This work was supported by grants from the National Institutes of Health (AI079442 and AI091918) awarded to S.E.F.D.

1. Preparation of Selective Agar Media (BHI/L+G) to Inhibit Intestinal Microbiota
<ol>
 <li>Weigh out 26 g Brain Heart Infusion Agar (Difco), 7.5 g LiCl and 5.0 g glycine and place in a 1 liter flask. Add 500 ml of deionized water.</li>
 <li>Heat on a magnetic stirrer until the agar boils, stirring continuously. Take the flask off the heat, let the bubbles settle briefly, and then bring back to a boil again.</li>
 <li>Autoclave for 30 min at 121 &deg;C under 16-19 psi on a liquid cycle. Note: leave the stir bar in the flask.</li>
 <li>Equilibrate the media to 55 &deg;C in a water bath or let cool at RT. Do not allow the solution to get cooler than 55 &deg;C or crystals will form in the agar. The crystals do not inhibit L. monocytogenes growth or affect the selective properties of the media. However, the crystals will be similar in appearance to small colonies and will make it difficult to count bacterial CFU.</li>
 <li>Gently mix the agar for 1 min using a magnetic stirrer. Avoid the formation of air bubbles. Pour the agar into Petri dishes (~ 25 ml/plate). 
 Note: This media does not support the growth of all L. monocytogenes strains. For example, L. monocytogenes EGDe grows well on this agar, with colonies visible within 36-48 hr, but 10403s does not grow on this media.</li>
</ol>
2. Preparation of the Inoculum
<ol>
 <li>Cut sliced white bread (Kroger) into small (2-3 mm) cubes using sterile scissors ora sterile scalpel blade. Do not use the crusts. Store individual pieces in microcentrifuge tubes at -20 &deg;C until the day of the infection.</li>
 <li>Using a sterile scalpel, cut small (0.5-1 cm) chunks of salted butter (Kroger) and place each in a sterile microcentrifuge tube. Each tube will contain enough butter to prepare 50- 60 bread pieces. Store at -20 &deg;C until needed.</li>
 <li>Grow L. monocytogenes in BHI broth shaking at 30 &deg;C until the culture reaches an OD600 of ~0.8-1.0. Prepare 500 &mu;l aliquots in microcentrifuge tubes, taking care to vortex the sample frequently so all of the tubes contain the same number of bacteria. Store at -80 &deg;C.</li>
 <li>To titer the bacterial aliquots, thaw a tube on ice, then add 500 &mu;l to 9.5 ml BHI broth. Incubate for 1.5 hr standing at 30 &deg;C. Plate serial dilutions on BHI agar. Expect a titer of ~ 1-5 x 108 CFU/ml. Note: If homogeneous aliquots are prepared, can expect each of the tubes to yield this titer when prepared in a similar manner with a variance of 2 to 4-fold.</li>
 <li>To infect mice, prepare an aliquot of L. monocytogenes as described in step 2.4. About 20 min before the L. monocytogenes culture is ready, thaw the bread pieces at RT. Melt an aliquot of butter at 55 &deg;C and pre-warm a small volume of PBS at 55 &deg;C. Prepare enough bread pieces so that there is at least one per mouse to be infected and at least one extra piece for determining the titer of the actual inoculum.</li>
 <li>Based on the predetermined titer of the bacterial aliquot, calculate the total volume of L. monocytogenes culture needed to prepare the inocula for the required number of bread pieces. Pellet the bacteria by centrifuging at 14,000 x g for 10 min Aspirate all BHI broth and resuspend the bacteria in a small volume of pre-warmed PBS (2 &mu;l/bread piece).</li>
 <li>Vortex the melted butter, add to bacteria (using a total volume equal to 3 &mu;l/bread piece) and mix thoroughly. Note: Do not attempt to prepare more than 10-15 bread pieces at one time or the butter will solidify.</li>
 <li>Working quickly, pipette 5 &mu;l of the bacterial suspension onto a single bread piece in a microcentrifuge tube. The solution should be completely absorbed by the bread piece.</li>
