This work was supported by the DBT-Ramalingaswami Fellowship awarded to Amitesh Anand.

The details of all the reagents and equipment used for the study are listed in the Table of Materials.
1. Sauton&#39;s media preparation
<ol>
	<li>Prepare 50 mL of 2.5% potassium dihydrogen phosphate solution by carefully weighing 1.25 g of potassium dihydrogen phosphate and dissolving it in 50 mL of deionized water.</li>
	<li>Prepare 50 mL of 2.5% magnesium sulfate solution by weighing 2.56 g of magnesium sulfate and dissolving it in 50 mL of deionized wate...

Developing and Assessing Pellicle Biofilms in Mycobacteria

<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>

The multicellular lifestyle of microbes was described almost a century ago; however, clinical studies remain sparse, mostly due to the lack of robust methods14. Methods described in works on biofilm biology are often difficult to adapt. Here, the detailed methodology, aided by demonstrations of critical steps, is expected to improve the reproducibility of the protocols.
The method of biofilm production described in this article is scalable, requiring a proportional incr...

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.

<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

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.

Author Spotlight: Advancing Mycobacterial Biofilm Protocols for Enhanced Bacterial Metabolism Research

Growing Mycobacterial Biofilm as a Model to Study Antimicrobial Resistance

Our research focuses on bacterial metabolism, especially energy metabolism, which is crucial for growth and survival. Despite extensive biochemical and structural data, systems level understanding and adaptive mechanisms remain unclear. We aim to deepen this knowledge and to address antimicrobial resistance.Our protocol enhances the physiological relevance of lab experiments by using biofilms, which mimics the natural bacterial growth better than the planktonic methods. Adapting microbacterial biofilms will offer a great system to understand the gene's lifestyle, and it'll also offer a screening system for antimicrobials. We are not the first ones to produce mycobacterial biofilms.There are several groups using this as a model. Our motivation behind producing this protocol was to simplify the procedures as much as possible so many labs can adapt this system as a model.

Biofilm pellicles become visible to the naked eye from the third day onwards. Although biofilm grows on Sauton&#39;s media without 2% glucose, an improvement was observed in the reticulation when it was added. We obtained 10.48 mg &#177; 3.13 mg (n = 4) of biofilm dry weight from each well of a 24-well plate with 1.5 mL of Sauton&#39;s media (supplemented with 2% glucose) grown for four days. In Figure 2, biofilm development was visible from day 3 to day 6. It starts forming a film with slig...

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.

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

Culture of Mycobacterium smegmatis for Biofilm Production

To begin, with a serological pipette dispense two milliliters of autoclave lysogeny broth media into two sterile 14-milliliter test tubes. inside a laminar hood. Add 20 microliters of filter sterilized 5%Tween 80 to the test tubes with a micro pipette.Next, add the cryostock of Mycobacterium smegmatis into a tube with an inoculation loop. Incubate the tubes for 48 hours in a shaker incubator at 300 rotations per minute and 37 degrees Celsius. To prepare the secondary cultures, dispense two milliliters of autoclave lysogeny broth media into two sterile 14-milliliter test tubes.Then use a micro pipette to add 20 microliters of filter sterilized 5%Tween 80 to the test tubes. Next, with a micro pipette, transfer 20 microliters of the primary culture to one of the tubes. Incubate the cultures in a shaking incubator at 300 rotations per minute and 37 degrees Celsius until they reach an OD600 of 0.6 to 0.8.

Setting Up and Estimating the Pellicle Biofilm Development in Mycobacterium smegmatis Cultures

To begin, in a laminar hood, dispense six milliliters of Soutan's media supplemented with 2%glucose into the wells of a six-well plate. Inoculate the wells with 120 microliters of the secondary cultures of mycobacterium smegmatis. Keep one un-inoculated well as a media control.Next, pipette the culture up and down with a 1-milliliter micropipette for even mixing. Cover the lid and carefully seal the plate with paraffin film. Incubate the plate in a static incubator at 37 degrees Celsius for seven days.For a visual estimation of biofilm growth, place the plates in a gel documentation system under epi white light illumination. To measure dry weight, weigh a sheet of blotting paper. Extract the biofilm from the six-well plate and transfer it onto the paper with a spatula.Carefully collect most of the biofilm from the culture. Place the biofilm on the blotting paper. Place the blotting paper in an incubator until the biofilm dries completely.Then measure the combined weight of the paper and biofilm. Biofilm pellicles were visible from Day 3 onwards. Reticulation of the cultures improved with the addition of 2%glucose to Soutan's media.Biofilm development was visible between days 3 to 6. A film with slight reticulation was observed on Day 3. This film matured by Day 5 and disintegrated from Day 6.

313 Views.  Tata Institute of Fundamental Research.  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.

Developing and Assessing Pellicle Biofilms in Mycobacteria

Summary