Growth of Mycobacterium tuberculosis Biofilms

Summary

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Abstract

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis ¹, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms ^2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) ^5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms ⁷.

In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin ^8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere ⁹. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc²7000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen ⁹. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.

Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Protocol

1. Growing biofilms of M. tuberculosis in a 250mL screw capped bottle

1. Media preparation: Dissolve 0.5g of KH₂PO₄, 0.5g of MgSO₄, 4g of L-Asparagine, 2g of Citric acid, 0.05g of Ferric Ammonium Citrate, 60mL of glycerol in 900mL of water. Adjust the pH to 7.0 with NaOH. Autoclave, cool and just prior to starting the experiment, add sterile ZnSO₄ to a final concentration of 0.1% w/v. Since mc²7000 is a pantothenate auxotroph this strain also requires pantothenic acid at 10μg/mL of final concentration.

Note: This is a standard composition of Sauton'....

Discussion

Tuberculosis (TB), caused by the infection of Mycobacterium tuberculosis, remains a major threat to the global public health. Nearly one third of the world's population is estimated to be asymptomatically infected by the pathogen, about 9 million new cases show up in clinic every year with symptoms of active TB and about 1.7 million die of the infection every year ¹¹. The huge burden of the disease is primarily contributed by lack of a vaccine and a highly complicated chemotherapy that involves a mult.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Incubator	VWR international	Model # 1923/25
Polystyrene culture bottles	Fisher Scientific	03-374-300
12-well tissue culture plate	VWR international	62406-165
50-mL conical tubes	VWR international	89039-660
Rocker	Thermo Fisher Scientific, Inc.	57019-662
Chromatographic refrigerator	VWR international	55702-520
petri dish	VWR international	25384-342
KH2PO4 (monobasic)	EMD Millipore	PX1565-1
MgSO4	Fisher Scientific	M65-500
L-asparagine	Sigma-Aldrich	A4284-100G
citric acid	Sigma-Aldrich	C1857-100G
ferric ammonium citrate	Sigma-Aldrich	F5879-100G
glycerol	EMD Millipore	GX0185-5
NaOH	Sigma-Aldrich	S8045-500G
ZnSO4	Sigma-Aldrich	Z4750-500G
D-pantothenic acid	Sigma-Aldrich	P2250-25G
Difco Middlebrook 7H9 Broth	BD Biosciences	271310
Middlebrook OADC Enrichment	BBL	212351
Tween-80	Fisher Scientific	T164-500
250mL storage bottle	Corning	430281
12 well plates	Falcon BD	353043
rifampicin	Sigma-Aldrich	R3501-1G
methanol	JT Baker	9070-05
10mlLsyringe	BD Biosciences	301604
1-200μL pipet tips	VWR international	89079-458
parafilm M	VWR international	PM-996
15mL centrifuge tube	Greiner Bio-One	188-285
Difco Mycobacteria 7H11 Agar	BD Biosciences	283810
NaCl	Fisher Scientific	BP358-1
KCl	Sigma-Aldrich	P9333-500G
Na2HPO4 (dibasic)	Sigma-Aldrich	S0876-500G