Biofilm Formation Assay in Pseudomonas syringae
丁香假单胞菌的生物膜形成实验   

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Molecular Plant Microbe Interactions
Dec 2018

 

Abstract

Pseudomonas syringae is a model plant pathogen that infects more than 50 plant species worldwide, thus leading to significant yield loss. Pseudomonas biofilm always adheres to the surfaces of medical devices or host cells, thereby contributing to infection. Biofilm formation can be visualized on numerous matrixes, including coverslips, silicone tubes, polypropylene and polystyrene. Confocal laser scanning microscopy can be used to visualize and analyze biofilm structure. In this study, we modified and applied the current method of P. aeruginosa biofilm measurement to P. syringae, and developed a convenient protocol to visualize P. syringae biofilm formation using a borosilicate glass tube as the matrix coupled with crystal violet staining.

Keywords: Biofilm (生物膜), Pseudomonas syringae (丁香假单胞菌), Plant pathogen (植物病原菌), Borosilicate glass (硼硅酸盐玻璃), Crystal violet (结晶紫), Visualization (可视化)

Background

Most Pseudomonas strains secrete exopolysaccharides, such as alginate, which is an important matrix molecule for biofilm formation (Hentzer et al., 2001; Nivens et al., 2001). Biofilm formed by the human pathogen P. aeruginosa plays important roles in its virulence and antibiotic resistance, and contributes to acute or chronic infections (Donlan and Costerton, 2002).

To date, various methods have been reported for biofilm characterization and quantification. Originally, biofilms were detected in microtiter plates made of polystyrene or polypropylene (O'Toole and Kolter, 1998; Merritt et al., 2005). During the growth of P. aeruginosa on a surface, the expression of genes involved in extracellular polysaccharide synthesis is induced (Davies et al., 1993; Davies et al., 1995), which promotes the adherence of cells to the surface. Crystal violet specifically stains the bacterial cells, and has been developed as a widely used dye for bacterial biofilm (George et al., 1998). Some recent studies have analyzed biofilm structure in a flow chamber coupled with confocal laser scanning microscopy (Sternberg and Tolker-Nielsen, 2006; Chua et al., 2016).

Biofilms formed by P. syringae strains have also been found in plant tissues (Osman et al., 1986; Fakhr et al., 1999; Preston et al., 2001). Alginate produced by P. syringae is an important polymer for P. syringae biofilm formation and contributes to its virulence and fitness, indicating its importance in plant-pathogen interaction (Preston et al., 2001; Engl et al., 2014). The formation of P. syringae and P. fluorescens biofilms can also be measured using crystal violet staining in microwell plates (Carezzano et al., 2017; Zhu et al., 2018; Patange et al., 2019).

In this study, we modified and applied the current method of P. aeruginosa biofilm measurement to P. syringae (Kong et al., 2015; Zhao et al., 2016; Shao et al., 2018). We present an economic, rapid and visual biofilm detection protocol that combines the use of borosilicate glass tubes and crystal violet staining methods, which have been efficiently used in our recent studies (Wang et al., 2018; Wang et al., 2019; Xie et al., 2019) for visualizing the biofilm of the model plant pathogen P. syringae.

Materials and Reagents

  1. 10 ml Borosilicate glass tube (ISOLAB, catalog number: 077.02.003)
  2. 14 ml sterile tube (SPL Lifescience, catalog number: 40014)
  3. Filter (PALL Lifesciences, catalog number: AP-4219)
  4. Strains P. syringae pv. phaseolicola 1448A (Psph) (Xiao et al., 2007) and rhpS deletion mutant (ΔrhpS) (Xie et al., 2019)
  5. NaOH (UNI-CHEM, catalog number: 1310-73-2) 
  6. MgSO4·7H2O (Aladdin, catalog number: 10025-84-0)
  7. K2HPO4 (Aladdin, catalog number: 7758-11-4) 
  8. BactoTM Proteose peptone No.3 (AOBOX, catalog number: 01-049)
  9. Rifampin (Aladdin, catalog number: 13292-46-1)
  10. Agar (MP Biomedicals, catalog number: 9002-18-0)
  11. Crystal violet (Beijing Dingguo, catalog number: 548-62-9)
  12. Glycerol (Beijing Bailingwei, catalog number: 262536)
  13. 100% ethanol (Honeywell, catalog number: 32221-2.5L)
  14. King's B (KB) (see Recipes)

