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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    • "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.
    Full-text · Article · May 2014 · BMC Microbiology
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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.
    Full-text · Article · Feb 2014 · PLoS ONE
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    • "RNA-seq has been applied to prokaryotic transcriptome studies [32] [33] and some efforts have been made to use next-generation sequencing in clinical microbiology to test the properties of growing bacterial pathogens [34]. Global transcriptomic profile studies in E. coli and Mycobacterium tuberculosis dormant persister sub-population have been previously carried out, being reported the dormant state was associated with changes in transcription of genes involved in metabolism, biosynthetic pathways or oxidative stress [23] [35]. "
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    ABSTRACT: The proportion of dormant bacteria within Staphylococcus epidermidis biofilms may determine its inflammatory profile. Previously, we have shown that S. epidermidis biofilms with higher proportions of dormant bacteria have reduced activation of murine macrophages. RNA-sequencing was used to identify the major transcriptomic differences between S. epidermidis biofilms with different proportions of dormant bacteria. To accomplish this goal, we used an in vitro model where magnesium allowed modulation of the proportion of dormant bacteria within S. epidermidis biofilms. Significant differences were found in the expression of 147 genes. A detailed analysis of the results was performed based on direct and functional gene interactions. Biological processes among the differentially expressed genes were mainly related to oxidation-reduction processes and acetyl-CoA metabolic processes. Gene set enrichment revealed that the translation process is related to the proportion of dormant bacteria. Transcription of mRNAs involved in oxidation-reduction processes was associated with higher proportions of dormant bacteria within S. epidermidis biofilm. Moreover, the pH of the culture medium did not change after the addition of magnesium, and genes related to magnesium transport did not seem to impact entrance of bacterial cells into dormancy.
    Full-text · Article · Feb 2014 · Applied Microbiology and Biotechnology
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