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Preparation of Candida albicans Biofilms Using an in vivo Rat Central Venous Catheter Model
使用体内大鼠中心静脉导管制备白色念珠菌生物膜   

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参见作者原研究论文

本实验方案简略版
PLOS Pathogens
Aug 2012

Abstract

In vivo biofilms grown on medical devices are necessary to understand the interactions of the fungal biofilm and the host environment in which it is most commonly found. This protocol describes a way to grow Candida albicans biofilms on the interior lumen of central venous catheters surgically implanted into rats, which mimics quite well the clinical cases of biofilms found on human central venous catheters. These infected catheters can then be studied via a multitude of different experiments, including cell counting by plating, imaging the catheters under light or electron microscopy, or comparing the relative content of in vivo biofilms to in vitro biofilms and planktonic cultures. These biofilms also provide enough high quality RNA for transcriptional profiling.

Keywords: Candida (念珠菌), Biofilm (生物膜), In vivo model (体内模型), Catheter infection (导管感染)

Materials and Reagents

  1. 350 g Specific-pathogen-free male Sprague-Dawley rats (Harlan)
  2. Plate containing grown Candida albicans colonies
  3. Ethylene oxide
  4. 4 way Large Bore (lipid resistant) Stopcock (Baxter, catalog number: 2C62047 )
  5. Xylazine (Sigma-Aldrich, catalog number: X1126 )
  6. Ketamine (Sigma-Aldrich, catalog number: K2753 )
  7. Bacitracin (Sigma-Aldrich, catalog number: B0125 )
  8. Bacto-peptone (BD Biosciences, catalog number: 211677 )
  9. Bacto-yeast extract (BD Biosciences, catalog number: 212750 )
  10. Betadine (SWIFT, catalog number: 159935 )
  11. Difco agar (BD Biosciences, catalog number: 214530 )
  12. Difco dextrose (BD Biosciences, catalog number: 215530 )
  13. Heparin 1,000 U/ml (Sigma-Aldrich, catalog number: H3393 )
  14. Intramedic PE 160 polyethylene tubing (BD Biosciences, catalog number: 427430 )
  15. Needle holder (World Precision Instruments, catalog number: 14109 )
  16. Silk braided sutures (size 2-0) (Thermo Fisher Scientific, catalog number: 14-516-130 )
  17. Size med rat jacket (Braintress Scientific, catalog number: RJ-M )
  18. Sodium chloride (Thermo Fisher Scientific, catalog number: S671-3 )
  19. Sterile surgical draping materials (autoclaved hospital towels)
  20. Surgical button attached to wire rat tether (Instech Solomon, catalog number: LW95S )
  21. Uridine (Sigma-Aldrich, catalog number: U3750 )
  22. Xylazine/ketamine anesthetic (see Recipes)
  23. YPD + uridine media (see Recipes)
  24. Heparinized saline (see Recipes)
  25. 0.85% saline solution (see Recipes)
  26. YPD + uridine plates (see Recipes)

Equipment

  1. Iris scissors (Braintree Scientific, catalog number: SC128 )
  2. Metzenbaum scissors (Braintree Scientific, catalog number: SC126 )
  3. Micro scissors (Braintree Scientific, catalog number: SC152 )
  4. Hartman Mosquito-Hemostatic forceps (World Precision Instruments, catalog number: 15920 )
  5. Adson forceps (Braintree Scientific, catalog number: FC028 )
  6. Micro-Tissue forceps (Braintree Scientific, catalog number: FC145 , FC146 , FC147 )
  7. Scalpels (Bladex, catalog number: 22-080-087 )
  8. 35 mm Surgical staples and associated stapler (Ethicon Endo-Surgery, catalog number: TR35B )
  9. Animal Care Facilities
  10. Gas sterilization chamber
  11. Hemocytometer
  12. Shaking incubator with adjustable temperatures and speeds
  13. Vein scissors

Procedure

  1. Cut polyethylene tubing into 54 cm segments and gas sterilize using ethylene oxide.
  2. Anesthetize rats with roughly 0.5 ml intraperitoneal (i.p.) injection with 1 mg/kg of xylazine/ketamine mixture.
    Note: Exact volume will depend on the size of the rat.

