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Evaluation of Burkholderia cepacia Complex Bacteria Pathogenicity Using Caenorhabditis elegans
利用秀丽隐杆线虫评估洋葱伯克霍尔德菌复合体的致病性   

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参见作者原研究论文

本实验方案简略版
PLOS ONE
Nov 2015

Abstract

This protocol describes two biological assays to evaluate pathogenicity of Burkholderia cepacia complex (Bcc) strains against the nematode Caenorhabditis elegans. Specifically, these two assays allow one to identify if the under-investigated Bcc strains are able to kill the nematodes by intestinal colonization (slow killing assay, SKA) or by toxins production (fast killing assay, FKA). The principal differences between the two assays rely on the different killing kinetics for worms.

Keywords: Burkholderia cepacia complex strains (洋葱伯克氏菌菌株), Caenorhabditis elegans (秀丽隐杆线虫), Animal model (动物模型)

Background

The Burkholderia cepacia complex (Bcc) occupies a critical position among Gram-negative multi-drug resistant bacteria. It consists of at least 20 closely related species. Many Bcc strains are multi drug and pandrug-resistant opportunistic human pathogens caused problematic lung infections in immune-compromised individuals, including cystic fibrosis (CF) patients. The use of non-vertebrate host model can be useful for dissecting virulence and pathogenicity determinants as well as identifying novel therapeutic targets (Kothe et al., 2003).

There are a good number of assays for detecting Bcc virulence against a large panel of host models, in liquid or in solid surface. However, some of those are mostly focused on phenotypic observations, which are difficult to detect and have a low reproducibility (Cardona et al., 2005). Herein, we developed two assays based on the analysis of surviving worms, which is a more reproducible and allows easy and fast comparison among the Bcc strains tested. In addition, these assays permit the detection of death mechanisms of Bcc towards nematode.

These killing assays allow us to identify bacterial strains that are able to colonize the nematode intestine and produce diffusible toxins capable of killing the host.

Materials and Reagents

  1. 15 ml Falcon tubes (Corning, Falcon®, catalog number: 352095 )
  2. 3.5 cm diameter agar plates (Corning, catalog number: 430165 )
  3. Caenorhabditis elegans
  4. Burkholderia cepacia complex (Bcc) strains
  5. E. coli OP50
  6. NaOH
  7. Bleach (Aurora)
  8. NGM agar
  9. PGS agar
  10. Sodium chloride (NaCl)
(Sigma-Aldrich, catalog number: S9888 )
  11. Tryptone
(Conda, catalog number: 1612 )
  12. Yeast extract (Conda, catalog number: 1702 )
  13. Peptone (Conda, catalog number: 1602 )
  14. European agar (Conda, catalog number: 1800 )
  15. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
  16. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
  17. Cholesterol (Sigma-Aldrich, catalog number: C3045 )
  18. Glucose (Conda, catalog number: 1900 )
  19. Sorbitol (Sigma-Aldrich, catalog number: S1876 )
  20. Potassium phosphate monobasic (KH2PO4)
(Sigma-Aldrich, catalog number: P5655 )
  21. Sodium phosphate dibasic (Na2HPO4)
(Sigma-Aldrich, catalog number: S5136 )
  22. LB broth (see Recipes)
  23. NGM agar medium (see Recipes)
  24. PGS agar medium (see Recipes)
  25. M9 buffer (see Recipes)

