Activity and interactions of levofloxacin, linezolid, ethambutol and amikacin in three-drug combinations against Mycobacterium tuberculosis isolates in a human macrophage model

Multidrug resistance is a problem in the management of tuberculosis, creating an urgent need for new regimens including currently available drugs. Macrophage models allow an evaluation of the effect of drugs against intracellular bacilli. The effect of the following different drug combinations against six multidrug-resistant and six drug-susceptible clinical isolates of Mycobacterium tuberculosis multiplying inside the human macrophage THP-1 cell line was studied: levofloxacin/linezolid/ethambutol; levofloxacin/amikacin/ethambutol; and levofloxacin/linezolid/amikacin. Macrophages were lysed, seeded onto Middlebrook 7H11 plates and CFU were counted after 21 days of incubation. The interaction of the drugs in combination was interpreted by the effect of the combination compared with the most active single drug alone. The antimicrobial activity of the drugs was evaluated comparing the log(10)CFU/well of the isolate with and without the drug. Drug concentrations within infected macrophages and in extracellular medium were simultaneously determined by chromatography. The levofloxacin/linezolid/amikacin and levofloxacin/linezolid/ethambutol combinations showed antagonism against most of the isolates (91.7%) after a 4-day protocol, whereas levofloxacin/amikacin/ethambutol displayed indifference. Levofloxacin alone and levofloxacin/amikacin/ethambutol were the most potent antimicrobials, presenting reductions up to 5.49 log(10) and 5.86 log(10), respectively. The drug penetration percentages ranged from 5.46% to 11.10%. Intracellular concentrations for the drug alone compared with those for the drugs in combination were not significantly different. All of the combinations tested against M. tuberculosis-infected macrophages showed antimicrobial activity, with combinations including linezolid and levofloxacin showing an antagonistic effect that may be explained by efflux transporters or changes in the macrophage environment.