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In vitro Dynamic Model of a Catheterized Bladder and Biofilm Assay
膀胱导管插入和生物膜试验的体外动态模型   

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Abstract

Biofilm formation on catheters is thought to contribute to persistence of catheter-associated urinary tract infections (CAUTI) which represent the most frequent nosocomial infections. Understanding of factors relevant for CAUTI pathogenesis and evaluation of new therapeutics or interference strategies requires a model system that mirrors the physico-chemical conditions prevailing in a catheterized human bladder. The described in vitro dynamic model of a catheterized bladder enables to emulate many of the characteristics of a catheterized human bladder albeit in the absence of a bladder epithelium. A minor modification compared to the original model system (Stickler, et al., 1999) allows temperature maintenance of the top 10 cm of the catheter, thereby enabling reproducible monitoring of biofilm formation on the internal catheter surface.

Materials and Reagents

  1. NaCl (Carl Roth, catalog number: 9265.1 )
  2. 70% ethanol
  3. dH2O
  4. NaCl (Carl Roth, catalog number: 9265.1)
  5. Tryptone (Carl Roth, catalog number: 8952.3 )
  6. Yeast extract (Carl Roth, catalog number: 2362.2 )
  7. Agar-agar (Carl Roth, catalog number: 5210.2 )
  8. Na2SO4 (Carl Roth, catalog number: 8560.1 )
  9. CaCl2.2H2O (Carl Roth, catalog number: CN93.1 )
  10. MgCl2.6H2O (Carl Roth, catalog number: 2189.1 )
  11. NaCl (Carl Roth, catalog number: 9265.1)
  12. Na3C6H5O7.2H2O (Carl Roth, catalog number: 3580.1 )
  13. (COONa)2 (Carl Roth, catalog number: 4267.1 )
  14. KH2PO4 (Carl Roth, catalog number: 3904.1 )
  15. KCl (Carl Roth, catalog number: 6781.1 )
  16. NH4Cl (Carl Roth, catalog number: K298.2 )
  17. Urea (Carl Roth, catalog number: X999.3 )
  18. Tryptic soya broth/CASO medium (Carl Roth, catalog number: X938.1 )
  19. Gelatine (Carl Roth, catalog number: 4582.2 )
  20. NaOH (Carl Roth, catalog number: 6771.1 )
  21. LB medium (see Recipes)
  22. LB agar (see Recipes)
  23. Artificial or human urine (see Recipes)

Equipment

  1. Peristaltic pump for urine filtration (e.g. Heidolph Instruments GmbH, model: Pump drive 5006 )
  2. 10 L polycarbonate carboy for urine reservoir (e.g. Thermo Fisher Scientific, Nalgene®, 10 L PC with polypropylene cap with three inlets)
  3. SARTORIUS P Midi Cap filter cartridge (SARTORIUS, catalog number: 5235307H9 )
  4. Peristaltic multi-channel pump for urine feed (Watson-Marlow Pumps Group, model: 205U )
  5. Borosilicate Glass bladder models (Figure 1) (Georg Becker Laboreinrichtungen GmbH & Co KG, www.laborbecker.at)


    Figure 1. Glass bladder model scheme and dimensions. Schematic representation including length and inner diameter (ID) indications A and custom-made model B. Relevant parts are indicated: Artificial bladder compartment (1), temperature control compartment (2), connection for incoming (3) and outgoing (4) temperature control water circuit, artificial urethra for catheter insertion (5).

