Anti-Tuberculosis Activity of Three Carbapenems, Clofazimine and Nitazoxanide Using a Novel Ex Vivo Phenotypic Drug Susceptibility Model of Human Tuberculosis

Abstract

We evaluated a novel physiological 3-D bioelectrospray model of the tuberculosis (TB) granuloma to test the activity of a known anti-TB drug, clofazimine; three carbapenems with potential activity, including one currently used in therapy; and nitazoxanide, an anti-parasitic compound with possible TB activity (all chosen as conventional drug susceptibility was problematical). PBMCs collected from healthy donors were isolated and infected with M. tuberculosis H37Rv lux (i.e., luciferase). Microspheres were generated with the infected cells; the anti-microbial compounds were added and bacterial luminescence was monitored for at least 21 days. Clavulanate was added to each carbapenem to inhibit beta-lactamases. M. tuberculosis (MTB) killing efficacy was dose dependent. Clofazimine was the most effective drug inhibiting MTB growth at 2 mg/L with good killing activity at both concentrations tested. It was the only drug that killed bacteria at the lowest concentration tested. Carbapenems showed modest initial activity that was lost at around day 10 of incubation and clavulanate did not increase killing activity. Of the carbapenems tested, tebipenem was the most efficient in killing MTB, albeit at a high concentration. Nitazoxanide was effective only at concentrations not achievable with current dosing (although this might partly have been an artefact related to extensive protein binding).

Keywords: anti-microbial drug resistance; drug susceptibility testing; three-dimensional bioelectrospray; tuberculosis.

Figure 1

Luminescence detection at different time points for clofazimine in TB-infected PMBCs obtained from donor 1. AC: alginate collagen; PMBCs: peripheral blood mononuclear cells; Mtb: Mycobacterium tuberculosis; DMSO: dimethyl sulfoxide; Clof 2: Clofazimine 2 mg/L; Clof 32: Clofazimine 32 mg/L.

Figure 2

Luminescence detection at different time points for nitazoxanide in TB-infected PMBCs obtained from donor 1. AC: alginate collagen; PMBCs: peripheral blood mononuclear cells; Mtb: Mycobacterium tuberculosis; DMSO: dimethyl sulfoxide; Nita8: Nitazoxanide 8 mg/L; Nita 64: Nitazoxanide 64 mg/L.

Figure 3

Luminescence detection at different time points for meropenem in TB-infected PMBCs obtained from donor 1. AC: alginate collagen; PMBCs: peripheral blood mononuclear cells; Mtb: Mycobacterium tuberculosis; DMSO: dimethyl sulfoxide; Mero 2: Meropenem 2 mg/L; Mero 32: Meropenem 32 mg/L; Clav 2.5: clavulanic acid 2.5 mg/L.

Figure 4

Luminescence detection at different time points for faropenem in TB-infected PMBCs obtained from donor 1. AC: alginate collagen; PMBCs: peripheral blood mononuclear cells; Mtb: Mycobacterium tuberculosis; DMSO: dimethyl sulfoxide; Faro 2: Faropenem 2 mg/L; Faro 32: Faropenem 32 mg/L; Clav 2.5: clavulanic acid 2.5 mg/L. Standard deviation included but are very small, i.e., within the size of the points on the curve.

Figure 5

Luminescence detection at different time points for tebipenem in TB-infected PMBCs obtained from donor 1. AC: alginate collagen; PMBCs: peripheral blood mononuclear cells; Mtb: Mycobacterium tuberculosis; DMSO: dimethyl sulfoxide; Tebi 2: Tebipenem 2 mg/L; Tebi 64: Tebipenem 64 mg/L; Clav 2.5: clavulanic acid 2.5 mg/L.