Malachite Green Interferes with Postantibiotic Recovery of Mycobacteria

Abstract

The genus Mycobacterium comprises slow-growing species with generation times ranging from hours to weeks. The protracted incubation time before colonies appear on solid culture medium can result in overgrowth by faster-growing microorganisms. To prevent contamination, the solid media used in laboratories and clinics for cultivation of mycobacteria contain the arylmethane compound malachite green, which has broad-spectrum antimicrobial activity. Malachite green has no impact on the plating efficiency of mycobacteria when cells are grown under normal conditions. However, we found that malachite green interfered with colony formation when bacteria were preexposed to antibiotics targeting cell wall biogenesis (isoniazid, ethionamide, ethambutol). This inhibitory effect of malachite green was not observed when bacteria were preexposed to antibiotics targeting cellular processes other than cell wall biogenesis (rifampin, moxifloxacin, streptomycin). Sputum specimens from tuberculosis patients are routinely evaluated on solid culture medium containing high concentrations of malachite green. This practice could lead to underestimation of bacterial loads and overestimation of chemotherapeutic efficacy. Mycobacterium tuberculosis grows very slowly, with a population doubling time of ϳ22 h. Consequently, detection of M. tuberculosis by outgrowth of CFU on solid culture medium requires weeks or months (17). Despite being labor-intensive and time-consuming, enumeration of CFU by plating sputum cultures on solid medium continues to be the "gold standard" for evaluating the effectiveness of antituberculosis chemotherapy (6,19). Contamination by faster-growing microorganisms is prevented by supplementing the medium with compounds that suppress the growth of common species in the human flora.In clinical practice, sputum samples from tuberculosis patients are routinely plated on Lowenstein-Jensen or Middlebrook 7H11 agar. These media contain high concentrations of malachite green, a diamino-triphenylmethane dye with broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria (1). The mechanistic basis of malachite green's antimicrobial activity is not understood, nor is it clear why mycobacteria can survive and replicate in the presence of high concentrations of this compound (1,12). This paper shows that malachite green interferes with the recovery of mycobacteria on solid culture medium following exposure to certain antibiotics. This inhibitory effect was specific to antibiotics that target cell wall biogenesis (isoniazid, ethionamide, ethambutol) and was not observed with antibiotics that target other cellular processes (rifampin, moxifloxacin, streptomycin). These observations may have implications for clinical practices because underestimation of bacterial loads in patients undergoing chemotherapy could result in overestimation of therapeutic efficacy. MATERIALS AND METHODSBacteria and culture conditions. Wild-type Mycobacterium smegmatis (strain mc 2 155) and M. tuberculosis (strain Erdman) were s...

Classic reaction kinetics can explain complex patterns of antibiotic action

“…In contrast to previous studies attributing the post-antibiotic effect to biological mechanisms (7, 8), we show here that it may be explained by chemical processes alone. Our modeling framework makes testable predictions regarding the strength of the post-antibiotic effect that may be used to design antibiotics that can be given less frequently.…”

“…Predictions of optimal dosing intervals are complicated by the fact that for some bacteria/antibiotic combinations, bacterial growth remains suppressed after removal of the antibiotic. This ‘post-antibiotic effect’ is not easily predicted, and has generally been attributed to bacterial stress responses induced by exposure to antibiotics (7, 8). …”

Methylcitrate cycle defines the bactericidal essentiality of isocitrate lyase for survival of Mycobacterium tuberculosis on fatty acids

“…Mtb viability was determined by using liquid cultures manipulated under experimentally identical conditions as filter-grown cultures used for metabolomic profiling, which we had demonstrated to be microbiologically similar (25). Colony forming units (CFU) were determined by plating on m7H9 with Bacto Agar because of the apparent toxicity of malachite green to ICL-deficient Mtb with supplements; 0.2% glycerol, 0.2% dextrose, 0.5 g/L BSA, and 0.085% NaCl (26).…”

Rapid, Semiquantitative Assay To Discriminate among Compounds with Activity against Replicating or Nonreplicating Mycobacterium tuberculosis

“…For example, activated charcoal may sequester a component in the agar or in the 7H11-OADC medium in which the agar is dissolved, and that component might otherwise synergize with certain test agents to kill M. tuberculosis during the postantibiotic recovery phase of a CFU-based assay. For example, malachite green, a common component in 7H11-based agar, can potentiate the postantibiotic effect of cell-wall-active drugs on mycobacteria ( 56 ); perhaps activated charcoal can neutralize this effect. We have observed that M. tuberculosis bacilli form smaller colonies on 7H11-OADC plates containing activated charcoal, which suggests that the activated charcoal may bind essential nutrients, albeit not to an extent that abrogates the ability to form colonies.…”