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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Strains and culture conditions
Note: M. tuberculosis strain CDC1551 is used in this study, but any M. tuberculosis strain can be used in the same manner.
<ol>
	<li>Grow the bacteria in M-OADC-TW (7H9 broth supplemented with 0.5% glycerol, 10% oleic acid dextrose complex without catalase, and 0.05% Tween-80) medium standing at 37 &#176;C to an OD600 of 0.5 (~ 0.2 x 107 colony-forming unit or CFU).</li>
	<li>Dilute the culture in M-OADC-TW (series of 1:10 dilutions of the bacteria). Plate the dilutions of the bacteria (105, 106, 107, 108) in triplicate onto selective 7H11 plates to allow determination of CFU.</li>
	<li>Incubate the plates for four weeks at 37 &#176;C or until colonies can be accurately counted.</li>
	<li>Centrifuge the bacterial inoculum at 8,534 x g for 5 min to obtain a pellet. Wash the pellet once with 10 mL of saline (0.9% NaCl) and re-suspend the pellet in 15 mL of saline (0.9% NaCl).</li>
</ol>
2. Aerosol infection of mice using a Madison Chamber
<ol>
	<li>Allow mice to acclimate to the new surroundings for a week.</li>
	<li>Weigh the mice before loading them into the chamber.</li>
	<li>Plug three cords into the power strip: the main chamber power, the vacuum pump, and the air compressor, in that order.</li>
	<li>Carefully unscrew the glass jar. Bring the glass jar to the biosafety cabinet and add the challenge inoculum suspended in 15 mL of saline (0.9% NaCl) to achieve ~ 104 - 106 CFU of bacteria in the lungs.</li>
	<li>Close the lid of the glass jar in the biosafety cabinet. Attach the jar to the nebulizer unit and adjust the vertical stainless steel tube so that the lower (intake) end is about a quarter of an inch below the level of the fluid in the jar.</li>
	<li>Load all the animals needed for the experiment (the chamber can hold up to 90 mice) into the chamber and close all the latches on the door.</li>
	<li>Check the main (room) air flow meter. Ensure that the center of the float (ball) runs about 50 L/min as measured on the scale on the left.</li>
	<li>Press the Start button on the control panel of the chamber. Set the airflow rate through the compressor air flow meter (the smaller meter on the left) as 4 L/min on the scale. Check visually to ensure that the challenge inoculum is being nebulized.</li>
	<li>After 15 min, when the red light on the front of the control panel appears and an audible signal indicates the end of the run, press the Reset button on the lower right corner of the control panel to reset the timers.</li>
	<li>Hold down the small red button on the door of the chamber to release the vacuum.</li>
	<li>Open the chamber door and remove the animals. Place the mice back into their cages.</li>
	<li>Remove the glass nebulizer jar and place it in a sealed, leak-proof transport container. Place the container inside the bio-safety cabinet and discard the challenge suspension into a designated waste container.</li>
	<li>Place the used nebulizer jar inside a biohazard bag and seal the bag for transport to the autoclave.</li>
	<li>At the end of the infection procedure, spray the inside of the chamber with buffered phenol and 70% ethanol and allow the chamber to sit for 10 min.</li>
	<li>Then, wipe down all accessible interior surfaces very thoroughly. Dispose of all contaminated paper towels, etc. in the biohazard trash. Autoclave the trash, waste containers, and used nebulizer jars.</li>
	<li>Place the animals back in the containment room until the imaging time-point.</li>
	<li>On the day of imaging, transfer the animals in a secondary container to the imaging room.</li>
</ol>
3. Animal anesthesia
<ol>
	<li>Anesthetize the mice with isoflurane using a customized gas anesthesia system.</li>
	<li>Weigh each of the two charcoal filter canisters located on top of the anesthesia unit.</li>
	<li>Replace with a new canister if the weight is 50 g above the initial weight.</li>
	<li>Check the vaporizer unit to ensure sufficient isoflurane for the procedure.</li>
	<li>Place the nose cone holder inside the imaging chamber. Place the number of nose cones required for the procedure and seal the remaining openings with the nose cone blockers.</li>
	<li>Turn on the oxygen supply from the high-pressure cylinder and set it at 55 psi.</li>
	<li>Turn on the evacuation pump located in front of the anesthesia unit and set it to 8 L/min.</li>
	<li>Turn on the oxygen toggle located in front of the anesthesia unit.</li>
	<li>Turn on the gas flow to the anesthesia induction chamber to set the flow of gas at 1.5 L/min. Turn the gas flow off.</li>
	<li>Turn on the gas flow to the imaging chamber to set the flow of gas at 0.25 L/min. Turn the gas flow off.</li>
	<li>Turn on the isoflurane vaporizer and set it at 2 - 2.5%. Adjust the isoflurane level according to the number and weight of animals being used for the experiment by rotating the dial on the isoflurane vaporizer (2 - 2.5% for a mouse weighing ~20 g; 4% for a guinea pig weighing 300 g).</li>
	<li>Place the mice in the anesthesia chamber and close the lid. Turn on the gas flow to the anesthesia induction chamber.</li>
	<li>Leave the mice in the anesthesia induction chamber for 5 - 10 min until completely anesthetized.</li>
	<li>Once the mice are anesthetized, apply an optical ointment to the eyes to protect them while imaging.</li>
	<li>Place the mice in ventral or sternal recumbency such that their noses are placed in the nose cone to facilitate the anesthesia of mice during the imaging procedure.</li>
</ol>
4. Reporter enzyme fluorescence (REF) imaging
<ol>
	<li>Inject the substrate (20 &#181;M, 2.5 &#181;L/g of weight) by intraperitoneal injection into the infected as well as the control mice. The mice are under anesthesia inside the imaging chamber for less than 1 minute.</li>
	<li>Start the imaging system.</li>
	<li>Initialize the system by clicking on Initialize. Wait until the temperature bar turns green. 
	NOTE: The imaging chamber consists of a heated platform to maintain the body temperature of the animal while imaging.</li>
	<li>For imaging acquisition setting, select Fluorescent | Trans-illumination for whole animal or Epi-illumination for lung tissues, Structure and Overlay in Acquisition Control Panel.</li>
	<li>Set the Field of view to B for a single mouse and the lamp level to high.</li>
	<li>Set the exposure time to auto, medium binning, 2 - 3 for the f/stop, and excitation filter at 745 nm and emission filters from 780 nm to 840 nm.</li>
	<li>For sequence setup, click on Sequence Setup, and select 9 - 12 Trans-illumination points in the lung area.</li>
	<li>Click on Acquire for image acquisition.</li>
	<li>Place the mouse back into the cage post-imaging.</li>
	<li>Monitor the mice until fully recovered or sacrifice them for quantifying CFU if imaging at a time-point of the experiment. Perform a modified Karnofsky score to monitor the well-being of mice post-imaging.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Isoflurane</td>
			<td>VETONE</td>
			<td>501027</td>
			<td></td>
		</tr>
		<tr>
			<td>CNIR800</td>
			<td>Custom synthesized</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fatal Plus solution</td>
			<td>Vortech Pharmaceutical Ls, Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7H9 Middlebrook broth</td>
			<td>BD</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>OADC Middlebrook enrichment</td>
			<td>BD</td>
			<td>212351</td>
			<td></td>
		</tr>
		<tr>
			<td>Sporcidin</td>
			<td></td>
			<td>RE-1284F</td>
			<td></td>
		</tr>
		<tr>
			<td>7H11 Middlebrook Agar</td>
			<td>BD</td>
			<td>212203</td>
			<td></td>
		</tr>
		<tr>
			<td>Madison Chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Spectrum</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td></td>
		</tr>
		<tr>
			<td>XGI-8-gas Anesthesia System</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Living Imaging software</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent nose cones</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M. tuberculosis strain CDC1551</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Female BALB/C mice, 5-7 weeks</td>
			<td>Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

