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.
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.
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 future animal and other in vivo studies.
While these methods serve an important role for evaluating killing action, they do not capture the cells' phenotypic response to pharmacological treatment. By incorporating scanning electron microscopy (SEM) with standard killing kinetic studies, a more thorough characterization of the antibiotic direct effects is possible.5,6,7 Here, we present a method where SEM is used as a means to profile the efficacy of antibiotic treatment.
1. Isolating C. difficile from Different Environmental or Clinical Sources
2. Culturing C. difficile and Killing Kinetic Procedures
3. Preparing Samples for Scanning Electron Microscopy
4. Imaging C. difficile Cells on a Scanning Electron Microscope
5. Image Processing and Analysis
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 exterior. Functionally, vegetative cells grow and divide rapidly and are responsible for the virulence of C. difficile infections by secreting toxins, whereas spores are normally dormant with little activity. Hence, antibiotics are mostly active against vegetative cells, while spores are usually resistant to drug treatment, due to several layers protecting the core with DNA, and lack of physiologic activity. Due to these facts, the majority of the morphologic analysis is focused on the vegetative cells.11,12
An antibiotic killing curve is the standard methodology to demonstrate antibiotic efficacy against growing bacteria. Concentrations used with C. difficile strain R20291 were based on the MICs values, 1 µg/mL for vancomycin and 0.51 µg/mL for metronidazole.10 As shown in Figure 2, the control cells grow and reach a plateau, whereas the treated cells decrease in total colony forming units (CFU) to the limit of detection (LOD) demonstrating bactericidal effect. As demonstrated, vancomycin (Figure 2A) and metronidazole (Figure 2B) are effective at killing C. difficile at supraMIC concentrations (4xMIC). One may notice that the antibiotic killing curves look similar, yet the drugs have different mechanisms of action (vancomycin prevents cell wall synthesis while metronidazole affects DNA replication). Therefore, we propose that antibiotic killing curves may not have enough of a discriminatory power to provide detailed differences between these antibiotics. We used microscopy to provide more discrimination.
Because of its microscopic size, C. difficile is not possible to image without high magnification. This has provided an opportunity for the use of scanning electron microscopy (SEM), an imaging method that can obtain resolution at the nanometer scale, to assess the detailed effects of antibiotic treatment directly on the cells. To demonstrate the utility of this approach, cells were imaged before and after drug treatment to determine how the morphology changed (Figure 3A). As demonstrated, some of the cells' walls were affected, as in the case of vancomycin, and some of the cells were smaller in size, as was the case for metronidazole. To test whether cell size was affected, we analyzed cell length using the program FIJI. Vegetative cell size can vary some in the control case, but most are roughly 6 µm in length (Figure 3B). When considering many cells from the control and treated settings, one can quickly notice that the cell length is preferentially affected in metronidazole but is not affected by vancomycin treatment (Figure 3C).
Figure 1: Differentiating Between Cell Types by Scanning Electron Microscopy. Shown are representative images of Clostridium difficile vegetative and spore cells. Vegetative cells are rod structures that are long and flagellated. In contrast, spore cells are small and have a rough and bumpy coat. Spores are pseudo-colored red for image purposes only. Scale bars are shown on the images. Please click here to view a larger version of this figure.
Figure 2: Antibiotic Time-Kill Curves. SupraMIC killing curves were carried for four different antibiotics: vancomycin (A), metronidazole (B). These antibiotics had significant killing action against C. difficile strain R20291 and approached the limit of detection (LOD) for counting colony forming units (CFU). Please click here to view a larger version of this figure.
Figure 3: Examining the Pharmacological Effects of Antibiotic Treatment. Images of treated and non-treated cells were taken at multiple time points throughout the killing kinetic studies (A). Shown is a representative example of a control vegetative cell that was measured for length (B). Treated and non-treated cells were measured and compared (C). Experiments were performed in at least duplicate and > 17 cells were measured per group. *P< 0.05 compared to control and vancomycin treatment groups. Please click here to view a larger version of this figure.
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 roughly 2 h in duration after collecting the cells, but can be much more discriminatory than typical killing kinetic studies alone. While learning to use an SEM can be technically demanding, preparation of samples is relatively simple and fast. We believe these protocols outlined here will provide an objective approach to evaluating the pharmacological action of antibiotic treatment.
Considering the similarities among the antibiotic killing curves (Figure 2), we sought to determine whether there were differences among the cell's response to pharmacological treatment. Using SEM, we determined that metronidazole had effects on cell length at supraMIC concentrations of the drug. While these phenotypes have not been reported previously, they are consistent with the antibiotics' mechanisms of actions and suggest an effect on cell metabolism and growth. In contrast, vancomycin had significant effects on the cell wall, which is also consistent with its mechanism of action.13 While there are similarities among the killing kinetics between these antibiotics, it is apparent from the SEM images that there are significant phenotypic differences. Creating an antibiotic phenotype library will allow for a more thorough characterization of drugs that may not have a clear mechanism of action identified, as is the case of ridinilazole.
Because this method is technically challenging, modifications may need to be made in order to address the scientific question including consistent cell preparation and alterations in sputtering of gold coating. Too much coating may blur any effects to the exterior of the cells, but not enough coating can result in charging of the beam and distort the images. To avoid this, prepare samples with different amounts of gold coating to optimize sputtering time. Lastly, consistent methodology between studies will allow inter-experimental comparisons to be made.
A limitation of using SEM is that it is restricted to observing cell morphology changes; therefore, functional studies are necessary to confirm any suspected response. Because of this, we believe that this imaging protocol can be used as a means of directing functional studies. Despite this limitation, immunogold labeling can be done using SEM to confirm any suspected changes in protein trafficking or aggregation; however, we have not yet conducted these experiments.
Due to the active pipeline of new antibiotics for C. difficile treatment, a more thorough evaluation of drug action is necessary. As presented here, SEM offers a unique, high-throughput, and reliable opportunity for characterizing pharmacological action. By imaging many different antibiotic effects among different strains of C. difficile, we will be able to understand why some antibiotics are more efficacious than others against specific pathogenic strains like the 027/NAP1/BI epidemic strain.14 Moreover, SEM analysis may be helpful to discriminate antibiotic effects between ribotypes or strains and could be used to study other bacterial species demonstrating its broad applicability.
The authors have nothing to disclose.
These experiments have been supported by research grants from Merck and Co. and Summit, PLC.
cotton gauze | Caring | PRM21408C | |
NaCl | Macron | 7532 | |
50mL 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 | 75x25mm |
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 |