Equipment

  1. Centrifuge
  2. 20 °C chamber
  3. 37 °C shaking incubator
  4. Dissecting microscope
  5. Platinum loop

Software

  1. Graph-pad Prism 5 software

Procedure

  1. Day 1
    1. Synchronize worms with bleaching protocols: use C. elegans plates with many gravid hermaphrodites (Stiernagle, 2006). Wash the plates with sterile H2O.
    2. Collect the liquid in a sterile 15 ml Falcon tube. Add H2O to a total volume of 3.5 ml.
    3. Mix 0.5 ml 5 N NaOH with 1 ml bleach. Make this solution fresh just before use! Add to the centrifuge tube with the worms.
    4. Vortex the tube for a few seconds. Repeat vortexing every 2 min for a total of 10 min.
    5. Spin the tube in a centrifuge for 30 sec at 1,300 x g to pellet the released eggs.
    6. Aspirate to 0.1 ml.
    7. Add sterile H2O to 5 ml. Vortex for a few seconds.
    8. Repeat steps A6 and A7.
    9. Transfer the eggs in the remaining 0.1 ml of liquid to the edge of a clean NGM plate seeded with an E. coli OP50 lawn and incubate at 20 °C.
      Note: If you use C. elegans mutants that do not change their intrinsic nature under high temperature, you can incubate eggs at 25 °C as some mutants grow slower than WT.
    10. Inoculate Bcc strains in 15 ml Falcon tubes containing 3 ml of LB and incubate the tubes at 37 °C for 24 h in shaking condition (220 rpm).
      Note: As controls, use E. coli OP50 in the place of Bcc strains.

  2. Day 2
    1. Normalize Bcc cultures at 1.7 OD/ml and seed 50 μl of the culture on 3.5 cm diameter plates containing 3 ml of NGM agar (slow killing assay, SKA) or PGS agar (fast killing assay, FKA). Incubate the plates O/N at 37 °C.
      Note: Incubation of Bcc strains at 37 °C should never exceed 16 h. Do not store Bcc seeded plates at 4 °C, as many of these pathogens are very sensitive to temperature and this can cause variation. 
    2. Check the developmental stage of worms. After 24 h worms should be at larval stage L2 or L3 when grown at 20 °C.

  3. Day 3
    1. Wash synchronized worms at larval stage 4 (L4) off plates with M9 buffer and collect them in 15 ml Corning tubes. 

    2. Wash worms 2-3 times with M9 buffer to remove residual bacterial cells. 

    3. Spot 30-40 L4 worms on the plates seeded with Bcc strains (5 replicas). 

      Note: Before adding the worms, plates should be kept at room temperature to cool them down after the incubation at 37 °C. 

    4. Count worms at time 0. Incubate the plates at 20 °C and perform daily count of surviving worms up to day 5 (for FKA) and day 6 (for SKA).
      Note: A worm is considered dead when it no longer responds to gentle touch with a platinum wire. 

    5. At the end of the experiment, calculate the average percentage of surviving worms. 

    6. Evaluate Bcc pathogenicity. The virulence ranking (VR) ranges from 0 to 3 and it is based on the percentage of surviving worms after the period of observation. A Bcc strain is considered to be non-pathogenic (VR = 0) when the percentage of worms alive at the conclusion of the period of observation ranges from 100% to 80%; VR = 1 corresponds to a percentage of worms alive between 79% to 50%; VR = 2 corresponds to a percentage of worms alive between 49% to 6%; finally, the VR is considered 3 when the percentage of surviving worms was ≤ 5% (Figure 1).


      Figure 1. Kaplan-Meier survival plots for L4 stage WT worms fed with E. coli OP50 (solid lines), Burkholderia metallica on NGM (dashed lines), Burkholderia metallica on PGS (dotted lines). n: Number of worms at day 0. The pathogenicity of Bcc strain B. metallica on SKA (n = 80) was compared with the ability on FKA (n = 113).

Data analysis

Kaplan-Meier survival curves can be generated and analyzed using Graph-pad Prism 5 software. Comparisons vs. control for both the C. elegans are performed using Fisher’s exact test to account for possible non-Normality in the data. Bonferroni-Holm correction of P-values is used to account for the multiple comparisons performed. More details can be found in Tedesco et al. (2015).

