The in vivo imaging facility at Imperial College was funded by the MRC.

1. Mice Preparation
<ol>
	<li>Purchase or breed enough 18-20 g C57BL/6J mice required for the study.</li>
	<li>If mice are purchased from an external supplier, following transport to the facility, habituate mice for 1 week on autoclaved food and water to stabilize the gut microbiota.</li>
	<li>Weigh, ear notch, and separate the required number of mice per cage.</li>
	<li>The day prior to infection, before performing any anesthesia, check that there is adequate oxygen and isoflurane for the duration of the procedure. If necessary, add more isofluorane or replace the oxygen cylinder. Subsequently, check the weight of the anaesthetic scavengers, if they have gained more than 50 g since their first use, discard them and replace with fresh scavengers.</li>
 <li>Anaesthetise mice using 3% isofluorane in the XGI-8 Anaesthesia System and depilate using VEET for 7 min.</li>
	<li>Notes:
	<ul>
		<li>Thorough depilation is essential, especially in black mice as melanin in the fur significantly attenuates the bioluminescent signal3,15.</li>
 <li>Depilation of mice usually lasts 8-10 days before the fur starts to regrow. To perform 4D multimodality imaging over longer periods, depilate mice when required so that the area of interest is nude for the imaging sessions. </li>
		<li>Mice are housed in a Biosafety Level 2 (BSL-2) containment facility.</li>
	</ul>
	</li>
</ol>
2. Bacterial Cell Preparation
<ol>
	<li>Prepare enough Luria Burtani broth and agar plates supplemented with Kanamycin [50 &mu;g ml-1] required for the study and store at 4 &deg;C until required.</li>
	<li>The day before the infection thaw a cryovial of C. rodentium strain ICC180 and immediately add one cryobead to 15 ml LB broth supplemented with Kanamycin [50 &mu;g ml-1] and culture overnight at 220 rpm, 37 &deg;C. As a control for medium contamination, prepare an additional uninoculated 15 ml LB broth supplemented with Kanamycin [50 &mu;g ml-1] and culture at 220 rpm, 37 &deg;C.</li>
	<li>The next morning (~16 hr), check the medium control for turbidity as a sign of bacterial growth. If the control is clear, transfer the ICC180 culture to a falcon tube and centrifuge at 4,000 rpm. Subsequently, wash the bacterial pellet in PBS. Then resuspend in 1.5 ml PBS (1/10th of the original volume of LB supplemented with Kanamycin) and mix thoroughly to create the bacterial inoculum.</li>
	<li>To verify that ICC180 has been cultured properly and is bioluminescent prior to infection of the mice, image the inoculum in the IVIS Spectrum CT using BLI and the auto setting in the Living Image 4.3.1 wizard as described previously1. 
	 Note:
	<ul>
		<li>Strain ICC180 is a bioluminescent derivative of wild type C. rodentium ICC1692. This bacterial strain has the luxCDABE operon including a Kanamycin resistance gene inserted into a pseudogene of xylE in the chromosome2.</li>
		<li>Carry out all bacterial culturing using standard aseptic techniques and if required either purchase sterile consumables/reagents or autoclave prior to use.</li>
		<li>In step 2.4, the bioluminescence reading (p/s/cm2/sr) can be used to estimate bacterial numbers prior to the infection of mice.</li>
	</ul>
	</li>
</ol>
3. Infection of Mice with Bioluminescent C. rodentium and Assessment of Bacterial Colonization
<ol>
	<li>Prior to infection, check that there is adequate oxygen and isoflurane for the duration of the procedure. If necessary, add more isofluorane or replace the oxygen cylinder. Subsequently, check the weight of the anaesthetic scavengers, if they have gained more than 50 g since their first use, discard them and replace with fresh scavengers.</li>
 <li>Anaesthetise mice with 3% isofluorane using an XGI-8 Anaesthesia System to provide humane restraint.</li>
	<li>Mix the bacterial inoculum thoroughly and draw 0.2 ml (approximately 5 x 109 cfu ICC180) into the syringe.</li>
	<li>Remove mice from the anesthesia system one at a time and scruff them by firmly grasping the skin behind the neck with your thumb and forefinger.</li>
	<li>Push the gavage needle towards the roof of the mouth, over the tongue, and down the esophagus and inject the bacterial inoculum into the stomach.</li>
	<li>Following oral gavage of the mice, monitor their gait and breathing for signs of distress which can be a sign that the bacterial inoculum has been delivered into the lungs. Euthanize any mice that have been inoculated in the lungs.</li>
