Characterization and Transcriptome Analysis of Mycobacterium tuberculosis Persisters

Antimicrobial Discovery Center and Department of Biology, Northeastern University, Boston, Massachusetts, USA.
mBio (Impact Factor: 6.79). 04/2011; 2(3):e00100-11. DOI: 10.1128/mBio.00100-11
Source: PubMed


Tuberculosis continues to be a major public health problem in many parts of the world. Significant obstacles in controlling the epidemic are the length of treatment and the large reservoir of latently infected people. Bacteria form dormant, drug-tolerant persister cells, which may be responsible for the difficulty in treating both acute and latent infections. We find that in Mycobacterium tuberculosis, low numbers of drug-tolerant persisters are present in lag and early exponential phases, increasing sharply at late exponential and stationary phases to make up ~1% of the population. This suggests that persister formation is governed by both stochastic and deterministic mechanisms. In order to isolate persisters, an exponentially growing population was treated with d-cycloserine, and cells surviving lysis were collected by centrifugation. A transcriptome of persisters was obtained by using hybridization to an Affymetrix array. The transcriptome shows downregulation of metabolic and biosynthetic pathways, consistent with a certain degree of dormancy. A set of genes was upregulated in persisters, and these are likely involved in persister formation and maintenance. A comparison of the persister transcriptome with transcriptomes obtained for several in vitro dormancy models identified a small number of genes upregulated in all cases, which may represent a core dormancy response.

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Available from: Iris Keren, Oct 05, 2015
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    • "Since the first report by Bigger in 1944 [1], bacterial persister cells have been described for a number of different species, including Escherichia coli[14], Staphylococcus aureus[14,15], Pseudomonas aeruginosa[16], and Mycobacterium tuberculosis[17,18]. For most of these bacterial species persister cells have also been found in biofilms, which contribute to recalcitrant and/or recurrent infections after antibiotic therapy [4,19-25]. "
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    ABSTRACT: Background Persister cells constitute a subpopulation of dormant cells within a microbial population which are genetically identical but phenotypically different to regular cells. Notably, persister cells show an elevated tolerance to antimicrobial agents. Thus, they are considered to represent a microbial ‘bet-hedging’ strategy and are of particular importance in pathogenic bacteria. Results We studied the ability of the zoonotic pathogen Streptococcus (S.) suis to form multi-drug tolerant variants and identified persister cells dependent on the initial bacterial growth phase. We observed lower numbers of persisters in exponential phase cultures than in stationary growth phase populations. S. suis persister cells showed a high tolerance to a variety of antibiotics, and the phenotype was not inherited as tested with four passages of S. suis populations. Furthermore, we provide evidence that the persister phenotype is related to expression of genes involved in general metabolic pathways since we found higher numbers of persister cells in a mutant strain defective in the catabolic arginine deiminase system as compared to its parental wild type strain. Finally, we observed persister cell formation also in other S. suis strains and pathogenic streptococcal species. Conclusions Taken together, this is the first study that reports multi-drug tolerant persister cells in the zoonotic pathogen S. suis.
    BMC Microbiology 05/2014; 14(1):120. DOI:10.1186/1471-2180-14-120 · 2.73 Impact Factor
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    • "This happens through a partially understood mechanism where individual cells can stochastically enter into a transient state characterized by arrested or reduced growth [5], [6]. This phenomenon appears to be widely spread among prokaryotic microorganisms and from its first reports in the late 1944 in Staphylococcus aureus [7], to the identification of high persistence strains in Escherichia coli in the late 80’s [8], [9], persistence occurrence has been reported in several other bacterial strains, including human pathogens such as Pseudomonas aeruginosa and the etiologic agent of tuberculosis Mycobacterium tuberculosis [3], [10]–[12]. Persister cells tolerance to antibiotic agents and their presence in biofilms has been associated with chronic recalcitrant bacterial infections [3], [13]. The study of bacterial persistence is therefore of great importance to public health. "
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    ABSTRACT: Bacterial persistence, where a fraction of a population presents a transient resistance to bactericidal substances, has great medical importance due to its relation with the appearance of antibiotic resistances and untreatable bacterial chronic infections. The mechanisms behind this phenomenon remain largely unknown in spite of recent advances, in great part because of the difficulty in isolating the very small fraction of the population that is in this state at any given time. Current protocols for persister isolation have resulted in possible biases because of the induction of this state by the protocol itself. Here we present a novel protocol that allows rapid isolation of persister cells both from exponential and stationary phase. Moreover, it is capable of differentiating between type I and type II persister cells, which should allow the field to move beyond its current state of studying only one type. While this protocol prompts a revision of many of the current results, it should greatly facilitate further advances in the field.
    PLoS ONE 02/2014; 9(2):e88660. DOI:10.1371/journal.pone.0088660 · 3.23 Impact Factor
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    • "To give an example in vivo, in the lungs of guinea pigs treated for 6 weeks with 15 mg/kg of TMC-207, 1 persisting Mtb out of about 20,000 bacilli was recovered after treatment.30In vitro, low numbers of Mtb persisters are present in early exponential phase, but their number increases sharply up to 1% of the population at late exponential and stationary phases.36 Thus, their proportion likely depends by specific conditions including the age of a culture, the length of drug exposure, the type and concentration of antibiotics.35 "
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    ABSTRACT: Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which kills about 2 million people annually. Furthermore, 2 billion people worldwide are latently infected with this organism, with 10% of them reactivating to active TB due to re-growth of nonreplicating (dormant) Mtb residing in their tissues. Because of the huge reservoir of latent TB it is important to find novel drugs/drug combinations killing dormant bacilli (microaerophiles, anaerobes and drug-tolerant persisters) surviving for decades in a wide spectrum of granulomatous lesions in the lungs of TB patients. Antibiotic treatment of drug-susceptible TB requires administration of isoniazid, rifampin, pyrazinamide, ethambutol for 2 months, followed by isoniazid and rifampin for 4 months. To avoid reactivation of dormant Mtb to active pulmonary TB, up to 9 months of treatment with isoniazid is required. Therefore, a strategy to eliminate dormant bacilli needs to be developed to shorten therapy of active and latent TB and reduce the reservoir of people with latent TB. Finding drugs with high rate of penetration into the caseous granulomas and understanding the biology of dormant bacilli and in particular of persister cells, phenotypically resistant to antibiotics, will be essential to eradicate Mtb from humans. In recent years unprecedented efforts have been done in TB drug discovery, aimed at identifying novel drugs and drug combinations killing both actively replicating and nonreplicating Mtb in vitro, in animal models and in clinical trials in humans.
    Mediterranean Journal of Hematology and Infectious Diseases 11/2013; 5(1):e2013072. DOI:10.4084/MJHID.2013.072
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