Biofilm Assays on Fibrinogen-coated Silicone Catheters and 96-well Polystyrene Plates
在纤维蛋白原包覆的硅胶导管和96孔聚苯乙烯板上进行生物膜检测   

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Elizabeth Libby Elizabeth Libby
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PLOS Pathogens
Sep 2018

 

Abstract

Biofilm formation is a well-known bacterial strategy that protects cells from hostile environments. During infection, bacteria found in a biofilm community are less sensitive to antibiotics and to the immune response, often allowing them to colonize and persist in the host niche. Not surprisingly, biofilm formation on medical devices, such as urinary catheters, is a major problem in hospital settings. To be able to eliminate such biofilms, it is important to understand the key bacterial factors that contribute to their formation. A common practice in the lab setting is to study biofilms grown in laboratory media. However, these media do not fully reflect the host environment conditions, potentially masking relevant biological determinants. This is the case during urinary catheterization, where a key element for Enterococcus faecalis and Staphylococcus aureus colonization and biofilm formation is the release of fibrinogen (Fg) into the bladder and its deposition on the urinary catheter. To recapitulate bladder conditions during catheter-associated urinary tract infection (CAUTI), we have developed a fibrinogen-coated catheter and 96-well plate biofilm assay in urine. Notably, enterococcal biofilm factors identified in these in vitro assays proved to be important for biofilm formation in vivo in a mouse model of CAUTI. Thus, the method described herein can be used to uncover biofilm-promoting factors that are uniquely relevant in the host environment, and that can be exploited to develop new antibacterial therapies.

Keywords: Biofilm, Urine, Infection, Enterococcus faecalis, Fibrinogen, CAUTI, Catheter

Background

Enterococcus faecalis is a leading cause of nosocomial infections, most notably infective endocarditis (IE) and catheter-associated urinary tract infections (CAUTI) (Arias et al., 2012; Chirouze et al., 2013; Flores-Mireles et al., 2015). Since these diseases are mainly biofilm-associated, a better understanding of how E. faecalis forms biofilms within the host can enable us to develop novel antibacterial therapies (Dunny et al., 2014).

The most common method to evaluate bacterial biofilm formation is the microplate biofilm assay, where bacteria are typically grown in microplate wells filled with laboratory media prior to analysis (Azeredo et al., 2017). However, there is increasing evidence that assays performed in laboratory growth media do not fully recapitulate conditions found within the host, and potentially overlook important bacterial factors required during infection (Nallapareddy and Murray, 2008; Guiton et al., 2013; Flores-Mireles et al., 2014; Colomer-Winter et al., 2017 and 2018; Xu et al., 2017). This is exemplified by studies investigating how E. faecalis forms biofilms on urinary catheters, a crucial step during persistent CAUTI (Nielsen et al., 2012; Guiton et al., 2013; Flores-Mireles et al., 2014, 2016a and 2016b). Early studies using animal models showed that E. faecalis forms robust biofilms on indwelling urinary catheters, and it was hypothesized that bacterial attachment occurred, at least in part, via Ebp, the endocarditis-and-biofilm-associated pilus (Nielsen et al., 2012). This hypothesis was substantiated by the finding that ebp deletion mutants were deficient in biofilm formation in vitro (in tryptic soy broth supplemented with 0.25% glucose [TSBG]) and in vivo, and were highly attenuated in animal models (Singh et al., 2007; Nallapareddy et al., 2011; Nielsen et al., 2012; Guiton et al., 2013; Sillanpaa et al., 2013; Flores-Mireles et al., 2014). However, the compelling body of work showing that Ebp-mediated biofilm formation is important during CAUTI contrasted with the observation that E. faecalis did not form biofilms in urine ex vivo (Flores-Mireles et al., 2014). This posed a significant paradox since urine is the environment that bacteria encounter during infection in the urinary tract. The paradox was resolved by the key finding that Ebp binds to fibrinogen (Nallapareddy et al., 2011) and that the host releases fibrinogen into the bladder as a result of catheter-associated inflammation (Flores-Mireles et al., 2014). Indeed, addition of fibrinogen to urine enhanced enterococcal biofilm formation ex vivo and enabled the discovery that E. faecalis cells attach to urinary catheters primarily via Ebp-fibrinogen interactions (Flores-Mireles et al., 2014). While this method successfully d the results found in vivo, it ultimately confirmed the critical role of fibrinogen to enterococcal pathogenesis and led to the development of a vaccine therapy (Flores-Mireles et al., 2014, 2016a and 2016b). Similarly, the assay was later used to probe the importance of recapitulatemanganese uptake to enterococcal biofilm formation in urine (Colomer-Winter et al., 2018).

The method described (Figure 1) herein highlights the importance of developing assays that closely mimic the host environment to be able to study bacterial processes that are critical during infection. This concept is not restricted to the urinary tract or to E. faecalis, as it could be generally applied to studies of bacterial pathophysiology within the vertebrate host, like for example the oral cavity or the cardiovascular system.

