颅内 Subarachnoidal 感染对猪链球菌生物膜在小鼠感染模型中作用的研究
1College of Veterinary Medicine,Nanjing Agricultural University, 2Key Lab of Animal Bacteriology,Ministry of Agriculture, 3OIE Reference Lab for Swine Streptococcosis, 4School of Medicine,Tsinghua University, 5Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses
Summary
本文描述了小鼠颅内 subarachnoidal 感染的途径, 研究了生物膜在猪链球菌脑膜炎中的作用。这种感染模型也适用于研究其他细菌性脑膜炎的发病机制和新药对细菌性脑膜炎的疗效。
Abstract
猪链球菌不仅是全球猪的主要细菌病原体, 而且是一种新兴的人畜共患病剂。在人类和猪中, 脑膜炎是感染猪的主要表现。适当的感染模型是了解病原体致病机制的基本工具。研究了小鼠感染的几种途径, 探讨了猪感染的发病机制。然而, 腹腔, 鼻腔和静脉感染的途径不适合研究猪的表面成分在脑膜炎直接在脑中的作用, 如生物膜胞外基质。虽然 intracisternal 接种已被用于猪的感染, 但精确注射部位尚未描述。本文用小鼠模型描述了颅内 subarachnoidal 感染的途径, 探讨了生物膜在猪链球菌脑膜炎中的作用。通过位于 bregma 3.5 毫米延髓的注射部位, 将猪的浮游细胞或生物膜状态细胞直接注入小鼠蛛网膜下腔。用生物膜状态细胞注射小鼠脑组织中 TLR2 和细胞因子的病理学分析和 mRNA 表达明显表明,猪的生物膜在猪的脑膜炎中起着决定性的作用。这种感染途径明显优于其他感染途径, 允许研究宿主-细菌相互作用。此外, 它允许细菌成分对宿主免疫应答的影响直接在大脑被评估, 并模仿细菌进入中枢神经系统。这种感染途径可以扩展, 以调查其他细菌引起的脑膜炎机制。此外, 还可用于检测药物对细菌性脑膜炎的疗效。
Introduction
猪链球菌(猪) 是全世界猪的主要细菌病原体, 造成严重疾病, 包括脑膜炎、肺炎、败血症、心内膜炎和关节炎1。它也是一个新兴的人畜共患病的特工。到目前为止, 据报道, 九型血清型可能导致人类感染, 包括血清型 2, 4, 5, 9, 14, 16, 21, 24, 312,3,4。在人类和猪中, 脑膜炎是感染猪的主要临床征象之一。在越南和泰国,美国猪是5成人脑膜炎的主要病因。微生物生物膜是粘在一起的微生物, 并集中在界面上;它们对细菌的毒力、不同环境下的生存和耐药性5至关重要。生物膜通常被一个细胞外基质所包围, 通常含有多糖、蛋白质和 DNA6。后者能够诱发宿主炎症反应和细胞因子的产生7。在以前的研究中, 生物膜的形成已被用于链球菌脑膜炎。生物膜有助于罗非鱼鱼模型中的链球菌脑膜炎链球菌, 生物膜的形成已经通过腹腔内接种8在体内的脑组织和脑膜表面周围显露出来。在脑膜炎期间,肺炎链球菌处于生物膜状状态, 而这种生物膜状态下的细菌更有效地诱发了老鼠感染模型9中的脑膜炎。此外, 在我们以前的研究中, 在小鼠脑中与猪的相关的生物膜状态通过生存分析导致细菌毒力10。然而, 直接证据的生物膜参与的猪链球菌脑膜炎需要进一步的调查。
采用腹腔 (ip)11、鼻腔 (i.n.)12、静脉注射 (静脉注射)13和 intracisternal (中) 感染14条的方法, 研制出了猪鼠感染动物模型.15,16. 然而, 感染的 ip、i.n. 和静脉注射途径不适合于直接在脑部研究在脑膜炎中猪的表面成分的作用。这些包括生物膜胞外基质。虽然在美国猪感染中使用了内源接种, 但是这些文件中没有描述精确的注射部位。与此相反, 颅内 subarachnoidal 接种的注射部位的立体定向坐标在前一项研究中已经明确描述过17。这允许容易地承认接种点和更加简单的实验性协议。此外, 颅内 subarachnoidal 感染的途径, 模拟细菌进入中枢神经系统从鼻窦或中耳17, 中耳和脑膜炎的关系, 由猪.已经证明了马德森et al18。此外, 通过应用颅内 subarachnoidal 感染的小鼠, 我们已经证明了猪的小 RNA rss04 导致脑膜炎在我们以前的研究10。
在本研究中, 采用颅内 subarachnoidal 感染途径对小鼠进行了研究, 探讨了生物膜在猪链球菌脑膜炎中的作用。通过这种感染途径, 小鼠感染了浮游细胞或猪的生物膜状态细胞。生物膜对小鼠脑组织中 TLR2 和细胞因子表达的组织病理学分析清楚地表明,猪的生物膜对脑膜炎有促进作用。
Protocol
小鼠感染实验由中国江苏省实验动物监测委员会批准, 并在南京农业大学实验动物中心 (许可证编号: SYXK (Su) 2017-0007) 上进行。 1. 细菌的制备 注:猪血清2型毒株 P1/7 是从患病的猪与脑膜炎19分离出来的。P1/7 菌株生长在托德-休伊特汤 (THB, 公式每公升 THB: 心脏输液, 3.1 克; neopeptone, 20.0 克; 葡萄糖, 2.0 克; 氯化钠, 2.0 克; 磷酸二钠, 0.4 克; …
Representative Results