Notes

  1. These protocols can be very useful for the identification of virulent Bcc strains and have a high reproducibility. However, slight variation in percentage of mortality can be observed, as the nematode is an in vivo model. To minimize those variations, worms and Bcc strains should be always maintained in the same conditions, using the same incubation times and temperatures.
  2. The principal concern relies on nematode’s larval stage. Only worms at larval stage 4 should be used. This is because worms at larval stage L3 or L2 can be more susceptible to pathogens, while older worms carry eggs, which can hamper the counting process.
  3. Timing is central. Please plan your experiment so that the experiment could be done using plates and animals that were prepared at the same time.
  4. Bcc strains can display different VR in the two assays. In our study we found a high variation in pathogenicity: strain B. metallica LMG 24068 and B. stabilis LMG 14294 had a maximum score in both assays, while B. cepacia LMG 1222 had VR = 3 in FKA and VR = 1 in SKA. B. multivorans LMG 13010 instead showed no pathogenicity at all VR = 0 in both assays.

Recipes

  1. LB broth (1 L)
    10 g NaCl
    10 g tryptone
    5 g yeast extract
    Add H2O to 1 L
    Sterilize by autoclaving.
  2. NGM agar medium (1L)
    2.5 g peptone
    17 g European agar
    2.9 g NaCl
    Add H2O to 1 L
    Sterilize by autoclaving.
    After solution cools down, add 1 ml autoclaved/sterile 1 M MgSO4, 1 ml autoclaved/sterile, 1 M CaCl2, 25 ml autoclaved/sterile 1 M KPO4, 1 ml cholesterol 5 mg/ml dissolved in 100% ethanol.
  3. PGS agar medium (1 L)
    12 g peptone
    12 g glucose
    27.25 g sorbitol
    17 g European agar
    2.9 g NaCl
    Add H2O to 1 L
    Sterilize by autoclaving.
    After solution cools down, add 1 ml autoclaved/sterile 1 M MgSO4, 1 ml autoclaved/sterile, 1 M CaCl2, 25 ml autoclaved/sterile 1 M KPO4, 1 ml cholesterol 5 mg/ml dissolved in 100% ethanol.
  4. M9 buffer (1 L)
    3 g KH2PO4
    6 g Na2HPO4
    5 g NaCl
    Add H2O to 1 L
    Sterilize by autoclaving.
    After solution cools down, add 1 ml autoclaved/sterile 1 M MgSO4.

Acknowledgments

Nematode strains used in this work were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Burkholderia cepacia complex strains, used in this work, were kindly provided by Prof. Peter Vandamme, University of Gent, Belgium. This work was supported by the EU-KBBE 2012-2016 project PharmaSea, grant No. 312184.

References

  1. Cardona, S. T., Wopperer, J., Eberl, L. and Valvano, M. A. (2005). Diverse pathogenicity of Burkholderia cepacia complex strains in the Caenorhabditis elegans host model. FEMS Microbiol Lett 250(1): 97-104.
  2. Kothe, M., Antl, M., Huber, B., Stoecker, K., Ebrecht, D., Steinmetz, I. and Eberl, L. (2003). Killing of Caenorhabditis elegans by Burkholderia cepacia is controlled by the cep quorum-sensing system. Cell Microbiol 5(5): 343-351.
  3. Stiernagle, T. (2006). Maintenance of C. elegans. WormBook.
  4. Tedesco, P., Visone, M., Parrilli, E., Tutino, M. L., Perrin, E., Maida, I., Fani, R., Ballestriero, F., Santos, R., Pinilla, C., Di Schiavi, E., Tegos, G. and de Pascale, D. (2015). Investigating the role of the host multidrug resistance associated protein transporter family in Burkholderia cepacia complex pathogenicity using a Caenorhabditis elegans infection model. PLoS One 10(11): e0142883.

