JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Immunology and Infection

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Published: May 06, 2018
doi:
10.3791/57322
,
1Mucosal Immunology and Biology Research Center, Division of Pediatric Gastroenterology and Nutrition,Massachusetts General Hospital, 2Department of Pediatrics,Harvard Medical School

Summary

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Abstract

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

Introduction

Biofilm formation is an important bacterial survival strategy induced during harsh environmental conditions. Exposure to bactericidal compounds like antibiotics or changes in nutrient or oxygen availability induces a stressed state in bacteria that can be alleviated through biofilm formation. A biofilm is characterized by bacterial attachment to a surface or other bacteria and is accompanied by the secretion of an EPS matrix primarily composed of polysaccharides1,2,3. Biofilm formation is a dynamic process in which a cascade of events culminates in formation of a mature adherent bacterial community1,2,3. Bacteria produce adhesins to facilitate early attachment while shifting adhesin gene expression profiles to strengthen attachment during biofilm maturation. Simultaneously, EPS production occurs to coat the bacterial community in a matrix to protect the cells from the initial stressor. Bacteria contained within the biofilm are slow growing; and as such, renders most antibiotics ineffective. Furthermore, the slow growth conserves energy until conditions change to favor bacterial growth1,2,3. After the harsh conditions have passed, bacteria disperse the biofilm and resume a planktonic lifestyle1,2,3. Traditionally, biofilms are observed on surfaces and represent a persistent clinical challenge due to infection reservoirs present on catheters and in-dwelling devices1,2,3.

Biofilm formation was recently described for several enteric pathogens; bacteria that infect the small intestine or colon4. For Shigella species, infection occurs in the human colon after a transit through the majority of the gastrointestinal tract. During passage through the small intestine, Shigella is exposed to bile; a lipid-degrading detergent secreted into the intestine to facilitate digestion of lipids while simultaneously killing most bacteria5. Enteric pathogens have a unique ability to resist the bactericidal effects of bile6. Our recent analysis utilized in vivo-like combinations of glucose and bile salts to demonstrate robust biofilm formation in S. flexneri as well as other species of Shigella, pathogenic Escherichia coli, and Salmonella4. Previously, Salmonella enterica serovar Typhi was shown to form a bile-induced biofilm due to unique colonization of the gallbladder during chronic infection7,8,9,10. Additionally, prior research with Vibrio11 and Campylobacter12 demonstrated biofilm formation in response to bile. Therefore, the analyses extended the bile-induced biofilm formation observations to other pathogens and help to establish demonstration of a conserved enteric pathogen response to bile. Unlike chronic biofilms in which bacterial gene transcription is limited and cell senescence can occur1,2,3, we propose that the enteric bile-induced biofilm is more transient in nature. This transient, virulent biofilm is hallmarked by a rapid disassembly (as seen in the dispersion assay) and enhanced virulence gene expression observed in the biofilm population4,6.

As biofilm formation is a multifaceted, dynamic process and the use of bile salts as an initiating factor has only been recently described for most enteric pathogens, the tools and techniques used are unique and creative applications of traditional methods. Thus, presented here are three complimentary strategies to quantify several important characteristics of bile salt-induced biofilm formation, including bacterial adherence, production of the EPS matrix, and dispersion of viable bacteria from the biofilm. These techniques have been utilized primarily for research with Shigella; and therefore, evaluation of other enteric pathogens may require optimization. Nevertheless, positive data from all three assays support identification of biofilms and establish reproducible protocols for bile salt-induced biofilm formation.

