The work was carried out with financial support from the National Institute of Health and American Lung Association.

1. Growing biofilms of M. tuberculosis in a 250mL screw capped bottle
<ol>
 <li>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 al...

<ol>
	<li>Saltini, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+and+diagnosis+of+tuberculosis.">Chemotherapy and diagnosis of tuberculosis.</a> Respir. Med. 100, 2085-2097 (2006).</li><li>Hall-Stoodley, L., Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+and+dispersal+and+the+transmission+of+human+pathogens.">Biofilm formation and dispersal and the transmission of human pathogens.</a> Trends Microbiol. 13, 7-10 (2005).</li><li>Costerton, J. W., Stewart, P. S., Greenberg, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+a+common+cause+of+persistent+infections.">Bacterial biofilms: a common cause of persistent infections.</a> Science. 284, 1318-1322 (1999).</li><li>Blankenship, J. R., Mitchell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+build+a+biofilm:+a+fungal+perspective.">How to build a biofilm: a fungal perspective.</a> Curr Opin Microbiol. 9, 588-594 (2006).</li><li>Henke, J. M., Bassler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+social+engagements.">Bacterial social engagements.</a> Trends Cell Biol. 14, 648-656 (2004).</li><li>Kolter, R., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+for+all+and+all+for+one.">One for all and all for one.</a> Science. 280, 226-227 (1998).</li><li>Branda, S. S., Vik, S., Friedman, L., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+the+matrix+revisited.">Biofilms: the matrix revisited.</a> Trends Microbiol. 13, 20-26 (2005).</li><li>Ojha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GroEL1:+a+dedicated+chaperone+involved+in+mycolic+acid+biosynthesis+during+biofilm+formation+in+mycobacteria.">GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria.</a> Cell. 123, 861-873 (2005).</li><li>Ojha, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Mycobacterium+tuberculosis+biofilms+containing+free+mycolic+acids+and+harbouring+drug-tolerant+bacteria.">Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria.</a> Mol. Microbiol. 69, 164-174 (2008).</li><li>Ojha, A. K., Trivelli, X., Guerardel, Y., Kremer, L., Hatfull, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+hydrolysis+of+trehalose+dimycolate+releases+free+mycolic+acids+during+mycobacterial+growth+in+biofilms.">Enzymatic hydrolysis of trehalose dimycolate releases free mycolic acids during mycobacterial growth in biofilms.</a> J. Biol. Chem. 285, 17380-17389 (2010).</li><li>Dye, C., Lonnroth, K., Jaramillo, E., Williams, B. G., Raviglione, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+tuberculosis+incidence+and+their+determinants+in+134+countries.">Trends in tuberculosis incidence and their determinants in 134 countries.</a> Bull World Health Organ. 87, 683-691 (2009).</li><li>Jindani, A., Dore, C. J., Mitchison, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bactericidal+and+sterilizing+activities+of+antituberculosis+drugs+during+the+first+14+days.">Bactericidal and sterilizing activities of antituberculosis drugs during the first 14 days.</a> Am. J. Respir. Crit. Care Med. 167, 1348-1354 (2003).</li><li>Carter, G., Wu, M., Drummond, D. C., Bermudez, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+biofilm+formation+by+clinical+isolates+of+Mycobacterium+avium.">Characterization of biofilm formation by clinical isolates of Mycobacterium avium.</a> J. Med. Microbiol. 52, 747-752 (2003).</li><li>Hall-Stoodley, L., Lappin-Scott, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+by+the+rapidly+growing+mycobacterial+species+Mycobacterium+fortuitum.">Biofilm formation by the rapidly growing mycobacterial species Mycobacterium fortuitum.</a> FEMS Microbiol. Lett. 168, 77-84 (1998).</li><li>Alibaud, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-dependent+regulation+of+mycolic+acid+cyclopropanation+in+saprophytic+mycobacteria:+role+of+the+Mycobacterium+smegmatis+1351+gene+(MSMEG_1351)+in+CIS-cyclopropanation+of+alpha-mycolates.">Temperature-dependent regulation of mycolic acid cyclopropanation in saprophytic mycobacteria: role of the Mycobacterium smegmatis 1351 gene (MSMEG_1351) in CIS-cyclopropanation of alpha-mycolates.</a> J. Biol. Chem. 285, 21698-21707 (2010).</li></ol>

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...

