Biofilm Assay for Clostridioides difficile with Applications for Drug Discovery

Summary

Biofilms of the bacterial pathogen Clostridioides difficile and their significance in disease are not well understood. Recent findings have suggested a role in recurrence, underscoring their importance. Here, we describe the adaptation of a biofilm assay coupled to a metabolic readout with applications for drug discovery against biofilms.

Abstract

Clostridioides difficile is a gastrointestinal bacterial pathogen able to take advantage of a dysbiotic microbiota environment to proliferate, secrete toxins, and damage the intestinal epithelium. A subset of C. difficile infection (CDI) patients will experience antibiotic (15%-30%) or fecal microbiota transplant (FMT) (<10%) treatment failure. Therefore, the development of additional therapeutic interventions is of critical importance. The role of C. difficile biofilms in recurrence is unclear. However, biofilms in other organisms are responsible for chronic and relapsing disease, suggesting this could also be the case in recurrent CDI. We hypothesize that biofilms of C. difficile present a valuable therapeutic target. The goal of the protocol presented here is to adapt a biofilm formation assay for the identification of repositionable compounds with activity against established C. difficile biofilms. The protocol refines a robust and reproducible assay for forming biofilms, couples it to a metabolic assay, and applies it to drug discovery. This protocol outlines the biofilm formation assay, biomass and metabolic activity readouts, drug susceptibility testing, drug screening of a repositioning library, and representative results.

Introduction

Clostridioides difficile is a spore-forming, anaerobic bacterial pathogen capable of inflicting severe damage to the human gastrointestinal (GI) tract by producing toxins. C. difficile is responsible for nearly half a million cases yearly, with ~30,000 fatalities (CDC, 2015). C. difficile infection (CDI) treatment adds a significant cost to the already strained healthcare system (~$4.8 billion)^1,2,3. Individuals most at risk are those with immunosuppression, antibiotic exposure, and/or the elderly, all populations that continue to significantly expa....

Protocol

1. Preparing cells for the biofilm assay

CAUTION: Clostridioides difficile is a human pathogen and requires BSL-2 containment.

NOTE: Spores utilized for this study were acquired from Dr. Carol Kumamoto at Tufts University School of Medicine. C. difficile spore stocks were prepared as previously described^43,44,45. Spores are then aliquoted as 10 µL volumes into PCR tubes for streaking out plates. All media and Phosphate Buffer Saline (PBS) required for the experiment should be pre-red....

Results

The aim of the protocol described above is to adapt and validate the 96-well plate biofilm formation model as a drug screening platform for the identification of repositionable compounds with activity against established biofilms of C. difficile. The impact of distinct cell densities (10⁴, 10⁵, 10⁶, and 10⁷ cells/mL) on biofilm formation was determined, and biofilm formation was quantified by Crystal violet staining as previously descri.......

Discussion

C. difficile biofilm formation and the role it plays in disease are not well understood. Recent findings suggest a role for biofilms in disease relapse^28,34. Biofilm formation is a significant virulence factor^22,23,24,25 and in bacterial infections, such as those caused by Staphylococcus aureus and Pseudomonas aeru.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ACETIC ACID SAF-CT ACS 500ML	Fisher Scientific	A38S500
AGAR BACTERIOLOGICAL GR 1KG	Fisher Scientific	AC443570010
BioTek Synergy H1 plate reader	Fisher Scientific	BTH1MG
BOTTLE YEAST EXTRACT 500G	Fisher Scientific	DF0127179
BRAIN HEART INFUSION BROTH	Fisher Scientific	CM1135R
Crystal Violet USP grade 100G	VWR	97061-850	For staining biofilms for biomass assays
CytoOne 96-well TC plate, flat bottom, clear, indiv.wrapped withlids, 50/case	USA Scientific	CC7682-7596
D-(+)-Glucose, anhydrous	Fisher Scientific	50712744
Dimethyl Sulfoxide for Molecular Biology (DMSO)	Millipore Sigma	D8418-250ML
FIDAXOMICIN 250MG	Fisher Scientific	AC468312500
L-CYSTEINE 98+% 1KG	Fisher Scientific	AAA104350B	For C. difficile media
METHANOL 99.8 ACROSEAL 1LT	Fisher Scientific	AC364390010
METRONIDAZOLE, 99% 5GR	Fisher Scientific	AC210340050
Phosphate buffer saline (PBS) 10X	Fisher Scientific	BP3991
PrestoBlue Cell Viability Reagent	Fisher Scientific	A13262
SILVERSEAL OPAQUE ADHES 100/CS	Fisher Scientific	7000379	Used to seal mother and daughter drug plates before storage
SODIUM TAUROCHOLATE 100MG	VWR	100291-598
VANCOMYCIN	Fisher Scientific	BP29581