  6. Water bath with connections for external water circle (e.g. LAUDA, model: E10 )
  7. Tripod or other holders for mounting of glass bladder models (e.g. Bochem Instrumente GmbH, Portable table frame TG, catalog number: TG500 )
  8. Silicone stopper (35 mm) with 6 mm decentralized hole
  9. Glass tube (15 cm, 6 mm outer diameter = OD)
    Note: We use cut 1 ml glass pipettes (BRAND).
  10. Silicone tubings: 2 m (ID 3 mm, OD 5 mm)
  11. Silicone tubings: 1 m (ID 9 mm)
  12. Silicone tubings: 1 m (ID 5-6 mm)
  13. Tygon tubings (ID 8 mm) (VWR International, catalog number: 228-1294 )
  14. Tube Connectors (PP, 3-5mm - 6-10 mm) (Bartelt, catalog number: 37.559 )
  15. T-connectors (PP) for tubings (ID 3 mm) (Carl Roth, catalog number: E763.1 )
  16. Urinary Catheter (all silicone Ch14) (BARD, catalog number: 153509 )
  17. 10 ml syringe
  18. Drainage bags (e.g. SARSTEDT AG, catalog number: 74.5220.005 )
  19. Clamps (e.g. VWR International, catalog number: 229-0072 )
  20. Ultrasonic cleaner (BANDELIN electronic, model: Sonorex RK100H )
  21. Vortex (e.g. VWR International, catalog number: 444-0206 )
  22. Recommended (air vents as breathing barrier for urine reservoir flask) (e.g. VWR International, catalog number: 28144-160 )

Procedure

  1. Preparation (Day 1)
    1. Overnight cultures (ONC) of test strains in LB medium (1 ml) are grown at 37 °C/180 rpm in an incubator shaker for 16 h.
    2. Autoclave bladder models, media bottle, filter cartridge, tubings and connectors prepared for the number of models you want to run (either in beakers or in aluminium foil).
    3. Depending on the time frame of the experiment, artificial urine (~700 ml/day/bladder model) is prepared according to Stickler et al. (1999) and filtered through a 0.45 µm filter cartridge (Sartobran P) into an autoclaved bottle (Figure 2).


      Figure 2. Assembly of filter sterilization for large volumes of artificial urine. The same procedure is applicable for pooled human urine. Relevant visible parts are marked according to equipment list.

  2. Inoculation (Day 2)
    1. Prepare a culture of test strains: 0.5 ml ONC in 20 ml sterile artificial urine incubated for 2-4 h at 37 °C/180 rpm.
    2. Install the heating water cycle (37 °C) and connect the urine supply tubings. Insert the tubing in the peristaltic pump (Figure 3 and Figure 4; Figure 5 for schematic representation).


      Figure 3. Connection of water cycle and urine supply. Water bath is directly connected to the bladder models (not shown), the urine is pumped from the reservoir using a peristaltic pump (use a channel for each model). Relevant visible parts are marked according to equipment list.


      Figure 4. Bladder model system with three bladders after connection of tubings prior to catheter insertion. Relevant visible parts are marked according to equipment list.


      Figure 5. Schematic representation of the closed system necessary for setup of a catheterized bladder model. For simplicity, one bladder without mounting equipment or water bath are shown. Relevant parts are marked according to equipment list.

    3. Insert a sterile Foley-catheter in each bladder and inflate the retention balloon with 10 ml H2O. Connect each catheter with the drainage bag.
    4. Fill the urine supply system and the bladders using the peristaltic pump to a level just underneath the eye hole of the catheter (Figure 6; Note: Check that the catheters are tight so that the bladders are not leaking!) Stop the urine flow and clamp of the tubings after the pump.


      Figure 6. Close up of artificial bladder model after catheter insertion and filling A and after cultivation B. Relevant parts are indicated.

    5. Measure OD600 of the cultures. Inoculate each model with an appropriate amount of cells for your experiment (depending on the infection phase you are interested the inoculum can range e.g. from 1 x 106 to 1 x 109 cfu). To do so remove the corresponding volume of urine from the models and replace it with the same volume of culture.
    6. Remove the clamp and start the urine flow 30-60 min after inoculation (~30 ml/h, corresponds to 1.7 rpm using Watson-Marlow 205U using 5 mm silicone tubings).