Protocol

1. Preparation of Reagents Bile salts medium: To prepare tryptic soy broth (TSB) containing 0.4% bile salts (weight/volume), resuspend 200 mg of bile salts in 50 mL autoclaved TSB. Filter sterilize using a 0.22 µm filter. Make fresh medium weekly. Notes: The bile salts routinely used is a 1:1 mixture of sodium cholate and sodium deoxycholate isolated from ovine and bovine gallbladders. As demonstrated previously4, the presence of glucose was required for bile salt-induced biofilm formation. TSB has added glucose relative to Luria-Bertani (LB) broth; and therefore, was sufficient to induce biofilm formation in Shigella and the other enteric pathogens analyzed. Depending on the bacteria to be analyzed, different glucose concentrations or different sugar requirements might be needed. 0.5% w/v crystal violet in water: Dissolve 2.5 g of crystal violet in 500 mL distilled water. Filter sterilize using a 0.22 µm filter. Concanavalin A (ConA) conjugated to fluorescein isothiocyanate (FITC): Reconstitute the stock in 1x PBS. Dilute the 10 mg concentrated stock with 400 μL of 1x PBS to a final concentration of 25 µg/mL, and protect from light. PBS + Glucose: Dissolve 0.2 g glucose in 10 mL 1x PBS (2% w/v glucose final). Filter sterilize using a 0.22 µm filter. Make fresh on the day of use. PBS + Bile Salts: Dissolve 40 mg in 10 mL 1x PBS (0.4% w/v bile salts final). Filter sterilize using a 0.22 µm filter. Make fresh on the day of use. PBS + glucose and bile salts: Dissolve 40 mg bile salts and 0.2 g glucose in 10 mL 1x PBS (0.4% w/v bile salts and 2% w/v glucose final). Filter sterilize using a 0.22 µm filter. Make fresh on the day of use. Prepare LB agar plates. Formaldehyde/glutaraldehyde fix: Add 810 µL formaldehyde (37% stock solution, 3% final concentration) and 125 µL glutaraldehyde (25% stock solution, 0.25% final concentration) to 14 mL 1x PBS. Mix thoroughly and store at 4 °C. The fix should be cold for proper use. Caution: The fix is toxic and requires hazardous waste disposal. Antifade mountant solution: Use antifade mountant solution containing 4,6-diamidino-2-phenylindole (DAPI) stain to inhibit photobleaching of immunofluorescent microscopy samples while fluorescently staining the DNA of the bacteria. 2. Preparation of Bacteria Grow overnight cultures of the bacterial strains to be tested by inoculating 3 mL of TSB with a single, well-isolated colony in a sterile culture tube. Incubate at 37 °C with shaking at 225 rpm for overnight incubation (16 – 24 h). NOTE: Strains should be restreaked from freezer stocks every 2 to 4 weeks, and maintained on plates no more than 2 weeks old. 3. Solid-phase Adherence Assay NOTE: This assay quantifies adherent bacteria using a 96-well plate method. Bacteria are grown statically in flat bottom plates. Washing is performed to remove non-adherent bacteria and adherent bacteria are stained with crystal violet. The crystal violet stain binds peptidoglycan in the bacterial cell wall and can be solubilized using ethanol. The number of adherent bacteria is determined based on crystal violet retention. Set up two 1.5 mL tubes. Label with TSB or TSB + bile salts (BS). Add 1 mL of TSB or TSB + BS to the respective tubes. Inoculate tubes with 20 µL of overnight culture (at a 1:50 dilution). In a sterile, clear, flat-bottomed, tissue culture-treated 96-well plate, add 130 µL/well of uninoculated control media to three wells to serve as the blank control. Set up three control wells for each media type (TSB and TSB + BS) to be tested. Add 130 µL/well of inoculated culture into three wells, and repeat until all experimental conditions are plated in triplicate. Incubate for 4 – 24 h at 37 °C statically. Using a plate reader, record the OD600. Set the control wells as 'blank.' Confirm the control medium is clear with no evidence of turbidity. If any turbidity is detected, discard the experiment. The OD600 values can be used to normalize the data if there are significant differences in growth rate between bacterial strains. Remove the culture medium using a vacuum line by gently tilting the plate and slowly aspirating the medium at the lower edge of the well. Be sure to collect all the culture medium without disrupting the adherent bacterial population located on the plastic surface. If EPS matrix was produced during the incubation time, the matrix will be visualized as a white precipitate. Do not disturb the EPS matrix. Gently wash the wells once with 200 µL sterile PBS. Remove the PBS wash using the vacuum line. Invert the plate to dry. Allow a minimum of 20 min to dry. NOTE: Since the biofilm must be thoroughly dried before staining, the protocol can be paused at this step for a few hours or even overnight. The added drying time will not alter the staining procedure while incomplete drying will affect quantification of the results and reproducibility. Add 150 µL of 0.5% crystal violet to each experimental and control well. Incubate for 5 min at room temperature (RT). Wash the wells once with 400 µL of distilled water. The added volume helps to remove residual crystal violet stain from the sides of the wells. Remove the wash with the vacuum line. Wash the wells five times with 200 µL of distilled water. Remove the wash with the vacuum line. NOTE: Thorough washing is important for quantification. When the blank wells are clear from the distilled water and do not contain any residual crystal violet stain, proceed to the next step. Invert the plate to dry, protected from light. Ensure complete drying of the plate as noted above. Destain the wells with 200 µL of 95% ethanol. Incubate the plate on the shaker for 30 min. To avoid evaporation, particularly at higher temperatures, perform this step at 4 °C. Using a plate reader, record the OD540. NOTE: The wavelength for maximum absorbance of crystal violet is near 590 nm. The literature reports a range from OD540 - OD5954,13,14,15,16 for crystal violet absorbance; thus choose a wavelength available based on the plate reader. 