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Ex vivo Model of Human Aortic Valve Bacterial Colonization
人类主动脉瓣膜细菌定植离体模型   

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Abstract

The interaction of pathogens with host tissues is a key step towards successful colonization and establishment of an infection. During bacteremia, pathogens can virtually reach all organs in the human body (e.g., heart, kidney, spleen) but host immunity, blood flow and tissue integrity generally prevents bacterial colonization. Yet, patients with cardiac conditions (e.g., congenital heart disease, atherosclerosis, calcific aortic stenosis, prosthetic valve recipients) are at a higher risk of bacterial infection. This protocol was adapted from an established ex vivo porcine heart adhesion model and takes advantage of the availability of heart tissues obtained from patients that underwent aortic valve replacement surgery. In this protocol, fresh tissues are used to assess the direct interaction of bacterial pathogens associated with cardiovascular infections, such as the oral bacterium Streptococcus mutans, with human aortic valve tissues.

Keywords: Streptococcus mutans(变形链球菌), Collagen(胶原), Adherence(粘附), ex vivo(离体), Aortic heart valve(主动脉瓣膜), Cardiovascular infection(心血管感染)

Background

The oral pathogen Streptococcus mutans is considered the major etiological agent in dental caries and can also be associated with extra-oral infections such as infective endocarditis (IE) (Banas, 2004). IE is generally initiated by a lesion of the heart valve endothelium which leads to the formation a sterile thrombus mainly composed of platelets, inflammatory cells, fibrin and other extracellular matrix (ECM) proteins (e.g., collagen, laminin) (Que and Moreillon, 2011). Other cardiovascular malignancies, such as calcific stenosis and atherosclerosis, can also cause tissue damage leading to the exposure and remodeling of ECM proteins (Yetkin and Waltenberger, 2009). This environment then provides suitable targets for colonization by different pathogens capable of interacting with host components. Thus, the development of relevant tools and experimental models may allow us to understand better how pathogens interact with heart tissues. Based on a previous protocol established by Chuang-Smith et al., 2010 using aortic heart valves from pigs, we developed an ex vivo tissue adherence assay using human heart valves obtained from patients that underwent aortic valve replacement (Freires et al., 2016). While this model does not reproduce the immunological responses and other host factors associated with the disease, it provides a relatively inexpensive system to assess the capacity of a given organism to directly interact with human heart valve tissues. Furthermore, while this model requires a close collaboration with a cardiac surgery unit, this type of surgery (i.e., aortic valve replacement) is routinely performed at health science centers in developed countries (Yetkin and Waltenberger, 2009).

Materials and Reagents

  1. Sterile specimen containers (Fisher Scientific, catalog number: 16-320-730 )
  2. 12-well tissue culture plates (Corning, Falcon®, catalog number: 351143 )
  3. Sterile culture tubes (4 ml) (Fisher Scientific, catalog number: 14-956-3D )
  4. Microcentrifuge tubes (1.7 ml) (Fisher Scientific, catalog number: S348903 )
  5. Glass scintillation vials (20 ml) (Sigma-Aldrich, catalog number: Z253081 )
  6. Sterile culture tubes (15 ml) (Fisher Scientific, catalog number: 14-956-6D )
  7. Desired bacterial strain(s) (e.g., Streptococcus mutans OMZ175)
  8. Extirpated heart tissues
  9. EGM-MV Bullet Kit (Lonza, catalog number: CC-3125 )
  10. Gentamicin (Sigma-Aldrich, catalog number: G1397 )
  11. Brain heart infusion medium (BHI) (BD, BactoTM, catalog number: 237500 )
  12. Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14025092 )
  13. Erythromycin (Sigma-Aldrich, catalog number: E5389 )
  14. Kanamycin (Sigma-Aldrich, catalog number: K1377 )
  15. Sodium chloride (NaCl) (Avantor® Performance Materials, J.T. Baker®, catalog number: 3628-01 )
  16. Potassium chloride (KCl) (Avantor® Performance Materials, J.T. Baker®, catalog number: 3045-01 )
  17. Sodium phosphate dibasic (Na2HPO4) (Avantor® Performance Materials, J.T. Baker®, catalog number: 3827-01 )
  18. Potassium dihydrogen phosphate (KH2PO4) (Avantor® Performance Materials, J.T. Baker®, catalog number: 3246-01 )
  19. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  20. Glutaraldehyde (Sigma-Aldrich, catalog number: 340855 )
  21. Sodium cacodylate trihydrate (Sigma-Aldrich, catalog number: C0250 )
  22. Agar (Fisher Scientific, catalog number: BP1423 )
  23. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F0392 )
  24. Hydrocortisone
  25. Bovine brain extract
  26. Human recombinant epidermal growth factor
  27. 1x phosphate buffer solution (PBS) (see Recipes)
  28. Fixative solution (see Recipes)