  3. Quantification of colonization
    1. Stop the urine flow, mix or sample the bladder content and carefully remove it to completion (transfer the bladder content to a 50 ml tube). You can estimate the volume by using a balance (1 g ~ 1 ml).
    2. After disconnection of the drainage tubing from the catheter, slowly deflate the retention ball. Carefully remove the catheter from the bladder and transfer it to a sterile surface. Cut away the tip of the catheter (including the eyehole) and transfer it to a microcentrifuge tube. Rinse the internal surface of the remaining catheter to remove unattached cells from bladder content with 2 ml of 0.9 % NaCl solution or sterile urine.
    3. Cut and transfer 1 cm catheter segments to 1,000 µl 0.9% NaCl solution.
    4. Sonicate the bladder contents and the saline solutions with the catheter pieces for 5 min (room temperature, 35 kHz, one round), then vortex for additional 2 min.
    5. Dilute suspensions in 0.9% NaCl solution (bladder to 10-6, catheter suspensions to 10-5).
    6. Plate 50-100 µl of dilutions on LB agar plates (with or without selection).
    7. Following incubation calculate the colony counts to receive cfu/ml bladder content and cfu/cm catheter. In competition experiments, determine the competitive index (the ratio of harvested CFU of e.g. the mutant strain relative to e.g. the parental strain, divided by their ratio in the inoculum).

Representative data



Figure 7. Effect of random mutations on competitive bladder and catheter colonization. Dynamic catheterized bladder models were inoculated with equal amounts of Proteus mirabilis HI4320 wild type and transposon mutant cells HI4320mut1 and HI4320mut2, respectively. Following irrigation with artificial urine for 24 h, population distribution in the residual urine of the artificial bladder and on the internal catheter surface was determined based on CFU. CI values indicate the ratio between the mutant type and the wild type strain in the output (bladder suspension or catheter) divided by the ratio of the two strains in the inoculum. Symbols represent CI values obtained in individual experiments, the line indicates the mean CI value.

Notes

  1. Due to high variation observed with the in vitro model system due to varying efficiency of biofilm disintegration, experiments need to be performed in triplicate. For comparison of fitness of two strains (e.g. wild type and mutant), competition experiments (e.g. inoculating two strains in the same artificial bladder) are recommended (Reisner et al., 2014 ).
  2. Statistical Analysis: Independent challenges and co-challenge experiments are routinely analyzed using paired t-test. If a data set does not fulfill necessary criteria for a parametric test, the nonparametric Wilcoxon matched-pair test can be applied.

Recipes

  1. Luria-Bertani broth (LB) and agar (1 L) (Bertani, 1951)
    Mix 10 g Tryptone, 5 g NaCl and 5 g Yeast Extract
    Add dH2O to 1 L
    For LB agar add agar to a final concentration of 1.5 %
    Sterilize by autoclaving and store at room temperature
  2. Artificial urine (Stickler et al., 1999)
    Add the following substances and dissolve in pre-warmed dH2O (max. 40 °C):

    g/L
    Na2SO4
    2.30
    CaCl2.2H2O
    0.65
    MgCl2.6H2O
    0.65
    NaCl
    4.60
    Na3C6H5O7.2H2O
    0.65
    (COONa)2
    0.02
    KH2PO4
    2.80
    KCl
    1.60
    NH4Cl
    1.00
    Urea
    25.00
    Adjust pH by addition of NaOH tablets to 6.1
    Add gelatine (5 g/L) and stir with heating to dissolve (max 40 °C liquid temperature)
    Add autoclaved tryptic soya broth (TSB, 1 g /L) to filtered urine

Acknowledgments

Establishment of the protocol in the lab was funded by the Austrian Science Fund FWF: P21434-B18 (to A.R.). The protocol is adapted and modified from a previous protocol (Stickler et al., 1999).

References

  1. Bertani, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62(3): 293-300.
  2. Stickler, D. J., Morris, N. S. and Winters, C. (1999). Simple physical model to study formation and physiology of biofilms on urethral catheters. Methods Enzymol 310: 494-501.
  3. Reisner, A., Maierl, M., Jörger, M., Krause, R., Berger, D., Haid, A., Tesic, D. and Zechner, E. L. (2014). Type 1 fimbriae contribute to catheter-associated urinary tract infections caused by Escherichia coli. J Bacteriol 196(5): 931-939.