Materials and Reagents

  1. Bacterial growth and biofilm assay
    1. 500 ml Corning disposable sterile bottle-top filters with 0.22 µm Membrane (Fisher Scientific, catalog number: 09-761-112)
    2. 500 ml Reusable Glass Media Bottles with Cap (Fisher Scientific, catalog number: FB800500)
    3. 100 x 15 mm Petri Dish (Fisher Scientific, catalog number: S43570)
    4. 1 µl inoculating loops (DB Difco, catalog number: BD 220215)
    5. 15 ml Conical tube, for growing microaerophilic bacteria (Fisher Scientific, catalog number: 50-153-5104) 
    6. Disposable Round-Bottom rimless glass tubes (Fisher Scientific, catalog number: 14-962-15A) and cap (Fisher Scientific, catalog number: 14-957-91)
    7. Cuvettes, Standard: Polystyrene (Fisher Scientific, catalog number: 14-955-127)
    8. 1.5 ml microcentrifuge tubes (Fisher Scientific, catalog number: 02-682-002)
    9. Pipette tips
    10. pH strips (EMD Millipore, catalog number: 1.09542.0001)
    11. Catheter-Nalgene 50 silicone tubing (Nalgene Brand Products, catalog number: 80600030)
    12. 96-well polystyrene plates (Grenier Bio-One CellSTAR, catalog number: 655180)
    13. 96-well Microtitration plates (Corning, catalog number: 3788)
    14. Axygen Scientific microplate presterilized sealing tape (Fisher Scientific, catalog number: 14-222-044)
    15. Bacterial species: Enterococcus faecalis OG1RF (ATCC 47077)
    16. Double deionized water
    17. 1x Phosphate sodium saline (Sigma-Aldrich, catalog number: P3813)
    18. 0.1 N Hydrochloric acid solution (HCl) (Sigma-Aldrich, catalog number: 2104)
    19. 0.1 N Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: SX0607C)
    20. Bovine serum albumin (Sigma-Aldrich, catalog number: A7906)
    21. Agar, Bacteriological Grade (BD Difco, catalog number: B281230)
    22. Human fibrinogen free from plasminogen and von Willebrand factor (13-14 mg/ml–concentration varies with each batch)(Enzyme Research Laboratory, catalog number: FIB 3)
    23. Brain Heart Infusion broth (BHI) (BD Company, catalog number: B237500) or any other media for the requirements of your bacterial species 
    24. Pooled human urine (collected from at least three healthy female donors [Internal Review Board approval needed] or fresh urine commercially available).
      Note: Urine should be fresh and stored under refrigeration for no longer than 3 days.
    25. BHI liquid media (see Recipes)
    26. BHI-agar plates (see Recipes)
    27. Urine (see Recipes)

  2. Assessment of biofilm formation on catheters
    1. 12-well polystyrene microplate (Fisher Scientific, catalog number: 08-772-50)
    2. Aluminum foil
    3. 10% Neutral Buffered Formalin (Fisher Scientific, catalog number: 22-046-361)
    4. 20% Sodium azide solution (Sigma-Aldrich, catalog number: S-2002)
    5. Tween-20 (Sigma-Aldrich, catalog number: P1379)
    6. Methyl α-d-mannopyronoside (Sigma-Aldrich, catalog number: M6882)
    7. Rabbit anti-Enterococcus antibody (Abcam, catalog number: ab68540) or any other anti-Enterococcus antibody commercially available
    8. IRDye 680LT goat anti-rabbit (LI-COR Biosciences, catalog number: 926-68021)
    9. Immunostaining Solutions (see Recipes)
      1. Blocking Solution
      2. Wash Solution
      3. Dilution Buffer
      4. Primary antibody solution
      5. Secondary antibody solution

  3. Assessment of Biofilm on microplate
    1. Paper towel
    2. Reagent reservoir (Fisher Scientific, catalog number: 14-387-065)
    3. Crystal violet (Sigma-Aldrich, catalog number: C0775)
    4. Acetic acid (Sigma-Aldrich, catalog number: A6283)
    5. Solutions (see Recipes)
      1. 0.5% Crystal violet solution
      2. 33% of Acetic Acid

Equipment

  1. Forceps
  2. Heraeus Multifuge X3R Refrigerated Centrifuge (VWR, catalog number: 75004516) or any refrigerated centrifuge with similar features
  3. Spectra Max ABS Plus Spectrophotometer (Molecular Devices, catalog number: ABS PLUS) or any spectrophotometer with similar features
  4. Branson UltrasonicsTM BransonicTM CPX-sonicator (waterbath sonicator) (Fisher Scientific, catalog number: 15-337-419) or any waterbath sonicator with similar features
  5. Odyssey CLx imaging system (LI-COR, catalog number: 9140-01)
  6. Test tube racks (Fisher Scientific, catalog number: 14-809-62) or any standard test tube racks
  7. VWR Microbiological Incubator (VWR, catalog number: 51030017) or any standard microbiological incubator
  8. Biological Safety Cabinet with UV light (Thermo Fisher, catalog number: 1395) or any standard equipment with similar features
  9. Vortexer (Fisher brand, catalog number: 02-215-418) or any standard vortex
  10. Autoclave (Getinge, catalog number: 633LS) or any standard autoclave

Software

  1. CLX image studio (LI-COR, catalog number: 9140-510)
  2. GraphPad Prism (GraphPad Software LLC)

Procedure

The individual steps of this protocol are summarized in Figure 1.


Figure 1. Protocol layout

  1. Bacterial growth
    1. Streak the bacterial species on a BHI agar plate using a sterile inoculation loop.
    2. Incubate bacteria overnight at 37 °C.
    3. Add 10 ml of BHI media into a 15 ml conical tube.
    4. Using a sterile inoculation loop, pick a single E. faecalis colony to inoculate the media.
    5. Incubate the bacterial culture for 18 h at 37 °C under static conditions (target OD600 = 1.0).

  2. Catheter and microplate preparation
    Catheter
    1. Cut the silicone tubing in 1 cm pieces.
    2. Cut in half the 1 cm pieces (Figure 2).


      Figure 2. Preparation of 1 cm silicone pieces. A. Silicone tubing. B. 1 cm silicone pieces. C. 1 cm pieces in half.

    3. Put the resulting pieces in an open Petri dish.
    4. UV-sterilize the pieces overnight (Biological Safety Cabinet standard UV settings)
    5. Use sterile forceps to transfer each silicone piece into sterile 5 ml glass test tube and cap it.
      Note: Fibrinogen is a sticky protein. Therefore use of a glass tube will reduce binding of fibrinogen to the tube walls. 
    6. Thaw the fibrinogen stock solution at 37 °C, and bring the 1x PBS solution to 37 °C. Fibrinogen is soluble at body temperature; therefore, keep it at 37 °C until you add it to the silicone pieces. 
    7. Prepare a working solution of 100 µg/ml of fibrinogen in 1x PBS. 
    8. Add 1 ml of the fibrinogen solution to the test tube containing the silicone piece. 
    9. Incubate the silicone pieces at 4 °C overnight under static conditions to allow fibrinogen to coat the catheter.