	<li>Gavage uninfected mice with 200 &mu;l PBS as a vehicle control, as described in steps 3.1-3.6.</li>
	<li>To confirm that the oral gavage was performed correctly, image infected and mock infected mice in the IVIS Spectrum CT using BLI and the auto setting in the Living Image 4.3.1 wizard as described previously1.</li>
	<li>Determine the numbers of viable bacteria in the inoculum by serial dilution in PBS and spotting in triplicate onto LB agar supplemented with Kanamycin [50 &mu;g ml-1].</li>
	<li>Calculate the number of ICC180 cfu/ml by finding a dilution where there are approximately 5-50 colonies and count the number of colonies from each spot at that dilution and record the dilution that the colonies were counted on. Subsequently, calculate the mean number of cfu (from the three spots) and multiply this value by the dilution factor 50 (each spot is 20 &mu;l of the original 1 ml sample) and the dilution the colonies were counted on, giving cfu/ml.</li>
	<li>Monitor colonization of the mice daily by collecting feces from each mouse into a pre-weighed Eppendorf tube (weighed on a fine balance) and dilute 1/10 in PBS based on the weight of the sample (0.1 g ml-1).</li>
	<li>Determine the numbers of viable bacteria in the feces by serial dilution in PBS and spotting in triplicate onto LB agar supplemented with Kanamycin [50 &mu;g ml-1].</li>
	<li>Calculate the number of viable bacteria per gram of feces by following step 3.9, then multiply the number of cfu/ml by the faecal dilution factor in PBS (step 3.10) of 0.1 g ml-1 to give cfu/g.</li>
	<li>To determine that mice were inoculated with a pure ICC180 culture and that all of the colonies are bioluminescent. Take the agar plates from Step 3.8, assess the bacterial colony morphology either by eye or pick a representative colony from the plate and analyze by light microscopy. If necessary, bacterial identification can be performed by strain specific colony PCR to assess culture purity. Subsequently, image the plates in the IVIS Spectrum CT using BLI as described in step 3.7 and check that 100% of the colonies on the plate are bioluminescent. 
	Notes:
	<ul>
		<li>In step 3.4, when inserting the gavage needle, if you feel resistance do not force the gavage needle, as this causes the inoculum to go into the lungs. Instead, re-insert the needle and gently try again.</li>
		<li>Step 3.1 is optional and mice can be oral gavaged without anesthesia depending on the institutes animal welfare policy.</li>
		<li>Stools must be plated out the day that they are collected. C. rodentium will grow even if left at 4 &deg;C overnight in PBS and will cause the quantification of cfu/g stools to be incorrect.</li>
	</ul>
	</li>
</ol>
4. Daily Composite 3D Diffuse Light Imaging Tomography with &mu;CT Imaging (DLIT- &mu;CT) of Infected Mice
Animal welfare considerations: DLIT-&mu;CT involves DLIT optical imaging integrated with a fast, low radiation dose &mu;CT scan (~23 mGY for a two mouse scan, ~53 mGY for a single mouse scan) of an immobilized animal. This dose accumulates with each imaging session, so the aim is keep the dose as low as possible (and always well below the LD50/30) while still accomplishing the study. In some cases, if there is no sign of infection in a conventional BLI scan of mice, the &mu;CT scan can be avoided to further minimize dose. Even though dose is kept as low as possible, in prolonged studies if there is any concern about radiation exposure, mice will be culled at the first sign of detrimental symptoms or at the end of the &mu;CT imaging period.
<ol>
	<li>Prior to imaging, initialize the IVIS Spectrum CT and check that the X-ray safety interlocks are working and that the heated stage is at the correct temperature (37 &deg;C) for imaging. Subsequently, check the level of isoflourane in the anesthesia system and the weight of the anaesthetic scavengers. If necessary, add more isofluorane and if the scavengers have gained more than 50 g since their first use, discard them and replace with fresh scavengers.</li>
	<li>Set up the Spectrum CT so that the imaging parameters that run the automatic exposure feature in Living Image 4.3.1 are optimized for imaging bacterial luciferase. Select Edit &gt; Preferences &gt; Acquisition and Auto Exposure Window. Then select &gt; Range Values &gt; Exp. Time (seconds) and set the &gt; Max. to 300s. Finally, select &gt; Target Count (minimum) &gt; Luminescent and set to 10,000 counts.</li>