Equipment

  1. Biosafety cabinet class 2 (Nuaire, model: Labgard ES Energy Saver Class II, Type A2 , catalog number: NU-425-600)
  2. 3 mm skin biopsy punch (Acuderm, catalog number: P325 )
  3. Stainless steel forceps (Sigma-Aldrich, catalog number: Z168696 )
  4. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM MultifugeTM 1 S-R )
  5. Vortex
  6. Motorized pestle (Kimble Chase Life Science and Research Products, catalog number: 7495400000 )
  7. pH meter
  8. Rocker (Reliable Scientific, model: 55D )
  9. CO2 incubator (VWR; model: 2325 )
  10. Zeiss-Auriga focused ion beam field emission scanning electron microscope (FIB-FE-SEM)
  11. Gatan Erlangshen digital camera

Procedure

  1. Sample collection and preparation
    1. Aortic valve tissues that would otherwise be discarded should be aseptically removed from patients undergoing aortic valve replacement by a cardiac surgery specialist.
    2. Immediately after surgery, tissues should be placed in a sterile specimen container, kept on ice and immediately taken to the laboratory for processing.
  2. Tissue preparation
    1. In a biosafety cabinet, open the vial containing the heart tissue and assess the level of calcification, inflammation and damage prior to further processing. Ideally, valve sections should be obtained from smooth areas with no calcification, signs of inflammation or residual blood (Figure 1).
    2. With a biopsy punch, press firmly on the selected areas of the tissue to cut through and remove the section from the rest of the tissues (Figure 1).


      Figure 1. Selection of tissue from aortic valve tissues. After primary tissues are obtained, they should be immediately taken to the laboratory for evaluation and preparation. Only areas with no signs of calcification or inflammation are selected for sectioning with a 3 mm biopsy punch.

