一种用于生物技术相关金黄色葡萄球菌感染活体可视化和内膜分析的斑马鱼胚胎模型
Summary
本研究描述了一种斑马鱼胚胎模型, 用于基于荧光显微镜的生物物质相关感染的体内可视化和体内分析。该模型是一个很有前途的系统, 补充哺乳动物动物模型, 如研究体内生物材料相关感染的小鼠模型。
Abstract
生物材料相关感染 (bai) 是生物技术/医疗器械失效的主要原因。金黄色葡萄球菌是 bai 的主要病原菌之一。目前实验的 bai 哺乳动物动物模型, 如小鼠模型, 成本昂贵, 耗时, 因此不适合高吞吐量分析。因此, 需要新的动物模型作为体内调查 bai 的补充系统。在本研究中, 我们的目标是开发一个斑马鱼胚胎模型, 用于基于荧光显微镜的生物材料存在下细菌感染的体内可视化和体内分析。此外, 还研究了引发的巨噬细胞反应。为此, 我们使用荧光蛋白表达金黄色葡萄球菌和转基因斑马鱼胚胎, 在巨噬细胞中表达荧光蛋白, 并开发了一种单独或与微球一起向肌肉注射细菌的程序胚胎组织。为了监测细菌感染的进展, 随着时间的推移, 我们设计了一个简单而可靠的方法, 显微镜评分荧光细菌。显微镜评分结果表明, 所有具有20多个菌落形成单位 (cfu) 细菌的胚胎都能产生细菌的阳性荧光信号。为了研究生物材料对感染的潜在影响, 我们确定了具有和不具有10μm 聚苯乙烯微球 (ps10) 的金黄色葡萄球菌的 cfu 数, 作为胚胎中的生物材料模型. 此外, 我们使用在 imagej 中运行的 objectj 项目文件 “zbrafan-vapoest” 来量化随着时间的推移有和没有 ps10 的金黄色葡萄球菌感染的荧光强度。这两种方法的结果表明, 在微球感染的胚胎中, 金黄色葡萄球菌的数量高于在没有微球的胚胎中, 这表明在生物材料存在的情况下, 感染易感性增加。因此, 本研究表明斑马鱼胚胎模型在研究 bai 方面具有应用本文所开发的方法的潜力。
Introduction
各种医疗设备 (称为 “生物材料”) 越来越多地用于现代医学, 以恢复或取代人体部位 1.然而, 生物材料的植入使患者容易感染, 称为生物材料相关感染 (bai), 这是手术中植入物的主要并发症。金黄色葡萄球菌和表皮葡萄球菌是两个最常见的细菌物种, 负责 bai2,3,4,5,6。植入的生物材料形成了一个表面容易受到细菌生物膜形成的影响。此外, 植入的生物材料可能会使局部免疫反应出轨, 导致细菌清除效果降低。感染细菌的初始清除主要是通过浸润中性粒细胞进行的, 中性粒细胞在植入或植入的生物材料7的存在下, 这些中性粒细胞的杀菌能力大大降低。此外, 巨噬细胞在中性粒细胞最初的涌入后渗入组织, 会吞噬剩余的细菌, 但不能有效地杀死它们的内部, 由于疯狂的免疫信号, 这是一个综合存在的结果生物材料和细菌8。因此, 生物材料的存在可以促进细菌在细胞内生存 9,10,11,12,13和生物膜形成的植入物生物材料4,14。因此, bai 可能导致无法和需要更换植入的生物材料, 导致发病率和死亡率上升, 住院时间延长, 额外费用2,15.
目前正在制定越来越多的反审调处战略 2、16、17。在体内评估这些策略在相关动物模型中的有效性是必不可少的。然而, 传统的实验 bai 动物模型 (例如,小鼠模型) 通常成本高昂、耗时长, 因此不适合多种策略的高吞吐量测试18。基于生物光学的生物光学成像技术的最新发展–宿主细胞和细菌荧光标记可能使单个小动物能够持续监测 bai 进展和宿主-病原体-材料相互作用例如小鼠18、19、20、21.然而, 这种技术相对复杂, 尚处于起步阶段, 对 bai18 进行定量分析必须解决几个问题。例如, 需要高挑战剂量来显示细菌的定植。此外, 还必须解决在哺乳动物试验动物组织中的光散射和生物发光荧光信号的吸附问题.因此, 新的、经济高效的动物模型, 允许随着时间的推移进行体内可视化和定量分析, 是在体内研究 bai 的宝贵补充系统。
斑马鱼 (胚胎) 已被用作一种多功能的体内工具, 用于解剖宿主-病原体相互作用和几种细菌的感染发病机制, 如22分枝杆菌,铜绿假单胞菌23, 大肠杆菌24例,粪便肠球菌25例, 葡萄球菌26、27例。斑马鱼胚胎具有许多优点, 如光学透明度、相对较低的维护成本以及拥有与哺乳动物高度相似的免疫系统 28, 29.这使得斑马鱼胚胎成为一个高度经济、有生命的生物, 用于对感染进展和相关宿主反应进行体内可视化和分析 28,29.为了使细胞在体内的行为可视化, 转基因斑马鱼系具有不同类型的免疫细胞 (如巨噬细胞和中性粒细胞), 甚至具有荧光标记的亚细胞结构. ,29。此外, 斑马鱼的高繁殖率提供了开发高吞吐量测试系统的可能性, 该系统具有自动机器人注射、自动荧光定量和 rna 序列分析27的特点,30岁.
