Journal: Frontiers in Microbiology

Article Title: HupB, a nucleoid-associated protein, is critical for survival of Mycobacterium tuberculosis under host-mediated stresses and for enhanced tolerance to key first-line antibiotics

doi: 10.3389/fmicb.2022.937970

Figure Lengend Snippet: Loss of hupB makes Mtb highly sensitive to host-simulated stress conditions. WT (red), KO (green), and KO+ hupB (blue) were grown in the rich medium to ~0.8 OD 600 , washed, and subcultured at 0.05 OD 600 . When absorbance reached 0.2–0.3 OD 600 , they were washed and normalized to 0.25 OD 600 . Five-ml cultures of each strain were subjected to various forms of host-simulated stress. (A–C) pH of the rich medium (pH 7.4) was reduced to pH as denoted; (D) medium was changed to 1X TBST (2.5 mM Tris, 4.2 mM NaCl, and 0.05% Tween 80); (E) oxidative stress was induced with 10 mM H 2 O 2 ; (F) nitrosative stress was induced with 5 mM nitric oxide; (G) equal number (CFUs/ml) of the WT, KO, and KO+ hupB cultures were washed in RPMI and used for infecting PMA (phorbol 12-myristate-13-acetate)-differentiated THP-1 macrophages at 1:10 MOI. After 4 h of infection, extracellular bacteria were killed with amikacin (200 μg/ml), THP-1 macrophages lysed with 0.1% Triton-X-100 at different time points (days on X-axis). The lysates obtained were plated on 7H11 + OADC + cycloheximide (50 μg/ml), CFUs were enumerated, and standard deviation values were calculated and then plotted as Log 10 CFUs/ml (Y-axis). Student t -test was performed for comparative analyses, and significance was evaluated. * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001. The data are a representation of biological triplicates and technical duplicates.

Article Snippet: WT, KO, KO+ hupB , and KO+ hlp ( ) were initially grown in the rich medium (10 ml at 37°C and 150 rpm) to ~1 OD 600 , and then pelleted down (at 4,000 rpm and RT for 10 min), and the pellets washed in 10 ml of the fresh sterile rich medium, For colony morphology, primary cultures were subcultured to 0.05 OD 600 , allowed to grow to 0.2–0.3 OD 600 , and then equal numbers of cells (~ 1.6 × 10 5 CFUs/ml) were spotted on Middlebrook 7H11 agar supplemented with 1× OADC, 0.5% glycerol, and 50 μg/ml cycloheximide.

Techniques: Infection, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: HupB, a nucleoid-associated protein, is critical for survival of Mycobacterium tuberculosis under host-mediated stresses and for enhanced tolerance to key first-line antibiotics

doi: 10.3389/fmicb.2022.937970

Figure Lengend Snippet: Loss of hupB makes Mtb highly susceptible in vitro to reduced amount of INH and RIF. WT (red), KO (green), and KO+ hupB (blue) were separately grown in the rich medium to ~0.8 OD 600 , washed, and subcultured at 0.05 OD 600 . When absorbance reached 0.4–0.6 OD 600 , they were washed and then normalized to 0.1 OD 600 . Five-ml cultures were imparted stress with (A) isoniazid and (B) rifampicin for 7 and 14 days (separated by a broken black straight line) with varying concentrations (X-axis, denoted as “fold MIC” (minimum inhibitory concentration; MIC denoted as X) and by dilution of surviving bacteria plated on 7H11 + OADC + cycloheximide (50 μg/ml) and enumerated as Log 10 CFUs/ml (Y-axis). MIC for isoniazid and rifampicin is 2.91 μM and 6 nM, respectively. Three biological triplicates were set up, CFUs were enumerated by technical duplicates, numbers were averaged, and SD values calculated were curve-plotted. Student t -test was performed for comparative analyses, and significance was evaluated. * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001. The data are a representation of biological triplicates and technical duplicates. (C) Growth on days 7 and 14 of the three mycobacterial strains [used in (A) and (B) ] without antibiotics (no drug control).

Article Snippet: WT, KO, KO+ hupB , and KO+ hlp ( ) were initially grown in the rich medium (10 ml at 37°C and 150 rpm) to ~1 OD 600 , and then pelleted down (at 4,000 rpm and RT for 10 min), and the pellets washed in 10 ml of the fresh sterile rich medium, For colony morphology, primary cultures were subcultured to 0.05 OD 600 , allowed to grow to 0.2–0.3 OD 600 , and then equal numbers of cells (~ 1.6 × 10 5 CFUs/ml) were spotted on Middlebrook 7H11 agar supplemented with 1× OADC, 0.5% glycerol, and 50 μg/ml cycloheximide.

Techniques: In Vitro, Concentration Assay

Journal: F1000Research

Article Title: High monocyte to lymphocyte ratio is associated with impaired protection after subcutaneous administration of BCG in a mouse model of tuberculosis

doi: 10.12688/f1000research.14239.2

Figure Lengend Snippet: Approximately half of each lung ( A ) and spleen ( B ) was homogenised at the time of Mtb challenge (6 weeks after immunisation) and plated on 7H11 agar plates to determine viable BCG bacteria. Total CFU per organ are reported. Data sets are presented in order of decreasing protection. Each symbol represents one animal. In some instances, bacteria could not be detected and values were set to zero. p values were determined by using Kruskal-Wallis test and Dunn’s post test for multiple comparisons. Error bars represent the median and interquartile range. Multiplicity adjusted p values are reported. p <0.05 was considered statistically significant.

Article Snippet: Each entire homogenate was plated onto two 7H11 agar plates containing 10% OADC supplement (Yorlab, York, UK) and 0.5% glycerol.

Techniques: Bacteria