 <li>To determine the actual titer of the inoculum, add 1 ml sterile PBS to one of the bread pieces. Vortex vigorously for 1 min. Prepare serial dilutions and plate both BHI and BHI/L+G agar.</li>
 <li>Alternate procedure for high titer (&gt;109 CFU) inocula. If the bacterial pellet is too large, it can be difficult to suspend in such a small volume, and the resulting material is very viscous, like a paste, and some of the inoculum may stick to the sides of the microcentrifuge tube. In this case, it is better to inoculate the bread in a sterile 60 mm culture dish, and to offer the entire dish (without the lid) to the mouse (see below). Any excess inoculum that is not absorbed by the bread can then be eaten directly by the mouse. In general, it takes longer for the mice to eat all of the bread and lick the dish clean than the standard protocol described below, so this is only the preferred method when dealing with very high titer inocula.</li>
</ol>
3. Infection of Mice
<ol>
 <li>Purchase female BALB/c/By/J (stock #0010026) and C57BL/6/J (stock # 000644) from The Jackson Laboratory (Bar Harbor, ME) and use at 6-9 weeks of age.</li>
 <li>Fast the mice one day (24 hr) prior to infection by removing the food and placing the mice on raised (1 inch) wire flooring (#3 mesh; Allentown) to prevent coprophagy. Leave only enough bedding in the cage to absorb urine, but not enough to elevate shed feces. Allow unrestricted access to water.</li>
 <li>Infect the mice at the onset of the dark cycle, working in a laminar flow hood outfitted with a red light bulb. Transfer a single mouse to an empty (no bedding) cage. Using a sterile forceps, transfer the contaminated bread piece to the bottom of the empty cage. Typically, the mouse will pick up the bread piece and consume it entirely within 2-10 min. If the mouse does not eat the bread right away, return the cage to rack and leave mouse undisturbed for 20-30 min. The majority of animals will eat the bread within this time frame, however, the mice can be left undisturbed, without access to other food or water for up to 2 hr.</li>
 <li>After infection, return the mouse to its original cage and replenish the mouse chow. Continue to house the mice on raised wire flooring for the duration of the experiment.</li>
</ol>
4. Monitoring the Level of Bacteria Shed in Feces
<ol>
 <li>Label and pre-weigh microcentrifuge tubes to be used for collection of feces.</li>
 <li>Working quickly after retrieving a cage of mice, place each mouse in a 500 ml plastic beaker. Typically the mice will expel 1-4 stool pellets within 5 min.</li>
 <li>Use a sterile forceps to transfer feces to appropriately labeled tubes.</li>
 <li>Weigh each tube and calculate the weight of the feces by subtracting the weight of the empty tube. Typical weights for fecal pellets range from 10-30 mg.</li>
 <li>Add PBS to each tube (200 &mu;l/30 mg feces) and use a sterile toothpick to mash the fecal pellets.</li>
 <li>Vortex each tube for 30 sec, then prepare serial dilutions and plate on BHI/L+G agar. Count the number of colonies and calculate CFU/mg feces.</li>
</ol>
5. Processing of Infected Intestinal Tissues
<ol>
 <li>Euthanize the mice, aseptically harvest the stomach and intestines from each mouse as a single tissue and collect in empty 100 mm Petri dishes.</li>
 <li>Separate the small intestines, cecum, and colon and place in separate 60 mm dishes stored on ice. The small intestines can be further divided by length into three equal pieces approximating the duodenum, jejunum, and ileum to facilitate removal of the luminal contents. The tissue thirds can then be processed either together (for CFU per whole small intestines) or separately. Wet tissues with ~ 0.5 ml sterile PBS to keep pliable and prevent tears during handling.</li>
 <li>Fill a 10 ml syringe with 8 ml sterile PBS and attach a 25 g needle. Use sterile forceps to squeeze out the luminal contents into a waste beaker (or a sterile 50 ml tube if collecting the luminal bacteria). Flush 4 ml PBS through one end of the tissue, use the forceps to squeeze out the contents, and then flip the tissue over and repeat on the other end.</li>
