Name: establishing the minimal bactericidal concentration of an antimicrobial agent for planktonic cells (mbc-p) and biofilm cells (mbc-b)
Uploaded: 2014-01-02T20:00:00
Description: Watch this Scientific Journal Video about Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B at JoVE.com

作者要感谢李章、李贤志、阿龙·欣兹和克莱顿·霍尔对这份手稿的编辑帮助。这种检测最初是在达特茅斯盖塞尔医学院乔治·奥图尔的实验室中开发的。Mah博士实验室的研究得到了加拿大自然科学和工程研究理事会和加拿大囊性纤维化研究理事会的资助。

1. MBC-B
<ol><li>
种植生物膜（改编自奥图尔9）。
	<ol>
<li>在 37 °C 的丰富介质中种植野生型兴趣菌株和突变菌株，持续 16 小时。</li>
		<li>将饱和的隔夜培养物 1：100 稀释到新的耐药性检测介质中。 P. aeruginosa 的标准介质是 M63 最小介质，辅以硫酸镁和精氨酸（见 表 1）。这种介质刺激形成更坚固的生物膜。</li>
		<li>在 96 井微蒂特菜中加入每口井...

<ol>
	<li>Hoiby, N., Bjarnsholt, T., Givskov, M., Molin, S., Ciofu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+of+bacterial+biofilms.">Antibiotic resistance of bacterial biofilms.</a> Int. J. Antimicrob. Agents. 35 (4), 322-332 (2010).</li><li>Mah, T. F., 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=Mechanisms+of+biofilm+resistance+to+antimicrobial+agents.">Mechanisms of biofilm resistance to antimicrobial agents.</a> Trends Microbiol. 9 (1), 34-39 (2001).</li><li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-specific+antibiotic+resistance.">Biofilm-specific antibiotic resistance.</a> Future Microbiol. 7 (9), 1061-1072 (2012).</li><li>Mah, T. F., Pitts, B., Pellock, B., Walker, G. C., Stewart, P. S., 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=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426 (6964), 306-310 (2003).</li><li>Zhang, L., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Involvement+of+a+Novel+Efflux+System+in+Biofilm-Specific+Resistance+to+Antibiotics.">The Involvement of a Novel Efflux System in Biofilm-Specific Resistance to Antibiotics.</a> J. Bacteriol. 190 (13), 4447-4452 (2008).</li><li>Zhang, L., Hinz, A. J., Nadeau, J. P., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+tssC1+Links+Type+VI+Secretion+and+Biofilm-specific+Antibiotic+Resistance.">Pseudomonas aeruginosa tssC1 Links Type VI Secretion and Biofilm-specific Antibiotic Resistance.</a> J. Bacteriol. 193 (19), 5510-5513 (2011).</li><li>Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Biofilm-Specific+Antibiotic+Resistance+Gene,+ndvB,+is+Important+for+Expression+of+Ethanol+Oxidation+Genes+in+Pseudomonas+aeruginosa+Biofilms.">The Biofilm-Specific Antibiotic Resistance Gene, ndvB, is Important for Expression of Ethanol Oxidation Genes in Pseudomonas aeruginosa Biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</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>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug+tolerance+of+biofilms+and+persister+cells.">Multidrug tolerance of biofilms and persister cells.</a> Curr. Top. Microbiol. Immunol. 322, 107-131 (2008).</li><li>Merritt, J. H., Kadouri, D. 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=Growing+and+Analyzing+Static+Biofilms.">Growing and Analyzing Static Biofilms.</a> Curr. Protoc. Microbiol. 1, 1B.1.1-1B.1.17 (2005).</li><li>Ramey, B. E., Parsek, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1.+Growing+and+analyzing+biofilms+in+fermenters.">Chapter 1. Growing and analyzing biofilms in fermenters.</a> Curr. Protoc. Microbiol. 1, 1B.3.1-1B.3.14 (2005).</li></ol>

浮石细胞中的抗生素耐药性被定义为由于细胞的永久变化（如 突变）而增加抗生素的最低抑制浓度 （MIC）。迄今已查明的生物膜特异性抗性或耐受性机制是生物膜中野生类型基因表达的结果。因此，抗药性的经典定义不适用于生物膜。然而，另一组定义已经提出：耐药性机制阻止抗生素进入其目标，而耐受性机制关闭抗生素的目标10。生物膜特异性抗生素耐药性机制包括这两个定?...

抗生素耐药性检测最初是为了检测浮游生物（自由游泳）细菌培养物的耐药性而开发的。由于许多细菌感染涉及生物膜（表面连接细胞），我们有兴趣开发一种检测生物膜特异性抗生素耐药性的方法。然而，大多数抗生素耐药性检测不适合测量生物膜的耐药性。例如，确定最低抑制浓度（MIC）是确定浮石细菌培养物 1抗生素耐药性的黄金标准。这种检测需要将稀释的浮石培养物与稀释系列抗生素混合。 抑制浮石细胞可见生长的抗生素浓度是MIC。由于这种检测依赖于对生长的抑制，根据定义，它不能与生物膜培养物配合使用，这需要检查预生生物膜中细胞的抗生素敏感性。此处描述的 MBC-B 检测结果不是测量生长抑制，而是确定杀死生物膜中已有细胞的抗生素浓度。因此，这种检测旨在模仿已建立的生物膜感染的抗生素治疗，并提供一个更相关的视野 中的细菌抗生素耐药性。
由于生物膜通常比浮石培养物2-4更具抗生素耐药性，因此有必要设计出一种将生物膜的抗生素耐药性与浮石培养物直接相关的方法。因此，这种方法的另一个目标是能够直接比较浮石细胞和生物膜细胞之间的抗生素耐药性水平。此处描述的 MBC-P 和 MBC-B 分析使这一点成为可能，因为细胞是在类似条件下培养的。我们利用这种方法研究了几个对生物膜特异性抗生素耐药性很重要的基因。