简介

该协议描述了评价洋葱伯克霍尔德菌(Burkholderia cepacia)复合物(Bcc)菌株对线虫秀丽隐杆线虫的致病性的两种生物学测定。具体地,这两个测定允许人们通过肠道定殖(慢速杀灭测定,SKA)或通过毒素生产(快速杀灭测定,FKA)来鉴定未研究的Bcc菌株是否能够杀死线虫。两种测定法之间的主要差异依赖于对蠕虫的不同杀死动力学。

[背景] Burkholderia cepacia 复合体(Bcc)在革兰氏阴性多药耐药细菌中占据关键位置。它由至少20种密切相关的物种组成。许多Bcc株是多药物和抗药性的机会性人类病原体在免疫受损的个体(包括囊性纤维化(CF))患者中引起有问题的肺部感染。使用非脊椎动物宿主模型可以用于解剖毒性和致病性决定因素以及鉴定新的治疗靶点(Kothe等人,2003)。
   存在用于检测液体或固体表面中针对大组宿主模型的Bcc毒力的很多测定。然而,其中一些主要集中在表型观察,其难以检测并具有低重现性(Cardona等人,2005)。在这里,我们开发了基于对存活蠕虫的分析的两种测定,其是更可重复的,并允许在测试的Bcc菌株之间容易和快速的比较。此外,这些测定允许检测Bcc对线虫的死亡机制。
   这些杀伤测定允许我们鉴定能够定殖线虫肠并产生能杀死宿主的扩散毒素的细菌菌株。

关键字:洋葱伯克氏菌菌株, 秀丽隐杆线虫, 动物模型

材料和试剂

  1. 15ml Falcon管(Corning,Falcon ,目录号:352095)
  2. 3.5cm直径的琼脂平板(Corning,目录号:430165)
  3. Caenorhabditis elegans
  4. 洋葱伯克霍尔德菌(Burkholderia cepacia)复合物(Bcc)菌株
  5. E。大肠杆菌 OP50
  6. NaOH
  7. 漂白剂(极光)
  8. NGM琼脂
  9. PGS琼脂
  10. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  11. 胰蛋白胨(Conda,目录号:1612)
  12. 酵母提取物(Conda,目录号:1702)
  13. 蛋白胨(Conda,目录号:1602)
  14. 欧洲琼脂(Conda,目录号:1800)
  15. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M7506)
  16. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C1016)
  17. 胆固醇(Sigma-Aldrich,目录号:C3045)
  18. 葡萄糖(Conda,目录号:1900)
  19. 山梨醇(Sigma-Aldrich,目录号:S1876)
  20. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  21. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S5136)
  22. LB肉汤(见配方)
  23. NGM琼脂培养基(见配方)
  24. PGS琼脂培养基(见Recipes)
  25. M9缓冲区(请参阅配方)

设备

  1. 离心机
  2. 20°C室
  3. 37℃振荡培养箱
  4. 解剖显微镜
  5. 铂金回线

软件

  1. Graph-pad Prism 5软件

程序

  1. 第1天
    1. 将蠕虫与漂白协议同步:使用 C。 elegans板,具有许多妊娠雌雄同体(Stiernagle,2006)。用无菌H 2 O洗涤平板。
    2. 在无菌的15ml Falcon管中收集液体。加入H 2 O至总体积为3.5ml。
    3. 将0.5ml 5N NaOH与1ml漂白剂混合。使该溶液在使用前清新!添加到带有蠕虫的离心管中。
    4. 涡旋管几秒钟。每2分钟重复涡旋,共10分钟。
    5. 在离心机中以1300×g离心管中30秒以沉淀释放的蛋。
    6. 吸出至0.1 ml
    7. 将无菌H 2 O添加至5ml。漩涡几秒钟。
    8. 重复步骤A6和A7。
    9. 将剩余的0.1ml液体中的蛋转移到接种有E的干净NGM板的边缘。大肠杆菌 OP50草坪并在20℃下孵育 注意:如果你使用线虫突变体,在高温下不改变它们的固有性质,你可以在25°C孵化鸡蛋,因为一些突变体生长得比WT慢。
    10. 在含有3ml LB的15ml Falcon管中接种Bcc菌株,并在振荡条件(220rpm)下将管在37℃下孵育24小时。
      注意:作为对照,使用大肠杆菌OP50代替Bcc毒株
  2. 第2天
    1. 将1.7cc/ml的Bcc培养物标准化,并在含有3ml NGM琼脂(缓慢杀伤测定,SKA)或PGS琼脂(快速杀灭测定,FKA)的3.5cm直径平板上接种50μl培养物。孵育板O/N在37℃。
      注意:在37℃下孵育Bcc菌株不应超过16小时。不要将Bcc种子板存放在4°C,因为许多这些病原体对温度非常敏感,这可能导致变异。 
    2. 检查蠕虫的发育阶段。 24小时后,蠕虫应在幼虫阶段L2或L3,当生长在20°C。