    Microplate
    1. Dispense 100 µl of the 100 µg/ml fibrinogen solution (Step B7) into each well of the 96-well polystyrene plates (Grenier Bio-One CellSTAR). 
    2. Seal the plate with a sterile plate sealing tape.
    3. Incubate the microplate at 4 °C overnight to allow fibrinogen to coat the bottom of the well.

  3. Culture preparation
    1. Centrifuge the overnight culture (Step A5) for 10 min at 7,000 rpm (7,505 x g).
    2. Remove supernatant.
    3. Resuspend the bacterial pellet with 10 ml of 1x PBS solution. Then centrifuge again for 10 min at 7,000 rpm (7,505 x g). Wash the bacterial cells by resuspending the bacterial pellet with 10 ml of 1x PBS solution. Repeat this step 3 times.
    4. Dilute 1 ml of bacterial solution into 9 ml of 1x PBS solution (dilution 1:10). This step is necessary to ensure the accuracy of the measurement.
    5. Take 1 ml of the diluted solution and put it into a cuvette. 
    6. Measure optical density (OD600) by using the spectrophotometer.
      Note: Multiply the OD600 value by 10 (dilution factor) to obtain the final optical density of the culture. 
    7. Dilute the culture to a final OD600 of 1.0.
    8. Supplement fresh filter-sterilized urine (see Recipe 2) with 20 mg/ml of BSA. 
    9. Filter sterilize the supplemented urine using bottle top filter. 
    10. Inoculate 1:100 of normalized culture (Step C7) into the filter-sterilized urine supplemented with BSA. 

  4. Catheter Fg-dependent biofilm setup
    1. After overnight incubation at 4 °C, remove the test tubes containing the Fg-coated silicone pieces (Step B9). 
    2. Aspirate the fibrinogen solution gently using a 1,000 µl pipette. 
    3. Add 1 ml of the bacteria-containing urine (Step C10). As negative control, incubate three Fg-coated pieces with only BSA-supplemented urine (no bacteria).
    4. Incubate the tubes under static conditions at 37 °C for 24 h (or as needed). 

  5. Assessment of the catheter biofilm
    1. After overnight incubation at 4 °C, remove the Fg-coated plates (Step B12). 
    2. Peel off the plate sealing tape. 
    3. Aspirate the fibrinogen solution gently using a pipette.
    4. Add 200 µl of the bacteria-containing urine (Step C10). As negative control, incubate 8 Fg-coated wells (one column of the microplate) with only urine (no bacteria).
    5. Cover the plate with a sterile lid. 
    6. Incubate the microplate tubes under static conditions at 37 °C for 24 h (or as needed). 

  6. Assessment of the microplate biofilm
    1. Aspirate the bacterial culture from the tube using a 1,000 µl pipette.
    2. Remove the unbound bacteria by pipetting vigorously using 1 ml of 1x PBS at room temperature. Repeat this step 3 times.

    Assessment of biofilm formation by colony forming units 
    1. Transfer the silicone piece with sterile forceps (from Step F2) into a 15 ml conical tube. 
    2. Add 1 ml of 1x PBS. 
    3. To detach the bacterial biofilm from the catheter, vortex for 30 s (maximum vortex’s speed) at room temperature. 
    4. Put conical tube into the Branson ultrasonic bath for 5 min at room temperature (40 kHz frequency), and vortex for another 30 s.
    5. Take a 200 µl sample and serially dilute it with PBS (1:10).
    6. Plate dilutions on BHI agar plates.
    7. Incubate the plates at 37 °C overnight. 
    8. Quantify colony forming units (Figure 3).

    Assessment of biofilm formation by immunostaining
    1. After washing the silicone pieces (Step F2), transfer them to a 12-well plate.
    2. Fix the silicone pieces by adding 5 ml of 10% neutralizing formalin solution for 20 min.
    3. Remove the formalin
    4. Wash the pieces by adding 5 ml of PBS (repeat 3 times). All washes in this protocol are done under static conditions at room temperature unless otherwise noted.
    5. Block the pieces by adding 5 ml blocking solution for 1 h at room temperature (or at 4 °C overnight).
    6. Wash the pieces with 5 ml wash solution. Repeat this step 3 times.
    7. Add 5 ml of the primary antibody solution. 
    8. Incubate for 2 h at room temperature under static conditions.
    9. Wash the pieces with 5 ml wash solution. Repeat this step 3 times.
    10. Add 5 ml of the secondary antibody solution. Cover the plate with aluminum foil (secondary antibody is sensitive to light).
    11. Incubate for 1 h at room temperature under static conditions
    12. Wash the pieces with 5 ml wash solution. Repeat this step 3 times.
    13. Transfer the pieces into a new 12-well plate.
    14. Let them dry overnight.
    15. Visualize the biofilm using the Odyssey imager (detection at 700 nm near-infrared region) (Figure 4).

    Colorimetric assessment of biofilm formation by crystal violet 
    1. After washing the silicone pieces (Step F2), transfer them to a 12-well plate.
    2. Let pieces air dry. 
    3. Add 5 ml of 0.5% of crystal violet solution.
    4. Stain for 10 min under static conditions at room temperature.
    5. Wash the pieces with distilled water (repeat this step 3 times).
    6. Add 1 ml of 33% acetic acid to solubilize the crystal violet staining.
    7. Incubate for 15 min at room temperature under static conditions.
    8. Transfer 200 µl of the solubilized crystal violet into a new microplate.
    9. Quantify absorbance at 595 nm using a plate reader. As a blank, use the 33% acetic acid in water. Serial dilution may be required to fall into the linear range of the plate reader. 