    3. Use sterile forceps to collect the section and place it in individual wells of a 12-well plate containing 1 ml of supplemented EGM + gentamicin 300 µg ml-1.
    4. The number of sections that can be effectively obtained depends on the quality and the size of the tissue as well as on the type of experiment to be performed (see below).
    5. Incubate the 12-well plate containing the valve sections at 37 °C in a 5% CO2 atmosphere overnight (18-20 h).
  3. Bacterial attachment to aortic valve tissues
    1. On the same day of valve tissue processing, grow overnight bacterial cultures in triplicate. To grow S. mutans OMZ175, add 2 ml of BHI in a 4 ml sterile culture tube and inoculate by picking a single isolated colony from a freshly streaked plate.
    2. Incubate cultures at 37 °C in a 5% CO2 atmosphere overnight (18-20 h) without aeration.
    3. On the following day, wash the valve sections twice at room temperature with HBSS buffer to remove residual antibiotics. For this, add 1 ml of HBSS into two clean empty wells of the same plate and transfer the sections using sterile tweezers. Gently rock plate containing valve sections for 5 min and set the rocker to a setting of 40-50 motions per minute for each wash step.
    4. In the meantime, centrifuge the overnight cultures at 3,500 x g for 10 min at 4 °C, wash twice with sterile PBS and resuspend cultures with the initial culture volume.
    5. For each strain replicate, add 2.7 ml of fresh EGM without antibiotics to a clean 4 ml sterile culture tube and 300 µl of the bacterial culture making a 1:10 dilution of the bacterial suspension (approximately 1 x 107 CFU ml-1).
    6. Transfer washed valve sections to a new 12-well plate and add 1 ml of the bacterial suspension in EGM. In addition, take 100 µl of each replicate for serial dilution and plating of the initial culture CFU (T0).
    7. Incubate plate at 37 °C in a 5% CO2 atmosphere with gentle rocking for 90 min.
    8. After incubation, transfer each valve section into a 1.7 ml microcentrifuge tube containing 1 ml of HBSS and then vortex at medium intensity for 5 sec. This process should be performed three times in order to remove loosely bound bacteria.
    9. After washing, place each valve section in a 4 ml sterile culture tube containing 500 µl of PBS and homogenize the tissue for 2 min with a motorized pestle to detach tightly adhered bacteria.
    10. Take 100 µl aliquot from the homogenized valve suspension for serial dilution and plating of the final culture on BHI medium plates to determine the CFU (TF).
    11. Incubate inoculated plates at 37 °C in a 5% CO2 atmosphere. After 48 h, count colonies for CFU determination.
  4. Tissue preparation for analysis of adhesion by scanning electron microscopy (SEM)
    1. To visualize bacterial adhesion to heart valve sections by SEM, follow the inoculation protocol described above (steps 3a-3g).
    2. After incubation of tissue with bacteria, transfer each valve section into a 1.7 ml microcentrifuge tube containing 1 ml of HBSS and then vortex at medium intensity for 5 sec once.
      Note: It is important not to perform too many washes at this stage as the SEM sample preparation includes several washing steps.
    3. Upon washing, transfer the valve section to a labeled glass scintillation vial containing 15 ml of fixative solution.
    4. After the sample is fixed at 4 °C with gentle rocking for 48 h, it is ready for SEM analysis.
  5. Competition analysis of bacterial attachment
    1. On the same day of valve tissue processing, grow overnight bacterial cultures in triplicate. Unlike monoculture inoculations, this protocol requires marked strains so that they can be distinguished in CFU determination.
    2. For this study, S. mutans OMZ175 containing an erythromycin resistance cassette (ErmR) and S. mutans OMZ175 with the collagen binding protein gene (cnm) interrupted by a kanamycin resistance cassette (KanR) were used.
    3. To grow bacterial cultures, add 2 ml of BHI + Erm 10 µg ml-1 or BHI + Kan 1 mg ml-1 in a 4 ml sterile culture tube and inoculate by picking colonies from freshly streaked plates.
    4. Incubate cultures at 37 °C in a 5% CO2 atmosphere overnight (18-20 h) without aeration.
    5. On the following day, wash the valve sections twice at room temperature with HBSS buffer to remove residual antibiotics. For this, add 1 ml of HBSS into two clean empty wells of the same plate and transfer the sections using sterile tweezers. Gently rock plate containing valve sections for 5 min and set the rocker to a setting of 40-50 motions per minute for each wash step.
    6. In the meantime, centrifuge the overnight cultures at 3,500 x g for 10 min at 4 °C, wash twice with sterile PBS and resuspend cultures with the initial culture volume.
    7. For each strain replicate, add 2.7 ml of fresh EGM without antibiotics to a clean 4 ml sterile culture tube and 300 µl of the bacterial culture making a 1:10 dilution of the bacterial suspension (approximately 1 x 107 CFU ml-1).
    8. Transfer washed valve sections to a new 12-well plate and add 500 µl of the S. mutans OMZ175 (ErmR) and 500 µl of the S. mutanscnm (KanR) bacterial suspensions in EGM. In addition, take 100 µl of each mixed bacterial replicate for serial dilution and plating of the initial culture CFU (T0).
    9. Incubate the inoculated valve sections at 37 °C in a 5% CO2 atmosphere with gentle rocking for 90 min.
    10. After incubation, transfer each valve section into a 1.7 ml microcentrifuge tube containing 1 ml of HBSS and then vortex at medium intensity for 5 sec. This process should be repeated three times in order to remove loosely bound bacteria.
    11. After washing, place each valve section in a 4 ml sterile culture tube containing 500 µl of PBS and homogenize the tissue for 2 min with a motorized pestle to detach tightly adhered bacteria.
    12. Take 100 µl aliquot from the homogenized valve suspension for serial dilution and plating of the final culture on both BHI + Erm 10 µg ml-1 and BHI + Kan 1 mg ml-1 plates to determine the CFU (TF).
    13. Incubate inoculated plates at 37 °C in a 5% CO2 atmosphere. After 48 h, count colonies for CFU determination.

Data analysis

  1. For visualization of bacterial adherence, tissues should be thoroughly examined by SEM (Figure 2). The top and bottom parts of the tissues, which are the areas of the heart valve continuously exposed to arterial flow and are generally undamaged, are referred to as smooth surfaces. The edge of tissues, which are displaying collagen fibers due to rupture by the biopsy punch, is referred to as rough surfaces.


    Figure 2. Adhesion of S. mutans OMZ175 to human aortic valve sections. Adhesion of S. mutans to human aortic valve sections analyzed by SEM. Center picture is a low magnification (25x) image of the valve section (scale bar = 200 µm). Representative images of attached bacteria to rough (A) and smooth surfaces (B) were taken at a 5,000x magnification (scale bars = 1 µm). The ex vivo adhesion of S. mutans OMZ175 (black arrows) to human valve sections requires the collagen-binding protein Cnm as no bacteria is detected in the ∆cnm strain.