<table border="1">
	<tbody>
		<tr>
			<td>Equipment and supplies</td>
			<td>SUPPLIER</td>
			<td>CATALOG NUMBER</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>Model # 1923/25</td>
		</tr>
		<tr>
			<td>Polystyrene culture bottles</td>
			<td>Fisher Scientific</td>
			<td>03-374-300</td>
		</tr>
		<tr>
			<td>12-well tissue culture plate</td>
			<td>VWR</td>
			<td>62406-165</td>
		</tr>
		<tr>
			<td>50-mL conical tubes</td>
			<td>VWR</td>
			<td>89039-660</td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>Thermo Scientific</td>
			<td>57019-662</td>
		</tr>
		<tr>
			<td>Chromatographic refrigerator</td>
			<td>VWR</td>
			<td>55702-520</td>
		</tr>
		<tr>
			<td>petri dish</td>
			<td>VWR</td>
			<td>25384-342</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>REAGENT</td>
			<td>SUPPLIER</td>
			<td>CATALOG NUMBER</td>
		</tr>
		<tr>
			<td>KH2PO4 (monobasic)</td>
			<td>EMD</td>
			<td>PX1565-1</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher</td>
			<td>M65-500</td>
		</tr>
		<tr>
			<td>L-asparagine</td>
			<td>Sigma</td>
			<td>A4284-100G</td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>Sigma</td>
			<td>C1857-100G</td>
		</tr>
		<tr>
			<td>ferric ammonium citrate</td>
			<td>Sigma</td>
			<td>F5879-100G</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>EMD</td>
			<td>GX0185-5</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045-500G</td>
		</tr>
		<tr>
			<td>ZnSO4</td>
			<td>Sigma</td>
			<td>Z4750-500G</td>
		</tr>
		<tr>
			<td>D-pantothenic acid</td>
			<td>Sigma</td>
			<td>P2250-25G</td>
		</tr>
		<tr>
			<td>Difco Middlebrook 7H9 Broth</td>
			<td>Becton Dickinson</td>
			<td>271310</td>
		</tr>
		<tr>
			<td>Middlebrook OADC Enrichment</td>
			<td>BBL</td>
			<td>212351</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Fisher</td>
			<td>T164-500</td>
		</tr>
		<tr>
			<td>250mL storage bottle</td>
			<td>Corning</td>
			<td>430281</td>
		</tr>
		<tr>
			<td>12 well plates</td>
			<td>Falcon (BD)</td>
			<td>353043</td>
		</tr>
		<tr>
			<td>rifampicin</td>
			<td>Sigma</td>
			<td>R3501-1G</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>J.T. Baker</td>
			<td>9070-05</td>
		</tr>
		<tr>
			<td>10mlLsyringe</td>
			<td>Becton Dickinson</td>
			<td>301604</td>
		</tr>
		<tr>
			<td>1-200&mu;L pipet tips</td>
			<td>VWR</td>
			<td>89079-458</td>
		</tr>
		<tr>
			<td>parafilm M</td>
			<td>VWR</td>
			<td>PM-996</td>
		</tr>
		<tr>
			<td>15mL centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>188-285</td>
		</tr>
		<tr>
			<td>Difco Mycobacteria 7H11 Agar</td>
			<td>Becton Dickinson</td>
			<td>283810</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-1</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333-500G</td>
		</tr>
		<tr>
			<td>Na2HPO4 (dibasic)</td>
			<td>Sigma</td>
			<td>S0876-500G</td>
		</tr>
	</tbody>
</table>

growth of mycobacterium tuberculosis biofilms

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.

Watch this Scientific Journal Video about Growth of Mycobacterium tuberculosis Biofilms at JoVE.com

Growth of Mycobacterium tuberculosis Biofilms

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.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

growth-of-mycobacterium-tuberculosis-biofilms

Research

JoVE Journal

Immunology and Infection

23.5K visualizações.  University of Pittsburgh. Mycobacterium tuberculosis forma biofilmes de drogas tolerantes quando cultivadas em certas condições. Aqui descrevemos métodos para cultura de M. biofilmes de tuberculose e que determinam a freqüência de persistentes drogas tolerantes. Estes protocolos serão úteis para estudos posteriores sobre os mecanismos de tolerância à droga em M. tuberculose.