Fig 1.
KatG levels and peroxidase activity in the clonal Mtb strains.
A and B). Western blot using anti-KatG (from mouse clone IT-57) from culture supernatants and cytosol fractions of Mtb clonal strains cultured in GAS media respectively (1 and 2 represent the two culture replicates of each strain used). Recombinant KatG was used as a positive control. C). Standard curve for peroxidase activity using 3,3′,5,5′-Tetramethylbenzidine (TMB) as substrate and recombinant KatG. D). KatG peroxidase activity (derived from the standard curve, C) from laboratory and clinical Mtb pairs using recombinant KatG as reference. Non-paired t-test without assuming consistent standard deviation, *p<0.05.
Fig 2.
AhpC leves from soluble cellular fractions of Mtb cultures.
Western blot using anti-AhpCMtb [17] from culture supernatants and cytosolic fraction of the four strains used in the study grown in GAS media (1 and 2 represent the two culture replicates of each strain used). CFP of H37Rv from BEI was used as a positive control. *indicates the lane for the ladder.
Fig 3.
Comparison of bacterial growth in C57BL/6 mice after low dose aerosol infection.
A) and C). Lung CFU count of mice infected with Laboratory and Clinical clonal strain pairs respectively. B) and D). Spleen CFU counts of Laboratory and Clinical clonal strain pairs respectively. Non-paired t-test without assuming consistent standard deviation (SD).*p<0.05, **p<0.001. Each time point represents the mean values for five mice infected with each of the four strains with the respective standard deviation shown in the error bars.
Fig 4.
Comparison of bacterial growth rates in BACTEC media using MGIT™320.
Time to positivity (TTP) of each strain was determined by seeding BACTEC tubes with 10-fold serial dilutions. TTP assessed in the MGIT™320.
Fig 5.
Lung pathology scores for C57BL/6 mice infected with clinical and laboratory clonal strains with different INH susceptibility profiles.
Mean of the lung scores with standard error (SEM) for five infected mice with each strain differentiated by color. *p<0.05 with Kruskal-Wallis test and Dunn’s post-test.
Fig 6.
Dynamics and comparison of cytokine levels in mouse lung homogenates.
A. Comparison of pro-inflammatory cytokines levels throughout the infection with pairs of clinical and laboratory clonal Mtb strains (t-test, *p<0.05). Limit of detection (LD) for IFN-γ: 0.5pg/mL, TNF-α: 0.9 pg/mL, and IL-6: 1.4 pg/mL.) Pair comparison between mice infected B). IL-10 (LD: 16.8 pg/mL) and IL-2 (LD: 0.1 pg/mL) levels in mice infected with laboratory and clinical Mtb pairs. Bars represent the mean values of cytokine concentration for five mice and the error bars represent the standard deviation.