  3. 第3天
    1. 在幼虫阶段4(L4)用M9缓冲液洗涤同步的蠕虫,并将其收集在15ml Corning管中。
    2. 用M9缓冲液洗涤蠕虫2-3次以除去残留的细菌细胞。
    3. 点播30-40 L4蠕虫在播种Bcc菌株的板上(5个复制品)。
      注意:在添加蠕虫之前,板应保持在室温以在37℃孵育后将其冷却。
    4. 在时间0计数蠕虫。将板在20℃下孵育,并且每天计数存活的蠕虫直到第5天(对于FKA)和第6天(对于SKA)。
      注意:当它不再响应用铂丝轻轻触摸时,蠕虫被认为死亡。
    5. 在实验结束时,计算存活蠕虫的平均百分比。
    6. 评估Bcc致病性。毒力等级(VR)的范围从0到3,它是基于观察后存活的蠕虫的百分比。当在观察期结束时存活的蠕虫的百分比为100%至80%时,认为Bcc菌株是非致病性的(VR = 0); VR = 1对应于79%至50%的蠕虫存活的百分比; VR = 2对应于49%至6%的蠕虫活的百分比;最后,当存活蠕虫的百分比≤5%时,VR被认为是3(图1)

      图1.用E处理的L4阶段WT蠕虫的Kaplan-Meier存活图。在PGS(虚线)上的大肠杆菌 OP50(实线),在伯明翰金属上(虚线),的蠕虫在第0天.Bcc菌株的致病性B。在SKA(n = 80)上的能力与FKA(n = 113)上的能力进行比较。

数据分析

可以使用Graph-pad Prism 5软件产生并分析Kaplan-Meier存活曲线。比较两者的对照。 elegans 是使用Fisher精确检验进行的,以考虑数据中可能的非正态性。 Bonferroni-Holm校正 P 值用于说明执行的多重比较。更多细节可以在Tedesco 中找到。 (2015)。

笔记

  1. 这些方案对于鉴定毒性Bcc菌株非常有用,并且具有高重现性。然而,可以观察到死亡率百分比的轻微变化,因为线虫是体内模型。为了使这些变化最小化,蠕虫和Bcc株应当始终在相同的条件下使用相同的温育时间和温度来维持。
  2. 主要关注依赖于线虫的幼虫阶段。只有幼虫阶段4的蠕虫应该使用。这是因为幼虫阶段L3或L2的蠕虫可能更容易受病原体影响,而较老的蠕虫携带卵,这可能阻碍计数过程。
  3. 时间是中心。请计划您的实验,以便可以使用同时准备的板和动物进行实验。
  4. Bcc菌株在两种测定中可以显示不同的VR。在我们的研究中,我们发现了致病性的高变异性:菌株B。 metallica LMG 24068和 B。稳定 LMG 14294在两种测定中具有最大得分,而。 cepacia LMG 1222在FKA中具有VR = 3,在SKA中VR = 1。 B。 multivorans LMG 13010在两种测定中都没有显示出致病性,在所有VR = 0。

