These experiments have been supported by research grants from Merck and Co. and Summit, PLC.

1. Isolating C. difficile from Different Environmental or Clinical Sources
<ol>
	<li>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.</li>
	<li>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 at -80 &#176;C for further analyses.7,9,10</li>
	<li>Enrich the environmental swab samples in brain heart infusion (BHI) broth with 0.05% sodium taurocholate and place in an anaerobic chamber at 37 &#176;C for 5 days. Centrifuge 1 mL of the culture at 10,000 x g and resuspend the pellet in 100 &#181;L of ethanol.</li>
	<li>Plate the resuspended cells (50 &#181;L) onto cycloserinecefoxitin fructose agar (CCFA) plates and incubate in an anaerobic chamber at 37 &#176;C for 40 - 48 h. Store isolated colonies of C. difficile stock in cryovials at -80 &#176;C for further analyses.</li>
	<li>Test suspected C. difficile colonies using the latex agglutination reagent or PCR.</li>
</ol>
2. Culturing C. difficile and Killing Kinetic Procedures
<ol>
	<li>Grow purified environmental or clinical C. difficile strains on blood agar plates in an anaerobic chamber at 37 &#176;C for 48 h.</li>
	<li>Take one isolated colony with an inoculation loop, transfer it into 5 mL BHI medium in a 15 mL tube, and grow for 24 h in an anaerobic chamber at 37 &#176;C .</li>
	<li>Dilute the pre-cultures 1:100 to approximately 106 colony-forming units (CFU)/mL in fresh pre-reduced BHIS (BHI plus 5 g/L yeast extract and 1% L-cysteine) supplemented with 0.1% sodium taurocholate and the appropriate concentration of antibiotic (T0).</li>
	<li>Collect a 1 mL sample with a pipette at each time point (T0, T6, T24, T48) and plate/spread a small aliquot (100 &#181;L in serial dilutions) onto a blood agar plate. Let the cells grow on the blood agar plate for 48 h in an anaerobic chamber at 37 &#176;C and count the resulting number of colonies to determine CFU.</li>
</ol>
3. Preparing Samples for Scanning Electron Microscopy
<ol>
	<li>Collect 1 mL of cells from each time point in microcentrifuge tubes and centrifuge at 10,000 x g for 10 min. Discard the supernatant and wash cells in PBS.</li>
	<li>Centrifuge the samples at 10,000 x g for 10 min again and discard the supernatant. Re-dilute cells in 1 mL of 4% paraformaldehyde and incubate for 1 h at room temperature.</li>
	<li>Centrifuge samples again for 10 min at 10,000 x g and discard supernatant. Wash cells twice with distilled water and redilute in 100 &#181;L of distilled water. Adjust volume depending on the turbidity of the solution. 
	NOTE: Making multiple serial dilutions is a good idea.</li>
	<li>Label coverslips and add 40 &#181;L of the sample on it. Incubate for 15 min in a flow hood to evaporate the liquid and allow the cells to adhere to the coverslip. If liquid is still present, use a blower to remove the liquid.</li>
	<li>Place labeled coverslips with cells in a desk sputtering machine and tape down. Secure pure gold in the sputtering machine. Turn machine on and commence sputtering at low pressure (50 mTorr). Coat cells for 30 s at 80 mA, which translates to 20 nm of gold coating.</li>
	<li>Transfer coated cells to the scanning electron microscope.</li>
</ol>
4. Imaging C. difficile Cells on a Scanning Electron Microscope
<ol>
	<li>Vent the scanning electron microscope (SEM) properly by pressing the vent button on the computer software.</li>
	<li>Using carbon tape, secure the coated coverslips onto the metal stage. Once the SEM is vented, the door should easily open. Lock the metal stage into the SEM chamber by screwing it in.</li>
	<li>Click the PUMP button on the computer software. The SEM will be useable when the system reads &#34;Vac OK&#34;.</li>
	<li>Click on the SE detector under the detectors tab. Turn on the beam by clicking on the button that displays the voltage. Start imaging at a lower voltage (5 kV) before increasing voltage (up to 15 kV). An image will appear after the beam is turned on.</li>
	<li>Using the tracking function, find an area on the coated coverslips to image. Zoom into the region and find rod-shaped structures; these are clostridium difficile.</li>
	<li>Zoom in and focus the image to calibrate the system. This should be done at multiple working distances: 15, 9 and 5 mm. Image at a working distance of 5 mm.</li>
	<li>Switch on the ultra high resolution imaging mode and begin to focus and optimize astigmatism.</li>
	<li>Begin to focus at high magnification. Use the coarse and fine focus toggles for this. Adjust astigmatism as well to obtain a clearer image.
	<ol>
		<li>Adjust astigmatism at high magnification. To do this, adjust the astigmatism toggles (they look similar to the fine/coarse focus toggles) and check the image for clarity by digitally zooming in on the computer software.</li>
	</ol>
	</li>
	<li>se the slow scan function to collect a high quality image. Save the collected image as a .TIFF file, which will be used for analysis. Make sure the data bar is selected if measurements will be made during analysis.</li>
	<li>Collect images at different angles (up to 52&#176; by manually turning the angle directly on the SEM. Angled images tend to reveal more depth information.</li>
	<li>Change beam voltage depending on the cells that are being imaged. Keep this mostly between 5 - 15 kV for all experiments.</li>
	<li>After imaging is completed, turn the beam off and raise the working distance to 20 mm. The chamber can then be vented and the stage can be removed. Copy all of the images onto a drive for further analysis.</li>
</ol>
5. Image Processing and Analysis
<ol>
	<li>Download and install the freely available software, FIJI (http://fiji.sc), on to the computer.</li>
	<li>Open the image file in FIJI.</li>
	<li>Using the line function, precisely trace the scale bar.</li>
	<li>Click on the Analyze tab in the FIJI program and finally select the Select Scale function. A window will appear that will require the setting of the known distance based on the scale bar. Change the unit of length as well and click OK.</li>
	<li>Now that the program is calibrated for distance, use the line function to measure cell length. To obtain the length, use the line function to trace the cell in its entirety. Select the Analyze tab again and then click on Measure. The length should appear in the units denoted previously.</li>
</ol>

