Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Open Life Sciences

formerly Central European Journal of Biology

Editor-in-Chief: Ratajczak, Mariusz

IMPACT FACTOR 2018: 0.504
5-year IMPACT FACTOR: 0.583

CiteScore 2018: 0.63

SCImago Journal Rank (SJR) 2018: 0.266
Source Normalized Impact per Paper (SNIP) 2018: 0.311

ICV 2017: 154.48

Open Access
See all formats and pricing
More options …
Volume 3, Issue 4


Volume 10 (2015)

Exhibition of persistent and drug-tolerant L-form habit of Mycobacterium tuberculosis during infection in rats

Nadya Markova / Lilia Michailova / Mimi Jourdanova / Vesselin Kussovski / Violeta Valcheva / Igor Mokrousov / Tatyana Radoucheva
Published Online: 2008-09-30 | DOI: https://doi.org/10.2478/s11535-008-0032-7


A model for studying mycobacterial L-form formation in vivo was established to demonstrate the ability of M. tuberculosis to behave as a drug-tolerant L-form persister. Rats were infected by intranasal (i.n.) and intraperitoneal (i.p.) routes with 1×108 cells/ml of M. tuberculosis. At weekly intervals during a period of five weeks, samples from lung, spleen, liver, kidney, mesenterial and inguinal lymph nodes, broncho-alveolar and peritoneal lavage liquid were plated simultaneously on Löwenstein-Jensen (LJ) medium or inoculated into specially supplemented for L-forms Dubos broth (drug-free and drug-containing variants). The use of liquid media enabled isolation of mycobacterial L-form cultures during the whole period of experiment including the last two weeks, when tubercle bacilli were not isolated on LJ medium. An unique feature of mycobacterial L-forms was their ability to grow faster than the classical tubercle bacilli. Isolation and growth of L-form cultures in primary drug-containing media demonstrated their drug-tolerant properties. Electron microscopy of liquid media isolates showed that they consisted of morphologically heterogenous populations of membrane-bound and of variable sized L-bodies that completely lack cell walls. The identity of the isolated non-acid fast and morphologically modified L-forms as M. tuberculosis was verified by specific spoligotyping test. The results contribute to special aspects concerning the importance of mycobacterial L-form phenomenon for persistence and latency in tuberculosis, phenotypic drug tolerance, as well as for diagnosis of difficult to identify morphologically changed tubercle bacilli which are often mistaken for contaminants.

Keywords: Mycobacterium tuberculosis; L-forms; Persistence; Drug-tolerance; Rat infection

  • [1] Calmette A., Valti J., Virulent filterable elements of the tubercle bacillus, Ann. Med., 1926, 19, 553 Google Scholar

  • [2] Much H., Die Variation des Tuberkelbacillus in Form und Wirkung, Beitr. Klin. Tuber., 1931, 7, 60–71 (in German) http://dx.doi.org/10.1007/BF02079342CrossrefGoogle Scholar

  • [3] Mellon R.R., Fisher L.W., New studies on the filterability of pure cultures of the tubercle group of microorganisms, J. Infect. Dis., 1932, 51, 117–128 CrossrefGoogle Scholar

  • [4] Mattman L.H., Mycobacterium tuberculosis and atypicals, In: Mattman L.H., (Ed.) Cell wall deficient forms. Stealth Pathogens, 3rd ed., CRC Press, Inc., Boca Raton, FL, USA, 2001 Google Scholar

  • [5] Rastogi N., David H.L., Ultrastructural and chemical studies on wall-deficient forms, spheroplasts and membrane vesicles from Mycobacterium avium, J. Gen. Microbiol., 1981, 121, 71–79 CrossrefGoogle Scholar

  • [6] Mattman L.H., Tunstall L.H., Matthews W.W., Gordon D.L., L variation in mycobacteria, Am. Rev. Resp. Dis., 1960, 82, 202–211 Google Scholar

  • [7] Chandrasekhar S., Ratnam S., Studies on cell-wall deficient non-acid fast variants of Mycobacterium tuberculosis, Tuberc. Lung. Dis., 1992, 73, 273–279 http://dx.doi.org/10.1016/0962-8479(92)90132-4CrossrefGoogle Scholar

  • [8] Domingue G.J., Woody H.B., Bacterial persistence and expression of disease, Clin. Microbiol. Rev., 1997, 10, 320–344 Google Scholar

  • [9] Judge M.S., Mattman L.H., Cell wall-deficient mycobacteria in tuberculosis, sarcoidosis, and leprosy, In: Domingue G.J. (Ed.), Cell wall-deficient bacteria: basic principles and clinical significance, Addison-Wesley Publishing Co., Reading Mass, 1982 Google Scholar

  • [10] Ratnam S., Chandrasekhar S., The pathogenicity of spheroplasts of Mycobacterium tuberculosis, Am. Rev. Respir. Dis., 1976, 114, 549–554 Google Scholar

  • [11] Takahashi S., L phase growth of Mycobacteria. 1. Cell wall deficient form of Mycobacteria, Kekkaku, 1979, 54, 63–70 Google Scholar

  • [12] Zhang Y., Persistent and dormant tubercle bacilli and latent tuberculosis, Front. Biosci., 2004, 9, 1136–1156 http://dx.doi.org/10.2741/1291CrossrefGoogle Scholar