Equipment

  1. 1 ml pipette (Eppendorf, catalog number: 3123000063)
  2. Benchtop shaking incubator (Labwit Scientific, model: ZWYR-240)
  3. Constant temperature incubator (Labwit Scientific, model: ZXDP-B2120) 
  4. EvolutionTM 350 UV-Vis Spectrophotometer (Thermo Fisher Scientific, catalog number: 912A0959)
  5. SynergyTM 2 Multi-Mode Microplate Reader (BioTek)
  6. Test tube stand (ISOLAB, catalog number: 079.01.005)
  7. -80 °C freezer (Thermo Scientific, catalog number: 5IDTSX)

Software

  1. Microsoft Office Excel 2016 and GraphPad Prism 8.0.2.

Procedure

Notes:

  1. Select wild-type P. syringae pv. phaseolicola 1448A (Psph) as the model strain (Xiao et al., 2007) and rhpS deletion mutant (ΔrhpS) (Xie et al., 2019) as the test strain.
  2. Perform the step-by-step protocol described in Figure 1.
  3. Perform the entire procedure gently to avoid damaging the biofilm.
  4. Collect the liquid crystal violet and ethanol waste liquor in specially labeled containers for professional disposal by trained staff, as per the safety regulations at City University of Hong Kong.


    Figure 1. Schematic step-by-step protocol for visualizing P. syringae biofilm formation. A. The Psph strain was activated on King’s B (KB) plate and cultured in liquid medium. B. Then the cultures were inoculated at 1:500 dilutions into KB liquid medium and incubated statically to sampling points. C. Then stained the biofilm by using 0.1% crystal violet. D. Measure the biofilm production at OD590nm. Two biological replicates were showed.

  1. Bacterial growth
    1. Collect a Psph colony from the glycerol stock culture frozen at -80 °C and inoculate on a King’s B (KB) plate supplemented with rifampicin (25 μg/ml). Incubate the KB plate at 28 °C for 36 h in a constant temperature incubator.
    2. Collect a single colony from the cultured plates and inoculate into a sterile 10 ml tube containing 2 ml KB liquid medium supplemented with rifampicin (25 μg/ml). Incubate the tube in a benchtop shaking incubator at 28 °C for 12 h with constant shaking at 220 rpm.

  2. Biofilm formation
    Inoculate the Psph culture (1:500 dilutions) into 16 sterile 10 ml borosilicate glass tubes containing 2 ml KB liquid medium (supplemented with rifampicin) and ensure consistent initial dose. Incubate the Psph-containing glass tubes at 28 °C in a constant temperature incubator without shaking.

  3. Biofilm visualization
    1. Harvest the biofilm samples at different time points. For Psph in our study, the biofilms were harvested at 24, 48, 72 and 96 h. Gently discard the planktonic cells with a 1 ml pipette and wash the tubes three times with sterile distilled water. Avoid damaging the biofilm formed on the tube wall.
    2. Stain the biofilm forming bacteria with 2.5 ml 0.1% crystal violet for 20 min without shaking. Discard the dye and wash the tubes with sterile distilled water to remove the unbound dye. Dry the tubes and take photographs (Figure 2A).

  4. Biofilm measurement
    1. Elute the biofilm with 2 ml 100% ethanol and shake the tubes at 220 rpm for 20 min to ensure that the dye has dissolved completely. Take photographs (Figure 2B).
    2. Measure the eluted samples at OD590nm using a spectrophotometer (2 ml) or Synergy 2 Plate Reader (BioTek) (100 μl). If the sample concentrations are too high, diluted them before measuring. Use an equal volume of 95%-100% ethanol as the blank control. Psph biofilm formation is shown in Figure 2C. The ΔrhpS produces lower biofilm compared with Psph wild-type strain (Figure 3).