简介

导管上的生物膜形成被认为有助于导管相关尿路感染(CAUTI)的持久性,其代表最常见的医院感染。 理解与CAUTI发病机理相关的因素和评价新的治疗或干扰策略需要一个反映导尿人膀胱中存在的物理化学条件的模型系统。 所描述的导管插入的膀胱的体外动态模型使得能够模拟导管插入的人膀胱的许多特征,尽管在没有膀胱上皮的情况下。 与原始模型系统(Stickler,et al。,1999)相比较小的修改允许导管的顶部10cm的温度维持,从而能够可再生地监测内部导管表面上的生物膜形成。

材料和试剂

  1. NaCl(Carl Roth,目录号:9265.1)
  2. 70%乙醇
  3. dH 2 2 O
  4. NaCl(Carl Roth,目录号:9265.1)
  5. 胰蛋白胨(Carl Roth,目录号:8952.3)
  6. 酵母提取物(Carl Roth,目录号:2362.2)
  7. 琼脂(Carl Roth,目录号:5210.2)

  8. (Carl Roth,目录号:8560.1)
  9. (Carl Roth,目录号:CN93.1)
  10. (Carl Roth,目录号:2189.1)
  11. NaCl(Carl Roth,目录号:9265.1)
  12. Na 3 H 6 H 6 H 5 O 7 Na 2 SO 4 H 6 H 6 H 6 H 6 CH 4 CH 2 CH 3/Carbo Roth,目录号:3580.1)
  13. (COONa)2(Carl Roth,目录号:4267.1)

  14. (Carl Roth,目录号:3904.1)</b>
  15. KCl(Carl Roth,目录号:6781.1)
  16. NH 4 Cl(Carl Roth,目录号:K298.2)
  17. 尿素(Carl Roth,目录号:X999.3)
  18. 胰蛋白酶大豆肉汤/CASO培养基(Carl Roth,目录号:X938.1)
  19. 明胶(Carl Roth,目录号:4582.2)
  20. NaOH(Carl Roth,目录号:6771.1)
  21. LB介质(见配方)
  22. LB琼脂(见配方)
  23. 人工或人尿(见配方)

设备

  1. 用于尿液过滤的蠕动泵(例如,Heidolph Instruments GmbH,型号:Pump drive 5006)
  2. 用于尿液储存器的10L聚碳酸酯杯(例如Thermo Fisher Scientific,Nalgene ,具有三个入口的聚丙烯盖的10L PC)
  3. SARTORIUS P Midi Cap滤芯(SARTORIUS,目录号:5235307H9)
  4. 用于尿液进料的蠕动多通道泵(Watson-Marlow Pumps Group,型号:205U)
  5. 硼硅酸盐玻璃膀胱模型(图1)(Georg Becker Laboreinrichtungen GmbH& Co KG, www.laborbecker.at


    图1.玻璃囊模型方案和尺寸包括长度和内径(ID)适应症A和定制模型B.示意图相关部分:人工膀胱隔室 (1),温度控制室(2),输入连接(3)和输出(4)温度控制水回路,用于导管插入的人造尿道(5)。
  6. 带有外部水圈连接的水浴(如 LAUDA,型号:E10)
  7. 用于安装玻璃气囊型号的三脚架或其他支架(例如,Bochem Instrumente GmbH,便携式台架TG,目录号:TG500)
  8. 硅胶塞(35 mm),带6 mm分散孔
  9. 玻璃管(15cm,外径6mm = OD)
    注意:我们使用1ml玻璃移液管(BRAND)。
  10. 硅胶管:2 m(ID 3 mm,OD 5 mm)
  11. 硅胶管:1 m(ID 9 mm)
  12. 硅胶管:1 m(ID 5-6 mm)
  13. Tygon管(ID 8mm)(VWR International,目录号:228-1294)
  14. 管连接器(PP,3-5mm-6-10mm)(Bartelt,目录号:37.559)
  15. T型连接器(PP)(ID 3 mm)(Carl Roth,目录号:E763.1)
  16. 尿道导管(所有硅胶Ch14)(BARD,目录号:153509)
  17. 10毫升注射器
  18. 排水袋(如 SARSTEDT AG,目录号:74.5220.005)
  19. 夹子(如 VWR International,目录号:229-0072)
  20. 超声波清洗机(BANDELIN电子,型号:Sonorex RK100H)
  21. Vortex(例如,VWR International,目录号:444-0206)
  22. 推荐(作为尿液储存瓶的呼吸屏障的通气孔)(例如VWR International,目录号:28144-160)