在本研究中, 我们的目标是开发一个斑马鱼胚胎模型的生物材料相关感染使用荧光成像技术。为此, 我们开发了一种在生物材料微球存在的情况下将细菌 (金黄色葡萄球菌) 注入斑马鱼胚胎肌肉组织的程序。我们使用金黄色葡萄球菌rn4220 表达 mcherry 荧光蛋白 (s. aureus-mcherry), 如其他地方所描述的另一种金黄色葡萄球菌菌株10,31。采用转基因斑马鱼系 (mpeg1:uas变为 kaede), 在巨噬细胞32和蓝色荧光聚苯乙烯微球中表达了 kaede 绿色荧光蛋白。在之前的一项研究中, 我们已经证明, 肌肉注射微球到斑马鱼胚胎, 以模仿生物材料植入是可行的33。为了定量分析 bai 的进展和相关细胞浸润在单个胚胎随着时间的推移, 我们使用了 “zobravies-vantest” 项目文件, 该文件在 “objectj” (imagej 的插件) 中操作, 以量化的荧光强度细菌居住和巨噬细胞浸润在微球注射部位附近 33.此外, 我们还确定了胚胎中存在和不存在微球的菌落形成单位 (cfu) 的数量, 以研究生物材料对感染的潜在影响。我们目前的研究表明, 随着这里开发的方法, 斑马鱼胚胎是一个有前途的, 新的脊椎动物模型, 用于研究体内生物技术相关感染。
Protocol
Representative Results
Discussion
生物材料相关感染 (bai) 是一种严重的临床并发症。更好地了解 bai 在体内的发病机制, 将为改进 bai 的预防和治疗提供新的见解。然而, 目前实验的 bai 动物模型, 如小鼠模型是昂贵的, 劳动密集型的, 需要受过复杂手术技术培训的专业人员。因此, 这些模型不适合高吞吐量分析。由于对斑马鱼胚胎模型的要求不那么复杂, 一般费用低于小鼠模型, 本研究评估了斑马鱼胚胎是否可以作为一种新的脊椎动?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究得到了 bifas 生物医疗材料方案国际金融和药物治疗项目的财政支持, 并得到了荷兰经济事务部的共同资助。作者要感谢澳大利亚莫纳什大学的 graham lieschke 教授提供斑马鱼转基因线 (mpeg1: galni-uas:kaede)。
Materials
| Tryptic soya agar | BD Difco | 236950 | Media preparation unit at AMC |
| Tryptic soya broth | BD Difco | 211825 | |
| Polyvinylpyrrolidone40 | Applichem | A2259.0250 | |
| 10 µm diameter polystyrene microspheres (blue fluorescent) | Life technology/ThemoFisher | F8829 | |
| Glass microcapilary (1 mm O.D. x 0.78 mm I.D.) | Harvard Apparatus | 30-0038 | |
| Micropipette puller instrument | Sutter Instrument Inc | Flaming p-97 | |
| Light microscope LM 20 | Leica | MDG33 10450123 | |
| 3-aminobenzoic acid (Tricaine) | Sigma-Aldrich | E10521-50G | |
| Agarose MP | Roche | 11388991001 | |
| Stereo fluorescent microscope LM80 | Leica | MDG3610450126 | |
| Microloader pipette tips | Eppendorf | 5242956.003 | |
| Micromanipulator M3301 with M10 stand | World Precision Instruments | 00-42-101-0000 | |
| FemtoJet express micro-injector | Eppendorf | 5248ZO100329 | |
| Microtrube 2ml pp | Sarstedt | 72.693.005 | |
| Zirconia beads | Bio-connect | 11079124ZX | |
| MagNA lyser | Roche | 41416401 | |
| MSA-2 plates (Mannitol Salt Agar-2) | Biomerieux | 43671 | Chapmon 2 medium |
| Methyl cellulose 4000cp | Sigma-Aldrich | MO512-250G | |
| Chloramphenicol | Sigma-Aldrich | C0378 | |
| Gyrotory shaker (for bacterial growth) | New Brunswick Scientific | G10 | |
| Zebrafish incubator | VWR | Incu-line | |
| Cuvettes | BRAND | 759015 | |
| Centrifuge | Hettich-Zentrifugen | ROTANTA 460R | |
| Spectrometer | Pharmacia biotech | Ultrospec®2000 | |
| Forceps | Sigma-Aldrich | F6521-1EA | |
| 48 well-plates | Greiner bio-one | 677180 | |
| 96 well-plates | Greiner bio-one | 655161 | |
| Petri-dish | Falcon | 353003 | |
| Petri-dish | Biomerieux | NL-132 | |
| ImageJ | Not applicable | Not applicable | link: https://imagej.nih.gov/ij/download.html |
| GraphPad 7.0 | Prism | Not applicable |
Riferimenti
- Williams, D. F. On the nature of biomaterials. Biomaterials. 30, 5897-5909 (2009).