      Figure 2. Visualization and quantification of biofilm in Psphwild-type strain. A. Biofilm samples were grown from 24 to 96 h. Biofilm adhered to borosilicate glass tubes at different time points were stained with crystal violet. B. The crystal violet bound to the biofilm on the wall of the tubes was eluted by ethanol. C. The elution samples were measured at OD590nm by using spectrophotometer or Synergy 2 Plate Reader (BioTek). Psph wild-type strain produced more biofilm at 96 h than 24 h. * represents P-value < 0.05. *** represents P-value < 0.001. Error bars indicate S.D. among four biological replicates. Two biological replicates were shown.


      Figure 3. The ΔrhpS strain produced less biofilm than did that in Psph wild-type strain. A. Biofilm produced by the Psph wild-type and the ΔrhpS strain were visualized using borosilicate glass tubes and stained with crystal violet at 96 h. B. The crystal violet bound to the biofilm on tube wall was eluted by ethanol. C. The Psph wild-type strain produced more biofilm than the ΔrhpS strain (P-value = 0.000136). *** represents P-value < 0.001. Error bars indicate S.D. among four biological replicates. Two biological replicates were shown.

Data analysis

Student’s t-tests were performed using Microsoft Office Excel 2016. Quantitative data (OD590nm) were collected from four biological replicates in the figures and tables (Tables 1 and 2).

Table 1. Biofilm production of Psph wild-type strain presented in Figure 2C. Two-sample equal variance was calculated by the following one-tailed Student’s t-test formula in Excel = TTEST (array1, array2, tails, type). Array 1 is the first data set. Array 2 is the second data set. Tails show the number of distribution tails (1 for the one-tailed distribution, 2 for two-tailed distribution). Type is the kind of t-test to perform (1 for paired, 2 for two-sample equal variance, and 3 for two-sample unequal variance. For example, we used TTEST (B2:E2, B3:E3, 1, 2), TTEST (B2:E2, B4:E4, 1, 2) and TTEST (B2:E2, B5:E5, 1, 2) for biofilm production at 48 h, 72 h, 96 h, compared to 24 h respectively. The P-values were showed in Excel H3, H4 and H5 respectively. * represents P-value < 0.05. ** represents P-value < 0.01. *** represents P-value < 0.001. Means and S.D. are shown in columns F and G. All experiments were repeated four times.


Table 2. Biofilm production of the Psph wild-type and the ΔrhpS strain presented in Figure 3C. Two-sample equal variance was calculated by the following Two-tailed Student’s t-test formula in Excel = TTEST (B2:E2, B3:E3, 2, 2). The P-values were showed in Column H3. * represents P-value < 0.05. ** represents P-value < 0.01. *** represents P-value <0.001. Means and S.D. are shown in Column F and G. All experiments were repeated four times.


In sum, the results showed that the wild-type Psph strain produced more biofilm in 48 h (P < 0.05), 72 h (P < 0.001) and 96 h (P < 0.001) than at 24 h (Figure 2 and Table 1). Besides, the ΔrhpS strain produced less biofilm than Psph wild-type strain (P < 0.001) at the same time point (96 h) (Figure 3 and Table 2).

Recipes

  1. King's B (KB) (King et al., 1954)
    BactoTM Proteose peptone No.3
    20.0 g/L
    K2HPO4
    1.5 g/L
    Glycerol
    15 ml/L
    Dissolve in 993.75 ml ddH2O and adjust the pH to 7.2. Add 15 g/L agar for solidified media and autoclave
    Dissolve 24.637 g MgSO4·7H2O in 100 ml ddH2O and sterilize this stock solution (1 M) using sterile filter (0.45 µm)
    Then, add 6.25 ml MgSO4·7H2O (1 M) (final concentration 1.5 g/L) into the autoclaved King's B medium when the temperature drops to 40 °C-50 °C

Acknowledgments

This project has been funded in Health Medical Research Fund [17160022 to X.D.]; National Natural Science Foundation of China Grants [31670127 to X.D.].
  This protocol was adapted from O'Toole et al. (1998) Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Molecular Microbiology, 30: 295-304.

Competing interests

Conflict of interest statement: None declared.