  7. Assessment of the microplate biofilm
  1. After incubation (Step E6), remove the culture by inverting the plate. 
  2. Wash the unbound cells by submerging the plate in a small container full of water. Shake the plate gently and shake out the water. Repeat this step 3 times.
  3. Dry the plate by tapping gently on a paper towel to remove residual liquid.
  4. Let the plate air dry.
  5. Add 200 µl of 0.5% of crystal violet solution.
  6. Stain for 10 min under static conditions at room temperature.
  7. Rinse by submerging the plate into the container full of water. Once submerged, shake the plate vigorously, dump the liquid, and tap the plate on a paper towel to remove the residual liquid. Repeat this step 3 times.
  8. Add 200 µl of 33% acetic acid to solubilize the crystal violet staining.
  9. Incubate for 15 min at room temperature under static conditions.
  10. Transfer 200 µl of the solubilized crystal violet into a new microplate.
  11. Quantify absorbance at 595 nm using a plate reader. As a blank use the 33% acetic acid in water. Serial dilution may be required to fall into the linear range of the plate reader.

Data analysis

  1. Analysis of biofilm-associated CFU
    1. Log10 transform final bacterial counts recovered from each catheter (n ≥ 3) (Figure 3).
    2. Plot data on a linear scale and include the arithmetic mean. 
    3. Depending on the amount of groups you want to compare, analyze the log10-transformed data with either a two-tailed Mann-Whitney U test or a one-way ANOVA followed by an appropriate post-test comparison.
      Note: Log10 transformation is optional. An alternative way to plot and analyze the data would be to plot untransformed data on a log scale with the geometric mean and the geometric standard deviation.


      Figure 3. Representative data of biofilm measured by colony forming units (CFU). Comparison of biofilm formation between E. faecalis OG1RF (wild-type strain) and a manganese uptake system triple mutant strain (ΔefaΔmntH1ΔmntH2), which is a biofilm deficient mutant. Two-tailed Mann-Whitney U tests were performed to determine significance between two groups (***P < 0.0002).

  2. Analysis of biofilm formation assessed by immunostaining or crystal violet
    1. Plot the relative fluorescence to the negative control (Figure 4) or absorbance (relative to the negative control) for each group (n ≥ 3) on a linear scale and include the arithmetic mean and the standard deviation.
    2. Analyze the data with a Mann-Whitney U test.
      Notes:
      1. The biofilm formation assays on catheters often do not follow a normal distribution as determined by the D’Agostino-Pearson test. Thus, a nonparametric analysis such as the Mann-Whitney U test is more appropriate than the parametric ANOVA test. 
      2. Outliers can be identified using the ROUT method (Q = 1% recommended) and can be removed from the final analysis if confirmed.


      Figure 4. Representative images and data of biofilm analyzed by immunostaining and crystal violet. A. Visualization of biofilm formation on catheter pieces by E. faecalis OG1RF and a manganese uptake system triple mutant strain (ΔefaΔmntH1ΔmntH2). Catheter controls are those Fg-coated catheters that were incubated with only urine (no bacteria). B. Immunostaining analysis of biofilm formation between OG1RF WT and deficient mutant by plotting fluorescence relative to catheter controls. C. CV staining to quantify and compare biofilm formation between OG1RF WT and deficient mutant by plotting absorbance relative to catheter controls. Two-tailed Mann-Whitney U tests were performed to determine significance between two groups (***P < 0.0002).

Recipes

  1. Media
    1. BHI liquid media
      Add 37 g of BHI broth powder into 1 L of distilled water
      Mix until solution is clear
      Autoclave for 20 min
      Let media cool down to room temperature prior to use
    2. BHI-agar plates
      Add 37 g of BHI broth powder and 15 g of agar into 1 L of distilled water
      Mix until solution is clear
      Autoclave for 20 min
      Let media cool down to a temperature of 45 °C
      Add antibiotics if needed and mix
      Keep media warm and pour 25 ml of media into Petri dishes
  2. Urine
    1. Pool the urine from at least three healthy female donors (or get commercially available pooled urine). Use the same urine batch for consistency during the study
    2. Centrifuge pooled urine for 5 min at 7,000 rpm (7,505 x g) to remove precipitates
    3. Measure pH using pH strips and adjust to 6.5 using HCl solution or NaOH solutions as needed
    4. Filter sterilize the pooled urine using bottle top filters. Preferentially use fresh urine every time. In case, it is not used right away, store urine at 4 °C for no longer than 3 days. Repeat centrifugation for 5 min at 7,000 rpm (7,505 x g) if formation of precipitates is observed
  3. Solutions
    1. Blocking Solution
      1x PBS with 1.5% BSA and 0.1% Sodium Azide. Keep solution at 4 °C
    2. Wash Solution
      1x PBS with 0.05% Tween-20. Keep solution at room temperature
    3. Dilution Buffer
      1x PBS with 0.05% Tween-20, 0.1% BSA, and 0.5% methyl α-d-mannopyronoside. Keep solution at 4 °C
    4. Primary antibody solution
      Add 1:500 of rabbit anti-Streptococcus group D antigen antisera into dilution buffer. Prepare fresh every time
    5. Secondary antibody solution
      Add 1:10,000 of goat anti-rabbit IRDye 680LT into dilution buffer. Prepare fresh every time
    6. 0.5% Crystal violet solution
      Add 0.5 mg into 100 ml of distilled water. Filter sterilize the solution. Keep solution at room temperature
    7. 33% of Acetic Acid
      Add 330 ml of Acetic acid into 670 ml of distilled water. Keep solution at room temperature

Acknowledgments

C.C.W. was supported by American Heart Association GSA Predoctoral Fellowship 16PRE29860000, J.A.L. was supported by National Institute of Allergy and Infectious Disease AI135158, A.L.F.M. was supported by startup funds from the University of Notre Dame.
Special acknowledgment to previous studies done by Nallapareddy et al. (2011) and Flores-Mireles et al. (2014).

Competing interests

The authors declare no competing financial interests.