<ol>
	<li>Lessa, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+Clostridium+difficile+infection+in+the+United+States.">Burden of Clostridium difficile infection in the United States.</a> N Engl J Med. 372 (9), 825-834 (2015).</li><li>Vickers, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ridinilazole:+a+novel+therapy+for+Clostridium+difficile+infection.">Ridinilazole: a novel therapy for Clostridium difficile infection.</a> Int J Antimicrob Agents. 48 (2), 137-143 (2016).</li><li>Shah, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+infection:+update+on+emerging+antibiotic+treatment+options+and+antibiotic+resistance.">Clostridium difficile infection: update on emerging antibiotic treatment options and antibiotic resistance.</a> Expert Rev Anti Infect Ther. 8 (5), 555-564 (2010).</li><li>Ambrose, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+EMA+guideline+for+antimicrobial+development.">New EMA guideline for antimicrobial development.</a> Lancet Infect Dis. 12 (4), 265-266 (2012).</li><li>Bass&#232;res, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+on+toxin+production+and+cell+morphology+in+Clostridium+difficile+by+ridinilazole+(SMT19969),+a+novel+treatment+for+C.+difficile+infection.">Impact on toxin production and cell morphology in Clostridium difficile by ridinilazole (SMT19969), a novel treatment for C. difficile infection.</a> J Antimicrob Chemother. 71 (5), 1245-1251 (2016).</li><li>Endres, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+imaging+the+pharmacological+effects+of+antibiotic+treatment+on+Clostridium+difficile.">A novel method for imaging the pharmacological effects of antibiotic treatment on Clostridium difficile.</a> Anaerobe. 40, 10-14 (2016).</li><li>Endres, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+Effects+of+Surotomycin+Treatment+on+Clostridium+difficile+Toxin+A+and+B+Production,+Immune+Response,+and+Morphological+Changes.">Evaluating the Effects of Surotomycin Treatment on Clostridium difficile Toxin A and B Production, Immune Response, and Morphological Changes.</a> Antimicrob Agents Chemother. 60 (6), 3519-3523 (2016).</li><li>Alam, M. J., Anu, A., Walk, S. T., Garey, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+potentially+pathogenic+Clostridium+difficile+contamination+in+household+environs.">Investigation of potentially pathogenic Clostridium difficile contamination in household environs.</a> Anaerobe. 27, 31-33 (2014).</li><li>Aitken, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+the+Endemic+Setting,+Clostridium+difficile+Ribotype+027+Is+Virulent+But+Not+Hypervirulent.">In the Endemic Setting, Clostridium difficile Ribotype 027 Is Virulent But Not Hypervirulent.</a> Infect Control Hosp Epidemiol. , 1-6 (2015).</li><li>Basseres, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+on+toxin+production+and+cell+morphology+in+Clostridium+difficile+by+ridinilazole+(SMT19969),+a+novel+treatment+for+C.+difficile+infection.">Impact on toxin production and cell morphology in Clostridium difficile by ridinilazole (SMT19969), a novel treatment for C. difficile infection.</a> J Antimicrob Chemother. 71 (5), 1245-1251 (2016).</li><li>Walters, B. A., Roberts, R., Stafford, R., Seneviratne, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relapse+of+antibiotic+associated+colitis:+endogenous+persistence+of+Clostridium+difficile+during+vancomycin+therapy.">Relapse of antibiotic associated colitis: endogenous persistence of Clostridium difficile during vancomycin therapy.</a> Gut. 24 (3), 206-212 (1983).</li><li>Chilton, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+effect+of+oritavancin+on+Clostridium+difficile+spore+germination,+outgrowth+and+recovery.">Evaluation of the effect of oritavancin on Clostridium difficile spore germination, outgrowth and recovery.</a> J Antimicrob Chemother. 68 (9), 2078-2082 (2013).</li><li>Ofosu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+infection:+a+review+of+current+and+emerging+therapies.">Clostridium difficile infection: a review of current and emerging therapies.</a> Ann Gastroenterol. 29 (2), 147-154 (2016).</li><li>McDonald, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epidemic,+toxin+gene-variant+strain+of+Clostridium+difficile.">An epidemic, toxin gene-variant strain of Clostridium difficile.</a> New Eng J Med. 353 (23), 2433-2441 (2005).</li></ol>