References

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简介

摘要: Pseudomonas syringae 是一种模式植物病原体,可感染全球50多种植物,从而导致显着的产量损失。 Pseudomonas 生物膜总是粘附在医疗器械或宿主细胞的表面上,从而导致感染。 生物膜形成可以在许多基质上可视化,包括盖玻片,硅胶管,聚丙烯和聚苯乙烯。 共聚焦激光扫描显微镜可用于可视化和分析生物膜结构。 在这项研究中,我们修改并应用了 P的当前方法。 铜绿假单胞菌生物膜测量 P. syringae ,并开发了一个方便的协议来可视化 P. 使用硼硅酸盐玻璃管作为基质与结晶紫染色结合,形成丁香科生物膜。

背景:大多数 Pseudomonas 菌株分泌外多糖,如海藻酸盐,藻酸盐是生物膜形成的重要基质分子(Hentzer et al。,2001; Nivens et al。,2001)。生物膜由人类病原体 P形成。铜绿假单胞菌在其毒力和抗生素抗性中起重要作用,并导致急性或慢性感染(Donlan和Costerton,2002)。

迄今为止,已经报道了用于生物膜表征和定量的各种方法。最初,在由聚苯乙烯或聚丙烯制成的微量滴定板中检测到生物膜(O'Toole和Kolter,1998; Merritt 等人,2005)。在 P的增长期间。表面上的铜绿假单胞菌诱导了细胞外多糖合成中涉及的基因的表达(Davies et al。,1993; Davies et al。,1995),促进细胞粘附于表面。结晶紫特别污染细菌细胞,并已被开发为广泛使用的细菌生物膜染料(George et al。,1998)。最近的一些研究已经分析了流动室中的生物膜结构以及共聚焦激光扫描显微镜(Sternberg和Tolker-Nielsen,2006; Chua 等人,2016)。

生物膜由 P形成。在植物组织中也发现了丁香科植物菌株(Osman et al。,1986; Fakhr et al。,1999; Preston et al。,2001)。由 P产生的藻酸盐。丁香科是 P的重要聚合物。丁香科生物膜形成并有助于其毒力和适应性,表明其在植物 - 病原体相互作用中的重要性(Preston et al。,2001; Engl et al。, 2014)。 P的形成。丁香科和 P.荧光素生物膜也可以使用微孔板中的结晶紫染色来测量(Carezzano et al。,2017; Zhu et al。,2018; Patange 等人,,2019)。

在这项研究中,我们修改并应用了 P的当前方法。铜绿假单胞菌生物膜测量 P. syringae (Kong et al。,2015; Zhao et al。,2016; Shao et al。,2018)。我们提出了一种经济,快速和可视化的生物膜检测方案,结合使用硼硅酸盐玻璃管和结晶紫染色方法,这些方法已在我们最近的研究中有效地使用(Wang et al。,2018; Wang et al。,2019; Xie et al。,2019),用于可视化模型植物病原体 P的生物膜。丁香。

关键字:生物膜, 丁香假单胞菌, 植物病原菌, 硼硅酸盐玻璃, 结晶紫, 可视化

材料和试剂

  1. 10毫升硼硅酸盐玻璃管(ISOLAB,目录号:077.02.003)
  2. 14毫升无菌管(SPL Lifescience,目录号:40014)
  3. 过滤器(PALL Lifesciences,目录号:AP-4219)
  4. 菌株 P.丁香科 pv。 phaseolicola 1448A( Psph )(Xiao et al。,2007)和 rhpS 缺失突变体(Δ rhpS )(谢等人,2019)
  5. NaOH(UNI-CHEM,目录号:1310-73-2)&nbsp;
  6. MgSO 4 ·7H 2 O(阿拉丁,目录号:10025-84-0)
  7. K 2 HPO 4 (阿拉丁,目录号:7758-11-4)&nbsp;
  8. Bacto TM 蛋白胨第3号(AOBOX,目录号:01-049)
  9. 利福平(阿拉丁,目录号:13292-46-1)
  10. 琼脂(MP Biomedicals,目录号:9002-18-0)
  11. 水晶紫(北京定国,目录号:548-62-9)
  12. 甘油(北京百灵威,目录号:262536)
  13. 100%乙醇(霍尼韦尔,目录号:32221-2.5L)
  14. King's B(KB)(见食谱)