References

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  17. Xu, W., Flores-Mireles, A. L., Cusumano, Z. T., Takagi, E., Hultgren, S. J. and Caparon, M. G. (2017). Host and bacterial proteases influence biofilm formation and virulence in a murine model of enterococcal catheter-associated urinary tract infection. NPJ Biofilms Microbiomes 3: 28.

简介

生物膜形成是众所周知的细菌策略,其保护细胞免受恶劣环境的影响。在感染期间,生物膜群落中发现的细菌对抗生素和免疫反应不太敏感,通常允许它们在宿主生态位中定居并持续存在。毫不奇怪,医疗设备上的生物膜形成,例如导尿管,是医院环境中的主要问题。为了能够消除这种生物膜,重要的是要了解导致它们形成的关键细菌因子。实验室环境中的常见做法是研究在实验室培养基中生长的生物膜。然而,这些培养基不能完全反映宿主环境条件,可能掩盖相关的生物学决定因素。在导尿术期间就是这种情况,其中粪肠球菌和金黄色葡萄球菌定植和生物膜形成的关键因素是纤维蛋白原(Fg)释放到膀胱及其沉积中在导尿管上。为了概括导管相关尿路感染(CAUTI)期间的膀胱状况,我们开发了一种纤维蛋白原涂层导管和尿液中的96孔板生物膜测定。值得注意的是,在这些体外测定中鉴定的肠球菌生物膜因子被证明对于CAUTI小鼠模型中体内体内生物膜形成是重要的。因此,本文描述的方法可用于揭示在宿主环境中独特相关的生物膜促进因子,并且可用于开发新的抗细菌疗法。
【背景】粪肠球菌是医院感染的主要原因,最明显的是感染性心内膜炎(IE)和导管相关性尿路感染(CAUTI)(Arias et al。,2012; Chirouze et al。,2013; Flores-Mireles et al。,2015)。由于这些疾病主要与生物膜相关,因此更好地了解 E。粪肠球菌在宿主体内形成生物膜可以使我们开发新的抗菌疗法(Dunny et al。,2014)。

评估细菌生物膜形成的最常用方法是微孔板生物膜测定,其中细菌通常在分析之前在填充有实验室培养基的微孔板中生长(Azeredo 等人,2017)。然而,越来越多的证据表明,在实验室生长培养基中进行的分析并未完全概括宿主体内发现的条件,并可能忽略感染期间所需的重要细菌因子(Nallapareddy和Murray,2008; Guiton 等。 ,2013; Flores-Mireles et al。,2014; Colomer-Winter et al。,2017和2018; Xu et al。,2017 )。研究 E的研究就是一个例子。粪便在导尿管上形成生物膜,这是持续性CAUTI期间的关键步骤(Nielsen et al。,2012; Guiton et al。,2013; Flores-Mireles et al。,2014,2016a和2016b)。使用动物模型的早期研究显示 E.粪肠球菌在留置导尿管上形成强健的生物膜,并假设细菌附着至少部分是通过Ebp发生的,即心内膜炎 - 和 - iofilm-associated p ilus(Nielsen et al。,2012)。 ebp 缺失突变体在体外生物膜形成(在补充有0.25%葡萄糖的胰蛋白酶大豆肉汤[TSBG])和中缺乏 ebp 缺失突变体的发现证实了这一假设。体内,并且在动物模型中高度减毒(Singh et al。,2007; Nallapareddy et al。,2011; Nielsen et al。 ,2012; Guiton et al。,2013; Sillanpaa et al。,2013; Flores-Mireles et al。,2014 )。然而,在CAUTI期间,显示Ebp介导的生物膜形成的重要工作主要与 E的观察结果形成对比。粪肠球菌不能在体外离体形成生物膜(Flores-Mireles et al。,2014)。这提出了一个重要的矛盾,因为尿液是细菌在泌尿道感染期间遇到的环境。 Ebp与纤维蛋白原结合的关键发现解决了悖论(Nallapareddy et al。,2011),并且由于导管相关炎症,宿主释放纤维蛋白原进入膀胱(Flores-Mireles et al。,2014)。实际上,向尿液中添加纤维蛋白原可以增强肠道内生物膜形成离体,并且能够发现 E.粪肠球菌细胞主要通过Ebp-纤维蛋白原相互作用附着于导尿管(Flores-Mireles et al。,2014)。虽然这种方法成功地发现了 in vivo ,但它最终证实了纤维蛋白原在肠球菌发病机制中的关键作用,并导致了疫苗疗法的发展(Flores-Mireles et al。,2014,2016a和2016b)。类似地,该测定随后用于探测重新吸收锰对尿中肠球菌生物膜形成的重要性(Colomer-Winter 等,,2018)。

本文描述的方法(图1)强调了开发密切模拟宿主环境的测定的重要性,以便能够研究在感染期间关键的细菌过程。这个概念不仅限于泌尿道或 E.粪肠球菌,因为它通常可用于脊椎动物宿主内的细菌病理生理学研究,例如口腔或心血管系统。