KWG has received past and current research support from Merck &amp; Co. and Summit, PLC.

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&#39;s pharmacological action. Using the protocols outlined here, we have demonstrated that imaging the cell&#39;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&#39;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&#39; 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.

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&#39; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>cotton gauze&#160;</td>
			<td>Caring</td>
			<td>PRM21408C</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Macron</td>
			<td>7532</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion (BHI)&#160;</td>
			<td>Criterion</td>
			<td>C5141</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Alfa Aesar</td>
			<td>A10389</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>Criterion</td>
			<td>C741</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium taurocholate</td>
			<td>Alfa Aesar</td>
			<td>A18346</td>
			<td></td>
		</tr>
		<tr>
			<td>anaerobic chamber</td>
			<td>Coy</td>
			<td></td>
			<td>vinyl anaerobic chamber</td>
		</tr>
		<tr>
			<td>cycloserinecefoxitin fructose agar (CCFA) plates</td>
			<td>Anaerobe systems</td>
			<td>AS-213</td>
			<td></td>
		</tr>
		<tr>
			<td>blood agar plates</td>
			<td>Hardy diagnostics</td>
			<td>A-10</td>
			<td></td>
		</tr>
		<tr>
			<td>latex agglutination reagent</td>
			<td>Oxoid</td>
			<td>DR1107A</td>
			<td>C. diff test kit</td>
		</tr>
		<tr>
			<td>microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>222363204</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>50-259-98</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>J. Melvin freed brand</td>
			<td>7525M</td>
			<td>75x25mm</td>
		</tr>
		<tr>
			<td>flow hood</td>
			<td>Labconco</td>
			<td>Class II type A2&#160;</td>
			<td>biosafety cabinet</td>
		</tr>
		<tr>
			<td>desk sputtering machine</td>
			<td>Denton Vacuum</td>
			<td>Desk II</td>
			<td></td>
		</tr>
		<tr>
			<td>tape</td>
			<td>Plastic Core</td>
			<td>05072-AB</td>
			<td>SPI Double Sided Adhesive Carbon Tape</td>
		</tr>
		<tr>
			<td>gold</td>
			<td>Denton Vacuum</td>
			<td>TAR001-0158</td>
			<td>2.375&#8221; Diameter x .002&#8221; Thick Gold foil</td>
		</tr>
		<tr>
			<td>scanning electron microscope</td>
			<td>FEI</td>
			<td>XL-30</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol to characterize the morphological changes of clostridium difficile in response to antibiotic treatment

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 (&#62; 50% reduction in cell length for each antibiotic; P&#60; 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&#39;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 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 &#181;g/mL for vancomycin and 0.51 &#181;g/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&#39; 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 &#181;m 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).
<img alt="Figure 1" src="/files/ftp_upload/55383/55383fig1.jpg" /> 
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. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55383/55383fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55383/55383fig2.jpg" /> 
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). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55383/55383fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55383/55383fig3.jpg" /> 
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 &#62; 17 cells were measured per group. *P&#60; 0.05 compared to control and vancomycin treatment groups. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55383/55383fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment at JoVE.com

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

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.

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

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

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Immunology and Infection

8.9K Views.  University of Houston College of Pharmacy. 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.

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

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...