<meta charset="utf8"></head><body>Développement et évaluation des biofilms pelliculaires chez les mycobactéries

<ol>
	<li>Davey, M. E., O'toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biofilms:+From+ecology+to+molecular+genetics.">Microbial biofilms: From ecology to molecular genetics.</a> Microbiol Mol Biol Rev. 64 (4), 847-867 (2000).</li><li>Rumbaugh, K. P., Sauer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+dispersion.">Biofilm dispersion.</a> Nat Rev Microbiol. 18 (10), 571-586 (2020).</li><li>Davies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+biofilm+resistance+to+antibacterial+agents.">Understanding biofilm resistance to antibacterial agents.</a> Nat Rev Drug Discov. 2 (2), 114-122 (2003).</li><li>Hern&#225;ndez-Jim&#233;nez, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+vs.+planktonic+bacterial+mode+of+growth:+Which+do+human+macrophages+prefer.">Biofilm vs. planktonic bacterial mode of growth: Which do human macrophages prefer.</a> Biochem Biophys Res Commun. 441 (4), 947-952 (2013).</li><li>Nadell, C. D., Drescher, K., Foster, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+structure,+cooperation+and+competition+in+biofilms.">Spatial structure, cooperation and competition in biofilms.</a> Nat Rev Microbiol. 14 (9), 589-600 (2016).</li><li>Jo, J., Price-Whelan, A., Dietrich, L. 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=Gradients+and+consequences+of+heterogeneity+in+biofilms.">Gradients and consequences of heterogeneity in biofilms.</a> Nat Rev Microbiol. 20 (10), 593-607 (2022).</li><li>Keating, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+modifies+cell+wall+carbohydrates+during+biofilm+growth+with+a+concomitant+reduction+in+complement+activation.">Mycobacterium tuberculosis modifies cell wall carbohydrates during biofilm growth with a concomitant reduction in complement activation.</a> Cell Surf. 7, 100065 (2021).</li><li>Ojha, A. K., et al. <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 (1), 164-174 (2008).</li><li>Dietrich, L. E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+community+morphogenesis+is+intimately+linked+to+the+intracellular+redox+state.">Bacterial community morphogenesis is intimately linked to the intracellular redox state.</a> J Bacteriol. 195 (7), 1371-1380 (2013).</li><li>Kulka, K., Hatfull, G., 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.">Growth of Mycobacterium tuberculosis biofilms.</a> J Vis Exp. (60), e3820 (2012).</li><li>Wiegand, I., Hilpert, K., Hancock, R. E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agar+and+broth+dilution+methods+to+determine+the+minimal+inhibitory+concentration+(MIC)+of+antimicrobial+substances.">Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances.</a> Nat Protoc. 3 (2), 163-175 (2008).</li><li>Anand, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyketide+quinones+are+alternate+intermediate+electron+carriers+during+mycobacterial+respiration+in+oxygen-deficient+niches.">Polyketide quinones are alternate intermediate electron carriers during mycobacterial respiration in oxygen-deficient niches.</a> Mol Cell. 60 (4), 637-650 (2015).</li><li>. Growth Curves: Generating Growth Curves Using Colony Forming Units and Optical Density Measurements Available from: <a target="_blank" href="https://www.jove.com/v/10511/growth-curves-cfu-and-optical-density-measurements">https://www.jove.com/v/10511/growth-curves-cfu-and-optical-density-measurements</a> (2019)</li><li>H&#248;iby, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+personal+history+of+research+on+microbial+biofilms+and+biofilm+infections.">A personal history of research on microbial biofilms and biofilm infections.</a> Pathog Dis. 70 (3), 205-211 (2014).</li><li>Rajewska, M., Maci&#261;g, T., Jafra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+source+and+surface+type+influence+the+early-stage+biofilm+formation+by+rhizosphere+bacterium+Pseudomonas+donghuensis+P482.">Carbon source and surface type influence the early-stage biofilm formation by rhizosphere bacterium Pseudomonas donghuensis P482.</a> bioRxiv. , (2023).</li><li>O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtiter+dish+biofilm+formation+assay.">Microtiter dish biofilm formation assay.</a> J Vis exp. (47), e2437 (2011).</li><li>Chakraborty, P., Bajeli, S., Kaushal, D., Radotra, B. D., Kumar, A. <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+in+the+lung+contributes+to+virulence+and+drug+tolerance+of+Mycobacterium+tuberculosis.">Biofilm formation in the lung contributes to virulence and drug tolerance of Mycobacterium tuberculosis.</a> Nat Comm. 12 (1), 1606 (2021).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#181;M PVDF syringe filter</td>
			<td>Axiva</td>
			<td>SFNY04 R</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL tips</td>
			<td>Genetix</td>
			<td>GXM-611000 C</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L tips</td>
			<td>Genetix</td>
			<td>GXM-6110 C</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L tips</td>
			<td>Genetix</td>
			<td>GXM-61200C</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well polypropylene plates</td>
			<td>Tarsons</td>
			<td>980010</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber tubes</td>
			<td>Tarsons</td>
			<td>546051</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Hospharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet A II</td>
			<td>MSET</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting paper</td>
			<td>Any suitable vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma</td>
			<td>251275</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvettes</td>
			<td>Bio-Rad</td>
			<td>2239955</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferric ammonium citrate</td>
			<td>Sigma</td>
			<td>F5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel documentation system</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Beads</td>
			<td>Sigma</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>49139</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Inoculation loops</td>
			<td>Genaxy</td>
			<td>HS81121C</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspargine</td>
			<td>Sigma</td>
			<td>A0884</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-agar</td>
			<td>Himedia</td>
			<td>M1151</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-media</td>
			<td>Himedia</td>
			<td>M575</td>
			<td></td>
		</tr>
		<tr>
			<td>M. smegmatis mc2155 cryo-stock</td>
			<td>ATCC</td>
			<td>700084</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Tarsons</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Tarsons</td>
			<td>460020</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Labman Scientific Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Reader</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene test tubes</td>
			<td>Genaxy</td>
			<td>GEN-14100-PS</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P5379</td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>MedchemExpress</td>
			<td>HY-B0272</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>SPL Life Sciences</td>
			<td>95210</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker Incubator</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Static Incubator</td>
			<td>CARON</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 10 mL syringe</td>
			<td>Becton Dickinson</td>
			<td>309642</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 50 mL syringe</td>
			<td>Becton Dickinson</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Sigma</td>
			<td>P1754</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing balance</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc sulfate</td>
			<td>Sigma</td>
			<td>Z0251</td>
			<td></td>
		</tr>
	</tbody>
</table>