关键字

材料和试剂

  1. 细菌生长和生物膜测定
    1. 500毫升康宁一次性无菌瓶盖过滤器,0.22微米膜(Fisher Scientific,目录号:09-761-112)
    2. 带瓶盖的500毫升可重复使用玻璃介质瓶(Fisher Scientific,目录号:FB800500)
    3. 100 x 15 mm培养皿(Fisher Scientific,目录号:S43570)
    4. 1μl接种环(DB Difco,目录号:BD 220215)
    5. 15毫升锥形管,用于培养微需氧菌(Fisher Scientific,目录号:50-153-5104)&nbsp;
    6. 一次性圆底无框玻璃管(Fisher Scientific,目录号:14-962-15A)和盖(Fisher Scientific,目录号:14-957-91)
    7. 比色皿,标准:聚苯乙烯(Fisher Scientific,目录号:14-955-127)
    8. 1.5 ml微量离心管(Fisher Scientific,目录号:02-682-002)
    9. 移液器吸头
    10. pH条(EMD Millipore,目录号:1.09542.0001)
    11. Catheter-Nalgene 50硅胶管(Nalgene Brand Products,目录号:80600030)
    12. 96孔聚苯乙烯板(Grenier Bio-One CellSTAR,目录号:655180)
    13. 96孔微量滴定板(康宁,目录号:3788)
    14. Axygen Scientific微孔板预灭菌密封胶带(Fisher Scientific,目录号:14-222-044)
    15. 细菌种类:粪肠球菌 OG1RF(ATCC 47077)
    16. 双去离子水
    17. 1x磷酸钠盐水(Sigma-Aldrich,目录号:P3813)
    18. 0.1 N盐酸溶液(HCl)(Sigma-Aldrich,目录号:2104)
    19. 0.1 N氢氧化钠(NaOH)(Sigma-Aldrich,目录号:SX0607C)
    20. 牛血清白蛋白(Sigma-Aldrich,目录号:A7906)
    21. 琼脂,细菌级(BD Difco,目录号:B281230)
    22. 不含纤溶酶原和血管性血友病因子的人纤维蛋白原(13-14 mg / ml浓度随每批次而异)(酶研究实验室,目录号:FIB 3)
    23. 脑心浸液肉汤(BHI)(BD公司,目录号:B237500)或任何其他媒介,以满足您的细菌物种的需求&nbsp;
    24. 合并的人尿(从至少三名健康女性捐献者[需要内部审查委员会批准]或商业上可获得的新鲜尿液中收集)。
      注意:尿液应该是新鲜的,并且在冷藏下保存不超过3天。
    25. BHI液体培养基(见食谱)
    26. BHI-琼脂平板(见食谱)
    27. 尿(见食谱)

  2. 评估导管上的生物膜形成
    1. 12孔聚苯乙烯微孔板(Fisher Scientific,目录号:08-772-50)
    2. 铝箔
    3. 10%中性缓冲福尔马林(Fisher Scientific,目录号:22-046-361)
    4. 20%叠氮化钠溶液(Sigma-Aldrich,目录号:S-2002)
    5. Tween-20(西格玛奥德里奇,目录号:P1379)
    6. 甲基α-d-甘露糖苷(Sigma-Aldrich,目录号:M6882)
    7. 兔抗肠球菌抗体(艾博抗(上海)贸易有限公司,目录号:ab68540)或其他商业上可获得的抗肠球菌抗体
    8. IRDye 680LT山羊抗兔(LI-COR Biosciences,目录号:926-68021)
    9. 免疫染色解决方案(见食谱)
      1. 阻止解决方案
      2. 洗液
      3. 稀释缓冲液
      4. 一抗溶液
      5. 二抗溶液

  3. 生物膜在微孔板上的评估
    1. 纸巾
    2. 试剂储存器(Fisher Scientific,目录号:14-387-065)
    3. 结晶紫(Sigma-Aldrich,目录号:C0775)
    4. 乙酸(Sigma-Aldrich,目录号:A6283)
    5. 解决方案(见食谱)
      1. 0.5%结晶紫溶液
      2. 33%的醋酸

设备

  1. 钳子
  2. Heraeus Multifuge X3R冷冻离心机(VWR,目录号:75004516)或具有类似功能的任何冷冻离心机
  3. Spectra Max ABS Plus分光光度计(Molecular Devices,目录号:ABS PLUS)或具有类似功能的任何分光光度计
  4. Branson Ultrasonics TM Bransonic TM CPX-sonicator(waterbath sonicator)(Fisher Scientific,目录号:15-337-419)或具有类似功能的任何水浴超声波仪
  5. Odyssey CLx成像系统(LI-COR,目录号:9140-01)
  6. 试管架(Fisher Scientific,目录号:14-809-62)或任何标准试管架
  7. VWR微生物培养箱(VWR,目录号:51030017)或任何标准微生物培养箱
  8. 带紫外线灯的生物安全柜(Thermo Fisher,目录号:1395)或具有类似功能的任何标准设备
  9. Vortexer(Fisher品牌,目录号:02-215-418)或任何标准涡流
  10. 高压灭菌器(Getinge,目录号:633LS)或任何标准高压灭菌器

软件

  1. CLX image studio(LI-COR,目录号:9140-510)
  2. GraphPad Prism(GraphPad Software LLC)

程序

该协议的各个步骤总结在图1中。


图1.协议布局

  1. 细菌生长
    1. 使用无菌接种环将细菌种类划线在BHI琼脂平板上。
    2. 在37°C孵育细菌过夜。
    3. 将10ml BHI培养基加入15ml锥形管中。
    4. 使用无菌接种环,选择一个 E. f aecalis 殖民地接种媒体。
    5. 将细菌培养物在37℃下在静态条件下孵育18小时(目标OD 600 = 1.0)。

  2. 导管和微孔板制备
    导管
    1. 切割1厘米的硅胶管。
    2. 切成1厘米的一半(图2)。


      图2. 1 cm硅胶片的制备。 A.硅胶管。 B. 1厘米硅胶片。 C. 1厘米的碎片。

    3. 将得到的碎片放在开放的培养皿中。
    4. 隔夜紫外线杀菌(生物安全柜标准紫外线设置)
    5. 使用无菌镊子将每个硅胶片转移到无菌的5毫升玻璃试管中并盖上盖子。
      注意:纤维蛋白原是一种粘性蛋白质。因此,使用玻璃管会降低纤维蛋白原与管壁的结合。
    6. 在37℃下解冻纤维蛋白原储备溶液,并将1x PBS溶液置于37℃。纤维蛋白原在体温下可溶;因此,将其保持在37°C,直到将其添加到硅胶片中。&nbsp;
    7. 在1x PBS中制备100μg/ ml纤维蛋白原的工作溶液。&nbsp;
    8. 将1毫升纤维蛋白原溶液加入含有硅胶片的试管中。&nbsp;
    9. 将硅胶片在4℃下在静态条件下孵育过夜,以使纤维蛋白原包被导管。