设备

  1. 1毫升移液器(Eppendorf,目录号:3123000063)
  2. 台式振动培养箱(Labwit Scientific,型号:ZWYR-240)
  3. 恒温培养箱(Labwit Scientific,型号:ZXDP-B2120)&nbsp;
  4. Evolution TM 350紫外 - 可见分光光度计(赛默飞世尔科技,目录号:912A0959)
  5. Synergy TM 2多功能微孔板读板机(BioTek)
  6. 试管架(ISOLAB,目录号:079.01.005)
  7. -80°C冰箱(Thermo Scientific,目录号:5IDTSX)

软件

  1. Microsoft Office Excel 2016和GraphPad Prism 8.0.2。

程序

注意:

  1. 选择野生型 P. syringae pv。 phaseolicola 1448A( Psph )作为模型菌株(Xiao 等 ,2007)和 rhpS 缺失突变体(Δ rhpS )(Xie 等。 ,2019)作为试验菌株。
  2. 执行图1中描述的分步协议。
  3. 轻轻地执行整个过程以避免损坏生物膜。
  4. 根据香港城市大学的安全规定,由经过培训的员工将液晶紫和乙醇废液收集在特别贴有标签的容器中进行专业处理。


    图1.可视化的示意性逐步协议 P.丁香科 生物膜形成。 A. Psph 菌株在King's B(KB)平板上被激活并在液体培养基中培养。 B.然后将培养物以1:500稀释度接种到KB液体培养基中并静态孵育至取样点。 C.然后用0.1%结晶紫染色生物膜。 D.测量OD 590nm 处的生物膜产量。显示了两个生物学重复。

  1. 细菌生长
    1. 从在-80℃冷冻的甘油储备培养物中收集 Psph 菌落,并接种在补充有利福平(25μg/ ml)的King's B(KB)平板上。将KB板在28℃下在恒温培养箱中孵育36小时。
    2. 从培养的平板中收集单个菌落,并接种到含有2ml KB液体培养基的无菌10ml管中,所述液体培养基补充有利福平(25μg/ ml)。将试管在台式振荡培养箱中于28℃孵育12小时,并以220rpm持续摇动。

  2. 生物膜形成
    将 Psph 培养物(1:500稀释液)接种到含有2ml KB液体培养基(补充有利福平)的16个无菌10ml硼硅酸盐玻璃管中,并确保一致的初始剂量。将含有 Psph 的玻璃管在28°C的恒温培养箱中孵育,不要摇晃。

  3. 生物膜可视化
    1. 在不同时间点收获生物膜样品。对于我们研究中的 Psph ,在24,48,72和96小时收获生物膜。用1ml移液管轻轻丢弃浮游细胞,并用无菌蒸馏水洗涤管三次。避免损坏管壁上形成的生物膜。
    2. 用2.5ml 0.1%结晶紫染色形成生物膜的细菌20分钟而不摇动。丢弃染料并用无菌蒸馏水洗涤管以除去未结合的染料。干燥管并拍照(图2A)。

  4. 生物膜测量
    1. 用2ml 100%乙醇洗脱生物膜,并以220rpm摇动管20分钟以确保染料完全溶解。拍照(图2B)。
    2. 使用分光光度计(2ml)或Synergy 2 Plate Reader(BioTek)(100μl)在OD 590nm 处测量洗脱的样品。如果样品浓度过高,请在测量前将其稀释。使用等体积的95%-100%乙醇作为空白对照。 Psph 生物膜形成如图2C所示。与 Psph 野生型菌株相比,Δ rhpS 产生较低的生物膜(图3)。


      图2. Psph 野生型菌株中生物膜的可视化和定量。 A.生物膜样品生长24至96小时。在不同时间点粘附到硼硅酸盐玻璃管上的生物膜用结晶紫染色。 B.用乙醇洗脱与管壁上的生物膜结合的结晶紫。 C.通过使用分光光度计或Synergy 2 Plate Reader(BioTek)在OD 590nm 下测量洗脱样品。 Psph 野生型菌株在96小时后产生更多的生物膜,而不是24小时。 *代表 P -value&lt; 0.05。 ***表示 P - 值&lt; 0.001。误差棒表示S.D.四个生物学重复。显示了两个生物学重复。