  2. To calculate the competitive index (CI), CFUs must be determined and applied in the following formula:



    where, Inoculum = T0; Substrate = TF.
  3. Values are then plotted on a column scatter plot and analyzed using one-way ANOVA with Bonferroni post hoc comparisons to determine substrate preferences among strain pairs. A CI value of 1 represents no difference in adherence between strains; a CI value lower than 1 represents less binding by test strain compared to the reference strain; a CI value higher than 1 represents more binding by the test strain compared to the reference strain.
    A representative graph showing the competitive index (CI) of S. mutans OMZ175 and its ∆cnm counterpart is shown below. Each point in the graph represents an individual experiment using different tissue samples. The variability comes from the nature of the tissues since specimens are not exactly the same for every patient (Figure 3).


    Figure 3. CI analysis of S. mutans OMZ175 and its ∆cnm counterpart. The CI values were calculated from CFU at T0 and TF for both strains using the CI formula described above. The mean CI was higher than 1 indicating that S. mutans OMZ175 outcompetes its ∆cnm counterpart. Expression of Cnm in OMZ175 enhances the bacterial capacity to adhere human aortic valve tissues.

Notes

  1. All protocols involving human subject must be previously approved by an Institutional Review Board. This protocol was established using discarded tissues from patients undergoing cardiac surgery for aortic valve replacement due to calcific stenosis. Patients younger than 18 years old, pregnant women and HIV-positive patients were excluded from the original study.
  2. All tissues should be individually and thoroughly evaluated prior to the ex vivo binding analysis. This is due to the different levels of calcification, inflammation and tissue damage in each patient.
  3. Tissues should be processed within 6 h of obtaining them. If required, tissues may be processed and placed in EGM + gentamicin 300 µg ml-1 for up to 72 h before performing colonization assay.
  4. For this study, tissue visualization by SEM was performed by the University of Rochester Medical Center Electron Microscopy Core. Samples were analyzed using a Zeiss-Auriga focused ion beam field emission scanning electron microscope (FIB-FE-SEM) and the images were captured using the attached Gatan Erlangshen digital camera.
  5. The number of input bacteria (total number of bacterial cells added to the tissues) for the competition experiment was previously determined to be approximately similar for S. mutans OMZ175 and its ∆cnm counterpart. Similarly, CFU input should be determined for any bacterial strains to be tested prior to competition analysis. To maintain reproducibility, we suggest adjusting bacterial cultures to specific OD600 nm to match the number of CFUs between strains.

Recipes

  1. 1x phosphate buffer solution (PBS)
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    2 mM KH2PO4
  2. Fixative solution
    4% paraformaldehyde
    2.5% glutaraldehyde
    0.1 M sodium cacodylate
  3. EGM-MV Bulletkit
    500 ml EGM complete medium
    5% fetal bovine serum
    500 mg hydrocortisone
    6 mg bovine brain extract
    0.1% human recombinant epidermal growth factor

Acknowledgments

We would like to thank Michael Swartz and Peter Knight from the University of Rochester for sample collection. This work was supported by the NIH-NIDCR (R01 DE022559). A.A.-R. was supported by NIH-NHLBI (F31 HL124951). I.A.F. was supported by the São Paulo Research Foundation (FAPESP, Brazil, 2013/25080-7). This protocol is a modified version from a study by Chuang-Smith et al., 2009.

References

  1. Banas, J. A. (2004). Virulence properties of Streptococcus mutans. Front Biosci 9: 1267-1277.
  2. Chuang-Smith, O. N., Wells, C. L., Henry-Stanley, M. J. and Dunny, G. M. (2010). Acceleration of Enterococcus faecalis biofilm formation by aggregation substance expression in an ex vivo model of cardiac valve colonization. PLoS One 5(12): e15798.
  3. Freires, I. A., Aviles-Reyes, A., Kitten, T., Simpson-Haidaris, P. J., Swartz, M., Knight, P. A., Rosalen, P. L., Lemos, J. A. and Abranches, J. (2016). Heterologous expression of Streptococcus mutans Cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence. Virulence 3: 1-12.
  4. Que, Y. A. and Moreillon, P. (2011). Infective endocarditis. Nat Rev Cardiol 8: 322-336.
  5. Yetkin, E. and Waltenberger, J. (2009). Molecular and cellular mechanisms of aortic stenosis. Int J Cardiol 135: 4-13.