  3. Shave the neck, midscapular space, and anterior chest of the rats to be catheterized and scrub with betadine.
  4. Prep a sterile surgical space containing the rat in a supine position.
  5. Fill the catheter piece with 540 μl of heparin (100 U/ml) via a 2 way stopcock. Once filled, place stopcock in the locked position to ensure sterility.
  6. Create a vertical incision just right of the midline of the anterior neck and horizontal cut in the midscapular region. Tunnel the catheter subcutaneously from the midscapular region to the vertical incision (secure subcutaneous button end with staple at midscapular area leaving rat wire tether exposed).
  7. Use blunt surgical dissection to find and expose jugular vein and tie off cranial end.
  8. Make a small longitudinal incision in the wall of the internal jugular vein with vein scissors.
  9. Place catheter in incision and advance along the vein until approximately 2 cm above the right atrium. Check for proper placement by opening stopcock and checking for flash of blood flowing inside catheter.
  10. Secure catheter to the vein with 2-0 silk ties. Close stopcock.
  11. Close incision site with surgical staples.
  12. Treat surgical sites with bacitracin and place rat jacket on.
  13. Rats are allowed to rest for 24 h to recover from the catheter placement and their recovery from surgery assessed according to standard animal care protocols.
    Note: Throughout rest of study, check on health of rats every 8hr and flush catheters with heparinized saline once a day.
  14. During 24 h rest period for the rat, 5 ml of YPD + uridine culture in a 15 ml conical tube is inoculated with a single colony of Candida albicans growing on a plate. Ideally, colony should have grown large enough to be picked easily (usually takes ~ 2 days) but not so old that it starts to lose its circular shape (usually after about 10 days of growth). Most Candida strains will grow well on YPD + uridine agar plates, but any medium that allows the strain to be studied to grow well can be used.
  15. Inoculum is incubated overnight at 30 °C and 200 rpm.
  16. Cell concentration of overnight culture is determined by cell counting on a hemocytometer.
  17. Inoculum diluted to 1 x 106 cells/ml in 0.85% saline.
    Viable cell counts are confirmed by plating unused inoculum on YPD + uridine plates.
  18. 540 μl of diluted inoculum is added to the intraluminal portion of the catheter (via stopcock) and allowed to adhere for 6 h.
  19. Inoculum is then removed through stopcock in the open position and the catheter volume is filled with 100 U/ml heparinized saline. The stopcock is then closed.
  20. Microbes within the catheters are then allowed to grow for 48 h.
    Note: If desired, drugs can be administered into the catheter lumen (via stopcock) after 24 h of growth to test in vivo drug susceptibility of biofilms.
  21. At the end of the growth period, rats are sacrificed according to animal safety protocols. Catheters are removed from the rats in a sterile surgical environment with all sterile tools and supplies, and cut into roughly 1-2 cm long segments.
  22. At this point, protocols diverge based on the nature of the in vivo experiment being conducted.

Recipes

  1. Xylazine/ketamine anesthetic
    Combine 20 mg/ml xylazine and 50 mg/ml ketamine in a 1:3 vol/vol ratio
  2. YPD + uridine media
    Bacto-yeast extract (1%)
    10 g
    Bacto-peptone (2%)
    20 g
    Dextrose (2%)
    20 g
    Uridine
    0.08 g
    Distilled water
    1,000 ml
    Combine all ingredients and aliquot into 100 ml bottles. Autoclave bottles on liquid cycle to sterilize.
  3. Heparinized saline
    Dilute heparin to 100 U/ml in 0.85% saline solution
  4. 0.85% saline solution
    Sodium Chloride
    4.25 g
    Distilled water
    500 ml
    Autoclave on liquids cycle.
  5. YPD + uridine plates
    Bacto-yeast extract (1%)
    10 g
    Bacto-peptone (2%)
    20 g
    Dextrose (2%)
    20 g
    Bacto-agar (2%)
    20 g
    Uridine
    0.08 g
    Distilled water
    1,000 ml
    Combine all ingredients in 2 L flask and autoclave on liquids cycle to sterilize before pouring plates.

References

  1. Andes, D., Nett, J., Oschel, P., Albrecht, R., Marchillo, K. and Pitula, A. (2004). Development and characterization of an in vivo central venous catheter Candida albicans biofilm model. Infect Immun 72(10): 6023-6031.
  2. Taff, H. T., Nett, J. E., Zarnowski, R., Ross, K. M., Sanchez, H., Cain, M. T., Hamaker, J., Mitchell, A. P. and Andes, D. R. (2012). A Candida biofilm-induced pathway for matrix glucan delivery: implications for drug resistance. PLoS Pathog 8(8): e1002848.