	<li>Insert the one mouse imaging platform into the Spectrum CT and plumb it into the anesthesia.</li>
	<li>Switch on the X-ray safety interlock and initialize the IVIS.</li>
	<li>Once the IVIS Spectrum CT has been initialized, anaesthetise the mice with 3% isofluorane using an XGI-8 Anaesthesia System to provide humane restraint.</li>
	<li>To determine whether it's necessary to perform a DLIT-&mu;CT scan, pre-image the mice using BLI as described previously1, in the one mouse imaging platform. If a robust signal is obtained the mouse is suitable for DLIT imaging. Leave the anaesthetised mouse in the one mouse imaging platform after the BLI scan ready for DLIT- &mu;CT.</li>
	<li>Use the Imaging Wizard in the Living Image software 4.3.1 to set-up the DLIT-&mu;CT scan. The Imaging Wizard automatically determines the FOV, exposure time, F-stop and binning for the DLIT and the FOV, exposure time, filter sets and binning for the &mu;CT imaging to give the best signal to noise ratio. When prompted by the Imaging Wizard, include only the 560, 580, 600, and 620 nm emission filters and select the one mouse &mu;CT scan. Then click &gt; Acquire to start the imaging.</li>
	<li>Return the anaesthetised mouse to its cage after imaging, remove the one mouse imaging platform from the Spectrum CT and disinfect using 1% Tri-gene. If needed, replace the foam pad that the mouse is positioned on to minimize the risk of cross-contamination between mice.</li>
	<li>Image mice daily up to day 8 post infection (P.I.) using DLIT-&mu;CT, as described in steps 4.1-4.8, to generate the longitudinal imaging data required to make a 4D movie of infection. 
	Note:
	<ul>
		<li>In step 4.7, we recommend the use of the 560-620 nm emission filters to image bacterial bioluminescence. Although the peak of bacterial bioluminescence is around 485 nm, due to tissue optics (the significant attenuation of blue/green photons by murine tissue), it is important to use the orange/red photons for 3D reconstructions as they facilitate the calculation of signal depth.</li>
		<li>The 'auto' exposure settings in Living Image 4.3.1 for DLIT need to be adjusted to optimize the amount of photons captured and the maximum imaging time used for each filter set.</li>
		<li>It is only necessary to perform the BLI pre-imaging described in step 4.6 when the researcher is not sure if there is a detectable bioluminescent signal, for example when using a new infection model.</li>
	</ul>
	</li>
</ol>
5. 3D Reconstruction of DLIT- &mu;CT Imaging Data
<ol>
	<li>Open Living Image software 4.3.1 and select &gt; Browse and open the folder containing the DLIT-&mu;CT files.</li>
	<li>Open the DLIT-&mu;CT file in the Living Image Browser, for example C. rodentium Day 1 P.I. by clicking Load.</li>
	<li>In the Tool Palette select &gt; Surface Topography &gt; Nude mouse, adjust the threshold, and then click &gt; Generate Surface. If the surface reconstruction is successful a surface outline will be displayed in the 3D view window.</li>
	<li>To view the rendered &mu;CT scan in the 3D view window, hide the surface outline (Click &gt; 3D optical tools and unselect &gt; Display Surface Object), or decrease the surface opacity (Click &gt; 3D optical tools and adjust the &gt; opacity slider bar).</li>
	<li>To provide detailed anatomical localization of the 3D DLIT reconstruction it is necessary to alter the 3D volumetric data (&mu;CT scan) so that only the mouse's skeleton is visible and that it has the optimum contrast. Click &gt; 3D Multimodality tools and select &gt; Logarithmic Histogram, Gradient Illumination, Quality and Denoise. Finally, adjust the Histogram slider to the right and watch the 3D volumetric data change contrast. Keep adjusting the data until only the bones are clearly visible. If desired, change the color for the transfer function points on the histogram.</li>
	<li>Next select &gt; DLIT 3D Reconstruction in the Tool Palette and make sure that only the 560, 580, 600, and 620 nm filter sets, which are required for imaging bacterial luciferase in vivo, have been selected. Click &gt; Start. The Data Preview Window menu will now appear showing the spectrally filtered BL signals from the mouse that was imaged in 2D. These signals are automatically thresholded by the software, but can be adjusted manually if necessary using tabs at the bottom of the menu. The threshold determines the minimum data intensity (represented as a percentage of the maximum signal in the image) which will be included as data in the reconstruction.</li>