简介

病原体与宿主组织的相互作用是成功定居和建立感染的关键步骤。在菌血症期间,病原体几乎可以达到人体内的所有器官(例如心脏,肾脏,脾脏),但是宿主的免疫力,血流和组织完整性通常防止细菌定植。然而,患有心脏病(例如先天性心脏病,动脉粥样硬化,钙化性主动脉狭窄,人工瓣膜受体)的患者处于较高的细菌感染风险。该方案从已建立的远端猪心脏粘连模型改编而成,并且利用从经历主动脉瓣置换手术的患者获得的心脏组织的可用性。在该方案中,使用新鲜组织来评估与心血管感染相关的细菌病原体(例如变形链球菌)与人主动脉瓣组织的直接相互作用。

背景 口腔病原体变形链球菌被认为是龋齿中的主要病原体,也可以与感染性心内膜炎(IE)等口腔外感染有关(Banas,2004)。 IE通常由心脏瓣膜内皮的病变引发,其导致形成主要由血小板,炎性细胞,纤维蛋白和其它细胞外基质(ECM)蛋白(例如胶原)组成的无菌血栓,层粘连蛋白)(Que和Moreillon,2011)。其他心血管疾病,如钙化狭窄和动脉粥样硬化,也可能导致组织损伤导致ECM蛋白的暴露和重塑(Yetkin和Waltenberger,2009)。然后,该环境为能够与宿主组分相互作用的不同病原体提供适合的定植靶标。因此,相关工具和实验模型的开发可能使我们更好地了解病原体与心脏组织的相互作用。基于由Chuang-Smith等人于2010年使用来自猪的主动脉心脏瓣膜建立的先前方案,我们开发了一种使用人心脏瓣膜从组织粘附测定经历主动脉瓣置换的患者(Freires等人,2016)。虽然该模型不再现与疾病相关的免疫应答和其它宿主因素,但它提供了相对便宜的系统来评估给定生物体与人心脏瓣膜组织直接相互作用的能力。此外,虽然这种模式需要与心脏手术单位密切合作,但这种类型的手术(主动脉瓣置换术)也常规地在发达国家的健康科学中心进行(Yetkin和Waltenberger,2009 )。

关键字:变形链球菌, 胶原, 粘附, 离体, 主动脉瓣膜, 心血管感染

材料和试剂

  1. 无菌标本容器(Fisher Scientific,目录号:16-320-730)
  2. 12孔组织培养板(Corning,Falcon ®,目录号:351143)
  3. 无菌培养管(4ml)(Fisher Scientific,目录号:14-956-3D)
  4. 微量离心管(1.7ml)(Fisher Scientific,目录号:S348903)
  5. 玻璃闪烁瓶(20ml)(Sigma-Aldrich,目录号:Z253081)
  6. 无菌培养管(15ml)(Fisher Scientific,目录号:14-956-6D)
  7. 所需的菌株(例如,例如,变形链球菌OMZ175)
  8. 推荐的心脏组织
  9. EGM-MV子弹套件(Lonza,目录号:CC-3125)
  10. 庆大霉素(Sigma-Aldrich,目录号:G1397)
  11. 脑心浸液(BHI)(BD,Bacto TM,目录号:237500)
  12. Hank的平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:14025092)
  13. 红霉素(Sigma-Aldrich,目录号:E5389)
  14. 卡那霉素(Sigma-Aldrich,目录号:K1377)
  15. 氯化钠(NaCl)(Avantor Performance Materials,J.T.Baker ,目录号:3628-01)
  16. 氯化钾(KCl)(Avantor Performance Materials,J.T.Baker ,目录号:3045-01)
  17. 磷酸二氢钠(Na 2 HPO 4)(Avantor,Performance Materials,JTBaker,目录号: 3827-01)
  18. 磷酸二氢钾(KH 2 O 3 PO 4)(AvantorPerformance Materials,JT Baker,目录号: 3246-01)
  19. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  20. 戊二醛(Sigma-Aldrich,目录号:340855)
  21. 三水合二钠(Sigma-Aldrich,目录号:C0250)
  22. 琼脂(Fisher Scientific,目录号:BP1423)
  23. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F0392)
  24. 氢化可的松
  25. 牛脑提取物
  26. 人重组表皮生长因子
  27. 1x磷酸缓冲溶液(PBS)(参见食谱)
  28. 固定溶液(参见食谱)