 <li>To quantify the number of Listeria in the luminal contents, centrifuge the pooled flushes for 20 min at 12,000 x g, suspend the pellet in 0.5 - 1.0 ml sterile water.</li>
 <li>To quantify the total number of cell-associated amount Listeria, open each washed tissue by cutting longitudinally with sterile scalpel blade, and then make several lateral cuts to slice the tissue into smaller fragments. Note: this step is essential to ensure that the intestinal tissue does not wrap around the homogenizer blade. Transfer the intestinal pieces to a 15 ml centrifuge tube containing 2 ml sterile water.</li>
 <li>Sterilize a tissue homogenizer (Fisher PowerGen 1000) by placing the probe in 70% ethanol at 60% power for 30 sec. Wait for ethanol to air dry, or process in sterile water for 10 sec. Homogenize each tissue for 1 min. Repeat treatment with sterile water between each sample, and use 70% ethanol between sample groups.</li>
 <li>Prepare serial dilutions of each sample in sterile water and plate on BHI/L+G agar.</li>
</ol>
6. Processing of Infected Mesenteric Lymph Nodes
<ol>
 <li>Harvest lymph nodes aseptically, place in a sterile 60 mm dish on ice, and use sterile forceps to remove all attached fat. Expect to find 3-6 nodes per mouse.</li>
 <li>Prepare wire mesh screens by cutting 1.5-2 inch square pieces of stainless steel #80 mesh. Make a small cut in each corner, and fold down the four sides, creating a raised bed. Dip in 95% ethanol and then flame to sterilize, and place in a 60 mm Petri dish containing 0.75 ml sterile water. After each use, the screens can be recycled by soaking in 70% ethanol for 20 min, scrubbing with a brush to remove embedded tissues, boiling for 20-30 min in 2N NaOH, and then flushing extensively with water.</li>
 <li>Use the plunger from a sterile 3 ml syringe to mash the nodes through the mesh screen. Be sure to push down on the screen to the bottom of the dish so it makes contact with the water in the dish.</li>
 <li>Pass 0.75 ml sterile water through the screen and then pipette up and down several times to rinse the screen. Transfer cell suspension to a microcentrifuge tube.</li>
 <li>Vortex each sample vigorously for 30 sec to lyse the cells. Prepare serial dilutions and plate on either BHI/L+G agar.</li>
</ol>
7. Processing of Infected Spleens, Livers, &amp; Gall Bladders
<ol>
 <li>Grasp spleen with sterile forceps and liberate by making two cuts, one at each end. Place in a 15 ml tube containing 2.5 ml sterile water. (Note: tubes with round bottoms may be easier to use with the homogenizer.)</li>
 <li>Locate the gall bladder (a small yellow fluid-filled sac attached to the liver) and snip carefully with sterile scissors to detach from liver without bursting. Transfer to a microcentrifuge tube containing 1 ml sterile water.</li>
 <li>Aseptically remove the liver, making sure to remove all lobes, and transfer to a 15 ml centrifuge tube containing 2.5 ml of sterile water.</li>
 <li>Homogenize spleens and livers as described above for 30 sec on 60% power. Forgall bladders, use sterile scissors to tear apart in the microcentrifuge tube, and then vortex vigorously for 30 sec.</li>
 <li>Prepare serial dilutions and plate each sample on either BHI or BHI/L+G agar.</li>
</ol>
8. Processing of Infected Brains
<ol>
 <li>Working from the back side of the mouse, cut the skin and tissue above the neck, and across both ears at a 45&deg; angle. Use a sterile forceps to peel off the U-shaped flap of skin by pulling towards the nose and expose the skull and facial bones.</li>
 <li>Immobilize the skull by holding the bony ridge between the eyes with a forceps, and use scissors to make shallow cuts across the lateral edges of the skull. Be careful to cut only the bone and not the brain tissue. Next, cut the bony ridge between the eyes. Use a forceps to hold the top of the skull and pull backwards (toward the neck) to expose the brain.</li>
