An ex vivo culture model for screening drug activity against in vivo phenotypes of Mycobacterium tuberculosis

Abstract

Since the activity of drugs against Mycobacterium tuberculosis grown in microbiological culture can differ from their activity against bacteria present in infected tissues, compounds with optimal activity against in vivo phenotypes may be overlooked in drug-discovery programmes that rely on in vitro screens. The authors have investigated the use of an ex vivo cell-culture model to assess the action of drugs on M. tuberculosis in an environment resembling that encountered during infection. Mycobacterial viability in the ex vivo model was shown to be regulated by the cell-mediated immune system, with growth inhibited by CD4 M T cells at an early stage of infection in BCGvaccinated mice, and at a later stage after infection in naive mice. Screening of drugs in the ex vivo model demonstrated a window of pyrazinamide susceptibility that coincides with the onset of the T-cell-mediated immune response in naive or vaccinated mice. It is proposed that pyrazinamide acts on a population of bacteria that are exposed to an acidic environment as a result of immune activation. Clinically, administration of pyrazinamide during the initial phase of treatment reduces the risk of relapse after 6 months, suggesting that the early pyrazinamide-susceptible population may contribute to the later pool of mycobacteria that persist during prolonged chemotherapy.

Alternative Luciferase for Monitoring Bacterial Cells under Adverse Conditions

“…Experiments with the luxAB reporter system in mycobacteria have shown that there is a strong dependence on changes in the availability of bacterial cofactors under different growth conditions (62,67). While this is advantageous in signaling a rapid response to the action of some drugs, a sharp decline in luminescence presents a limitation in studying nondividing bacteria in stationary-phase cultures.…”

“…M. tuberculosis research requires containment level 3 facilities, which coupled with a slow doubling time (nearly 24 h) makes studies using conventional microbiological techniques challenging. In our laboratory (35,62,67) and other laboratories (4,5,16,17,20,34) extensive use has been made of the bacterial and beetle luciferases as reporter genes in mycobacteria to determine cell numbers and viability. We have shown that the Gluc luciferase is expressed in the fast-growing organism Mycobacterium smegmatis and have characterized its performance under different stress conditions.…”

In vitro and ex vivo activity of peptide deformylase inhibitors against Mycobacterium tuberculosis H37Rv

“…Log10 CFU in lungs a Control 5.07 ± 0.15 INH (25) 4.04 ± 0.18*** Actinonin (50) 4.29 ± 0.14*** BB-3497 (20) 4.17 ± 0.23*** Hydroxylamine hydrochloride (50) 5.08 ± 0.14 Hydroxylamine hydrochloride (100) 4.84 ± 0.51 1,10-Phenanthroline (50) 4.94 ± 0.53 1,10-Phenanthroline (100) 5.08 ± 0.09 a Values are expressed as the mean ± standard deviation of three to five mice per group. The experiment was repeated two times with similar results.…”

“…Splenocytes were re-suspended in culture medium at a density of 1 × 10 6 cells/mL, seeded in 24-well tissue culture plates and incubated at 37 • C with 5% v/v CO 2 for 7 days. Stocks of inhibitors and drugs (actinonin/BB-3497/hydroxylamine hydrochloride/1,10-phenanthroline/ATDs) were prepared in RPMI medium and added to the splenocyte preparation before their distribution into 24-well tissue culture plates [25]. At different time intervals (0 days and 4 days), aliquots were taken from each well for CFU enumeration.…”

IFN-γ- and TNF-Independent Vitamin D-Inducible Human Suppression of Mycobacteria: The Role of Cathelicidin LL-37

“…PBMC (5 ϫ 10 5 ) were plated in triplicate on 48-well plates in RPMI 1640/10% FCS. Infection and subsequent handling of cultures was according to techniques described previously (31). MN preparations were obtained by adherence, and periodic assessment by FACS found them to be 82 Ϯ 4% CD14 and 76 Ϯ 7% CD11b positive.…”