<meta charset="utf8"></head><body>Mycobacteria의 Pellicle Biofilms 개발 및 평가

<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>저자 스포트라이트: 향상된 박테리아 대사 연구를 위한 Mycobacterial Biofilm 프로토콜 발전

항생제 내성을 연구하기 위한 모델로 Mycobacterial Biofilm 성장

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.

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

373 조회수.  Tata Institute of Fundamental Research. 우리의 연구는 박테리아 대사, 특히 성장과 생존에 중요한 에너지 대사에 중점을 둡니다. 광범위한 생화학 및 구조적 데이터에도 불구하고 시스템 수준의 이해와 적응 메커니즘은 여전히 불분명합니다. 우리는 이러한 지식을 심화하고 항생제 내성을 해결하는 것을 목표로 합니다.당사의 프로토콜은 플랑크톤 방법보다 자연적인 박테리아 성장?...