Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria

Abstract

Successful treatment of human tuberculosis requires 6-9 months' therapy with multiple antibiotics. Incomplete clearance of tubercle bacilli frequently results in disease relapse, presumably as a result of reactivation of persistent drug-tolerant Mycobacterium tuberculosis cells, although the nature and location of these persisters are not known. In other pathogens, antibiotic tolerance is often associated with the formation of biofilms--organized communities of surface-attached cells--but physiologically and genetically defined M. tuberculosis biofilms have not been described. Here, we show that M. tuberculosis forms biofilms with specific environmental and genetic requirements distinct from those for planktonic growth, which contain an extracellular matrix rich in free mycolic acids, and harbour an important drug-tolerant population that persist despite exposure to high levels of antibiotics.

Fig. 1

Growth of M. tuberculosis biofilms. A. M. bovis BCG or M. tuberculosis H37Rv was cultured in a Petri dish containing Sauton's medium for 5 weeks with either a standard loose-fitting lid or with a sealed parafilm covering. B. Bottle assay for the growth of M. tuberculosis mc²7000 biofilms. Bottle caps were kept either loose or tight for 5 weeks, or tight for the first 3 weeks and loosened for the last 2 weeks, as indicated. C. Gases present in either loosely capped or tightly capped bottles (as indicated) were examined by GC/MS. CO₂ (44 amu) accumulates in the tightly capped bottles.

Fig. 2

Environmental and genetic requirements for M. tuberculosis biofilms. A. M. tuberculosis mc²4640, a mutant deleted for genes encoding 2-oxoglutarate dehydrogenase, requires CO₂ for colony formation on solid media and is defective in biofilm formation. Tightly capping the bottle for the first 3 weeks or complementing with genes Rv2454c-Rv2455c (mc²4640/comp) partially restores biofilm formation; all cultures were grown for 5 weeks. B. Exogenous CO₂ (5%) is not sufficient to support full growth of M. tuberculosis biofilms. Biofilms were grown in air or air supplemented with 5% CO₂ as shown. C. Low iron (2 μM) and low zinc (none added) inhibit biofilm development with no effect on planktonic growth (see Fig. S1). D. M. tuberculosis strain mc²7025 defective in gene Rv1013 is strongly defective in biofilm formation, and complemented by plasmid pRv1013comp that contains the Rv1013 gene.

Fig. 3

Lipid analysis of planktonically grown cells and biofilms of M. tuberculosis. Total apolar and polar lipids from planktonic and biofilm cultures of M. tuberculosis mc²7000 resolved on 2D-TLC are solvent systems as described in the Experimental procedures (solvent A, Sol. A; solvent C, Sol. C; solvent D, Sol. D). Samples were labelled with ¹⁴C-acetate and equal amounts of total radioactivity were examined. The lipids are annotated as previously described (Besra, 1998): A–C, phthiocerol dimycoserosate family; MK, menaquinone; D and E, apolar mycolipenates of trehalose; F, free fatty acid; CF, cord factor; DAT, 2,3,-di-O-acyltrehaloses; SL, sulphatides. Spot 1 which is induced in biofilms is also marked.

Fig. 4

M. tuberculosis biofilms contain free mycolic acids. A. 1D-TLC of apolar lipids extracted from either M. tuberculosis mc²7000 cells or the mc²7025 mutant growing either planktonically or as biofilms resolved in solvent system C. Samples were labelled with ¹⁴C-acetate and equal amounts of total radioactivity were examined. B. Tween-80 solubilization of biofilm lipids. Biofilm samples were Tween-80 extracted and the Tween-soluble and Tween-insoluble fractions separated by TLC as above. The Tween-containing supernatant from planktonically grown cells was analysed similarly. C. ¹H-NMR spectrum of Spot 1. ¹H signals of protons associated with functional groups were assigned using known chemical shifts (Watanabe et al., 1999; X. Trivelli and Y. Guerardel, unpubl. obs.) and labelled (a to h) according to their relative positions. A slight de-shielding of Ha (Δδ + 0.06 p.p.m.) proton from 3.64 to 3.70 p.p.m. and Hb (Δδ + 0.03 p.p.m.) proton from 2.43 to 2.46 p.p.m. compared with their equivalent protons associated to a methyl-esterified carboxyl group is in agreement with the occurrence of a free mycolate. D. MALDI-TOF-MS analysis of native (top panel), -CH3 (middle panel) and -CD3 (bottom panel) esterified Spot 1. Signals correspond to [M + Na]⁺ adducts of a family of methoxy mycolates. E. Spot 1 (lane 1) was methyl-esterified (lane 2) and compared with methyl-esterified mycolic acids purified from Mtb cell wall (lane 3), by resolution on a TLC plate developed in petroleum ether : acetone (95:5) and visualized as in Fig. 4A. a, m and k indicate the alpha, methoxy and keto mycolic acids respectively.

Fig. 5

M. tuberculosis biofilms are drug-tolerant. M. tuberculosis mc²7000 or the biofilm-defective mc²7025 mutant were grown planktonically or as biofilms and treated with either isoniazid (A) or rifampicin (B); the percentages of viable cells remaining after different times of drug treatment are shown. (C) Percentage of surviving cells following 7 day treatment of M. tuberculosis biofilms with antibiotics at different concentrations.