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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.

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