	<li>Next select &gt; DLIT 3D Reconstruction in the Tool Palette and check that the optical properties used for the DLIT reconstruction are correct. Select &gt; Properties tab and make sure that the settings for Tissue Properties is set to mouse tissue, Source Spectrum is set to Bacteria.</li>
	<li>Click &gt; Reconstruct to perform the DLIT reconstruction.</li>
	<li>To generate the DLIT-&mu;CT 3D movie click &gt; Tools &gt; 3D animation and select &gt; Preset Animations &gt; CCW Y-axis and &gt; Total Duration &gt; 10 sec (25 frames per second). Once the settings for the 3D animation are correct press &gt; Record and save the DLIT-&mu;CT 3D reconstructions as .avi files labeled with experiment, group and time point in a separate folder. 
	Note:
	<ul>
		<li>It is important when performing 3D reconstructions of DLIT-&mu;CT data to use a PC with Living Image 4.3.1 and service package 1 installed and that the PC has a graphics card suitable for running the Multi-modality imaging software. This software can be downloaded, and GPU specifications are contained in the release notes (<a href="http://www.caliperls.com/support/software-downloads.htm" target="_blank">http://www.caliperls.com/support/software-downloads.htm</a> ).</li>
		<li>If the DLIT-&mu;CT 3D reconstructions appear backwards after being saved, your computer is running an old version of its graphics driver and this can be rectified by downloading an update from the manufacturers' website.</li>
		<li>In step 5, we present our preferred settings for generating 3D DLIT-&mu;CT reconstructions. However, there are multiple points during the 3D reconstructions where parameters that are not discussed in this protocol can be modified. For a comprehensive guide to 3D reconstructions using Living Image 4.3.1 please refer to the manufacturers' instruction manual.</li>
		<li>It is important to create the 3D reconstructions using identical settings in the multi-modality imaging tool so that they are comparable. In addition to this, when creating the DLIT-&mu;CT 3D reconstructions, it's important to make the videos using the same number of frames per second and total duration (length), otherwise the timing of the 4D video is not homogenous.</li>
	</ul>
	</li>
</ol>
6. Generation of a 4D Movie of C. rodentium Infection
<ol>
	<li>Clearly label all of the DLIT-&mu;CT 3D reconstructions with the correct experiment, time point and group in an easily accessible folder.</li>
	<li>Create a new file in Windows Live Movie maker and insert the DLIT-&mu;CT reconstructions in chronological order from day 1 P.I. using the tools tab from the folder prepared in step 6.1.</li>
	<li>Add captions to the start of every video describing the time point, for example Day 1 P.I. by selecting the appropriate DLIT-&mu;CT video and then selecting Home &gt; Add Caption. A text box appears on the screen, where the appropriate legend is added. Adjust the Font size, style, color, and justification as desired using identical tabs to Microsoft word in the software toolbar. Finally, move the caption to the top left hand corner of the video.</li>
	<li>Repeat step 7.3 for all of the DLIT-&mu;CT videos.</li>
	<li>Add a title page, for example C. rodentium infection days 1-8 by clicking Home &gt; Title. A text box appears on the screen where the appropriate title is added to a blank slide. Adjust the Font size, style, color, and justification as desired using identical tabs to Microsoft word in the software toolbar.</li>
	<li>Save the project as a movie maker project *mmp in an appropriate folder with the title of the movie. This step is essential if you want to amend the movie.</li>
	<li>Save the movie as a .wmv file. Click Movie maker menu &gt; Save movie &gt; for computer.</li>
	<li>Convert the movie maker file using a file converter to .avi or another suitable file type compatible with your computer. 
	Notes:
	<ul>
		<li>It is recommended that the 4D movies are generated on a PC with Windows Live Movie Maker installed (<a href="http://windows.microsoft.com/en-GB/windows7/products/features/movie-maker" target="_blank">http://windows.microsoft.com/en-GB/windows7/products/features/movie-maker</a>) and a good multi-media program capable of playing .wmv or .avi files types.</li>
	</ul>
	</li>
</ol>