简介

在医疗装置上生长的体内生物膜是必要的,以了解真菌生物膜和其中最常见的宿主环境的相互作用。 该协议描述了在手术植入大鼠中的中心静脉导管的内腔上生长白假丝酵母生物膜的方法,其相当好地模拟在人中心静脉导管上发现的生物膜的临床情况。 然后可以通过多种不同的实验研究这些感染的导管,包括通过电镀的细胞计数,在光学或电子显微镜下对导管成像,或者将体内生物膜的相对含量与 体外生物膜和浮游培养物。 这些生物膜还提供足够高质量的RNA进行转录分析。

关键字:念珠菌, 生物膜, 体内模型, 导管感染

材料和试剂

  1. 350g无特定病原体的雄性Sprague-Dawley大鼠(Harlan)
  2. 含有生长的白色念珠菌菌落的平板
  3. 环氧乙烷
  4. 4路大口径(耐脂质)止回阀(Baxter,目录号:2C62047)
  5. 甲苯噻嗪(Sigma-Aldrich,目录号:X1126)
  6. 氯胺酮(Sigma-Aldrich,目录号:K2753)
  7. 杆菌肽(Sigma-Aldrich,目录号:B0125)
  8. 细菌蛋白胨(BD Biosciences,目录号:211677)
  9. 细菌酵母提取物(BD Biosciences,目录号:212750)
  10. Betadine(SWIFT,目录号:159935)
  11. Difco琼脂(BD Biosciences,目录号:214530)
  12. Difco葡萄糖(BD Biosciences,目录号:215530)
  13. 肝素1000U/ml(Sigma-Aldrich,目录号:H3393)
  14. Intramedic PE 160聚乙烯管(BD Biosciences,目录号:427430)
  15. 针座(World Precision Instruments,目录号:14109)
  16. 丝编织缝线(尺寸2-0)(Thermo Fisher Scientific,目录号:14-516-130)
  17. 大鼠套(Braintress Scientific,目录号:RJ-M)
  18. 氯化钠(Thermo Fisher Scientific,目录号:S671-3)
  19. 无菌手术巾材料(高压灭菌医院毛巾)
  20. 手术按钮连接到线大鼠系绳(Instech Solomon,目录号:LW95S)
  21. 尿苷(Sigma-Aldrich,目录号:U3750)
  22. 甲苯噻嗪/氯胺酮麻醉剂(见配方)
  23. YPD +尿苷培养基(参见配方)
  24. 肝素化盐水(见配方)
  25. 0.85%盐水溶液(见配方)
  26. YPD +尿苷板(见配方)

设备

  1. 虹膜剪刀(Braintree Scientific,目录号:SC128)
  2. Metzenbaum剪刀(Braintree Scientific,目录号:SC126)
  3. 微型剪刀(Braintree Scientific,目录号:SC152)
  4. Hartman蚊子止血钳(世界精密仪器,目录号:15920)
  5. Adson镊子(Braintree Scientific,目录号:FC028)
  6. 微组织钳(Braintree Scientific,目录号:FC145,FC146,FC147)
  7. 手术刀(Bladex,目录号:22-080-087)
  8. 动物保健设施
  9. 气体灭菌室
  10. 血细胞计数器
  11. 以可调温度和速度摇动培养箱
  12. 静脉剪刀

