JoVE Journal > Immunology and Infection
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Abstract
Introduction
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Immunology and Infection

使用 COMSTAT 软件量化抗菌剂对 体外 生物膜结构的影响

Published: December 14, 2020
doi:
10.3791/61759
, , , ,
1Translational Medicine,Hospital for Sick Children, 2Microbiology, Department of Pediatric Laboratory Medicine,Hospital for Sick Children, 3Infectious Diseases, Department of Pediatrics,Hospital for Sick Children

Summary

抗菌诱导的 铜绿假单胞 菌生物膜结构变化在从囊性纤维化和慢性肺部感染患者培养的临床分离株中有所不同。在共聚焦显微镜之后，COMSTAT软件可用于量化单个分离株的生物膜结构（例如，表面积、厚度、生物量）的变化，以评估抗感染剂的功效。

Abstract

生物膜是微生物的聚集体，它们依赖于自产生的细胞外聚合物物质基质来保护和结构完整性。已知医院病原体 铜绿假单胞 菌采用生物膜生长模式，导致囊性纤维化 （CF） 患者发生慢性肺部感染。计算机程序COMSTAT是一种有用的工具，用于通过从三维共聚焦图像中提取数据来量化 铜绿假单胞菌 生物膜结构中抗菌诱导的变化。然而，软件的标准化操作较少得到解决，这对于生物膜行为的最佳报告和跨中心比较非常重要。因此，该协议的目的是提供一个简单且可重复的框架，用于通过COMSTAT量化不同抗菌条件下的 体外 生物膜结构。该技术使用铜 绿假单胞菌 分离物建模，以生物膜重复的形式生长，并暴露于妥布霉素和抗 Psl 单克隆抗体 Psl0096。循序渐进的方法旨在减少用户的歧义，并最大限度地减少忽视关键图像处理步骤的机会。具体而言，该协议强调消除与COMSTAT手动操作相关的主观变化，包括图像分割和选择适当的定量分析功能。尽管这种方法要求用户在运行COMSTAT之前花费额外的时间处理共聚焦图像，但它有助于最大限度地减少自动输出中错位的生物膜异质性。

Introduction

生物膜是微生物的聚集体，定向在自产生的细胞外聚合物物质 （EPS） 基质中。EPS 基质非常复杂，主要由细菌细胞、水、蛋白质、多糖、脂质和核酸1 组成，所有这些都使生物膜与自由生活的浮游细胞截然不同。生物膜EPS相互粘附在各种表面上。EPS 基质具有介导代谢物、遗传物质和用于细胞间信号转导和防御的化合物的细胞间交换的特性2。这些特性共同提供了生物膜结构完整性和抵御外部压力源的保护，有助于免疫逃避和抗菌素耐药性3.

铜绿假单胞菌是一种公认的医院病原体，已知它对抗菌药物采取逃避生物膜生长策略。一个典型的例子发生在患有隐性遗传疾病囊性纤维化 （CF） 的患者身上。生物膜在抗菌耐药性铜绿假单胞菌4 的发展中起着关键作用，并允许 CF 患者建立慢性肺部感染，导致肺功能加速下降和过早死亡5。因此，进行体外生物膜研究以测试抗生素和新型抗感染剂对从 CF 6,7 患者获得的铜绿假单胞菌分离株的疗效。生物膜形成后，将抗菌剂应用于结构外部，并使用共聚焦激光扫描显微镜 （CLSM） 生成生物膜段的高分辨率三维重建。通常的做法是使用计算机软件COMSTAT作为ImageJ的插件，以量化生物膜结构的变化8,9,10,11。

尽管COMSTAT可用于量化生物膜结构，但图像分析的可重复性和标准化问题较少得到解决。例如，在运行COMSTAT之前执行的图像处理程序是客观的，但在设置图像阈值12,13时包含主观性因素。以类似的方式，COMSTAT程序允许操作员选择从基本到高级的图像分割条件和参数，以及十种定量分析功能（例如，厚度分布、表面积、生物量、无量纲粗糙度系数）。众多的用户选项，再加上不同的操作人员专业知识水平，可能会导致对生物膜行为的误导性报告。

因此，该协议的目标是提出一种相对简单的方法，用于使用COMSTAT对体外生物膜结构进行定量比较。本文中，使用带腔盖玻片模型14 通过 CLSM 捕获来自铜绿假单胞菌分离物的生物膜片段的三维图像——这是一种用于执行可重复体外生物膜实验的成熟技术。利用 COMSTAT 作为 ImageJ 的插件，该方法使研究人员能够在不同条件下定量识别存在抗菌剂的情况下生物膜结构的变化。总体而言，该方法旨在消除与COMSTAT手动操作相关的主观差异，从而促进各中心协议的标准化。

Protocol

1. 细菌分离物收集 从 SickKids（多伦多）接受吸入妥布霉素根除治疗的 CF 儿科患者队列中获取 铜绿假单胞菌 分离株。在-80°C的柠檬酸甘油中冷冻分离物，并在使用前进行至少三次传代培养。 2. 体外 生物膜的形成 注：使用腔室盖玻片方法1 进行体外生物膜形成并进行修饰。该模型的整体工作流程如图 <strong …

Representative Results

从感染的CF患者中培养的 铜绿假单胞菌 分离物用于证明该方法在准确量化抗菌诱导 的体外 生物膜结构变化方面的优势。该模型的整体工作流程如图 1 所示。ImageJ 中的图像处理和 COMSTAT 分析过程如图 2 所示。 图 3 显示了 ImageJ 中用于图像分割的简单直方图阈值方法，该方法应用于 CLSM z 堆栈图像（另存为 OME-TIFF）。?…

Discussion

没有规定的方法来定量比较 体外 生物膜结构的三维图像，并且由于操作者之间的可变性，在这种情况下描述的程序通常难以标准化20。因此，该协议为寻求量化不同抗菌条件下 体外 生物膜结构变化的COMSTAT应用提供了一个简单且可重复的框架。该技术的优势是使用 CF 铜绿假单胞菌 分离物建模的，该分离物以生物膜重复的形式生长，并暴露于妥布霉素和抗 Psl…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢囊性纤维化基金会为这项研究提供资金。

Materials

Anti-Psl mAb, Psl0096 Medimmune
Blood Agar (TSA with 5 % Sheep Blood) Medium Fisher Scientific R01200
Eight-well Chambered Coverglass w/ non-removable wells Thermo Fisher Scientific 155411
Invitrogen SYTO 9 Green Fluorescent Nucleic Acid Stain Thermo Fisher Scientific S34854
LB BROTH (LENNOX), Liquid Autoclave Sterilized BioShop Canada LBL666
Tobramycin, 900 µg/mg Alfa Aesar by Thermo Fisher Scientific J66040 It is recommended to perform a minimal inhibitory concentration (MIC) test for every batch made to ensure quality control of antimicrobial potency
Quorum Volocity 6.3 Quorum Technologies Image analysis software
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Tags

BiofilmAntimicrobialPseudomonas AeruginosaCOMSTATCystic FibrosisTobramycinAnti-Psl Monoclonal AntibodyImage SegmentationBiofilm ArchitectureConfocal Microscopy

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Morris, A. J., Li, A., Jackson, L., Yau, Y. C. W., Waters, V. Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software. J. Vis. Exp. (166), e61759, doi:10.3791/61759 (2020).
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