<ol>
	<li>Chang, M. H., Cirillo, S. L., Cirillo, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Luciferase+to+Image+Bacterial+Infections+in+Mice.">Using Luciferase to Image Bacterial Infections in Mice.</a> J. Vis. Exp. (48), e2547 (2011).</li><li>Wiles, S., Pickard, K. M., Peng, K., MacDonald, T. T., Frankel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+of+the+murine+pathogen+Citrobacter+rodentium.">In vivo bioluminescence imaging of the murine pathogen Citrobacter rodentium.</a> Infect Immun. 74, 5391-5396 (2006).</li><li>Wiles, S., 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=Organ+specificity,+colonization+and+clearance+dynamics+in+vivo+following+oral+challenges+with+the+murine+pathogen+Citrobacter+rodentium.">Organ specificity, colonization and clearance dynamics in vivo following oral challenges with the murine pathogen Citrobacter rodentium.</a> Cell Microbiol. 6, 963-972 (2004).</li><li>Wiles, S., Dougan, G., Frankel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+a+'hyperinfectious'+bacterial+state+after+passage+of+Citrobacter+rodentium+through+the+host+gastrointestinal+tract.">Emergence of a 'hyperinfectious' bacterial state after passage of Citrobacter rodentium through the host gastrointestinal tract.</a> Cell Microbiol. 7, 1163-1172 (2005).</li><li>Fanning, S., 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=Bifidobacterial+surface-exopolysaccharide+facilitates+commensal-host+interaction+through+immune+modulation+and+pathogen+protection.">Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 2108-2113 (2012).</li><li>Doyle, T. C., Burns, S. M., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+for+integrated+studies+of+infection.">In vivo bioluminescence imaging for integrated studies of infection.</a> Cell Microbiol. 6, 303-317 (2004).</li><li>Collins, J. W., 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=Pre-treatment+with+Bifidobacterium+breve+UCC2003+modulates+Citrobacter+rodentium-induced+colonic+inflammation+and+organ+specificity+of+infection.">Pre-treatment with Bifidobacterium breve UCC2003 modulates Citrobacter rodentium-induced colonic inflammation and organ specificity of infection.</a> Microbiology. , 10-1099 (2012).</li><li>Contag, 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=Photonic+detection+of+bacterial+pathogens+in+living+hosts.">Photonic detection of bacterial pathogens in living hosts.</a> Mol. Microbiol. 18, 593-603 (1995).</li><li>Hardy, 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=Extracellular+replication+of+Listeria+monocytogenes+in+the+murine+gall+bladder.">Extracellular replication of Listeria monocytogenes in the murine gall bladder.</a> Science. 303, 851-853 (2004).</li><li>Szittner, R., Meighen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence,+expression,+and+properties+of+luciferase+coded+by+lux+genes+from+a+terrestrial+bacterium.">Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium.</a> J. Biol. Chem. 265, 16581-16587 (1990).</li><li>Andreu, N., Fletcher, T., Krishnan, N., Wiles, S., Robertson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+antituberculosis+drug+activity+in+vitro+and+in+macrophages+using+bioluminescence.">Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence.</a> J. Antimicrob. Chemother. 67, 404-414 (2012).</li><li>Kuo, C., Coquoz, O., Troy, T. L., Xu, H., Rice, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+in+vivo+bioluminescent+sources+based+on+multispectral+imaging.">Three-dimensional reconstruction of in vivo bioluminescent sources based on multispectral imaging.</a> J. Biomed. Opt. 12, 024007 (2007).</li><li>Cronin, M., 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=High+resolution+in+vivo+bioluminescent+imaging+for+the+study+of+bacterial+tumour+targeting.">High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting.</a> PLoS One. 7, e30940 (2012).</li><li>Mundy, R., MacDonald, T. T., Dougan, G., Frankel, G., Wiles, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrobacter+rodentium+of+mice+and+man.">Citrobacter rodentium of mice and man.</a> Cell Microbiol. 7, 1697-1706 (2005).</li><li>Curtis, A., Calabro, K., Galarneau, J. R., Bigio, I. J., Krucker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+variations+of+skin+pigmentation+in+C57BL/6+mice+affect+optical+bioluminescence+quantitation.">Temporal variations of skin pigmentation in C57BL/6 mice affect optical bioluminescence quantitation.</a> Mol. Imaging Biol. 13, 1114-1123 (2011).</li><li>Wiles, S., Crepin, V. F., Childs, G., Frankel, G., Kerton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+biophotonic+imaging+as+a+training+aid+for+administration+of+substances+in+laboratory+rodents.">Use of biophotonic imaging as a training aid for administration of substances in laboratory rodents.</a> Lab Anim. 41, 321-328 (2007).</li><li>Crepin, V. F., 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=Dissecting+the+role+of+the+Tir:Nck+and+Tir:IRTKS/IRSp53+signalling+pathways+in+vivo.">Dissecting the role of the Tir:Nck and Tir:IRTKS/IRSp53 signalling pathways in vivo.</a> Mol. Microbiol. 75, 308-323 (2010).</li><li>Willekens, I., 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+radiation+dose+in+micro-CT+with+optimization+of+the+scan+protocol.">Evaluation of the radiation dose in micro-CT with optimization of the scan protocol.</a> ContrastMedia Mol. Imaging. 5, 201-207 (2010).</li><li>Boone, J. M., Velazquez, O., Cherry, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-animal+X-ray+dose+from+micro-CT.">Small-animal X-ray dose from micro-CT.</a> Mol Imaging. 3, 149-158 (2004).</li><li>Sato, F., Sasaki, S., Kawashima, N., Chino, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+effects+of+whole+or+partial+body+x-irradiation+on+mice:+life+shortening.">Late effects of whole or partial body x-irradiation on mice: life shortening.</a> Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 39, 607-615 (1981).</li><li>Kohn, H. I., Kallman, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+strain+on+acute+x-ray+lethality+in+the+mouse.+II.+Recovery+rate+studies.">The influence of strain on acute x-ray lethality in the mouse. II. Recovery rate studies.</a> Radiat. Res. 6, 329-338 (1957).</li></ol>