      图3.Δ rhpS 菌株比 Psph 野生型菌株产生更少的生物膜。 A. 生成的生物膜使用硼硅酸盐玻璃管观察Psph 野生型和Δ rhpS 菌株,并在96小时用结晶紫染色。 B.用乙醇洗脱与管壁上的生物膜结合的结晶紫。 C. Psph 野生型菌株比Δ rhpS 菌株产生更多的生物膜( P - 值= 0.000136)。 ***表示 P - 值&lt; 0.001。误差棒表示S.D.四个生物学重复。显示了两个生物学重复。

数据分析

使用Microsoft Office Excel 2016进行学生的 t - 测试。从图和表中的四个生物学重复中收集定量数据(OD 590nm )(表1和2)。

表1.生物膜产生 Psph 野生型菌株如图2C所示。双样本等方差为由Excel = TTEST(array1,array2,tails,type)中的以下单尾学生 t - 测试公式计算得出。数组1是第一个数据集。数组2是第二个数据集。尾部显示分布尾部的数量(单尾分布为1,双尾分布为2)。类型是要执行的 t - 测试类型(1对于配对,2对于双样本等方差,3对于双样本不等方差。例如,我们使用TTEST(B2:E2, B3:E3,1,2),TTEST(B2:E2,B4:E4,1,2)和TTEST(B2:E2,B5:E5,1,2)在48小时,72小时,96小时生物膜生产与24小时相比, P - 值分别在Excel H3,H4和H5中显示。*代表 P - 值<0.05。**代表 P - 值<0.01。***代表 P - 值<0.001。平均值和SD显示在F和G列中。所有实验重复四次。 />

表2.生物膜产生 Psph 野生型和Δ rhpS 应变如图3C所示。双样本等方差由Excel = TTEST中的以下双尾学生 t - 测试公式计算得出( B2:E2,B3:E3,2,2)。 P - 值显示在第H3列中。 *代表 P -value&lt;&nbsp; 0.05。 **代表 P - 值&lt; 0.01。 ***表示 P - 值<0.001。手段和S.D.列F和G显示。所有实验重复四次。


总之,结果表明,野生型 Psph 菌株在48 h内产生更多的生物膜( P <0.05),72 h( P <0.001)和96小时( P <0.001)比24小时(图2和表1)。此外,Δ rhpS 菌株在同一时间点(96 h)产生的生物膜比 Psph 野生型菌株( P <0.001)少。 )(图3和表2)。

食谱

  1. King's B(KB)(King et al。,1954)Bacto TM 蛋白胨第3号
    20.0克/升
    K 2 HPO 4
    1.5克/升
    甘油
    15毫升/升 溶于993.75ml ddH 2 O并调节pH至7.2。加入15 g / L琼脂用于固化培养基和高压灭菌器 将24.637g MgSO 4 ·7H 2 O溶解在100ml ddH 2 O中,并使用无菌过滤器(0.45)对该储备溶液(1M)进行灭菌。 μm)
    然后,当温度降至40°时,将6.25 ml MgSO 4 ·7H 2 O(1M)(终浓度1.5 g / L)加入高压灭菌的King's B培养基中C-50°C

    致谢

    该项目由卫生医学研究基金资助[17160022至X.D.];国家自然科学基金资助项目[31670127至X.D.]。
    &NBSP;该方案改编自O'Toole 等。(1998)Flagellar和抽搐运动是铜绿假单胞菌生物膜发育所必需的。 Molecular Microbiology ,30:295-304。

    利益争夺

    利益冲突声明:未声明。

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引用:Shao, X., Xie, Y., Zhang, Y. and Deng, X. (2019). Biofilm Formation Assay in Pseudomonas syringae. Bio-protocol 9(10): e3237. DOI: 10.21769/BioProtoc.3237.
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