在实验条件下对生物膜的形成进行了 SEM 分析。如图 1所示, 浮游细胞 (图 1a) 与生物膜状态细胞之间的生物膜形成有显著差异 (图 1B)。SEM 分析表明, 生物膜细菌在团簇和多层, 它们被包裹在细胞外基质, 而浮游细菌的密度更低, 主要分散单独。 <p class="jove_content" fo:keep-together.within-…
Discussion
这里描述的颅内 subarachnoidal 感染途径与其他感染途径相比有明显的优势。它允许调查人员研究宿主-细菌相互作用和细菌成分对宿主免疫应答的影响直接在大脑, 这模仿细菌进入中枢神经系统。因此, 这种感染途径可以扩展, 以调查其他细菌引起的脑膜炎机制。此外, 还可用于检测药物对细菌性脑膜炎的疗效。
为了获得良好的结果, 使用这个模型, 以下关键步骤是明确的。生物膜…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国国家重点研究开发计划 (2017YFD0500102) 的资助;中国国家自然科学基金 [31572544];兽医病因生物学国家重点实验室 [SKLVEB2016KFKT005];上海农业应用技术发展项目, 中国 [G2016060201]。
Materials
| Todd Hewitt Broth(THB) | Becton, Dickinson and Company | DF0492078 | Dissolve 30 g of the powder in 1 L of purified water. Autoclave at 121° for 15 min. |
| Agar | DSBIO | 16C0050 | Dissolve 15 g of the powder in 1 L of THB. Autoclave at 121° for 15 min. |
| Milli-Q Reference Water Purification System | Merck KGaA | Z00QSVCUS | Without Dnase/ Rnase |
| NaCl | Tianjin Kemiou Chemical Reagent Co., Ltd | 10019318 | Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121° for 15 min. Use KOH to adjust pH to 7.4. |
| Na2HPO3 | Xilong Scientific Co., Ltd | 9009012-01-09 | Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121° for 15 min. Use KOH to adjust pH to 7.4. |
| KCl | Xilong Scientific Co., Ltd | 9009017-01-09 | Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121° for 15 min. Use KOH to adjust pH to 7.4. |
| KH2PO4 | Xilong Scientific Co., Ltd | 9009019-01-09 | Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121° for 15 min. Use KOH to adjust pH to 7.4. |
| KOH | Xilong Scientific Co., Ltd | 9009014-01-09 | Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121° for 15 min. Use KOH to adjust pH to 7.4. |
| Glycerol | Sionpharm Chemical Reagent Co., Ltd | 10010618 | Diluted with equal volumu of purified water, autoclave at 121° for 15 min |
| 4% paraformaldehyde | Sionpharm Chemical Reagent Co., Ltd | 80096675 | |
| 25% Glutaraldehyde | Sionpharm Chemical Reagent Co., Ltd | 30092436 | 10-fold diluted with purified water for fixation. |
| Ethanol | Sionpharm Chemical Reagent Co., Ltd | 10009218 | |
| Chloroform | Sionpharm Chemical Reagent Co., Ltd | 10006818 | |
| Spctrophotometre | DeNovix Inc. | DS-11+ | |
| Ultrasound cell crusher | NingBo Scientz Biotechnology Co.,Ltd | JY96-IIN | |