  • [13] Khomenko A.G., Kochemasova Z.N., Dykhno M.M., Zemskova Z.N., Dorozhkova I.R., Significance of L-form transformation and reversion of the causative agent in the epidemiology of tuberculosis, Zh. Mikrobiol. Epidemiol. Immunobiol., 1984, 3, 9–14 (in Russian) Google Scholar

  • [14] Michailova L., Kussovski V., Radoucheva T., Jourdanova M., Berger W., Rinder H., et al., Morphological variability and cell wall deficiency in M. tuberculosis “heteroresistant” strains, Int. J. Tuber. Lung. Dis., 2005, 9, 907–914 Google Scholar

  • [15] Cockerill F.R., Genetic methods for assessing antimicrobial resistance, Antimicrob. Agents Chemother., 1999, 43, 199–212 Google Scholar

  • [16] Mokrousov I., Otten T., Filipenko M.L., Vyazovaya A., Chrapov E., Limeschenko, et al., Detection of isoniazid-resistant Mycobacterium tuberculosis strains by multiplex allele-specific PCR assay targeting katG codon 315 variation, J. Clin. Microbiol., 2002, 40, 2509–2512 http://dx.doi.org/10.1128/JCM.40.7.2509-2512.2002CrossrefGoogle Scholar

  • [17] Mokrousov I., Otten T., Vyshnevsky B., Narvskaya O., Detection of embB306 mutations in ethambutol-susceptible clinical isolates of M. tuberculosis from Northwestern Russia, J. Clin. Microbiol., 2002, 40, 3810–3813 http://dx.doi.org/10.1128/JCM.40.10.3810-3813.2002CrossrefGoogle Scholar

  • [18] Van Embden J.D.A., Cave M.D., Crawford J.T., Dale J.W., Eisenach K.D., Giequel B., et al., Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology, J. Clin. Microbiol., 1993, 31, 406–409 Google Scholar

  • [19] Kamerbeek J., Schouls L., Kolk A., van Agterveld M., van Soolingen D., Kuijper S.S., et al., Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology, J. Clin. Microbiol., 1997, 35, 907–914 Google Scholar

  • [20] Wayne L.G, Sohaskey C.D., Nonreplicating persistence of Mycobacterium tuberculosis, Annu. Rev. Microbiol., 2001, 55, 139–163 http://dx.doi.org/10.1146/annurev.micro.55.1.139CrossrefGoogle Scholar

  • [21] Ulrichs T., Kaufmann S.H.E., Immunology and persistence, In: Kaufmann S.H.E, Hahn H. (Eds.), Mycobacteria and TB, Basel, Switzerland, Karger, 2003 Google Scholar

  • [22] Parrish N.M., Dick J.D., Bishai W.R., Mechanisms of latency in Mycobacterium tuberculosis, Trends Microbiol., 1998, 6, 107–112 http://dx.doi.org/10.1016/S0966-842X(98)01216-5CrossrefGoogle Scholar

  • [23] Honer zu Bentrup K., Russell D.G., Mycobacterial persistence: adaptation to a changing environment, Trends Microbiol., 2001, 9, 597–605 http://dx.doi.org/10.1016/S0966-842X(01)02238-7CrossrefGoogle Scholar

  • [24] Bishai W., Lipid lunch for persistent pathogen, Nature, 2000, 406, 683–685 http://dx.doi.org/10.1038/35021159CrossrefGoogle Scholar

  • [25] Dienes L., Weinberger H.J., The L forms of bacteria, Bacteriol. Rev., 1951, 15, 245–288 Google Scholar

  • [26] Prozorovski S.V., Kaz L.N., Kagan G.J., Bacterial L-forms (mechanisms of formation, structure, role in pathology), Medicine Publishing, Moscow, 1981 (in Russian) Google Scholar

  • [27] Markova N., Michailova L., Vesselinova A., Kussovski V., Radoucheva T., Nikolova S., et al., Cell wall-deficient forms (L-forms) of Listeria monocytogenes in experimentally infected rats, Zentralbl. Bakteriol., 1997, 286, 46–55 Google Scholar

  • [28] Michailova L., Stoitsova S., Markova N., Kussovski V., Jourdanova M., Dimova I., Interaction of alveolar macrophages with Staphylococcus aureus and induction of microbial L-forms during infection in rats, Int. J. Med. Micobiol., 2000, 290, 259–267 CrossrefGoogle Scholar

  • [29] Boris M., Teubner D., Shinefield H., Bacterial interference with L-forms, J. Bacteriol., 1969, 100, 791–795 Google Scholar

  • [30] Fodor M., Roger H.J., Antagonism between vegetative cells and L-forms of Bacillus lichniformis 6346, Nature, 1966, 211, 658–659 http://dx.doi.org/10.1038/211658a0CrossrefGoogle Scholar

About the article

Published Online: 2008-09-30

Published in Print: 2008-12-01

Citation Information: Open Life Sciences, Volume 3, Issue 4, Pages 407–416, ISSN (Online) 2391-5412, DOI: https://doi.org/10.2478/s11535-008-0032-7.

Export Citation

© 2008 Versita Warsaw. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

Srinivasan Vijay, Mukkayyan Nagaraja, Jees Sebastian, and Parthasarathi Ajitkumar
Archives of Microbiology, 2014, Volume 196, Number 3, Page 157
Nadya Markova, Georgi Slavchev, and Lilia Michailova
Human Vaccines & Immunotherapeutics, 2012, Volume 8, Number 6, Page 759
Nadya Markova
Bioscience Hypotheses, 2009, Volume 2, Number 6, Page 441

Comments (0)

Please log in or register to comment.
Log in