Full-length blot images in Supplementary Fig. . 1-VC ATc, 2-MDP1_#1-cKD, 3-MDP1_#2-cKD. Image is representative of three experiments.

Article Snippet: At indicated time points 100 µl of each culture was harvested, and 10-fold serial dilutions were made with Saline Tyloxapol Buffer (STB) to disrupt bacteria clots before plating 10 µl on Middlebrook 7H10/OADC agar supplemented with appropriate antibiotics.

Techniques: Sequencing, Bacteria, Cell Culture, Expressing, Western Blot