Drug Resistance in Mycobacterium Tuberculosis

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Tuberculosis (TB) is a potentially deadly disease caused by pathogenic bacteria, usually Mycobacterium tuberculosis. It has existed in humans since ancient times and had high mortality rates without adequate treatment options before the invention of antibiotics , specifically streptomycin in 1943, that were potent enough to kill the bacteria. [6] In the 1960’s, following a drastic reduction in TB rates around the world, people began to predict that the disease could be completely eradicated within a century. [5] However, this goal proved overly-optimistic as drug-resistant strains had begun to emerge since the first use of antibiotics to treat TB. At first this was mainly due to only using a single drug, streptomycin, to treat the infection, prompting the use of multi-drug therapy but in recent decades multi-drug resistant (MDR), extensively- drug resistant (XDR), and totally-drug resistant (TDR) strains of TB have emerged. [1] Many of these strains are effectively incurable, especially the XDR and TDR strains, even for patients with access to an array of anti-TB drugs. [1] Given their grave public health threat it is crucial to study the molecular biology of the intrinsic and acquired mechanisms of resistance in M. tuberculosis in order to develop new drugs that avoid these mechanisms.

Intrinsic Drug Resistance

M. tuberculosis possess a multitude of resistance mechanisms against a wide range of antibiotics, as far as its intrinsic mechanisms (as opposed to acquired mechanisms that are brought about by chromosomal mutations, as discussed below) they can be divided into two categories: passive and specialized resistance. [7]

Passive Resistance Mechanisms

The primary feature of passive resistance to antibiotics in M. tuberculosis is a highly impermeable cell wall. Hydrophobic chemicals are unable to enter this cell wall due to a layer of hydrophilic arabinogalactan [6]. This layer is then wrapped in hydrophobic mycolic acids which severely limits the entrance of hydrophilic molecules. The mycolic acids are added to arabinogalactan in the cell wall by group of mycolyltransferase enzymes. One gene that encodes one of the mycolytransferases is the FbpA gene and studies of FbpA mutants have led to the conclusion that there is a strong connection between mycolic acid content and antibiotic resistance. [6] Despite being considered Gram-positive, Mycobacterium cell wall layers create space that resembles the periplasm of Gram-negative bacteria. [6] Another piece of evidence supporting the impermeability of the Mycobacterial cell wall is the fact that the time it takes for β-lactam antibiotics to diffuse through the cell wall takes a hundred times longer than it does for Escherichia coli. [7]

Specialized Resistance Mechanisms

The cell wall of mycobacteria is only part of what makes this species innately resistant to such a broad range of antibiotics. Studies have shown that the mycobacterial cell envelope lets in enough hydrophilic antibiotics over time to potentially kill the cell; however, since the cell wall significantly hinders the entrance of antibiotics it is thought that it slows antibiotic accumulation enough to allow for various cellular systems to detoxify the invading drugs. [6] This is especially evident in the case of β-lactams, which work by inhibiting the assembly of peptidoglycan and ultimately causing cell death, as peptidoglycan is a crucial part of the bacterial cell wall. [7] While the mycobacterial cell wall greatly limits the rate at which β-lactams are able to accumulate in the cell, effectively protecting against certain antibiotics, such as carbapenems which are not very stable and degrade relatively quickly, they still would be able to high enough levels over time due to the slow rate at which M. tuberculosis undergoes cell division. [7] However, M. tuberculosis possesses certain enzymes called β-lactamases that effectively degrade β-lactam antibiotics. Clinical evidence and in vitro experiments involving successful treatments of both β-lactams and β-lactamase inhibitors, as well as β-lactamase-resistant- β-lactams have strengthened the claim that β-lactamases are the most important factor in mycobacterial resistance to β-lactams. [6]

• Modification of drug targets

• Efflux pumps

  o Figure 2. Shows role of efflux pumps in intrinsic resistance of M. tuberculosis to the β-lactam class of antibiotics, as well as vancomycin and bacitracin.