设备

  1. 生物安全柜2级(Nuaire,型号:Labgard ES节能二级,A2型,目录号:NU-425-600)
  2. 3 mm皮肤活检穿孔(Acuderm,目录号:P325)
  3. 不锈钢钳(Sigma-Aldrich,目录号:Z168696)
  4. 离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Multifuge TM 1 S-R)
  5. 涡流
  6. 电动杵(Kimble Chase Life Science and Research Products,目录号:7495400000)
  7. pH计
  8. 摇杆(可靠科学,型号:55D)
  9. CO 2培养箱(VWR;型号:2325)
  10. 蔡司 - Auriga聚焦离子束场发射扫描电子显微镜(FIB-FE-SEM)
  11. Gatan Erlangshen数码相机

程序

  1. 样品收集和准备
    1. 否则将被丢弃的主动脉瓣组织应从心脏手术专家接受主动脉瓣置换的患者中无菌除去。
    2. 手术后立即将组织置于无菌标本容器中,保存在冰上,并立即送至实验室进行处理。
  2. 组织准备
    1. 在生物安全柜中,打开包含心脏组织的小瓶,并在进一步加工之前评估钙化水平,炎症和损伤。理想情况下,瓣膜切片应从没有钙化,炎症征象或残留血液的平滑区域获得(图1)。
    2. 使用活检穿孔器,牢牢地按压在组织的选定区域以切割并从其余组织中移除部分(图1)。