4. EPS Matrix Detection NOTE: These complimentary assays quantify and visualize the EPS. In both, EPS is detected using a lectin to bind polysaccharides. The fluorescently-conjugated protein allows quantification (step 4.1) or visualization (step 4.2). Semi-quantitative detection of EPS Set up two 1.5 mL tubes. Label with TSB or TSB + BS. Add 1 mL of TSB or TSB + BS to the respective tubes. Inoculate tubes with 20 µL of overnight culture (a 1:50 dilution). In a sterile, black, flat-bottomed, tissue culture-treated 96-well plate, add 130 µL/well of uninoculated control media to three wells to serve as the blank control. Set up three control wells for each medium type to be tested. Add 130 µL/well of inoculated culture to the wells, and repeat until all experimental conditions are plated in triplicate. Incubate for 4 – 24 h at 37 °C statically. Transfer the culture medium to a clear 96-well plate with a pipette, ideally a multichannel pipette. Be cautious to collect all the culture medium without disrupting the adherent population and/or the EPS located on the plastic surface. Set aside. Fix the black plate for 15 min at RT using 200 µL/well of the formaldehyde/glutaraldehyde in 1x PBS. While the adherent population is fixing, evaluate the supernatant fraction from step 4.1.6. Using a plate reader, record the OD600. Set the control wells as 'blank.' Confirm the control medium is clear with no evidence of turbidity. If any turbidity is detected, discard the experiment. Alternatively, the entire assay can be performed in a black clear bottom plate. In those circumstances, the OD600 values can be recorded and the culture medium can be subsequently discarded prior to proceeding to step 4.1.7. The OD600 values can be used to normalize the data in the case there are significant differences in growth rate between bacterial strains. Remove the fix and dispose in the hazardous waste. Gently wash the wells twice with 200 µL/well of sterile PBS. Remove the PBS wash using the vacuum line by gently tilting the plate and slowly aspirating the wash at the lower edge of the well. Add 150 µL/well of 25 µg/mL ConA-FITC, and incubate for 15 min at RT. Gently wash twice with 200 µL of PBS. Add 150 µL of PBS to each well. Record the fluorescence at 488 nm. Confocal microscopy visualization of EPS production and calculation of biofilm thickness Set up two 1.5 mL tubes. Label with TSB or TSB + BS. Add 1 mL of TSB or TSB + BS to the respective tubes. Inoculate tubes with 20 µL of overnight culture (at a 1:50 dilution). Set up a sterile 24-well plate with 12 mm sterile round glass coverslips. Add 400 µL/well of uninoculated control media to three wells to serve as the blank control. Set up three control wells for each medium type to be tested. Add 400 µL of inoculated culture to wells in duplicate, and repeat until all experimental conditions are plated. Incubate for 4 – 24 h at 37 °C statically. Visually confirm growth in the TSB condition, growth and EPS precipitate (white) in the TSB + BS condition, and no growth and/or white precipitate in sterile control wells. Remove the supernatants. Fix for 15 min at RT using 200 µL/well of formaldehyde/glutaraldehyde solution in 1x PBS. Remove the fix and dispose in hazardous waste. Gently wash the wells twice with 200 µL/well of sterile PBS. Remove the PBS wash using the vacuum line. Add 150 µL/well of 25 µg/mL ConA-FITC and incubate for 15 min at RT. Gently wash twice with 200 µL/well of PBS. Mount with antifade mountant solution with DAPI. NOTE: The solution is a ready-made, glycerol-based mount solution containing DAPI for preserving the fluorescent label while simultaneously staining the DNA in bacterial cells for visualization as a counterstain. Follow the kit's directions and recommendations for incubation times prior to imaging samples. The DAPI staining procedure can be performed prior to applying antifade mountant solution that does not contain DAPI. Evaluate by confocal microscopy. On a confocal microscope, set the laser to 495 nm/519 nm excitation/emission for FITC visualization. Set a second laser to 360 nm/460 nm excitation/emission to visualize DAPI. Locate the biofilm by focusing on the DAPI channel. Use both the DAPI and FITC channels to determine the full thickness and set the upper and lower imaging borders. Record full thickness Z-stack images by capturing images every 0.25 µm after setting perimeters to the top and bottom of the z-stack. For more information on confocal microscopy, please refer to publications by Paddock et al.17,18,19,20 Reconstruct the 3D images in ImageJ (https://imagej.nih.gov/ij/) to visualize the full biofilm formation and calculate the biofilm thickness. The average thickness obtained for S. flexneri bile-salt induced biofilms was 14 µm4. 5. Dispersion Assay NOTE: In this assay, the disassembly of biofilm through bacterial dispersion is detected. Here, mature biofilms are established and subsequently (usually the next day), the media are replaced with PBS or supplemented PBS. The supernatant component is then assessed to quantitate the number of bacteria that have dissociated from the biofilm. Set up two 1.5 mL tubes. Label with TSB or TSB + BS. Add 1 mL of TSB or TSB + BS to the respective tubes. Inoculate tubes with 20 µL of overnight culture (at a 1:50 dilution). In a sterile, clear, flat-bottomed, tissue culture-treated 96-well plate, add 130 µL/well of uninoculated control media to three wells to serve as the blank control. Set up three control wells for each media type to be tested. Add 130 µL/well of inoculated culture to three wells, and repeat until all experimental conditions are plated in triplicate. Incubate the plate for 4 – 24 h at 37 °C statically. Before continuing, warm the PBS, PBS + glucose, PBS + bile salts, and PBS + bile salts + glucose to 37 °C. Prepare these reagents fresh daily. Using a plate reader, record the OD600. Set the control wells as 'blank.' Confirm the medium is clear with no evidence of turbidity. If any turbidity is detected, discard the experiment. The OD600 values can be used to normalize the data if there are significant differences in growth rate between bacterial strains. Remove the culture medium using a vacuum line. Be sure to collect all the culture medium without disrupting the adherent population located on the plastic surface. Gently wash the wells twice with 200 µL/well of sterile PBS. Remove the PBS wash using the vacuum line. Replace the wash with 130 µL/well of the following into three wells each: PBS, PBS + glucose, PBS + bile salts, and PBS + bile salts + glucose. These reagents must be pre-warmed to 37 °C. Incubate the plate for 30 min at 37 °C. Carefully remove the plate from the 37 °C incubator. Transfer the supernatants to a fresh, sterile 96-well plate. Using a 96-well plate or dilution block, prepare 10-fold (1:10) serial dilutions of the supernatant into sterile PBS. NOTE: The dilution series should range from undiluted to 10-6 depending on the bacterial strain to be tested. Pilot experiments can be performed to determine the optimal dilution range. Spot plate 5 µL of each dilution onto LB agar using a multichannel pipette. Incubate at 37 °C overnight. Count colonies on the next day and account for the dilution factor when determining the recovery colony forming units (CFU). To calculate the percent dispersion relative to the 1x PBS control (set at 100%), divide the recovery CFU from each treatment condition by the recovery CFU from the 1x PBS control sample. NOTE: Routine media plates for the bacterial strain of interest can also be used. For example, Shigella can be plated on Congo red plates. Ensure the plates are dry for appropriate spot-plating techniques.

