JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Results
Discussion
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Acknowledgements
Materials
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方法学研究<em>乙。枯草</em>生物膜作为模型的表征小分子抑制剂生物膜

Published: October 09, 2016
doi:
10.3791/54612
, ,
1Department of Molecular Genetics,Weizmann Institute of Science, 2Electron Microscopy Unit,Weizmann Institute of Science

Summary

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Abstract

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Introduction

多细胞细菌群落的自然和人为环境中发挥显著作用，可以是有益或有害的高度。这些多细胞集落被称为生物膜，其中所述个体的细胞包埋在自产生的细胞外聚合物（EPS）基质中。 EPS的强烈坚持的细胞，他们殖民表面。它们作为对机械和化学力盾，并创建相邻小区之间的紧密联系，促进蜂窝通信1。生物膜可以被看作是一个分化的社会，在那里细胞利用高度管制，编排流程在社区内协调其活动，以及跨品种2-5。从生长的浮游，自由生活模式到生物膜状态的转变通常与发育过程相关联。一个很好的例子是革兰氏阳性土壤细菌枯草芽孢杆菌 ，因此一个undomesticated应变作为一个强大的模式生物研究发展阶段，导致生物膜的形成。在这种细菌，游动细胞组织起来成执行特殊任务4显眼的多细胞结构。细胞的一组中，基质生产者分泌胞外多糖6中 ，淀粉样蛋白塔萨7,8，和表面疏水性蛋白质BSLA 9,10;所有这些参加EPS 11-13的组装。

考虑到自然和人为壁龛丰富生物膜和公认的致命伤害，他们可能会导致，有迫切需要找到办法，以防止它们的形成。小分子抑制剂可以在新的监管途径，酶和参与生物膜形成的结构蛋白的发现帮助，从而促进多社区装配的复杂过程的见解。作为B.枯草芽孢杆菌是一个生物充分研究模型成膜14,15，它可以被用来评估各种生物膜抑制剂的效果。这项研究铲球是评估生物膜的小分子抑制剂的反应键四种基本方法。首先，以确保这些抑制剂具有特定生物膜目标，从上生物膜形成的影响上浮游生长的作用的分离是至关重要的。大多数抗菌药物靶向的浮游生长期细胞，但该目标生物膜的生活方式分子是罕见的。另外，如不影响浮游生长的分子是没有毒性，它们能减少选择压力利于抗生素抗性突变体16。例如，当生物膜与D-氨基酸或某些其它细胞壁干扰分子治疗，他们要么干扰或拆卸，但这些抑制剂仅轻度影响浮游生长12,17。与此相反，许多抗生素大大削弱浮游生长，与L-ittle或生物膜形成17没有影响。

第二，建立一致的和健壮的试验框架研究小分子的作用是至关重要的。我们观察到的小分子抑制剂的活性浓度范围是对预培养条件，并用于研究这些小分子抑制剂的效果的实验装置的敏感。各种报告，特别是那些学习B.枯草芽孢杆菌，揭示了在浓度范围内的变化在该D-氨基酸抑制药膜的形成-浮细菌生物膜12,17-19。此处呈现的结果表明，下列因素考虑在活性浓度范围不同：预培养条件（对数12,17与晚平稳20生长阶段），在该预培养条件中使用的生长培养基（丰富，未定义[卢里亚肉汤，LB]对定义[味精- 甘油，MSgg]），接种率，特别是在接种前除去预培养基的。静态防护膜生长的温度显示出在小分子抑制剂D-亮氨酸，在这项研究中使用的代表性的D-氨基酸的活性范围不太重要的作用。

最后，一旦生物膜与特定的生物膜抑制剂，需要坚固的信息的方法来表征生物膜健身这些抑制剂的效果处理。 （1）的影响生物膜集落及其与抗菌剂抗性内对单个细胞：在这里，两种方法来独立地表征小分子抑制剂的效果进行详细说明。相对于自由生活的细菌21-23时生物膜细胞通常对抗生素更耐。虽然这种现象是多因素的EPS，以减少抗生素的渗透能力通常被认为是一个有吸引力的解释24 </suP>。此方法评估预建立生物膜细胞暴露于抗菌物质后的存活。 （2）向小规模上生物膜集落结构的影响，从大。生物膜菌落可以通过三维结构和所述EPS的存在表征。利用扫描电子显微镜，在细胞形态，生物膜集落结构和EPS的结构和数量的变化可以从大（毫米）被可视化的小规模（微米）。

Protocol

1.评估小分子抑制剂的影响薄皮生物膜集落形成制备定义生物膜诱导MSgg介质25的2倍的溶液未经氯化钙和氯化铁（III）六水合物。除菌过滤后，加入氯化钙。介质是准备直接使用，或者它可以被存储在4℃在黑暗中。 准备在实验当天的1倍MSgg稀释。 稀释2倍MSgg介质用无菌蒸馏水（药膜）或无菌的3％热（80℃）琼脂（生物膜）为1x和添加铁（III）六水合氯化至50微米（药膜?…

