Bacterial persistence, the ability of bacteria to survive high concentrations of antibiotics for extended periods of time, is an important contributing factor to therapy failure and development of chronic and recurrent infections. Several recent studies have suggested that this persistence is mediated primarily by (p)ppGpp, through its interactions with toxin-antitoxin modules and polyphosphates. In this study, we address whether these key players play a role in mycobacterial persistence against antibiotics. We targeted these specific pathways in Mycobacterium smegmatis by constructing deletion strains of (p)ppGpp synthetase/hydrolase (relA), polyphosphate kinases (ppk1 and ppk2), exopolyphosphatases (ppx1 and ppx2), and the lon protease. None of these mutant strains exhibited altered levels of persisters against isoniazid and ciprofloxacin, when compared with wild-type strain. Even under conditions in which the stringent response usually gets activated, these strains displayed wild-type persister levels. Interestingly, we also found that unlike Escherichia coli, maintaining M. smegmatis in exponential phase by repeated passaging does not eliminate persisters suggesting that at least against the antibiotics tested, stationary-phase dependent persisters (type I) are not the major contributors. Thus, our data demonstrate that multiple mechanisms of antibiotic persistence exist and that these vary widely among different bacterial species. © 2018 IUBMB Life, 70(9):836-844, 2018.

A major obstacle in the process of discovery of drugs against Mycobacterium tuberculosis is its extremely slow growth rate and long generation time (ϳ20 to 24 h). Consequently, determination of MICs and minimum bactericidal concentrations (MBCs) of potential drug candidates using current methods requires 7 days (resazurin-based MIC assay [REMA]) and 1 month (CFU enumeration), respectively. We employed a synthetic luciferase operon optimized for expression in high-GC-content bacteria and adapted it for use in mycobacteria. Using luminescence-based readouts, we were able to determine the MICs and bactericidal activities of approved tuberculosis (TB) drugs, which correlated well with currently used methods. Although luminescencebased readouts have been used previously to determine the MICs and bactericidal activities of approved TB drugs, in this study we adapted this assay to carry out a pilot screen using a library of 1,114 compounds belonging to diverse chemical scaffolds. We found that MICs derived from a 3-day luminescence assay matched well with REMA-based MIC values. To determine the bactericidal potencies of compounds, a 1:10 dilution of the cultures from the MIC plate was carried out on day 7, and the bactericidal concentrations determined based on time to positivity in 2 weeks were found to be comparable with MBC values determined by the conventional CFU approach. Thus, the luminescent mycobacterium-based approach not only is very simple and inexpensive but also allowed us to generate the information in half the time required by conventional methods.T uberculosis (TB) continues to be a major threat to public health worldwide, accounting for 1.3 million deaths annually, and is also a leading cause of death among people coinfected with HIV/AIDS. Despite the facts that the estimated number of people falling ill with tuberculosis each year is declining and that TB death rates dropped 45% over the last 2 decades, the rising incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB cases is alarming (1). Only half of MDR-TB cases are successfully treated, reflecting exceptionally high mortality rates. Moreover, 1/10 of MDR TB cases are redefined as XDR TB, as they are resistant to the majority of existing first-and second-line anti-TB drugs. The emergence of MDR and XDR TB strains compromises the existing treatment regimens and has prompted the search for new medicines. Therefore, an efficient, rapid, low-cost, highthroughput assay that would allow us to screen compound libraries and follow their effects on mycobacterial cells in real time would be of tremendous help.The extremely slow growth rates of pathogenic mycobacteria and therefore the long incubation times needed to assay antimycobacterial activity represent major obstacles in drug discovery. Plating of bacterial cultures on specialized mycobacterial agar medium for CFU enumeration continues to be the "gold standard" for evaluating the effectiveness of antituberculosis compounds, despite being very labor-intensive and time-consuming...

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...

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