    的 微孔板
    1. 将100μl的100μg/ ml纤维蛋白原溶液(步骤B7)分配到96孔聚苯乙烯板(Grenier Bio-One CellSTAR)的每个孔中。&nbsp;
    2. 用无菌板密封带密封板。
    3. 将微量培养板在4℃孵育过夜,使纤维蛋白原包被在孔的底部。

  3. 文化准备
    1. 将过夜培养物(步骤A5)以7,000rpm(7,505 xg )离心10分钟。
    2. 去除上清液。
    3. 用10ml 1x PBS溶液重悬细菌沉淀。然后在7,000rpm(7,505 x g )下再次离心10分钟。通过用10ml 1x PBS溶液重悬细菌沉淀来洗涤细菌细胞。重复此步骤3次。
    4. 将1 ml细菌溶液稀释到9 ml 1x PBS溶液中(1:10稀释)。该步骤对于确保测量的准确性是必要的。
    5. 取1 ml稀释溶液,放入比色皿中。&nbsp;
    6. 使用分光光度计测量光密度(OD 600 )。
      注意:将OD 600 值乘以10(稀释因子)以获得培养物的最终光密度。&nbsp;
    7. 将培养物稀释至最终OD 600 1.0。
    8. 用20毫克/毫升的BSA补充新鲜过滤灭菌的尿液(见配方2)。&nbsp;
    9. 使用瓶盖过滤器对补充的尿液进行过滤消毒。&nbsp;
    10. 将1:100标准化培养物(步骤C7)接种到补充有BSA的过滤灭菌尿液中。&nbsp;

  4. 导管依赖Fg的生物膜设置
    1. 在4°C温育过夜后,取出含有Fg涂层硅胶片的试管(步骤B9)。&nbsp;
    2. 使用1,000μl移液器轻轻吸出纤维蛋白原溶液。&nbsp;
    3. 加入1ml含细菌的尿液(步骤C10)。作为阴性对照,仅用BSA补充的尿液(无细菌)孵育三个Fg包被的片。
    4. 将管在37°C的静态条件下孵育24小时(或根据需要)。&nbsp;

  5. 评估导管生物膜
    1. 在4℃下孵育过夜后,除去涂有Fg的板(步骤B12)。&nbsp;
    2. 剥掉板密封胶带。&nbsp;
    3. 用移液管轻轻吸出纤维蛋白原溶液。
    4. 加入200μl含细菌的尿液(步骤C10)。作为阴性对照,仅用尿液(无细菌)孵育8 Fg包被的孔(一个微孔板柱)。
    5. 用无菌盖子盖住盘子。&nbsp;
    6. 将微孔板管在37°C的静态条件下孵育24小时(或根据需要)。&nbsp;

  6. 评估微孔板生物膜
    1. 使用1,000μl移液管从管中吸出细菌培养物。
    2. 通过在室温下使用1ml 1x PBS剧烈移液来除去未结合的细菌。重复此步骤3次。

    通过菌落形成单位评估生物膜形成&nbsp;
    1. 用无菌镊子(从步骤F2)将硅胶片转移到15ml锥形管中。&nbsp;
    2. 加入1毫升1x PBS。&nbsp;
    3. 为了从导管中分离细菌生物膜,在室温下涡旋30秒(最大涡旋速度)。&nbsp;
    4. 在室温(40kHz频率)下将锥形管放入Branson超声波浴中5分钟,并涡旋另外30秒。
    5. 取200μl样品,用PBS(1:10)连续稀释。
    6. BHI琼脂平板上的平板稀释液。
    7. 将板在37°C孵育过夜。&nbsp;
    8. 量化菌落形成单位(图3)。

    通过免疫染色评估生物膜形成
    1. 在洗涤硅氧烷片后(步骤F2),将它们转移到12孔板中。
    2. 通过加入5ml 10%中和的福尔马林溶液20分钟固定硅胶片。
    3. 去除福尔马林
    4. 加入5ml PBS洗涤碎片(重复3次)。除非另有说明,否则该方案中的所有洗涤均在室温下在静态条件下进行。
    5. 通过在室温下(或在4℃下过夜)加入5ml封闭溶液1小时来封闭碎片。
    6. 用5ml洗涤溶液洗涤碎片。重复此步骤3次。
    7. 加入5毫升一抗溶液。&nbsp;
    8. 在室温下在静态条件下孵育2小时。
    9. 用5ml洗涤溶液洗涤碎片。重复此步骤3次。
    10. 加入5毫升二抗溶液。用铝箔覆盖板(二抗对光敏感)。
    11. 在室温下在静态条件下孵育1小时
    12. 用5ml洗涤溶液洗涤碎片。重复此步骤3次。
    13. 将碎片转移到新的12孔板中。
    14. 让它们干燥过夜。
    15. 使用Odyssey成像仪(在700nm近红外区域检测)可视化生物膜(图4)。

    结晶紫生物膜形成的比色评估&nbsp;
    1. 在洗涤硅氧烷片后(步骤F2),将它们转移到12孔板中。
    2. 让碎片风干。&nbsp;
    3. 加入5毫升0.5%的结晶紫溶液。
    4. 在室温下在静态条件下染色10分钟。
    5. 用蒸馏水清洗碎片(重复此步骤3次)。
    6. 加入1毫升33%乙酸以溶解结晶紫染色。
    7. 在室温下在静态条件下孵育15分钟。
    8. 将200μl溶解的结晶紫转移到新的微孔板中。
    9. 使用读板器定量595nm处的吸光度。作为空白,使用33%的乙酸水溶液。可能需要连续稀释进入读板器的线性范围。&nbsp;