食谱

  1. LB肉汤(1L)
    10克NaCl
    10g胰蛋白胨
    5g酵母提取物
    将H <2> O添加到1 L
    通过高压灭菌消毒。
  2. NGM琼脂培养基(1L) 2.5克蛋白胨 17克欧洲琼脂
    2.9克NaCl
    将H <2> O添加到1 L
    通过高压灭菌消毒。
    在溶液冷却后,加入1ml高压灭菌的/无菌的1M MgSO 4,1ml高压灭菌/无菌,1M CaCl 2,25ml高压灭菌/无菌1M KPO 4, sub> 4,1ml胆固醇5mg/ml溶于100%乙醇中
  3. PGS琼脂培养基(1L) 12克蛋白胨
    12克葡萄糖 27.25g山梨醇 17克欧洲琼脂
    2.9克NaCl
    将H <2> O添加到1 L
    通过高压灭菌消毒。
    在溶液冷却后,加入1ml高压灭菌的/无菌的1M MgSO 4,1ml高压灭菌/无菌,1M CaCl 2,25ml高压灭菌/无菌1M KPO 4, sub> 4,1ml胆固醇5mg/ml溶于100%乙醇中
  4. M9缓冲液(1 L)
    3g KH sub 2 PO 4 sub
    6g Na 2 HPO 4
    5克NaCl
    将H <2> O添加到1 L
    通过高压灭菌消毒。
    溶液冷却后,加入1ml高压灭菌的/无菌的1M MgSO 4

致谢

本研究中使用的线虫菌株由CGC提供,CGC由NIH研究基础设施计划办公室(P40OD010440)资助。本研究中使用的洋葱伯克霍尔德菌(Burkholderia cepacia)复合菌株由比利时根特大学Peter Vandamme教授友情提供。这项工作得到EU-KBBE 2012-2016项目PharmaSea,拨款号312184的支持。

参考文献

  1. Cardona,ST,Wopperer,J.,Eberl,L.and Valvano,MA(2005)。  复杂菌株在秀丽隐杆线虫宿主模型中的不同致病性。 FEMS Microbiol Lett > 250(1):97-104。
  2. Kothe,M.,Antl,M.,Huber,B.,Stoecker,K.,Ebrecht,D.,Steinmetz,I.和Eberl,L。(2003)。  通过 Burkholderia cepacia 杀死Caenorhabditis elegans 细胞 微生物 5(5):343-351。
  3. Stiernagle,T。(2006)。维护C。 elegans 。 WormBook 。
  4. Tedesco,P.,Visone,M.,Parrilli,E.,Tutino,ML,Perrin,E.,Maida,I.,Fani,R.,Ballestriero,F.,Santos,R.,Pinilla, Schiavi,E.,Tegos,G。和de Pascale,D。(2015)。  使用秀丽隐杆线虫感染模型研究宿主多药耐药相关蛋白转运蛋白家族在洋葱伯克霍尔德杆菌复合物致病性中的作用。 PLoS One 10(11):e0142883。
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Copyright: © 2016 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. Tedesco, P., Di Schiavi, E., Palma Esposito, F. and de Pascale, D. (2016). Evaluation of Burkholderia cepacia Complex Bacteria Pathogenicity Using Caenorhabditis elegans. Bio-protocol 6(20): e1964. DOI: 10.21769/BioProtoc.1964.
  2. Tedesco, P., Visone, M., Parrilli, E., Tutino, M. L., Perrin, E., Maida, I., Fani, R., Ballestriero, F., Santos, R., Pinilla, C., Di Schiavi, E., Tegos, G. and de Pascale, D. (2015). Investigating the role of the host multidrug resistance associated protein transporter family in Burkholderia cepacia complex pathogenicity using a Caenorhabditis elegans infection model. PLoS One 10(11): e0142883.
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