A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Summary

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Abstract

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (> 50% reduction in cell length for each antibiotic; P< 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug's MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

Introduction

Clostridium difficile is a gram positive, spore-forming bacterium, causing approximately 500,000 infections annually in the US and is considered a threat level urgent pathogen by the Centers for Disease Control and Prevention (CDC), the highest level of risk.¹ The past decade has seen considerable drug development in antimicrobials with activity against C. difficile.^2,3In vitro studies are a necessary component of the drug development process.⁴ Traditionally, in vitro susceptibility and time kill studies are used to validate fu....

Protocol

1. Isolating C. difficile from Different Environmental or Clinical Sources

1. Environmental isolates: Using a pre-sterilized cotton gauge (lightly wetted with 0.85% NaCl), swab the surface of any area of interest (floor, door, handle, shelf, etc.).⁸ Make sure to wear sterile gloves and place the swab in a sterilized tube after completed.
2. Clinical isolates (stool): Plate 10 to 100 mg of clinical stool samples onto cefoxitin-cycloserine-fructose agar (CCFA) using an inoculation loop and incubate under strict anaerobic conditions for 48 - 72 h. Store isolated colonies of C. difficile stock in cryovials....

Results

Clostridium difficile is a spore-forming bacterium and thus it is essential to determine the morphology differences between vegetative and spore cells prior to any functional analysis. Figure 1 demonstrates representative images of vegetative cells that were captured during the exponential phase of the growth curve and spore cells. As depicted, vegetative cells are long, smooth, rod-shaped structures whereas spores are small, oval structures that have a rough ext.......

Discussion

The goal of the current study was to create a high-throughput method for isolating C. difficile and testing antibiotic susceptibility using scanning electron microscopy (SEM) as a means for a more thorough characterization of the antibiotic's pharmacological action. Using the protocols outlined here, we have demonstrated that imaging the cell's phenotypic response to antibiotic treatment can reveal insight into the pharmacological action of the drug. In total, the imaging portion of this protocol takes r.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
cotton gauze	Caring	PRM21408C
NaCl	Macron	7532
50 mL tubes	Falcon	352098
Brain Heart Infusion (BHI)	Criterion	C5141
L-cysteine	Alfa Aesar	A10389
yeast extract	Criterion	C741
sodium taurocholate	Alfa Aesar	A18346
anaerobic chamber	Coy		vinyl anaerobic chamber
cycloserinecefoxitin fructose agar (CCFA) plates	Anaerobe systems	AS-213
blood agar plates	Hardy diagnostics	A-10
latex agglutination reagent	Oxoid	DR1107A	C. diff test kit
microcentrifuge tubes	Eppendorf	222363204
PBS	Gibco	10010-031
4% paraformaldehyde	Fisher Scientific	50-259-98
microscope slides	J. Melvin freed brand	7525M	75 x 25mm
flow hood	Labconco	Class II type A2	biosafety cabinet
desk sputtering machine	Denton Vacuum	Desk II
tape	Plastic Core	05072-AB	SPI Double Sided Adhesive Carbon Tape
gold	Denton Vacuum	TAR001-0158	2.375” Diameter x .002” Thick Gold foil
scanning electron microscope	FEI	XL-30