程序

  1. 将聚乙烯管切成54厘米的段,并使用环氧乙烷进行气体灭菌
  2. 麻醉大鼠与大约0.5毫升腹膜内注射1毫克/公斤甲苯噻嗪/氯胺酮混合物。
    注意:精确的体积将取决于大鼠的大小。
  3. 刮胡子的颈部,中间间隙和前胸部插入导管并用betadine擦洗。
  4. 准备一个无菌手术空间包含大鼠在仰卧位。
  5. 用540μl肝素(100U/ml)通过双向活栓填充导管片。 一旦填充,将活塞放在锁定位置,以确保无菌
  6. 创建一个垂直切口正好在前颈部的中线和水平切口在肩胛中区域。 从肩胛间区域到垂直切口(安全的皮下按钮末端,在中间肩胛部区域有钉缝,露出大鼠线系绳)将导管皮下地导入。
  7. 使用钝的手术解剖,找到并暴露颈静脉,绑住头端
  8. 用静脉剪刀在颈内静脉的壁上做一个小的纵向切口
  9. 将导管置于切口中,沿着静脉前进,直到右心房上方约2厘米。 通过打开活塞并检查导管内流动的血液闪烁来检查是否正确放置
  10. 用2-0丝扣将导管固定到静脉。 关闭旋塞。
  11. 用外科缝合器闭合切口部位。
  12. 用杆菌肽处理手术部位,并放上大鼠夹克
  13. 使大鼠休息24小时以从导管放置中恢复,并根据标准动物护理方案评估其从手术中恢复。
    注意:在整个研究过程中,每8小时检查一次大鼠的健康状况,每天用肝素化盐水冲洗导管。
  14. 在大鼠的24小时休息期间,在15ml锥形管中5ml的YPD +尿苷培养物接种在板上生长的白色念珠菌的单个菌落。理想情况下,菌落应该增长到足够容易被挑选(通常需要〜2天),但不是那么老,以至于开始失去其圆形(通常在约10天的生长后)。大多数假丝酵母菌株在YPD +尿苷琼脂平板上生长良好,但可以使用允许研究菌株生长良好的任何培养基。
  15. 将接种物在30℃和200rpm下温育过夜
  16. 过夜培养物的细胞浓度通过血细胞计数器上的细胞计数来确定
  17. 将接种物在0.85%盐水中稀释至1×10 6个细胞/ml 通过将未使用的接种物接种在YPD +尿苷平板上来证实存活细胞计数
  18. 将540μl稀释的接种物加入到导管的腔内部分(通过旋塞阀),并使其粘附6小时。
  19. 然后在打开位置通过活栓取出接种物,并用100U/ml肝素化盐水填充导管体积。然后关闭旋塞阀。
  20. 然后允许导管中的微生物生长48小时 注意:如果需要,可以在24小时的生长后将药物给予导管管腔(通过旋塞阀),以测试生物膜的体内药物敏感性。
  21. 在生长期结束时,根据动物安全方案处死大鼠。 使用所有无菌工具和用品在无菌手术环境中从大鼠中取出导管,并切割成大约1-2cm长的段。
  22. 在这一点上,协议基于正在进行的实验实验的性质而发散

食谱

  1. 甲苯噻嗪/氯胺酮麻醉剂
    以1:3体积/体积比混合20mg/ml甲苯噻嗪和50mg/ml氯胺酮
  2. YPD +尿苷培养基
    细菌酵母提取物(1%) 10克
    细菌蛋白胨(2%)
    20克
    葡萄糖(2%)
    20克
    尿嘧啶
    0.08克
    蒸馏水
    1000 ml
    将所有成分混合并等分至100 ml瓶中。 高压灭菌瓶上的液体循环灭菌。
  3. 肝素化盐水
    在0.85%盐水溶液中稀释肝素至100U/ml
  4. 0.85%盐水溶液
    氯化钠
    4.25克
    蒸馏水
    500 ml
    液体循环高压灭菌。
  5. YPD +尿苷板
    细菌酵母提取物(1%) 10克
    细菌蛋白胨(2%)
    20克
    葡萄糖(2%)
    20克
    细菌琼脂(2%)
    20克
    尿嘧啶
    0.08克
    蒸馏水
    1000 ml
    将所有成分在2L烧瓶中混合,高压灭菌器在液体循环中灭菌,然后倒入板中

参考文献

  1. Andes,D.,Nett,J.,Oschel,P.,Albrecht,R.,Marchillo,K.and Pitula,A。(2004)。 体内中心静脉导管的发育和表征假丝酵母 白蛋白生物膜模型。 Infect Immun 72(10):6023-6031。
  2. Taff,HT,Nett,JE,Zarnowski,R.,Ross,KM,Sanchez,H.,Cain,MT,Hamaker,J.,Mitchell,AP and Andes,DR(2012)。假丝酵母生物膜诱导的基质葡聚糖递送途径:对药物抗性的影响。 PLoS Pathog 8(8):e1002848。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Taff, H. T., Marchillo, K. and Andes, D. R. (2013). Preparation of Candida albicans Biofilms Using an in vivo Rat Central Venous Catheter Model . Bio-protocol 3(14): e823. DOI: 10.21769/BioProtoc.823.
  2. Taff, H. T., Nett, J. E., Zarnowski, R., Ross, K. M., Sanchez, H., Cain, M. T., Hamaker, J., Mitchell, A. P. and Andes, D. R. (2012). A Candida biofilm-induced pathway for matrix glucan delivery: implications for drug resistance. PLoS Pathog 8(8): e1002848.
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