程序

  1. 准备(第1天)
    1. 在LB培养基(1ml)中的试验菌株的过夜培养物(ONC)在37℃/180rpm下在恒温摇床中生长16小时。
    2. 高压灭菌器膀胱型号,介质瓶,滤芯,管道 和为您要运行的模型数量准备的连接器   在烧杯或铝箔)。
    3. 根据时间框架   实验,人工尿(约700ml /天/膀胱模型) 根据Stickler等人(1999)制备并通过0.45μm过滤   μm滤筒(Sartobran P)装入高压灭菌瓶(图2)。


      图2.大型过滤器消毒装配 体积的人造尿液。同样的程序适用于合并   人尿。 相关可见部件根据设备标记 列表。

  2. 接种(第2天)
    1. 准备测试菌株的培养物:0.5ml ONC在20ml无菌人造尿中,在37℃/180rpm温育2-4小时。
    2. 安装加热水循环(37°C)并连接尿液供应   管道。 将管道插入蠕动泵(图3和图 4;图5为示意图)。


      图3.连接 水循环和尿液供应。水浴直接连接到 膀胱模型(未示出),使用尿液从贮存器泵出  蠕动泵(每个型号使用一个通道)。相关可见 部件根据设备列表进行标记

      图4.膀胱 模型系统与三个气囊连接后的管道 导管插入。 相关的可视部件根据标记 设备列表。


      图5.关闭的示意图 系统设置导尿膀胱模型所需的 简单,一个膀胱没有安装设备或水浴 显示。 相关部件根据设备列表进行标记。

    3. 在每个膀胱插入无菌Foley导管和充气 保留气球用10ml H 2 O。 连接每个导管 排水袋。
    4. 填充尿液供应系统和膀胱使用   将蠕动泵的水平位于眼孔的正下方 导管(图6; 注意:检查导管是否紧,   气囊不泄漏!)停止尿液流动并夹住管道   泵后。


      图6.人工膀胱模型的关闭 在导管插入和填充A后和培养B后。相关   部件。

    5. 测量培养物的OD 600。 接种适当数量的细胞为每个模型 实验(取决于感兴趣的阶段你感兴趣的 接种物的范围可以是例如从1×10 6到1×10 9 pfu。 要这样做删除 相应体积的尿液从模型并替换它 相同体积的培养物。
    6. 取出夹子并启动尿液   流速30-60分钟后(约30ml/h,对应于1.7rpm 使用Watson-Marlow 205U,使用5mm硅树脂管)。

  3. 定殖定量
    1. 停止尿流,混合或取样膀胱内容,并仔细 将其移除至完成(将膀胱内容物转移至50ml管)。 您可以使用天平(1 g〜1 ml)估算音量。
    2. 在导管从导管断开连接后,缓慢 放气保持球。 小心地从中取出导管 膀胱并将其转移至无菌表面。 切掉的尖端 导管(包括眼孔)并将其转移至微量离心机 管。 冲洗剩余导管的内表面以除去 未附着的细胞用2ml的0.9%NaCl溶液从膀胱含量 或无菌尿液
    3. 切割和转移1厘米导管段到1,000微升0.9%NaCl溶液
    4. 超声膀胱内容物和盐溶液与 导管片5分钟(室温,35kHz,一轮),然后 再涡旋2分钟
    5. 在0.9%NaCl溶液中稀释悬浮液(膀胱至10μL,导管悬浮液至10μL-5μL)。
    6. 在LB琼脂平板(有或无选择)上稀释50-100μl稀释液
    7. 孵育后计算菌落计数以接受cfu/ml 膀胱内容物和cfu/cm导管。 在竞争实验中, 确定竞争性指数(所收获的CFU的比例) 突变体菌株相对于例如亲本菌株的比例除以它们 比例)。