Representative Results

In Figure 1, biofilm formation is induced in most of the six enteric pathogens tested following growth in media containing bile salts. A significant increase in adherent bacteria after bile salts exposure is observed in nearly all strains tested. The exception is enteroaggregative E. coli (EAEC); however, note the induced observation of the Δaaf mutant4. The results indicate that additional adherence mec…

Discussion

Analysis of biofilm formation is challenging due to the dynamic nature of biofilms and the variability between strains, materials, laboratories, and assays. Here, several strategies are presented to determine biofilm formation in enteric pathogens following bile salts exposure with experimental insight provided to promote reproducibility. There are additional considerations to ensure reproducibility. First and foremost, we recommend performing at least three independent experiments each with technical triplicates to conf…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Rachael B. Chanin and Alejandro Llanos-Chea for technical assistance. We thank Anthony T. Maurelli, Bryan P. Hurley, Alessio Fasano, Brett E. Swierczewski, and Bobby Cherayil for the strains used in this study. This work was supported by the National Institute of Allergy and Infectious Diseases Grant K22AI104755 (C.S.F.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Materials

Tryptic Soy Broth Sigma-Aldrich  22092-500G
Crystal Violet Sigma C6158-50
Concanavalin-A FITC Sigma C7642-10mg
Glucose Sigma G7021-1KG
Bile Salts Sigma B8756-100G 
LB Agar Sigma L7533-1KG
14 mL culture tubes, 17 x 100 mm, plastic, sterile Fisher 14-959-11B
Vectashield hard-set antifade with DAPI Vector Laboratories H-1500 
Formaldehyde Sigma-Aldrich  F1635-500
Gluteraldehyde Sigma-Aldrich  G6257
Flat-bottomed 96-well plates (clear) TPP 92696
Flat-bottomed 96-well plates (black) Greiner Bio-One  655076
Flat-bottomed 24-well plates (clear) TPP 92424
Glass coverslips 12mm, round Fisher 08-774-383
96-well plate reader Spectramax
Flourescent plate reader Biotek Synergy 2
Confocal or Fluorescent Microscope Nikon A1 confocal microscope
37°C Shaking Incubator New Brunswick Scientific Excella E25
37°C Plate Incubator Thermolyne Series 5000
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Bile SaltsBiofilm FormationEnteric PathogensBacterial PathogenesisShigella FlexneriCrystal Violet AssayQuantification96-well PlateStatic IncubationPBS Washing
Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

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Nickerson, K. P., Faherty, C. S. Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification. J. Vis. Exp. (135), e57322, doi:10.3791/57322 (2018).
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