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

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 polysaccharides^1,2,3. Biofilm formation is a dynamic process in which a cascade of events culminates in formation of a mature adher....

Protocol

1. Preparation of Reagents

1. 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 Shigell....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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