代表数据



图7.随机突变对竞争性膀胱和导管定殖的影响动物导尿管膀胱模型用等量的奇异变形杆菌HI4320野生型和转座子突变体细胞HI4320mut1和HI4320mut2接种, 分别。在用人造尿液灌洗24小时后,基于CFU测定人造膀胱的残余尿和内部导管表面上的群体分布。 CI值表示输出(膀胱悬浮液或导管)中突变型和野生型菌株之间的比例除以接种物中两种菌株的比例。符号表示在各个实验中获得的CI值,线表示平均CI值。

笔记

  1. 由于由于生物膜分解的不同效率,在体外模型系统中观察到高的变化,实验需要一式三份进行。用于比较两个菌株的适应性 (例如野生型和突变体),推荐竞争实验(例如,在同一人工膀胱中接种两个菌株)(Reisner等人 2014)。
  2. 统计分析:使用配对t检验常规地分析独立的攻击和共同攻击实验。 如果数据集不满足参数测试的必要标准,则可以应用非参数Wilcoxon匹配对测试。

食谱

  1. Luria-Bertani肉汤(LB)和琼脂(1L)(Bertani,1951)
    混合10g胰蛋白胨,5g NaCl和5g酵母提取物
    将dH <2> O添加到1 L
    对于LB琼脂,加入琼脂至最终浓度为1.5%
    通过高压灭菌消毒并在室温下贮存
  2. 人工尿(Stickler等人,1999)
    加入以下物质并溶解在预热的dH 2 O(最高40℃)中:

    g/L
    Na 2 4
    2.30
    CaCl 2 2H O
    0.65
    MgCl 2 6H <2> O
    0.65
    NaCl
    4.60
    Na 3 H 6 H 6 H 5 O 7 Na 2 SO 4 H 6 H 6 H 6 H 6 CH 4 CH 2 CH 3/sub> O
    0.65
    (COONa) 2
    0.02
    KH 2 PO 4
    2.80
    KCl
    1.60
    NH 4 Cl
    1.00
    尿素
    25.00
    通过加入NaOH片调节pH至6.1
    加入明胶(5g/L),加热搅拌溶解(最高40℃液体温度)
    加入高压灭菌的胰蛋白酶大豆肉汤(TSB,1g/L)以过滤尿液

致谢

实验室中的方案的建立由奥地利科学基金FWF:P21434-B18(到A.R.)资助。 该协议根据先前的协议被修改和修改(Stickler等人,1999)。

参考文献

  1. Bertani,G。(1951)。 关于溶血发生的研究。 I.The mode of phage liberation by lysogenic in Escherichia coli 。 62(3):293-300。
  2. Stickler,D.J.,Morris,N.S.and Winters,C。(1999)。 简单的物理模型,用于研究尿道导管上生物膜的形成和生理学。 Methods Enzymol 310:494-501。
  3. Reisner,A.,Maierl,M.,Jöger,M.,Krause,R.,Berger,D.,Haid,A.,Tesic,D.and Zechner,E.L。 1型菌毛有助于导致大肠杆菌引起的导管相关性尿路感染。 >。 J Bacteriol 196(5):931-939。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Maierl, M., Jörger, M., Rosker, P. and Reisner, A. (2015). In vitro Dynamic Model of a Catheterized Bladder and Biofilm Assay. Bio-protocol 5(2): e1381. DOI: 10.21769/BioProtoc.1381.
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