Production and Free Access to this article is sponsored by PerkinElmer.
The authors Kevin P, Francis, Jeff Meganck and Chaincy Kuo are employees of Caliper- A PerkinElmer Company.
All animal experiments were performed in accordance with the Animals Scientific Procedures Act 1986 and were approved by the local Ethical Review Committee.

The 4D movie of bacterial infection provides a useful tool to visualize and interpret large amounts of multi-modality imaging data quickly and easily. This technique facilitates the detailed analysis of how an infection spreads through an individual mouse and can be used to investigate how deletion of host or bacterial genes or particular intervention strategies effect bacterial load, distribution, and localization during a longitudinal study7. These videos also provide useful teaching aids and a means of disseminating information to the public.
There are several critical steps in this protocol that could affect the quality of the data obtained from DLIT-&mu;CT imaging and the ability to compile a 4D video of infection. The most important part of this protocol is the successful and homogenous infection of mice with C. rodentium. It is essential that the mice used for the study are between 18-20 g and that the bacterial inoculums are freshly prepared and approximately 5 x 109 cfu, as described previously2,3. Prior to infection of the mice it is important to check that the inoculum is bioluminescent using the Spectrum CT and once the inoculum has been prepared, it must be continually homogenized before each mouse is gavaged to ensure that the mice receive similar infectious doses. The DLIT-&mu;CT imaging of mice has been optimized so that the auto exposure function in Living Image 4.3.1 software automatically determines the optimized imaging parameters for the signal to be well above the noise. However, the auto exposure function relies on user defined settings and parameters which need to be modified as described in the procedure. Failure to do this will result in poor images with a low number of photons collected that do not result in an obvious progression in the infection, as the Spectrum CT's factory settings for autoexposure are programmed for imaging tumors expressing firefly luciferase. Reconstructions performed using 560-620 nm give the best agreement between simulated and measured data and, therefore, are the more reliable data to include in the reconstruction.
A limitation to the use of DLIT-&mu;CT is that ionizing radiation from the &mu;CT scan causes sub-lethal radiation damage that is cumulative over a longitudinal study18. Sub-lethal radiation exposure can weaken the immune response, cause DNA damage, and apoptosis in internal organs19. Ultimately, cumulative sub-lethal radiation damage can cause death if the LD50/30 for ionizing radiation is exceeded, which is between 5 to 7 Gy depending on the mouse strain and age of the mice used18,20,21. Although some of the molecular damage from ionizing radiation can heal, since the overarching principle is to estimate dose conservatively, this is not typically accounted for in study planning. Instead, the aim is to stay as far below these limits as possible while still accomplishing the study goals. This is particularly important in this study because of the normal immune response to the infection, the frequency of imaging, and the fact that transgenic, immuno-comprised, or heavily infected animals may be more susceptible to ionizing radiation.
When planning the experiment to generate a 4D movie of infection, it is important to consider the length of the experiment, the number of &mu;CT scans required during this period and the LD50/30 for ionizing radiation for the mouse strain being used. Another potential limitation to DLIT-&mu;CT is the strength of the reporter expression within the bacterial strain being used, as this will affect bacterial detection limits and imaging times. It is highly recommended that researchers use validated bacterial strains that are fully virulent, but optimized for maximal lux operon expression, as demonstrated previously for BLI2,3.
One caveat to the current design of the 4D imaging is that each movie is comprised of individual DLIT-&mu;CT scans which have different photon scaling. This can make the images difficult to interpret if the changes to the localization of the BL foci, or its intensity are subtle, or if there is one intense BL focus surrounded by multiple weak foci. Therefore, for longitudinal visualizations, it is important to keep the color bars consistent across the time points.
The concept of a 4D movie of infection can be applied to any suitably labeled bacterial pathogen. Future development of this technique will aim to use fluorescence imaging tomography (FLIT) as well as DLIT to facilitate the investigation of host responses to infection using a combination of bioluminescent bacterial pathogens and injectable fluorescent near infrared probes to investigate host responses to infection. In addition to this, in this protocol we only describe the use of bioluminescent bacteria to create 4D movies of infection. However, in some instances it may be necessary to use fluorescent labeled bacteria, for example tagged with iRFP, so that the bioluminescence reporter can be used for investigating host genetics during infection. Importantly, the use of multi-modality imaging combining DLIT/FLIT-&mu;CT will allow us to non-invasively investigate multiple parameters during a bacterial infection, which will contribute significantly to the reduction, refinement, and replacement of the use of animals in scientific research as outlined in the NC3R's initiative (<a href="http://www.nc3rs.org.uk/" target="_blank">http://www.nc3rs.org.uk/</a>).

