针对相关生物膜<em>金黄色葡萄球菌</em>使用基于刃天青药物敏感性测定
Summary
Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.
Abstract
Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.
Introduction
致病微生物可在体内形成生物膜导致非急性慢性感染1。生物膜相关的感染是涉及异物（ 例如，人造骨替换，乳房植入物）或气管导管或导尿管2的安装植入医疗过程的严重危险因素。在这些情况下，抗感染治疗几乎总是必要的，因为基于生物膜的感染很少自行清除，甚至在免疫的个体。 金黄色葡萄球菌是在生物膜相关并发症牵连的最常观察到的病原体的使用期间发生的1微创医疗器械3。
不幸的是，生物膜的性质作为保护屏障，使他们对治疗比浮游细胞1,4-更耐磨，并预测临床疗效的评价是两个初始的关键部分药物开发以及耐药性监测。有越来越多的承认，实验室条件下，专注于浮游文化，可能无法忠实地代表现实世界的疾病5。进一步使问题复杂化，生物膜表型的复制是困难的，现有的生物膜模型繁琐和高之间和内部检测变异6受苦。因此，许多研究人员，通过必要性，默认为药物敏感性的浮游细胞的测定，从而潜在地忽略细菌毒力和疾病的一个重要方面。
在这里，我们描述了协议用 于测定细菌，特别是S.金黄色葡萄球菌 ，在利用96孔基于生物膜系统-7,8-预生长的生物膜。而对生物膜形成和挑战的协议基本上遵循制造商的建议，我们提出一种替代信息丰富的方法用于量化biof的可行性攻击后ILM。简要地说，细菌是在栓板，其中生物膜上附着在板的盖的突出栓形成中培养。经过生物膜形成，钉在一个新的盘子装满PBS去除浮游细胞的井轻轻蘸。栓柱盖，具有附着生物膜，被转移到一个新的挑战板，含有不同浓度的抗生素进行检测。第二次温育后，盖子被再次除去，洗净，并转移至含有刃天青染料，这里它们经受一个最终温育恢复板。刃天青转化动力学上能够记录或作为一个定义的恢复期后端点读数。此染料型定量生物膜的可行性的方法从繁琐CFU（菌落形成单位）有很大不同的原始协议7所述计数基于方法学。药物的挑战板和刃天青转化动力学OD 600测量结果作为可行性读浮游和生物膜细胞，分别出局，提供快速，可靠，且技术上简单的信息丰富的生物膜存活试验。
Protocol
Representative Results
Discussion
这里，我们已经描述了修饰的生物膜测定为S上确定测试抑制剂活性葡萄球菌生物膜注重生物膜有关的细胞的代谢状态。尽管所述生物膜起始和挑战的程序大多模仿制造商的建议，使用刃天青染料来检测和量化存活24小时抑制剂挑战生物膜相关的细胞显着地简化细胞回收（通过水浴超声处理），并随后列举的建议的过程通过的CFU计数的活细胞。该制造商推荐的程序涉及至少5个额外的操…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Saran Kupul for technical assistance. This work was supported in part by NIH grant R01-AI104952 to FW. Further support was provided by the University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR), an NIH funded program (P30 AI027767) that was made possible by the following NIH Institutes: NIAID, NIMH, NIDA, NICHD, NHLDI, NIA.
Materials
| Mueller-Hinton Medium | Oxoid | CM0405 | Follow recommendations of manufacturer |
| RPMI-medium | Corning | 17-105-CV | |
| CRPMI | Ref 9 | RPMI-1640 medium chelexed for 1h with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640 | |
| Chelex 100 Resin | Bio Rad | 142-2822 | |
| MBEC-plates | Innovotech | 19111 | |
| Resazurin Sodium Salt | Sigma | R7017 | 800µg/ml in DI water Filter sterile |
| Micro Plate Shaking Platform | Heidolph Titramax 1000 | ||
| Cytation 3 Plate Reader | Biotek | ||
| Gen5 software | Biotek | Recording and analysis of resazurin conversion | |
| Neocuproine | Sigma | N1501 | prepare 10 mM stock in 100% Ethanol, store at -80ºC |
| Copper sulfate | Acros Organics | 7758-99-8 | prepare a 100 mM stock solution in water, store at 4ºC |
| Cu-Neocuproine | Self-Made | Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration. | |
| Gentamicin | Sigma | G3632-1G |
References
- Bjarnsholt, T., Ciofu, O., Molin, S., Givskov, M., Hoiby, N. Applying insights from biofilm biology to drug development – can a new approach be developed. Nat Rev Drug Discov. 12, 791-808 (2013).
- Song, Z., et al. Prosthesis infections after orthopedic joint replacement: the possible role of bacterial biofilms. Orthop Rev (Pavia). 5, 65-71 (2013).
- Hall-Stoodley, L., Costerton, J. W., Stoodley, P. Bacterial biofilms: from the natural environment to infectious diseases). Nat Rev Microbiol. 2, 95-108 (2004).
- Bordi, C., de Bentzmann, S. Hacking into bacterial biofilms: a new therapeutic challenge. Ann Intensive Care. 1, 19 (2011).
- Fux, C. A., Shirtliff, M., Stoodley, P., Costerton, J. W. Can laboratory reference strains mirror ‘real-world’ pathogenesis?. Trends Microbiol. 13, 58-63 (2005).
- Kwasny, S. M., Opperman, T. J. Static biofilm cultures of Gram-positive pathogens grown in a microtiter format used for anti-biofilm drug discovery. Curr Protoc Pharmacol. Chapter 13, Unit 13A 18 (2010).
- Ceri, H., et al. The MBEC Assay System: multiple equivalent biofilms for antibiotic and biocide susceptibility testing. Methods Enzymol. 337, 377-385 (2001).
- Ceri, H., et al. The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clinical Microbiol. 37, 1771-1776 (1999).
- Baker, J., et al. Copper stress induces a global stress response in Staphylococcus aureus and represses sae and agr expression and biofilm formation. Appl Environ Microbiol. 76, 150-160 (2010).
- Haeili, M., et al. Copper complexation screen reveals compounds with potent antibiotic properties against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 58, 3727-3736 (2014).
- O’Brien, J., Wilson, I., Orton, T., Pognan, F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem. 267, 5421-5426 (2000).
- Mah, T. F. Establishing the minimal bactericidal concentration of an antimicrobial agent for planktonic cells (MBC-P) and biofilm cells (MBC-B). J Vis Exp. (83), e50854 (2014).
- Junker, L. M., Clardy, J. High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development. Antimicrob Agents Chemother. 51, 3582-3590 (2007).
- Shah, D., et al. Persisters: a distinct physiological state of E. coli. BMC Microbiol. 6, 53 (2006).
- Lewis, K. Riddle of biofilm resistance. Antimicrob Agents Chemother. 45, 999-1007 (2001).