 <li>Gently lift the brain from its cavity using a sterile forceps and place in a 15 ml centrifuge tube containing 1.5 ml sterile water.</li>
 <li>Homogenize for 20 sec on 60% power as described above. Prepare serial dilutions and plate on either BHI or BHI/L+G agar.</li>
</ol>
9. Supplemental Procedure: Fractionation of Intestinal Tissues
9.1 Isolation of Bacteria in the Mucus Fraction
<ol>
 <li>For each intestinal tissue, prepare 3 tubes containing 3 ml of 6 mM N-acetylcysteine (NAC; Sigma #A-9165).</li>
 <li>Place the flushed and longitudinally cut tissue in the first tube for 1-2 min, vigorously swirling every 20-30 sec. Using sterile forceps, pick up the tissue and gently squeeze against the side of the tube to remove excess liquid.</li>
 <li>Repeat step 9.1.2 twice more using the remaining NAC tubes. Remove the intestinal tissue and set aside for further processing.</li>
 <li>Pool the NAC washes (total of 9 ml), and centrifuge for 20 min at 12,000 x g.</li>
 <li>Resuspend the pellet in sterile water, vortex for 30 sec, prepare serial dilutions and plate on BHI/L+G.</li>
</ol>
9.2 Isolation of Bacteria in the Epithelial Cell (EC) Fraction
<ol>
 <li>Cut each tissue into small pieces with sterile scissors and place in a 50 ml tube containing 5 ml of RPMI (Invitrogen #21870) supplemented with 5% FBS (RP-5), 5 mM EDTA, and 1 mM DTT. Incubate shaking at 37 &deg;C for 20 min.</li>
 <li>Transfer the intestinal pieces to a fresh tube containing 5 ml RP5/EDTA/DTT and incubate shaking at 37 &deg;C for 20 min. Save the media from the first tube and set aside at 37 &deg;C. Repeat this process once more for a total of three 20 min incubations in RP5/EDTA/DTT. If proceeding to the lamina propria isolation step, transfer the remaining intestinal pieces into empty 50 ml tube.</li>
 <li>Combine the three RP5/EDTA/DTT washes (total volume of 15 ml) and centrifuge at low speed (1,200 x g) to pellet the cells.</li>
 <li>To quantify extracellular bacteria, collect the supernatant and centrifuge for 20 min at 12,000 x g. Resuspend the pellet in 0.5 - 1.0 ml sterile water and plate serial dilutions on BHI/L+G agar.</li>
 <li>To enumerate intracellular bacteria, resuspend the EC pellet in 5 ml of RP-5 containing 25 &mu;g/ml gentamicin. Incubate the single cell suspension for 30 min at 37 &deg;C plus 7% CO2 to kill any remaining extracellular L. monocytogenes. Wash the cells twice in PBS, suspend in 0.5 ml sterile water, and vortex for 30 sec to lyse the cells. Prepare serial dilutions in water and plate on BHI/L+G. 
 Note: The cells collected at this stage will also contain cells from the underlying Peyer's Patches. If desired, the visible Peyer's Patches can be removed from the intestinal tissues prior to flushing and cutting longitudinally.</li>
</ol>
9.3 Isolation of Bacteria in the Lamina Propria (LP) Fraction
<ol>
 <li>Rinse the excess DTT/EDTA from the intestinal pieces by adding 25 ml sterile PBS to the tube. Shake vigorously, transfer into a fresh tube and repeat twice.</li>
 <li>Transfer the intestinal pieces to a 50 ml tube containing 4 ml of digestion solution consisting of RP-5 supplemented with 1 mg/ml type IV collagenase and 40 &mu;g/ml DNAse I. Incubate shaking at 37 &deg;C for 40 min.</li>
 <li>Transfer the undigested pieces into a fresh tube containing 4 ml digestion solution and repeat once or twice until the tissue pieces are completely digested. Save each of the digestion solutions, which contain liberated LP cells, at 37 &deg;C.</li>
 <li>Centrifuge the pooled digestion solutions at low speed (1,200 x g) to pellet the cells. Process the supernatant and cell pellet separately as described above to enumerate extracellular and intracellular bacteria, respectively.</li>
</ol>