<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td style="width:160px;">
			1x&#160;M63
			</td>
			<td style="width:160px;">
			
			</td>
			<td style="width:160px;">
			
			</td>
			<td style="width:160px;">
			Prepare as a 5x&#160;M63 stock by dissolving 15g KH2PO4, 35g K2HPO4 and 10g (NH4)2SO4 in 1 L of water. This stock does not need to be autoclaved and can be stored at room temperature.&#160; Dilute 5x&#160;stock 1:5, autoclave, cool, then add the desired components.
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			KH2PO4
			</td>
			<td style="width:160px;">
			Fisher
			</td>
			<td style="width:160px;">
			P285-500
			</td>
			<td style="width:160px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			K2HPO4
			</td>
			<td style="width:160px;">
			Fisher
			</td>
			<td style="width:160px;">
			P288-500
			</td>
			<td style="width:160px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			(NH4)2SO4
			</td>
			<td style="width:160px;">
			Sigma
			</td>
			<td style="width:160px;">
			A5132
			</td>
			<td style="width:160px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Magnesium sulfate
			</td>
			<td style="width:160px;">
			Fisher
			</td>
			<td style="width:160px;">
			M63-500
			</td>
			<td style="width:160px;">
			Add to 1 mM final concentration.&#160; Prepare as a 1 M stock in water and autoclave.
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Tobramycin
			</td>
			<td style="width:160px;">
			Sigma
			</td>
			<td style="width:160px;">
			
			</td>
			<td style="width:160px;">
			Prepare 50 mg/m&#160;stock. Aliquot and store at -20&#176;C.
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Arginine
			</td>
			<td style="width:160px;">
			Sigma
			</td>
			<td style="width:160px;">
			A5131
			</td>
			<td style="width:160px;">
			Add to 0.4% final concentration.&#160; Prepare as a 20% stock in water and filter sterilize.&#160; This alternative carbon/energy source can replace glucose and casamino acids
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			96-well microtiter plates
			</td>
			<td style="width:160px;">
			Corning
			</td>
			<td style="width:160px;">
			3595
			</td>
			<td style="width:160px;">
			Sterile, flat-bottom, low evaporation
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Tranferpette (multichannel pipette)
			</td>
			<td style="width:160px;">
			BrandTech
			</td>
			<td style="width:160px;">
			2703610
			</td>
			<td style="width:160px;">
			8-channel, 20-200 &#956;l
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Multiprong device
			</td>
			<td style="width:160px;">
			Dan-Kar
			</td>
			<td style="width:160px;">
			MC48
			</td>
			<td style="width:160px;">
			48 prongs fit into &#189; of a 96-well microtiter plate
			</td>
		</tr>
	</tbody>
</table>

establishing the minimal bactericidal concentration of an antimicrobial agent for planktonic cells (mbc-p) and biofilm cells (mbc-b)

该协议允许浮石和生物膜耐药性之间的直接比较的细菌菌株，可以形成一个生物膜 体外。细菌被接种到96井微质板的井中。在浮游生物检测中，选择的抗菌剂的连续稀释被添加到细菌悬浮剂中。在生物膜检测中，一旦接种，细菌就会在一定的时间内形成生物膜。未连接的细胞从井中去除，媒体补充，并添加选择的抗菌剂的连续稀释。接触抗菌剂后，浮石细胞被检测为生长。对于生物膜检测，媒体刷新了缺乏抗菌剂的新鲜介质，生物膜细胞有留给恢复。生物膜细胞的生存能力在恢复期后被检测。抗菌剂的MBC-P被定义为杀死浮石培养细胞的药物浓度最低。 相比之下，菌株的 MBC-B 通过将预制生物膜暴露在 24 小时内抗菌剂浓度增加的程度来确定。MBC-B 被定义为杀死生物膜中细胞的抗菌剂的最低浓度。

进行了 MBC-P 和 MBC-B 检测，比较了 PA14 野生类型的灵敏度与 PA14 ∆ndvB。托布拉霉素被用作抗生素。结果与步骤 1.4.4（图 1）和步骤 2.3.4（图 2）相对应。PA14 和∆ndvB 接种到 MBC-P 和 MBC-B 三方检测中。在完成 MBC-B 协议的第 1.0-1.4 步和 MBC-P 协议的步骤 2.0-2.3 后，可行单元被镀在 LB agar 板上。细胞左侧显示托布拉霉素（μ?...

Watch this Scientific Journal Video about Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B at JoVE.com

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

建立浮星细胞（MBC-P）和生物膜细胞（MBC-B）抗菌剂的最小杀菌浓度

该协议允许使用 96 井微蒂板直接比较浮石和生物膜耐药性，以识别细菌菌株 ，从而在体外 形成生物膜。浮游生物或生物膜细菌暴露在首选抗菌剂的连续稀释中。活力被阿加板块的生长所测定。

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria ...

Research

JoVE Journal

Immunology and Infection

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

铜绿假单胞菌和酿酒酵母流细胞生物膜

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

科研

免疫与感染

In vitro Biofilm Formation in an 8-well Chamber Slide

In vitro Biofilm Formation in an 8-well Chamber Slide

在体外生物膜的形成以及8商会幻灯片

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

生物膜启动对病毒感染的细胞测定细菌和真菌

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â±

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

A High-throughput Shigella-specific Bactericidal Assay

A High-throughput Shigella-specific Bactericidal Assay

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

多能干细胞人类巨噬细胞的产生与表征

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...