  7. 评估微孔板生物膜
  1. 孵育后(步骤E6),翻转平板除去培养物。&nbsp;
  2. 通过将板浸入装满水的小容器中来洗涤未结合的细胞。轻轻摇动盘子并摇出水。重复此步骤3次。
  3. 轻轻敲打纸巾以除去残留的液体,使板干燥。
  4. 让盘子风干。
  5. 加入200μl0.5%结晶紫溶液。
  6. 在室温下在静态条件下染色10分钟。
  7. 通过将板浸入装满水的容器中进行冲洗。浸没后,剧烈摇动盘子,倾倒液体,然后用纸巾轻拍盘子以除去残留的液体。重复此步骤3次。
  8. 加入200μl33%乙酸以溶解结晶紫染色。
  9. 在室温下在静态条件下孵育15分钟。
  10. 将200μl溶解的结晶紫转移到新的微孔板中。
  11. 使用读板器定量595nm处的吸光度。作为空白,使用33%的乙酸水溶液。可能需要连续稀释以落入读板器的线性范围。

数据分析

  1. 生物膜相关CFU的分析
    1. 记录 10 转换从每个导管中回收的最终细菌计数(n≥3)(图3)。
    2. 以线性比例绘制数据并包括算术平均值。&nbsp;
    3. 根据您要比较的组的数量,使用双尾Mann-Whitney U检验或单向ANOVA以及相应的帖子分析log 10 转换后的数据 - 测试比较。
      注意:Log 10 转换是可选的。绘制和分析数据的另一种方法是使用几何平均值和几何标准差在对数刻度上绘制未转换的数据。


      图3.通过菌落形成单位(CFU)测量的生物膜的代表性数据。&nbsp; E之间生物膜形成的比较。粪便&nbsp; OG1RF(野生型菌株)和锰摄取系统三重突变菌株(ΔefaΔmntH1ΔmntH2),它是一种生物膜缺陷型突变体。进行双尾Mann-Whitney U检验以确定两组之间的显着性(*** P <0.0002)。

  2. 通过免疫染色或结晶紫评估生物膜形成的分析
    1. 在线性标度上绘制每组(n≥3)的阴性对照(图4)或吸光度(相对于阴性对照)的相对荧光,并包括算术平均值和标准偏差。
    2. 使用Mann-Whitney U检验分析数据。
      注意:
      1. 导管上的生物膜形成测定通常不遵循由D'Agostino-Pearson测试确定的正态分布。因此,非参数分析(如Mann-Whitney U检验)比参数ANOVA检验更合适。&nbsp;
      2. 可以使用ROUT方法识别异常值(建议Q = 1%),如果确认,可以从最终分析中删除。


      图4.通过免疫染色和结晶紫分析生物膜的代表性图像和数据。 A.通过 E可视化导管片上生物膜的形成。粪肠球菌 OG1RF和锰摄取系统三重突变株(ΔefaΔmntH1ΔmntH2)。导管控制是那些仅用尿液(无细菌)孵育的Fg涂层导管。 B.通过相对于导管对照绘制荧光,对OG1RF WT和缺陷突变体之间的生物膜形成进行免疫染色分析。 C.CV染色通过绘制相对于导管对照的吸光度来量化和比较OG1RF WT和缺陷突变体之间的生物膜形成。进行双尾Mann-Whitney U检验以确定两组之间的显着性(*** P <0.0002)。

食谱

  1. 媒体
    1. BHI液体媒体
      将37克BHI肉汤粉加入1升蒸馏水中 混合直至溶液澄清
      高压灭菌20分钟
      在使用前让介质冷却至室温
    2. BHI-琼脂平板
      将37g BHI肉汤粉和15g琼脂加入1L蒸馏水中 混合直至溶液澄清
      高压灭菌20分钟
      让介质冷却到45°C的温度
      如果需要添加抗生素并混合
      保持培养基温暖,将25毫升培养基倒入培养皿中
  2. 尿液
    1. 将来自至少三个健康女性供体的尿液汇集(或获得市售的混合尿液)。在研究期间使用相同的尿液批次以保持一致性
    2. 将合并的尿液以7,000 rpm(7,505 x g )离心5分钟以除去沉淀物
    3. 使用pH条测量pH,并根据需要使用HCl溶液或NaOH溶液调节至6.5
    4. 使用瓶顶过滤器对混合的尿液进行过滤消毒。每次优先使用新鲜尿液。如果不立即使用,将尿液储存在4°C不超过3天。如果观察到沉淀物形成,则以7,000rpm(7,505 x g )重复离心5分钟
  3. 解决方案
    1. 阻止解决方案
      1x PBS,含1.5%BSA和0.1%叠氮化钠。保持溶液在4°C
    2. 洗涤液
      含有0.05%Tween-20的1x PBS。保持溶液在室温下
    3. 稀释缓冲液
      含有0.05%Tween-20,0.1%BSA和0.5%甲基α-d-甘露糖苷的1x PBS。保持溶液在4°C
    4. 一抗解决方案
      将1:500的兔抗链球菌组D抗原抗血清加入稀释缓冲液中。每次都准备新鲜
    5. 二抗溶液
      将1:10,000的山羊抗兔IRDye 680LT加入稀释缓冲液中。每次都准备新鲜
    6. 0.5%结晶紫溶液
      将0.5毫克加入100毫升蒸馏水中。过滤消毒溶液。保持溶液在室温下
    7. 33%的醋酸
      将330毫升乙酸加入670毫升蒸馏水中。保持溶液在室温下

致谢

C.C.W.得到了美国心脏协会GSA Predoctoral Fellowship 16PRE29860000,J.A.L。的支持。由国家过敏和传染病研究所AI135158,A.L.F.M。提供支持。得到了圣母大学的启动资金支持。
对Nallapareddy 等人(2011)和Flores-Mireles 等人(2014)所做的先前研究的特别肯定。

利益争夺

作者声明没有竞争性的经济利益。

参考

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引用:Colomer-Winter, C., Lemos, J. A. and Flores-Mireles, A. L. (2019). Biofilm Assays on Fibrinogen-coated Silicone Catheters and 96-well Polystyrene Plates. Bio-protocol 9(6): e3196. DOI: 10.21769/BioProtoc.3196.
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