Representative Results

该薄膜法是一种方法来研究B的严格监管和动态过程枯草多细胞。除此之外，薄膜法适合于测试的范围内任一起动机前置条件或小分子的浓度在单细胞培养multidish板在一项实验。然而，B.枯草薄膜的形成是在培养前的条件是敏感的（ 例如 ，预培养的生长培养基和其生长阶段），接种比率并在除去预培养基的。因此，我们首先筛选了一个实验装置，使我们能够再现由D-亮氨…

Discussion

枯草杆菌形式健壮和高度结构化的生物膜无论在液体（药膜）和固体培养基上（集落）。因此，它可作为一种理想的模式生物表征特定生物膜抑制剂的作用方式。在固体培养基中，细胞形成具有独特的功能，不在药膜明显，像皱纹从中心到边缘的辐射的多细胞结构。因此，菌膜和菌落互补系统学习B.枯草多细胞。

这项研究的目标是开发一个B.生物膜（药膜…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Electron microscope imaging was conducted at the Electron Microscopy Unit of the Weizmann Institute of Science, supported in part by the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging. This research was also supported by the ISF I-CORE grant 152/1, Mr. and Mrs. Dan Kane, Ms. Lois Rosen, by a Yeda-Sela research grant, by the Larson Charitable Foundation, by Ruth and Herman Albert Scholars Program for New Scientists, by the Ilse Katz Institute for Materials Sciences and Magnetic Resonance Research grant, by the Ministry of Health grant for alternative research methods, and by the France-Israel Cooperation – Maimonide-Israel Research Program. IKG is a recipient of the Rowland and Sylvia Career Development Chair.

Materials

Luria Broth, Lennox Difco 240230
Bacto Agar Difco 214010
potassium phosphate monobasic  Sigma, 136.09 g/mol P0662-500G
potassium phosphate dibasic  Fisher Scientific, 174.18 g/mol BP363-1
3-(N-morpholino)propanesulfonic acid Fisher Scientific, 209.27 g/mol BP308-500
magnesium chloride hexahydrate  Merck, 203.30 g/mol  1.05833.0250
calcium chloride anhydrous J.T. Baker, 110.98 g/mol 1311-01
manganese(II) chloride tetrahydrate Sigma, 197.91 g/mol 31422-250G-R
iron(III) chloride hexahydrate  Sigma, 270.30 g/mo) F2877-500G
zinc chloride anhydrous  Acros Organics, 136.29 g/mol 424592500
thiamine hydrochloride Sigma, 337.27 g/mol T1270-100G
L-tryptophan Fisher Scientific, 204.1 g/mol BP395-100
L-phenylalanine Sigma, 165.19 g/mol P5482-100G
L-threonine Sigma, 119.12 g/mol T8625-100G
glycerol anhydrous Bio-Lab Itd 712022300
L-glutamic acid monosodium salts hydrate  Sigma, 169.11 g/mol G1626-1KG
D-leucine Sigma, 169.11 g/mol 855448-10G
ethanol anhydrous Gadot 830000054
razor blade Eddison NA
circular cellulose filter papers Whatman, 90 mm 1001-090
glutaraldehyde EMS (Electron Micoscopy Science), 25% in water 16220
paraformaldehyde  EMS, 16% in water 15710
sodium cacodylate Merck, 214.05 g/mol  8.2067
calcium chloride 2-hydrate Merck, 147.02 g/mol  1172113
stub-aluminium mount EMS, sloted head 75230
carbon adhesive tape EMS 77825-12
Shaker 37°C New Brunswick Scientific Innowa42 NA
Centrifuge Eppendorf table top centrifuge 5424 NA
Digital Sonifier, Model 250, used with Double Step Microtip Branson NA
Incubator 30 °C Binder NA
Incubator 23 °C Binder NA
Filter System, 500 ml, polystyrene Cornig Incorporated NA
Rotary Shaker – Orbitron Rotatory II Boekel NA
S150 Sputter Coater  Edwards NA
CPD 030 Critical Point Dryer BAL-TEC NA
Environmental Scanning Electron Microscope XL30 ESEM FEG Philips (FEI) NA
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Tags

B. Subtilis BiofilmsSmall Molecule Biofilm InhibitorsBiofilm FormationBacillus SubtilisPseudomonas AeruginosaXanthomonas CitriScanning Electron MicroscopyPellicle FormationBiofilm GrowthMSgg MediumStarter CultureOptical Density12-well Cell Culture PlateAgar Plate
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Bucher, T., Kartvelishvily, E., Kolodkin-Gal, I. Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors. J. Vis. Exp. (116), e54612, doi:10.3791/54612 (2016).
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