| Centrifuge | Hitachi Koki Co., Ltd | CT15RE | |
| Refrigerator | Aucma Co., Ltd | DW-86L500 | |
| Scanning electron microscope | Zeiss | EVO-LS10 | |
| FastRNA Pro Green Kit | MP Biomedicals | #6045-050 | |
| FastPrep-24 Instrument | MP Biomedicals | 116005500 | |
| Instrument for PCR | SensoQuest GmbH | 1124310110 | |
| QuantStudio 6 Flex | Thermo Fisher Scientific | 4485689 | |
| SYBR Premix Ex Taq II | Takara Biomedical Technology (Beijing) Co., Ltd | RR820A | |
| PrimeScript RT reagent kit with gDNA Eraser | Takara Biomedical Technology (Beijing) Co., Ltd | RR047A | |
| Fully Enclosed Tissue Processor | Leica Biosystems Nussloch GmbH | ASP200S | |
| Heated Paraffin Embedding Module | Leica Biosystems Nussloch GmbH | EG1150H | |
| Semi-Automated Rotary Microtome | Leica Biosystems Nussloch GmbH | RM2245 | |
| Water bath for paraffin sections | Leica Biosystems Nussloch GmbH | HI1210 | |
| Autostainer XL | Leica Biosystems Nussloch GmbH | ST5010 | |
| Agilent 2100 | Agilent Technologies | G2939A | |
| Optical microscope | Olympus | BX51 |
Referencias
- Gottschalk, M., Xu, J., Calzas, C., Segura, M. Streptococcus suis: a new emerging or an old neglected zoonotic pathogen?. Future Microbiology. 5, 371-391 (2010).
- Goyette-Desjardins, G., Auger, J. P., Xu, J., Segura, M., Gottschalk, M. Streptococcus suis, an important pig pathogen and emerging zoonotic agent-an update on the worldwide distribution based on serotyping and sequence typing. Emerging Microbes & Infections. 3 (6), e45 (2014).
- Kerdsin, A., et al. Emergence of Streptococcus suis serotype 9 infection in humans. Journal of Microbiology, Immunology and Infection. 50 (4), 545-546 (2017).
- Hatrongjit, R., et al. First human case report of sepsis due to infection with Streptococcus suis serotype 31 in Thailand. BMC Infect Diseases. 15, 392 (2015).
- Fittipaldi, N., Segura, M., Grenier, D., Gottschalk, M. Virulence factors involved in the pathogenesis of the infection caused by the swine pathogen and zoonotic agent Streptococcus suis. Future Microbiology. 7 (2), 259-279 (2012).
- Hall-Stoodley, L., Costerton, J. W., Stoodley, P. Bacterial biofilms: from the natural environment to infectious diseases. Nature Reviews Microbiology. 2 (2), 95-108 (2004).