- Busscher, H. J., et al. Biomaterial-Associated Infection: Locating the Finish Line in the Race for the Surface. Science Translational Medicine. 4, 153rv10 (2012).
- Otto, M. Staphylococcus epidermidis – the ‘accidental’ pathogen. Nature Reviews Microbiology. 7, 555-567 (2009).
- Moriarty, T. F., et al. Orthopaedic device-related infection: current and future interventions for improved prevention and treatment. EFORT Open Reviews. 1, 89-99 (2016).
- Campoccia, D., Montanaro, L., Arciola, C. R. The significance of infection related to orthopedic devices and issues of antibiotic resistance. Biomaterials. 27, 2331-2339 (2006).
- Schierholz, J. M., Beuth, J. Implant infections: a haven for opportunistic bacteria. Journal of Hospital Infection. 49, 87-93 (2001).
- Zimmerli, W., Lew, P. D., Waldvogel, F. A. Pathogenesis of foreign body infection. Evidence for a local granulocyte defect. Journal of Clinical Investigation. 73 (4), 1191-1200 (1984).
- Boelens, J. J., et al. Biomaterial-associated persistence of Streptococcus epidermidis in pericatheter macrophages. Journal of Infectious Diseases. 181 (4), 1337-1349 (2000).
- Broekhuizen, C. A. N., et al. Tissue around catheters is a niche for bacteria associated with medical device infection. Critical Care Medicine. 36, 2395-2402 (2008).
- Riool, M., et al. Staphylococcus epidermidis originating from titanium implants infects surrounding tissue and immune cells. Acta Biomaterial. 10, 5202-5212 (2014).
- Zaat, S. A. J., Broekhuizen, C. A. N., Riool, M. Host tissue as a niche for biomaterial-associated infection. Future Microbiology. 5, 1149-1151 (2010).
- Broekhuizen, C. A. N., et al. Staphylococcus epidermidis is cleared from biomaterial implants but persists in peri-implant tissue in mice despite rifampicin/vancomycin treatment. Journal of Biomedical Materials Research Part A. 85, 498-505 (2008).
- Broekhuizen, C. A. N., et al. Peri-implant tissue is an important niche for Staphylococcus epidermidis in experimental biomaterial-associated infection in mice. Infection and Immunity. 75, 1129-1136 (2007).
- Zimmerli, W., Sendi, P. Pathogenesis of implant-associated infection: the role of the host. Seminars in Immunopathology. 33, 295-306 (2011).
- Darouiche, R. O. Current concepts – Treatment of infections associated with surgical implants. New England Journal of Medicine. 350, 1422-1429 (2004).
- Riool, M., de Breij, A., Drijfhout, J. W., Nibbering, P. H., Zaat, S. A. J. Antimicrobial peptides in biomedical device manufacturing. Frontiers in Chemistry. 5, 63 (2017).
- Brooks, B. D., Brooks, A. E., Grainger, D. W., Moriaty, F. T., Zaat, S. A., Busscher, H. J. Antimicrobial medical devices in preclinical development and clinical use. Biomaterials Associated Infection. , 307-354 (2013).
- Sjollema, J., et al. The potential for bio-optical imaging of biomaterial-associated infection in vivo. Biomaterials. 31, 1984-1995 (2010).
- Suri, S., et al. In vivo fluorescence imaging of biomaterial-associated inflammation and infection in a minimally invasive manner. Journal of Biomedical Materials Research Part A. 103, 76-83 (2015).
- Zhou, J., Hu, W. J., Tang, L. P. Non-invasive characterization of immune responses to biomedical implants. Annals of Biomedical Engineering. 44, 693-704 (2016).