growing mycobacterial biofilm as a model to study antimicrobial resistance

Bacteria are able to survive as single-celled entities; however, in most physiologically relevant conditions, they organize into community mimetics. Biofilm is a widely recognized community organization of bacteria formed by aggregated cells encased in a self-produced matrix1. Such assembly possesses signatures of early multicellularity and provides higher stress resilience to bacterial systems. Biofilms are often tolerant to antimicrobials and are estimated to be responsible for almost 80% of microbial infections2,3.
Shake flask and plate-based cultures have traditionally been the usual practices for bacterial culturing. Their enormous acceptability and success can be attributed to their ease of handling, reproducibility, and scalability. However, the lack of physiological context limits the translational potential of the knowledge generated using such systems4. Therefore, biofilms are becoming an attractive model system to study bacterial pathophysiology. Biofilms provide a dynamic model system, closely mirroring natural conditions, allowing researchers to replicate physiological aspects such as nutrient gradients and spatial heterogeneity5,6.
The biofilm lifestyle is particularly pertinent in mycobacterial studies, as mycobacteria, including the notorious Mycobacterium tuberculosis, are adept biofilm formers7. Their ability to thrive within biofilms contributes to their persistence in host tissues during infections. It poses a formidable challenge in treating mycobacterial diseases, given the inherent antibiotic resistance associated with biofilm lifestyles8. Biofilms also provide an ideal model system to study mycobacterial metabolism, as they allow for the investigation of the unique metabolic adaptations and nutrient utilization strategies employed by mycobacteria within complex microbial communities9.
While biofilm is increasingly being accepted as a better model system for mycobacterial studies10, there is a need for consistent and reproducible standard operating procedures, especially for drawing parallels among studies conducted in different laboratories. The method outlined here describes biofilm formation procedures for a mycobacterial species, M. smegmatis. M. smegmatis is a more accessible model for studying mycobacterial biofilms, given its non-pathogenicity and faster biofilm formation kinetics. The method can be modified to suit applications like antimycobacterial screening, metabolite extraction, and omics studies.

<meta charset="utf8"></head><body>Pleins feux sur l’auteur : Faire progresser les protocoles de biofilm mycobactérien pour une recherche améliorée sur le métabolisme bactérien

Cultiver un biofilm mycobactérien comme modèle pour étudier la résistance aux antimicrobiens

Many bacteria thrive in intricate natural communities, exhibiting key attributes of multicellularity such as communication, cooperation, and competition. ...

growing-mycobacterial-biofilm-as-model-to-study-antimicrobial

Research

JoVE Journal

Biology

Growing Mycobacterial Biofilm as a Model to Study Antimicrobial Resistance

418 vues.  Tata Institute of Fundamental Research. Nos recherches portent sur le métabolisme bactérien, en particulier le métabolisme énergétique, qui est crucial pour la croissance et la survie. Malgré de nombreuses données biochimiques et structurales, la compréhension au niveau des systèmes et les mécanismes d’adaptation restent incertains. Notre objectif est d’approfondir ces connaissances et de lutter contre la résistance aux antimicrobiens.Notre protocole renforce la pertinence phy...

Vidéo: Pleins feux sur l’auteur : Faire progresser les protocoles de biofilm mycobactérien pour une recherche améliorée sur le métabolisme bactérien

Many bacteria thrive in intricate natural communities, exhibiting key attributes of multicellularity such as communication, cooperation, and competition. The most prevalent manifestation of bacterial multicellular behavior is the formation of biofilms, often linked to pathogenicity. Biofilms offer a haven against antimicrobial agents, fostering the emergence of antimicrobial resistance. The conventional practice of cultivating bacteria in shake flask liquid cultures fails to represent their proper physiological growth in nature, consequently limiting our comprehension of their intricate dynamics. Notably, the metabolic and transcriptional profiles of bacteria residing in biofilms closely resemble those of naturally growing cells. This parallelism underscores the significance of biofilms as an ideal model for foundational and translational research. This article focuses on utilizing Mycobacterium smegmatis as a model organism to illustrate a technique for cultivating pellicle biofilms. The approach is adaptable to various culture volumes, facilitating its implementation for diverse experimental objectives such as antimicrobial studies. Moreover, the method&#39;s design enables the qualitative or quantitative evaluation of the biofilm-forming capabilities of different mycobacterial species with minor adjustments.

This protocol describes a robust method for developing pellicle biofilm. The method is scalable to different culture volumes, allowing easy adoption for various experimental objectives. The method's design enables qualitative or quantitative assessment of the biofilm-forming potential of several mycobacterial species.