      图1.来自主动脉瓣组织的组织选择获得主要组织后,应立即将其送至实验室进行评估和准备。只有没有钙化或炎症迹象的区域选择用3 mm活检穿刺切片。

    3. 使用无菌镊子收集切片,并将其置于含有1ml补充EGM +庆大霉素300μg/ ml的12孔板的各个孔中。
    4. 可以有效获得的部分数量取决于组织的质量和尺寸以及要进行的实验类型(见下文)。
    5. 将含有阀部分的12孔板在37℃下在5%CO 2气氛中孵育过夜(18-20小时)。
  3. 细菌附着于主动脉瓣组织
    1. 在瓣膜组织处理的同一天,一式三份培养一夜细菌培养物。成长。变形菌 OMZ175,在4ml无菌培养管中加入2ml BHI,并通过从新鲜条纹板中挑选单个分离的菌落进行接种。
    2. 在37℃下在5%CO 2气氛中孵育培养物过夜(18-20小时),不通风。
    3. 第二天,用HBSS缓冲液在室温下清洗两次阀门部分以除去残留的抗生素。为此,将1ml HBSS加入同一板的两个干净的空孔中,并使用无菌镊子转移切片。轻轻摇动板上含有阀门5分钟,将每个洗涤步骤的摇杆设定为每分钟40-50次。
    4. 同时,在4℃下以3,500×g离心过夜培养物10分钟,用无菌PBS洗涤两次,并以初始培养体积重新悬浮培养物。
    5. 对于每个菌株重复,将2.7ml没有抗生素的新鲜EGM加入到干净的4ml无菌培养管中,并加入300μl细菌培养物,使细菌悬浮液的1:10稀释(约1×10 7 / sup > CFU ml -1 )。
    6. 将洗涤的阀切片转移到新的12孔板上,并在EGM中加入1ml细菌悬浮液。另外,每个重复使用100μl进行连续稀释和初始培养CFU(T <0>)的铺板。
    7. 在37℃下在5%CO 2气氛中孵育板,轻轻晃动90分钟。
    8. 孵育后,将每个瓣膜部分转移到含有1ml HBSS的1.7ml微量离心管中,然后以中等强度涡旋5秒。这个过程应该进行三次,以便去除松散结合的细菌
    9. 洗涤后,将每个瓣膜部分置于含有500μlPBS的4ml无菌培养管中,并用电动杵将组织匀浆2分钟,以分离紧密粘附的细菌。
    10. 从均质阀悬浮液中取100μl等分试样进行连续稀释,并在BHI培养基平板上铺板最终培养物,以确定CFU(T )。
    11. 将接种的板在37℃下在5%CO 2气氛中孵育。 48小时后,计数菌落进行CFU测定。
  4. 通过扫描电子显微镜(SEM)分析粘附的组织制备
    1. 通过SEM观察细菌对心脏瓣膜切片的粘附,按照上述接种方案(步骤3a-3g)。
    2. 在用细菌培养组织后,将每个瓣膜部分转移到含有1ml HBSS的1.7ml微量离心管中,然后以中等强度涡旋5秒。
      注意:在这个阶段,重要的是不要进行太多的洗涤,因为SEM样品制备包括几个洗涤步骤。
    3. 洗涤后,将阀部分转移到含有15ml固定液的标记玻璃闪烁瓶中
    4. 样品在4℃下用轻微摇摆固定48小时后,即可进行SEM分析
  5. 细菌附着的竞争分析
    1. 在瓣膜组织处理的同一天,一式三份培养一夜细菌培养物。与单一培养接种不同,该方案需要明显的菌株,以便在CFU测定中可以区分。
    2. 对于这项研究,包含红霉素抗性盒(Erm R )和S 的变体链OMZ175。使用由卡那霉素抗性盒(Kan R )中断的具有胶原结合蛋白基因(OMG175)的变体链霉素OMZ175。
    3. 为了培养细菌培养物,在4ml无菌培养管中加入2ml BHI + Erm10μg/ ml,或BHI + Kan 1mg ml -1,在4ml无菌培养管中,并通过从新鲜的条纹板挑选殖民地。
    4. 在37℃下在5%CO 2气氛中孵育培养物过夜(18-20小时),不通风。
    5. 第二天,用HBSS缓冲液在室温下清洗两次阀门部分以除去残留的抗生素。为此,将1ml HBSS加入同一板的两个干净的空孔中,并使用无菌镊子转移切片。轻轻摇动板上含有阀门5分钟,将每个洗涤步骤的摇杆设定为每分钟40-50次。
    6. 同时,在4℃下以3,500×g离心过夜培养物10分钟,用无菌PBS洗涤两次,并以初始培养体积重新悬浮培养物。
    7. 对于每个菌株重复,将2.7ml没有抗生素的新鲜EGM加入到干净的4ml无菌培养管中,并加入300μl细菌培养物,使细菌悬浮液的1:10稀释(约1×10 7 / sup > CFU ml -1 )。
    8. 将洗涤的阀部分转移到新的12孔板中,并加入500μl的S。变形菌 OMZ175(Erm R )和500μl的S。在EGM中的变体链细胞悬浮液(Δ)。另外,取100μl的每个混合细菌重复进行连续稀释和初始培养CFU(T <0>)的铺板。
    9. 将接种的阀门部分在37℃下在5%CO 2/2气氛中孵育90分钟。轻轻摇摆90分钟。
    10. 孵育后,将每个瓣膜部分转移到含有1ml HBSS的1.7ml微量离心管中,然后以中等强度涡旋5秒。该过程应重复三次以除去松散结合的细菌。
    11. 洗涤后,将每个瓣膜部分置于含有500μlPBS的4ml无菌培养管中,并用电动杵将组织匀浆2分钟,以分离紧密粘附的细菌。
    12. 从均质阀悬浮液中取100μl等分试样进行连续稀释,并将最终培养物在BHI + Erm10μg/ ml和BHI + Kan 1mg ml -1上进行铺板>板以确定CFU(T
    13. 将接种的板在37℃下在5%CO 2气氛中孵育。 48小时后,计数菌落进行CFU测定。

数据分析

  1. 为了细菌粘附的可视化,组织应通过SEM彻底检查(图2)。组织的顶部和底部是心脏瓣膜连续暴露于动脉血流并且通常未损伤的区域,被称为光滑表面。由于活检冲孔破裂而显示胶原纤维的组织边缘被称为粗糙表面。


    图2.粘合力变形菌 OMZ175与人类主动脉瓣切片。粘连。通过SEM分析人类主动脉瓣切片的变体链。中心图像是阀部分的低倍率(25倍)图像(比例尺= 200μm)。以5,000x放大倍率(比例尺=1μm)取代附着细菌至粗糙(A)和光滑表面(B)的代表性图像。 &lt; em&gt;离体粘附。变性链霉素OMZ175(黑色箭头)对人体瓣膜切片需要胶原蛋白结合蛋白Cnm,因为在Δ菌株中没有检测到细菌。

  2. 要计算竞争力指数(CI),必须按照以下公式确定和应用CFU:



    Inoculum = T 0 ;基板= T
  3. 然后将值绘制在列散点图上,并使用具有Bonferroni事后比较的单因素方差分析进行分析,以确定菌株对之间的底物偏好。 CI值为1表示菌株之间的粘附性无差异;与参考菌株相比,低于1的CI值表示测试菌株的结合度较小;与参考菌株相比,高于1的CI值表示测试菌株的结合度 一个代表性的图表显示了S的竞争力指数(CI)。变形菌 OMZ175及其相对应的Δ如下所示。图中的每一点代表使用不同组织样本的单独实验。变异性来自组织的性质,因为每个患者的样本不完全相同(图3)