Small animal models, in particular those utilizing mice, are routinely used to investigate bacterial pathogenesis or to test intervention strategies for infections, such as antibiotics, probiotics, prebiotics and vaccines1-7. The main experimental readouts from small animal infections are pathogen load, spatial and temporal localization of the infection, and changes to the immune response of the infected organism. In vivo optical imaging is a valuable tool for infectious disease research and can be used to monitor multiple experimental readouts through the use of reporter genes (luciferase, fluorescent proteins, beta-lactamase, etc), fluorescent dyes, nanoparticles or chemiluminescent probes targeted to a protein, biological process, or microorganism6.
Bioluminescence imaging (BLI) is an optical imaging modality used to monitor the colonization of small animals, such as mice and rats, by pathogenic bacteria3,6,8,9. Mice are infected with recombinant bacteria expressing a luciferase, such as the luxCDABE operon from Photorhabdus luminescens. These bacteria can then be detected through their light production using a CCD based in vivo imaging system3,6,9. Importantly, only metabolically active micro-organisms are bioluminescent (BL), meaning only viable bacterial cells are detected by this methodology 10,11. Using 2D BLI, the location of the BL source is inferred from the surface of the animal where the signal is emitted8. The exact anatomical localization of the BL foci in vivo has to be determined through the ex vivo analysis of organs3,6,9 In contrast, composite 3D diffuse light imaging tomography (DLIT) can be used to compile a quantitative 3D reconstruction of the BL source12. DLIT is performed by collecting BL images taken using defined narrow band-pass optical filters and subsequently inputting them into a diffuse optical tomography 3D reconstruction algorithm1,7,12,13.
Currently, multi-modality imaging is the only methodology available to get true non-invasive anatomical localization of bioluminescent foci in vivo without the need for ex vivo analysis. Recently, we used a combination of DLIT co-registered with &#956;CT imaging to evaluate Citrobacter rodentium (C. rodentium) colonization dynamics following prophylactic treatment with a probiotic bacterium7. C. rodentium is a murine specific enteric pathogen used to model human infection with enteropathogenic and enterhemorrhagic Escherichia coli14. C. rodentium infection causes colitis, typically associated with mild weight loss, diarrhea, polarized Th1 immune response and distinct pathological changes, including colonic crypt hyperplasia and attaching and effacing lesion formation14. In addition to this, C. rodentium pathogenesis has been thoroughly studied using BLI and its colonization dynamics in C57BL/6J mice are well documented, making this bacterium an ideal model microorganism for use with multi-modality imaging3,4,7.
This protocol is the first to outline a methodology for integrated DLIT-&#956;CT imaging of a bacterial infection using a single multimodality imaging platform, the IVIS Spectrum CT, and the generation of a 4D movie showing the true dynamics of this infection non-invasively.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Bioluminescent C. rodentium</td>
			<td>Frankel lab</td>
			<td>ICC180</td>
			<td>Wiles et al., 2004</td>
		</tr>
		<tr>
			<td>Veet</td>
			<td>Boots</td>
			<td>&nbsp;</td>
			<td>Optimal depilation time is 7 min. Depilation works better if the cream is rubbed in well.</td>
		</tr>
		<tr>
			<td>Isofluorane (100% v/v)</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Medical Oxygen</td>
			<td>BOC Medical</td>
			<td>&nbsp;</td>
			<td>Size F Cylinder. Note: an appropriate regulator is required.</td>
		</tr>
		<tr>
			<td>Luria Bertani broth</td>
			<td>Merck</td>
			<td>1.10285.0500</td>
			<td>25 g in 1L Demineralised water.</td>
		</tr>
		<tr>
			<td>Luria Bertani agar</td>
			<td>Merck</td>
			<td>1.10283.0500</td>
			<td>37 g in 1L Demineralised water.</td>
		</tr>
		<tr>
			<td>Kanamycin sulphate</td>
			<td>Sigma (Fluka)</td>
			<td>60615</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml Polypropylene conical Falcon tubes</td>
			<td>BD (Falcon)</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Universals</td>
			<td>Corning (Gosselin)</td>
			<td>E5633-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD (Plastipak)</td>
			<td>300013</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oral dosing needle (16G x 75 mm) curved</td>
			<td>Vet Tech</td>
			<td>DE005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microbanks (Cryovial)</td>
			<td>Pro-Lab Diagnostics</td>
			<td>PL.170/Y</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IVIS Spectrum CT</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>133577 Rev A/ Spectrum CT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>6kVA UPS</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>XGI-8 anesthesia system</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>118918</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>XAF-8 Anaesthesia filter charcoal</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>118999/00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Living Image v4.3.1 SP1</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Benchtop shaking incubator</td>
			<td>New Brunswick Scientific</td>
			<td>Innova 44</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

