Summary

Рост Микобактерий туберкулеза Биопленки

Published: February 15, 2012
doi:

Summary

Микобактерий туберкулеза лекарственных форм терпимо биопленки при культивировании в определенных условиях. Здесь мы опишем методы культивирования M. туберкулез биопленки и определения частоты наркотиков терпимо persisters. Эти протоколы будут полезны для дальнейших исследований в механизмах толерантности препарата в М. туберкулезом.

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. Рост биопленки М. туберкулезом в 250 мл винт крышками бутылки СМИ приготовления: Растворите 0,5 г KH 2 PO 4, 0,5 г MgSO 4, 4 г L-аспарагин, 2 г лимонной кислоты, 0,05 г железа цитрат аммония, 60 мл глицерина в 900 мл воды. Скорректировать значение рН до 7,0 с NaOH. Автоклав, остудить …

Discussion

Туберкулез (ТБ), вызванный в заражении микобактериями туберкулеза, остается серьезной угрозой для общественного здравоохранения. Почти одна треть населения в мире, по оценкам, бессимптомно инфицированных возбудителем, около 9 миллионов новых случаев заболевания появляются в кли?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Работа выполнена при финансовой поддержке Национального института здравоохранения и Американской легочной ассоциации.

Materials

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

References

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Cite This Article
Kulka, K., Hatfull, G., Ojha, A. K. Growth of Mycobacterium tuberculosis Biofilms. J. Vis. Exp. (60), e3820, doi:10.3791/3820 (2012).

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