    图3.S分析。变体链 OMZ175及其Δ对应 使用上述CI式,对于两个菌株,在T 0 O和T F F CFU处计算CI值。平均CI高于1,表示S。变形菌 OMZ175比较其对应的Δ cnm 。 OMZ175中Cnm的表达增强细菌粘附人类主动脉瓣组织的能力。

笔记

  1. 涉及人类受试者的所有议定书必须事先得到机构审查委员会的批准。该方案使用由于钙化狭窄进行主动脉瓣置换的心脏手术患者的丢弃组织建立。 18岁以下的患者,孕妇和艾滋病毒阳性患者被排除在原来的研究之外。
  2. 所有组织应在离体结合分析之前单独和彻底评估。这是由于每个患者的钙化,炎症和组织损伤程度不同。
  3. 组织应在6 h内进行处理。如果需要,可以在进行定殖测定之前将组织加工并放置在EGM +庆大霉素300μg/ ml中达72小时。
  4. 对于这项研究,SEM的组织可视化由罗切斯特大学医学中心电子显微镜核心进行。使用Zeiss-Auriga聚焦离子束场发射扫描电子显微镜(FIB-FE-SEM)分析样品,并使用附加的Gatan Erlangshen数码相机捕获图像。
  5. 针对竞争实验的输入细菌数量(添加到组织中的细菌细胞的总数)先前被确定为与S大致相似。变体链 OMZ175及其Δ对应物。同样地,在竞争分析之前,应该对待测试的任何细菌菌株确定CFU输入。为了保持重复性,我们建议将细菌培养物调整至特定的OD 600 nm以匹配菌株之间的CFU数量。

食谱

  1. 1x磷酸盐缓冲溶液(PBS)
    137 mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4
    2mM KH PO 4
  2. 固定解决方案
    4%多聚甲醛
    2.5%戊二醛
    0.1M二聚氰酸钠
  3. EGM-MV Bulletkit
    500毫升EGM完成培养基
    5%胎牛血清
    500毫克氢化可的松
    6毫克牛脑提取物
    0.1%人重组表皮生长因子

致谢

感谢罗切斯特大学的Michael Swartz和Peter Knight的样品收集。这项工作得到NIH-NIDCR(R01 DE022559)的支持。 A.A.-R.由NIH-NHLBI(F31 HL124951)支持。 I.A.F.得到了圣保罗研究基金会(FAPESP,Brazil,2013 / 25080-7)的支持。该协议是由Chuang-Smith等人,2009年的研究的修改版本。

参考

  1. Banas,JA(2004)。&nbsp; 变形链球菌。前面的Biosci 9:1267-1277。
  2. 创业史密斯,ON,Wells,CL,亨利 - 斯坦利,MJ和Dunny,GM(2010)。&nbsp; 通过心脏瓣膜定植的离体模型中的聚集物质表达来促进粪肠球菌生物膜形成。 PLoS One < / em> 5(12):e15798。
  3. Freares,IA,Aviles-Reyes,A.,Kitten,T.,Simpson-Haidaris,PJ,Swartz,M.,Knight,PA,Rosalen,PL,Lemos,JAand Abranches,J。(2016)一个class =“ke-insertfile”href =“https://www.ncbi.nlm.nih.gov/pubmed/?term=Heterologous+expression+of+Streptococcus+mutans+Cnm+in+Lactococcus+lactis+promotes+细胞内+入侵%2C +粘附+至+人类心脏+ + +组织和毒力+“。目标=“_ blank”>乳酸乳球菌中的变异链球菌Cnm的异源表达促进细胞内侵袭,对人心脏组织的粘附和毒力。毒力< / em> 3:1-12。
  4. Que,YA和Moreillon,P.(2011)。感染性心内膜炎 Nat Rev Cardiol 8:322-336。
  5. Yetkin,E.和Waltenberger,J.(2009)。&nbsp; Int J Cardiol 135:4-13。
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引用:Avilés-Reyes, A., Freires, I. A., Rosalen, P. L., Lemos, J. A. and Abranches, J. (2017). Ex vivo Model of Human Aortic Valve Bacterial Colonization. Bio-protocol 7(11): e2316. DOI: 10.21769/BioProtoc.2316.
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