- van Oosten, M., et al. Real-time in vivo imaging of invasive- and biomaterial-associated bacterial infections using fluorescently labelled vancomycin. Nature Communications. 4, 2584 (2013).
- Lesley, R., Ramakrishnan, L. Insights into early mycobacterial pathogenesis from the zebrafish. Current Opinion in Microbiology. 11, 277-283 (2008).
- Brannon, M. K., et al. Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos. Cellular Microbiology. 11, 755-768 (2009).
- Wiles, T. J., Bower, J. M., Redd, M. J., Mulvey, M. A. Use of zebrafish to probe the divergent virulence potentials and toxin requirements of extraintestinal pathogenic Escherichia coli. PLoS Pathogens. 5, e1000697 (2009).
- Prajsnar, T. K., et al. Zebrafish as a novel vertebrate model to dissect enterococcal pathogenesis. Infection and Immunity. 81, 4271-4279 (2013).
- Prajsnar, T. K., Cunliffe, V. T., Foster, S. J., Renshaw, S. A. A novel vertebrate model of Staphylococcus aureus infection reveals phagocyte-dependent resistance of zebrafish to non-host specialized pathogens. Cellular Microbiology. 10, 2312-2325 (2008).
- Veneman, W. J., et al. A zebrafish high throughput screening system used for Staphylococcus epidermidis infection marker discovery. BMC Genomics. 14, 255 (2013).
- Renshaw, S. A., Trede, N. S. A model 450 million years in the making: zebrafish and vertebrate immunity. Disease Model and Mechanism. 5, 38-47 (2012).
- Meijer, A. H., van der Vaart, M., Spaink, H. P. Real-time imaging and genetic dissection of host-microbe interactions in zebrafish. Cellular Microbiology. 16, 39-49 (2014).
- Spaink, H. P., et al. Robotic injection of zebrafish embryos for high-throughput screening in disease models. Methods. 62, 246-254 (2013).
- Riool, M., et al. A chlorhexidine-releasing epoxy-based coating on titanium implants prevents Staphylococcus aureus experimental biomaterial-associated infection. European Cells and Materials. 33, 143-157 (2017).
- Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A., Lieschke, G. J. Mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood. 117, E49-E56 (2011).
- Zhang, X., et al. The zebrafish embryo as a model to quantify early inflammatory cell responses to biomaterials. Journal of Biomedical Materials Research Part A. 105, 2522-2532 (2017).
- Traber, K., Novick, R. A slipped-mispairing mutation in AgrA of laboratory strains and clinical isolates results in delayed activation of agr and failure to translate delta- and alpha-haemolysins. Molecular Microbiology. 59, 1519-1530 (2006).
- Rosen, J. N., Sweeney, M. F., Mably, J. D. Microinjection of zebrafish embryos to analyze gene function. Journal of Visualized Experiments. (25), e1115 (2009).
- Benard, E. L., et al. Infection of zebrafish embryos with intracellular bacterial pathogens. Journal of Visualized Experiments. 61, 3781 (2012).
- Brand, M., Granato, M., Christiane, N. -. V., Dahm, R., Nüsslein-Volhard, C. Keeping and raising zebrafish. Zebrafish, A Practical Approach. , 7-37 (2002).
- Chaplin, W. T. P. . Development of a microinjection platform for the examination of host-biomaterial interactions in zebrafish embryos. , (2017).
- Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proceedings of the National Academy of Science of the United States of America. 99, 12651-12656 (2002).
- Witherel, C. E., Gurevich, D., Collin, J. D., Martin, P., Spiller, K. L. Host-biomaterial interactions in zebrafish. ACS Biomaterials Science & Engineering. 4, 1233-1240 (2018).
- Anderson, J. M. Biological responses to materials. Annual Review of Materials Research. 31, 81-110 (2001).
- Onuki, Y., Bhardwaj, U., Papadimitrakopoulos, F., Burgess, D. J. A review of the biocompatibility of implantable devices: current challenges to overcome foreign body response. Journal of Diabetes Science and Technology. 2, 1003-1015 (2008).
- Carvalho, R., et al. A High-Throughput Screen for Tuberculosis Progression. PLoS One. 6, e16779 (2011).
- Stockhammer, O. W., et al. Transcriptome analysis of Traf6 function in the innate immune response of zebrafish embryos. Molecular Immunology. 48, 179-190 (2010).
- Thisse, C., Thisse, B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nature Protocols. 3, 59 (2007).
- Prajsnar, T. K., et al. A privileged intraphagocyte niche is responsible for disseminated infection of Staphylococcus aureus in a zebrafish model. Cellular Microbiology. 14, 1600-1619 (2012).