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The authors declare that they have no competing financial interests. All experiments described here were performed in compliance with The Office for Laboratory Animal Welfare (OLAW) with the approval of the ICAUC at the University of Kentucky.

Inbred mice are not uniformly receptive to feeding at all times of the day, and their willingness to eat the contaminated bread will depend both on the strain type and the age of the mice 17. In our experience, 6-9 week old B6 mice are receptive to feeding at any time of day, but BALB mice will not consistently eat the bread piece unless it is offered during their dark cycle. The light cycle of the room used to house the animals can be altered so the dark phase coincides with the normal working day for laborat...

Listeria monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. The bacteria are resistant to many of the processes used to protect our food supply, such as drying, salting, or refrigeration 1,2 and infections are typically linked to processed, "ready-to-eat" foods that are not heated prior to consumption. In several previous outbreaks, the source of L. monocytogenes-contaminated food was identified, and the restricted group of exposed individuals was monitored closely 3-6. In those examples, clinical disease in otherwise healthy individuals varied from a mild, self-limiting gastroenteritis to more severe intestinal and systemic infections that required hospitalization. L. monocytogenes infections in immune compromised individuals, including both neonates and the elderly, have been associated with a high mortality rate (25-30%), even with antibiotic treatment 7,8. Little is known about the infectious dose for L. monocytogenes, or the host factors that govern susceptibility to infection after oral transmission, primarily due to the lack of a small animal model that recapitulates this wide range of infection outcomes.
The most widely used model of listeriosis is intravenous (i.v.) inoculation of mice. The i.v. model is highly reproducible, and has been extremely useful for studying both na&iuml;ve and memory T cell responses during infection 9,10. The drawback of the i.v. model is that it completely bypasses the intestinal phase of infection. After food borne transmission, the gut mucosa provides a barrier that presumably slows and limits the number of bacteria that can continually disseminate to peripheral tissues. In contrast, the entire inoculum can be found in the spleen and liver within minutes of i.v. administration, and this large bolus of organisms may overwhelm innate immune defenses in these tissues. Oral infection of mice by gavage is less commonly used, because large doses (109-1011 CFU) are typically required to achieve intestinal colonization 10. Also, intragastric (i.g.) inoculation with a feeding needle does not generate a reproducible period of gastrointestinal infection prior to systemic spread. Some labs have reported that L. monocytogenes reach the spleen and liver within 4-12 hr post infection (hpi), while others showed no systemic spread until 48 hpi 11-15. This lab-to-lab variation may be a consequence of the invasive nature of i.g. inoculation, which can result in minor trauma to the lining of the esophagus, and promote direct bloodstream invasion of the bacteria.
We recently developed a novel mouse model of oral L. monocytogenes infection that closely mimics all phases of human disease 16. Infection occurs when the mice ingest pieces of contaminated food, a process that is non-traumatic, and does not require specialized skills by laboratory investigators. For a discrete period of time (36-48 hr), L. monocytogenes reproducibly colonize only the gastrointestinal tract, thus allowing investigation of the mechanisms used by pathogenic Listeria to translocate across the gut mucosa and disseminate to peripheral tissues. Importantly, the model can be used to study differences in host innate resistance to infection. In a previous study, we showed that BALB/c/By/J mice were highly susceptible to food borne listeriosis, with exponential replication of L. monocytogenes occurring in the gut, spleen, liver, and gall bladder 16. In contrast, C57BL/6 mice were resistant to food borne infection, with only transient colonization of L. monocytogenes occurring in each of these tissues. An additional feature of the food borne model that closely mimics human disease is that natural dissemination to the brain occurred during the later stages of the infection (5-7 dpi).

<table border="1">
	<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Brain Heart Infusion Agar</td>
 <td>Difco</td>
 <td>BD-241830</td>
 <td></td>
 </tr>
 <tr>
 <td>Lithium chloride</td>
 <td>Sigma</td>
 <td>L9650</td>
 <td ></td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>Omnipur</td>
 <td>4840</td>
 <td ></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Gibco</td>
 <td>15575-038</td>
 <td ></td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Sigma</td>
 <td>D5545</td>
 <td ></td>
 </tr>
 <tr>
 <td>Collagenase, type IV</td>
 <td>Worthington</td>
 <td>LS004089</td>
 <td></td>
 </tr>
 <tr>
 <td>DNAse I</td>
 <td>Worthington</td>
 <td>LS002007</td>
 <td></td>
 </tr>
 <tr>
 <td>Diff-Quik</td>
 <td>Dade-Behring</td>
 <td>B4132-1A</td>
 <td></td>
 </tr>
 <tr>
 <td>PowerGen 1000 homogenizer</td>
 <td>Fisher</td>
 <td>14-261-06</td>
 <td></td>
 </tr>
 <tr>
 <td>stainless steel type 304 mesh #80</td>
 <td>Small Parts, Inc.</td>
 <td>CX-0080-C</td>
 <td></td>
 </tr>
 <tr>
 <td>Cytospin</td>
 <td>Statspin</td>
 <td>M801-22</td>
 <td></td>
 </tr>
	 </tbody>
 </table>

oral transmission of listeria monocytogenes in mice via ingestion of contaminated food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

L. monocytogenes colonies will be visible on BHI/L+G plates after 36-48 hr incubation at 37 &deg;C. The colonies have a smooth, dome-shaped creamy white appearance (Figure 1A). Growth will be inhibited for the majority of the intestinal microbiota, but it is common to see some colonies that are not L. monocytogenes, particularly when plating small intestine or colon directly without significant dilution (Figure 1B). Suspect colonies can be confirmed by plating on ChromA...

Watch this Scientific Journal Video about Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food at JoVE.com

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

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JoVE Journal

Immunology and Infection

19.1K Views.  University of Kentucky. This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

Video: Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...