4d multimodality imaging of citrobacter rodentium infections in mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

Infection of C57BL/6J mice with 5 x 109 cfu C. rodentium is a well described bacterial infection model and results in a self-limiting gastrointestinal infection that peaks between days 6-8 post infection and lasts between 3 to 4 weeks2,14. The infection is confined to the intestinal lumen by the murine immune system and as a consequence, bacteria are continually shed in the feces. Colonization of mice by C. rodentium can be monitored non-invasively by direct bacterial enumeration from feces, or bioluminescence imaging 2,3.
This manuscript outlines an optimized protocol for performing 3D and 4D multimodality imaging of C. rodentium within mice to monitor bacterial load and localization during infection. The results presented in Figure 1 demonstrate the controls that are required for successful 4D imaging. Prior to the infection of mice it is essential to determine that the bacterial inoculum used is bioluminescent (Figure 1A) and that following the oral gavage of a mouse with bioluminescent C. rodentium, that the signal can be observed in the stomach of the animal and not in the lungs (Figure 1B)16.
In addition to monitoring bacterial colonization using bioluminescence imaging, it's good practice to quantify bacterial numbers in feces; which is used as an indirect measurement of bacterial colonization of the gastrointestinal mucosa. Figure 2 demonstrates the typical bacterial load in feces taken from 8 C57BL6/J mice that have been infected with ~5 x 109 cfu ICC180 and monitored for 8 days P.I. Colonization increases from day 2 P.I. until day 6-7 where the infection peaks at ~5 x 1010 cfu/g, which is in line with previous reports2,3,17.
To emphasize the importance of evaluating the spread of infection in a single mouse, Figures 1b and 3 as well as Video 1 have been generated from the same mouse following C. rodentium infection, as described above. Daily DLIT-&mu;CT was used to evaluate the spatial distribution of bioluminescent bacteria within this mouse, using the skeleton as an anatomical reference. Figure 3 demonstrates the DLIT-&mu;CT reconstruction of a C57BL/6J mouse infected with ICC180 at 3 key time points P.I. At day 3 P.I. small bioluminescent foci can be observed in the colon, which exhibit a moderate increase in bioluminescence intensity by day 5 P.I. with little change in spatial distribution. At day 7 P.I. we observed a significant increase in bioluminescence and the bioluminescent foci spread across the entire colon.
Video 1 is a compilation of 3D DLIT- &mu;CT reconstructions from days 1-8 P.I. with ICC180 and illustrates C. rodentium within the proximal gastrointestinal tract between days 1-2 P.I. which spreads to the colon at day 3 P.I. From days 4-6 P.I. the bacterial numbers in the colon expand until peaking at day 7 P.I. where the bacterial foci covers the entire colon. At day 8 P.I. only two distinct bacterial foci are present in the proximal and distal colon. The ability to view the full 3D reconstruction at each time point in chronological order represents a powerful tool to analyse host pathogen interactions and is easier to interpret than a 3D still of the same dataset.
<img alt="Figure 1" src="/files/ftp_upload/50450/50450fig1.jpg" /> 
 Figure 1. 2D bioluminescence imaging of A) Bioluminescent C. rodentium ICC180 inoculum and B) a mouse following oral gavage with 200 &mu;l of the inoculum. Arrowhead (&gt;) illustrates bioluminescent C. rodentium in the stomach.
<img alt="Figure 2" src="/files/ftp_upload/50450/50450fig2.jpg" />
Figure 2. C. rodentium colonization dynamics. Quantification of C. rodentium colony forming units from feces for 8 days P.I.
<img alt="Figure 3" fo:content-width="3in" fo:src="/files/ftp_upload/50450/50450fig3highres.jpg" src="/files/ftp_upload/50450/50450fig3.jpg" /> 
Figure 3. Diffuse light imaging tomography-&mu;CT scan of bioluminescent C. rodentium infection from one mouse monitored at day 3, 5 and 7 P.I. Arrowhead (&gt;) illustrates colonic colonization. <a href="https://www.jove.com/files/ftp_upload/50450/50450fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
Video 1. <a href="https://www.jove.com/files/ftp_upload/50450/LAB_MEDIA_50450_Frankel_Video1.wmv" target="_blank">Click here to view video.</a> 4D movie of C. rodentium infection from one mouse monitored from days 1-8 P.I.

Watch this Scientific Journal Video about 4D Multimodality Imaging of Citrobacter rodentium Infections in Mice at JoVE.com

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

4d-multimodality-imaging-of-citrobacter-rodentium-infections-in-mice

Research

JoVE Journal

Immunology and Infection

12.9K Views.  Imperial College London. التصوير المتعدد طريقة هو نهج قيمة لدراسة الاستعمار الجرثومي في نماذج حيوانية صغيرة. هذا البروتوكول يحدد إصابة الفئران مع إضاءة الحيوية الليمونية rodentium ورصد الطولي للاستعمار الجرثومي باستخدام مركب ضوء منتشر 3D التصوير المقطعي التصوير مع μCT التصوير لخلق ...

Video: 4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...