- Fuxman Bass, J. I., et al. Extracellular DNA: a major proinflammatory component of Pseudomonas aeruginosa biofilms. Journal of Immunology. 184 (11), 6386-6395 (2010).
- Isiaku, A. I., et al. Biofilm is associated with chronic streptococcal meningoencephalitis in fish. Microbial Pathogenesis. 102, 59-68 (2017).
- Oggioni, M. R., et al. Switch from planktonic to sessile life: a major event in pneumococcal pathogenesis. Molecular Microbiology. 61 (5), 1196-1210 (2006).
- Xiao, G., et al. Streptococcus suis small RNA rss04 contributes to the induction of meningitis by regulating capsule synthesis and by inducing biofilm formation in a mouse infection model. Veterinary Microbiology. 199, 111-119 (2017).
- Dominguez-Punaro, M. C., et al. Streptococcus suis serotype 2, an important swine and human pathogen, induces strong systemic and cerebral inflammatory responses in a mouse model of infection. Journal of Immunology. 179 (3), 1842-1854 (2007).
- Seitz, M., et al. A novel intranasal mouse model for mucosal colonization by Streptococcus suis serotype 2. Journal of Medical Microbiology. 61 (Pt 9), 1311-1318 (2012).
- Busque, P., Higgins, R., Caya, F., Quessy, S. Immunization of pigs against Streptococcus suis serotype 2 infection using a live avirulent strain. Canadian Journal of Veterinary Research. 61 (4), 275-279 (1997).
- Williams, A. E., Blakemore, W. F. Pathology of Streptococcal meningitis following intravenous intracisternal and natural routes of infection. Neuropathology and Applied Neurobiology. 4 (4), 345-356 (1990).
- Dominguez-Punaro, M. C., et al. Severe cochlear inflammation and vestibular syndrome in an experimental model of Streptococcus suis infection in mice. European Journal of Clinical Microbiology & Infectious Diseases. 31 (9), 2391-2400 (2012).
- Auger, J. P., Fittipaldi, N., Benoit-Biancamano, M. O., Segura, M., Gottschalk, M. Virulence Studies of Different Sequence Types and Geographical Origins of Streptococcus suis Serotype 2 in a Mouse Model of Infection. Pathogens. 5 (3), (2016).
- Chiavolini, D., et al. Method for inducing experimental pneumococcal meningitis in outbred mice. BMC Microbiology. 4, 36 (2004).
- Madsen, L. W., Svensmark, B., Elvestad, K., Jensen, H. E. Otitis interna is a frequent sequela to Streptococcus suis meningitis in pigs. Veterinary Pathology. 38 (2), 190-195 (2001).
- Holden, M. T., et al. Rapid evolution of virulence and drug resistance in the emerging zoonotic pathogen Streptococcus suis. PLoS One. 4 (7), e6072 (2009).
- Dominguez-Punaro Mde, L., et al. In vitro characterization of the microglial inflammatory response to Streptococcus suis, an important emerging zoonotic agent of meningitis. Infection and Immunity. 78 (12), 5074-5085 (2010).
- Hassan, A., et al. Evaluation of different detection methods of biofilm formation in the clinical isolates. Brazilian Journal of Infectious Diseases. 15 (4), 305-311 (2011).
- Vanier, G., et al. New putative virulence factors of Streptococcus suis involved in invasion of porcine brain microvascular endothelial cells. Microbial Pathogenesis. 46 (1), 13-20 (2009).
- Takeuchi, D., et al. The contribution of suilysin to the pathogenesis of Streptococcus suis meningitis. Journal of Infectious Diseases. 209 (10), 1509-1519 (2014).
- Tenenbaum, T., et al. Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitro. Cellular Microbiology. 11 (2), 323-336 (2009).
Citar este artículo
Zhang, S., Gao, X., Xiao, G., Lu, C., Yao, H., Fan, H., Wu, Z. Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model. J. Vis. Exp. (137), e57658, doi:10.3791/57658 (2018).