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

The overall goal of this biofilm formation and assessment protocol is to determine the ability of enteric bacterial pathogens to form a biofilm, especially in conditions that mimic gastrointestinal transit and exposure to bile salts. The methods can help answer key questions in the bacterial pathogenesis field, such as the ability of enteric pathogens to survive the harsh bile salt conditions of the gastrointestinal tract. The main advantage of the techniques is that the combined results from the three assays support in-depth characterization of biofilm formation in response to bile salt exposure for each pathogen.

We first had the idea for the combined methods, given the need to fully quantify bile salt-induced biofilm formation in Shigella flexneri. Generally, individuals new to this method will struggle because aspects of the assay can limit reproducibility. So please pay attention to the critical steps, to ensure reproducibility.

Demonstrating the procedures will be members from my laboratory, Kourtney Nicherson, postdoctoral fellow, and Alejandro Llanos, clinical research fellow. First, label two 1.5 milliliter tubes as TSB and TSB plus BS.Then add one milliliter of TSB or TSB plus BS to the respective tubes. Next, inoculate the tubes with 20 microliters of overnight culture at a one to 50 dilution.

Then add 130 microliters of uninoculated control media to each of three wells in a sterile, clear, flat-bottomed, tissue culture-treated, 96-well plate. This will be the blank control. Add 130 microliters of inoculated culture into each of the three wells.

After adding the control and the inoculated culture in the respective wells, incubate the 96-well plate for four to 24 hours at 37 degrees Celsius, statically. Once the incubation is over, set the control well as blank on the plate reader. Next, aspirate the culture medium from each well.

Then gently wash the wells once with 200 microliters of sterile PBS. After washing, aspirate the PBS, and then invert the plate to dry for about 20 minutes. It's very important to ensure gentle removal of the media and gentle washing, to ensure that the EPS matrix is not disturbed.

Next, add 150 microliters of 0.5%crystal violet to each of the experimental and control wells. Then incubate the plate for five minutes at room temperature. After five minutes, wash the wells once with 400 microliters of distilled water.

Add 200 microliters of distilled water to wash the wells five times. After all the five washes, invert the plate and let it dry completely, protected from the light. In order to obtain consistent results, it is important that the samples are thoroughly dried before proceeding.

After the plate is completely dried, use 200 microliters of 95%ethanol to de-stain the wells. Then leave the plate on the shaker for 30 minutes at four degrees Celsius, in order to avoid evaporation. After 30 minutes, record the OD 540 on the plate reader.

For semi-quantitative detection of EPS, use a multichannel pipette to transfer the culture medium to a clear 96-well plate. Then use 200 microliters of fixing reagent to fix the black plate for 15 minutes at room temperature. Now record the OD 600 reading by setting the control well as the blank for standardization.

Then record the reading of the culture medium while the adherent population is fixing. Once the fixing is complete, remove the reagent and discard in the hazardous waste. Then use 200 microliters of sterile PBS to gently wash the wells twice.

After washing, aspirate the PBS. Next add 150 microliters of 25 micrograms per milliliter of ConA-FITC. Incubate the plate at room temperature for 15 minutes.

Post-incubation, gently wash the wells with 200 microliters of PBS. Then add 150 microliters of PBS to each well, and record the fluorescence at 488 nanometers. After preparing fresh reagents, warm the PBS, PBS with glucose, PBS with bile salts, and PBS with glucose and bile salts to 27 degrees Celsius.

Then record the OD 600 values from the overnight biofilm plate on the plate reader by setting the control well as blank. Next, aspirate the culture medium using a vacuum line, without disturbing the adherent population on the plastic surface. Gently wash the wells twice with 200 microliters of sterile PBS.

After washing with PBS, add 130 microliters of the pre-warmed PBS, PBS with glucose, PBS with bile salts, and PBS with bile salts and glucose to each of the three wells, then incubate the plate at 37 degrees Celsius for 30 minutes. After 30 minutes, remove the plate from the incubator. Pipette the supernatant out to a fresh sterile 96-well plate.

Finally, prepare one to 10 serial dilutions of the supernatant with PBS in the 96-well plate, or in a dilution block. Then use a multichannel pipette to transfer five microliters of each dilution on LB agar plates. Incubate the plates at 37 degrees Celsius overnight.

The next day, count the number of colonies. The bar plot is obtained to quantify the bile salt-dependent biofilm formation in enteric pathogens. This is done by plotting the OD 540 values of the individual strains in the presence and absence of bile salts in the medium.

The plot shows significant increase in the biofilm formation for most of the pathogen strains, except Enteroaggrative E.Coli. Next, the EPS produced during biofilm formation is detected by determining the amount of ConA-FITC retained by the EPS. The bar plot shows significantly increased fluorescence reading in the presence of bile salts.

This confirms EPS production for pathogen Shigella flexneri. Here, ConA-FITC-dependent staining of the EPS matrix is significantly high when exposed to the bile salts. This indicates the formation of bacterial biofilm, characterized by the polysaccharide-rich EPS matrix.

One X PBS and DAPI are used to show FITC-dependent specificity and presence of the bacterial DNA, respectively. Finally, the number of colony-forming units is counted to analyze the bile salt-dependent dispersion of bacteria from the biofilm. The plot shows that the bacteria readily disperse from the biofilms in PBS and PBS with glucose.

However, exposure of the bacteria to bile salts drastically inhibits its dispersion from the biofilms. These assays were developed to capture and quantify the transient virulent biofilms formed by enteric pathogens, particularly Shigella flexneri, in response to bile salt exposure in the small intestine prior to infection in the colon. Modifications to the assays, such as the base media types, composition of the bile salts used, and bacterial growth conditions, may need to be adjusted for different pathogens, depending on the site of infection within the gastrointestinal tract.

After watching this video, you should have a good understanding of how to perform each of the assays to thoroughly evaluate biofilm formation in your bacterial or mutant strain of interest. Remember that working with the pathogens and some of the solutions can be extremely hazardous. Precautions such as appropriate handling and disposal methods should always be considered when performing these assays.

Furthermore, fresh bile salt media preparation, proper controls, and ensuring experimental reproducibility are vital to the data analysis and confirmation of the bile salt-induced biofilm phenotype. The techniques, which are unique and creative high-throughput applications of traditional methods, will enable appropriate quantification of the multifaceted and dynamic process of bile salt-induced biofilm formation in enteric pathogens.