File:Journal.pone.0083006.g001.jpeg

Source: [1]

Subscript: H₂O
Superscript: Fe³⁺

Acquired Resistance

While in many other types of bacteria drug resistance is gained through horizontal gene transfer by plasmids or transposons, in M. tuberculosis all strains with acquired resistance that are currently known are through chromosomal mutations due to the selective pressure of antibiotics. [1] This selection of drug-resistant strains could not occur without the extensive and prolonged use of antibiotics necessary to treat the disease as those strains would have lower fitness under normal conditions. [7] Additionally recent studies, such as that by Kohanski et al. [10], have shown that sub-lethal doses of antibiotics can cause multi-drug resistance in E. Coli due to increased mutation rates by free radicals. (Figure 3.) This could very well apply to M. tuberculosis as well, suggesting that current TB drugs are not only selecting for drug-resistant strains but quite possibly creating them as well.

Promising New Drugs

Benzothiazinones (BTZ)

Dinitrobenzamide derivatives (DNB)

References

1. Almeida Da Silva PE, Palomino JC. Molecular basis and mechanisms of drug resistance in mycobacterium tuberculosis: Classical and new drugs. J Antimicrob Chemother 2011 JUL;66(7):1417-30.

2. Christophe T, Jackson M, Jeon HK, Fenistein D, Contreras-Dominguez M, Kim J, Genovesio A, Carralot J, Ewann F, Kim EH, et al. High content screening identifies decaprenyl-phosphoribose 2 ' epimerase as a target for intracellular antimycobacterial inhibitors. Plos Pathogens 2009 OCT;5(10):e1000645.

3. Crellin PK, Brammananth R, Coppel RL (2011) Decaprenylphosphoryl-β-D-Ribose 2′-Epimerase, the Target of Benzothiazinones and Dinitrobenzamides, Is an Essential Enzyme in Mycobacterium smegmatis. PLoS ONE 6(2): e16869. doi:10.1371/journal.pone.0016869 4. Dinesh N, Sharma S, Balganesh M. Involvement of Efflux Pumps in the Resistance to Peptidoglycan Synthesis Inhibitors in Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy. 2013;57(4):1941-1943. doi:10.1128/AAC.01957-12.

5. Mokrousov I, Otten T, Vyazovaya A, Limeschenko E, Filipenko M, Sola C, Rastogi N, Steklova L, Vyshnevskiy B, Narvskaya O. PCR-based methodology for detecting multidrug-resistant strains of mycobacterium tuberculosis beijing family circulating in russia. European Journal of Clinical Microbiology & Infectious Diseases 2003 JUN;22(6):342-8.

6. Nguyen L, Pieters J. Mycobacterial subversion of chemotherapeutic reagents and host defense tactics: challenges in tuberculosis drug development. Annu Rev Pharmacol Toxicol. 2009;49:427-53. doi: 10.1146/annurev-pharmtox-061008-103123. Review. PubMed PMID: 19281311.

7. Smith T, Wolff KA, Nguyen L. Molecular Biology of Drug Resistance in Mycobacterium tuberculosis. Current topics in microbiology and immunology. 2013;374:53-80. doi:10.1007/82_2012_279.

8. Zhao L, Sun Q, Zeng C, Chen Y, Zhao B, Liu H, Xia Q, Zhao X, Jiao W, Li G, et al. Molecular characterisation of extensively drug-resistant mycobacterium tuberculosis isolates in china. Int J Antimicrob Agents 2015 FEB;45(2):137-43. 9. Cui Z, Li Y, Cheng S, Yang H, Lu J, Hu Z, Ge B. Mutations in the embC-embA intergenic region contribute to mycobacterium tuberculosis resistance to ethambutol. Antimicrob Agents Chemother 2014 NOV;58(11):6837-43. 10. Kohanski MA, DePristo MA, Collins JJ. Sub-lethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis. Molecular cell. 2010;37(3):311-320. doi:10.1016/j.molcel.2010.01.003.

Edited by (Noah Knowlton-Latkin), a student of Nora Sullivan in BIOL168L (Microbiology) in The Keck Science Department of the Claremont Colleges Spring 2014.