title: L-form transformation phenomenon in Mycobacterium tuberculosis associated with drug tolerance to ethambutol.
abstract: Cell wall-deficient bacterial forms (L-forms) may occur along with resistance to factors that trigger their appearance. It is of interest to study the relationship between the L-form transformation of Mycobacterium tuberculosis and the exhibition of drug tolerance to ethambutol (EMB), an inhibitor of cell wall synthesis.
  
  
  
  L-form variant was produced from a sensitive EMB strain of M. tuberculosis through a cryogenic stress treatment protocol and was subsequently cultivated in Middlebrook 7H9 semisolid medium, containing EMB in a minimal inhibitory concentration of 2mg/L. Susceptibility to EMB of the parental strain and its L-form variant was evaluated phenotypically and using polymerase chain reaction-restriction fragment length polymorphism assay targeting a mutation in the embB306 gene fragment.
  
  
  
  In contrast to the sensitivity to EMB of the parental strain, its L-form variant showed phenotypic resistance to high concentrations of EMB (16mg/L), but the mutation in embB306 was not found. Electron microscopy observation of the L-form variant showed a heterogenic population of bacteria, with different degrees of cell wall deficiency, as well as cells of protoplastic type without cell walls. Of special interest were the observed capsule-like structures around the L-form cells and the biofilm-like matrix produced by the L-form population.
  
  
  
  We suggest that the expression of phenotypic resistance to EMB in M. tuberculosis can be associated with alterations or loss of cell walls in L-form bacteria, respectively, which results in a lack of a specific target for EMB action. In addition, production of capsule-like structures and biofilm matrix by L-forms could contribute to their resistance and survival in the presence of antibacterial agents.
authors.affiliation: Department of Infectious Microbiology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria.
authors.forename: Georgi
authors.initials: G
authors.lastname: Slavchev
authors.affiliation: Department of Infectious Microbiology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria.
authors.forename: Lilia
authors.initials: L
authors.lastname: Michailova
authors.affiliation: Department of Infectious Microbiology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria. Electronic address: nadya.markova@gmail.com.
authors.forename: Nadya
authors.initials: N
authors.lastname: Markova
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001431:Bacteriological Techniques; D002473:Cell Wall / Q000187:drug effects; D003470:Culture Media / Q000737:chemistry; D004361:Drug Tolerance; D004977:Ethambutol / Q000494:pharmacology*; D006801:Humans; D007740:L Forms / Q000166:cytology* / Q000187:drug effects*; D009169:Mycobacterium tuberculosis / Q000166:cytology* / Q000187:drug effects*
chemical_list: D000995:Antitubercular Agents; D003470:Culture Media; D004977:Ethambutol
keywords: Drug tolerance; Ethambutol; L-forms; Mycobacterium tuberculosis