TB Persistence is Futile, Time to End Them All

TB Persistance is Futile, Time to End Them All - Yanmin Hu<br><br>This presentation was delivered at the 2017 INTERTB symposium, held at St George’s, University of London. The full collection of outputs from the meeting can be accessed from the link referenced below.<br><br

Growth and survival of <i>M. tuberculosis</i> Δ<i>tpx</i> in resting and IFN-γ-activated macrophages.

<p>A. Infection in resting bone marrow derived macrophages from BALB/c mice. B. Infection in IFNγ activated bone marrow derived macrophages from BALB/c mice. These results are the means and standard deviation derived from one representative of three independent experiments. C. Infection in resting bone marrow derived macrophages from C57BL/6 mice. D. Infection in IFNγ activated bone marrow derived macrophages from C57BL/6 mice. E. Infection in resting bone marrow derived macrophages from iNOS KO mice. F. Infection in IFNγ activated bone marrow derived macrophages from iNOS KO mice. The results are the means and SDs derived from triplicate wells. The experiments have been reproductively repeated once. Solid square: WT <i>M. tuberculosis</i> H37Rv. Open square: YHΔ<i>tpx.</i> Solid triangle, YH<i>tpx</i>Comp.</p

Biochemical analyses of peroxidase activities in <i>M. tuberculosis</i> WT, <i>tpx</i> mutant and the complemented strain.

<p>Presence of hydrogen peroxide was measured by xylenol orange assay. The open bar, initial H₂O₂ at 1 mM. The solid bar, initial H₂O₂ at 0.5 mM. Control, H₂O₂ only. The data was repeated twice with similar results.</p

Inactivation of <i>tpx</i> gene renders the mutant more susceptible to oxidative and nitrosative stresses.

<p>Survival of YH<i>Δtpx</i> compared with WT and the complemented strains in response to H₂O₂ at 5 and 10 mM (A), DETA/NO at 1.25, 2.5 and 5 mM (B), paraquat at 10 and 20 mM (C) and GSNO at 5 and 10 mM (D). The data shown is a representative of three independent experiments. Data are represented as mean±SD of triplicate tests. The CFU counts in the Δ<i>tpx</i> are significantly lower using a t-test than in WT after exposure to both H₂O₂ and NO for 24 hours (p<0.0001 all concentrations for both stress conditions).</p

Survival of SCID mice (<i>n</i> = 6 per group) infected intravenously with <i>M. tuberculosis</i> H37Rv WT, YHΔ<i>tpx</i> and the complemented strains.

<p>Deletion of <i>tpx</i> gene led to no death of the mice over 60 days after infection.</p

Confirmation of the <i>M. tuberculosis tpx</i> gene deletion.

<p>A. Southern blotting analysis of DNA from the WT and the YHΔ<i>tpx.</i> which was digested with EcoRV and PvuI and hybridized with a probe synthesized to make the complemented construct. B. PCR amplification of DNA from the WT and the YHΔ<i>tpx.</i> 1. WT, 2. <i>tpx</i> mutant. The primers used were designed in the coding region of the <i>tpx</i> gene. 3. WT, 4. <i>tpx</i> mutant. The primers used were for the amplification of complemented construct. M, molecular weight marker (Invitrogen). The experiments were repeated twice, with identical results.</p

The MSC₅₀ and MIC of HT61 against clinically isolated MRSA, VISA and VRSA.

<p>The MSC₅₀ and MIC of HT61 against clinically isolated MRSA, VISA and VRSA.</p

Thin sectioned electron micrographs of <i>S. aurues</i> analyzed by transmission electron microscopy.

<p>The cells were fixed 10 minutes after HT61 treatment. A. normal <i>S. aureus</i> cells. B. HT61 at 10 µg/ml. C. HT61 at 20 µg/ml. D. HT61 at 40 µg/ml. The scale bar is 0.2 µm.</p

Effect of HT61 against MSSA and MRSA in a murine skin bacterial infection model.

<p>A. After tape-stripping the skin, log phase MSSA was applied onto the skin area. At different time points CFU counts of the bacteria were determined. The arrow indicates the point which the treatment was initiated. B. Treatment of HT61, Bactroban and placebo (control) against MSSA and C. Treatment of HT61, Bactroban and placebo (control) against MRSA. **, P<0.01. The data has been repeated twice.</p

Effects of HT61 and marketed antibiotics against stationary phase non-multiplying MSSA and MRSA.

<p>HT61 and the antibiotics were added to the non-multiplying cultures at different concentrations. CFU counts were carried out after 24 hours of incubation. A. Effects of HT61, amoxicillin/clavulanic acid, azithromyicin, levofloxacin, linezolid, daptomycin and mupirocin against MSSA. B. Effects of HT61, vancomycin, daptomycin and mupirocin against MRSA. These results were confirmed in two independent experiments.</p