reporter enzyme fluorescence imaging for in vivo tracking of bacterial infection

Source: Sharan, R., et al. <a href="https://app.jove.com/v/56801">Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence.</a> J. Vis. Exp. (2018).
This video demonstrates the reporter enzyme fluorescence (REF) imaging for in vivo detection of Mycobacterium tuberculosis infection. The infected mice are intraperitoneally injected with a substrate for REF imaging. Upon being hydrolyzed by bacterial &#946;-lactamase enzyme, the substrate emits fluorescence, the detection of which aids evaluation of infection.

Reporter Enzyme Fluorescence Imaging for In Vivo Tracking of Bacterial Infection

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Expose a mouse to aerosol droplets containing Mycobacterium tuberculosis.
Bacteria reach the lumen of alveoli &#8212; the tiny air sacs within the lungs &#8212; containing macrophages.
Macrophage pattern recognition receptors bind to the pathogen-associated molecular patterns on the bacteria, triggering their engulfment into a phagosome.
Engulfed bacteria secrete specialized proteins, which rupture the phagosome membrane. The bacteria escape into the cytoplasm and reproduce.
Post-infection, anesthetize the mouse. Intraperitoneally administer a reporter substrate of the bacterial &#946;-lactamase enzyme for reporter enzyme fluorescence or REF imaging.
The substrate reaches lung alveoli via circulation and enters the infected cells.
It penetrates the bacterial outer membrane to reach the periplasmic space containing &#946;-lactamase.
The substrate contains a &#946;-lactam ring connecting a fluorochrome and a quencher. Due to proximity, the fluorochrome&#39;s fluorescence is absorbed by the quencher.
&#946;-lactamase hydrolyzes the &#946;-lactam ring, releasing the fluorophore and resulting in fluorescence emission.
Image the fluorescence to assess mycobacterial infection.

reporter-enzyme-fluorescence-imaging-for-vivo-tracking-bacterial

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

102 Görüntüleme. Kaynak: Sharan, R., et al. Farelerde Mycobacterium tuberculosis'in Raportör Enzim Floresan ile Görüntülenmesi. J. Vis. Uzm. (2018). Bu video, Mycobacterium tuberculosis enfeksiyonunun in vivo tespiti için raportör enzim floresan (REF) görüntülemeyi göstermektedir. Enfekte farelere intraperitoneal olarak REF görüntüleme için bir substrat enjekte edilir. Bakteriyel β-laktamaz enzimi tarafından hidrolize edildikten sonra, substrat floresan yayar ve bunun tespiti enfeksiyonun değer...

Video: Bakteriyel Enfeksiyonun İn Vivo Takibi için Raportör Enzim Floresan Görüntüleme - Kavram

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Non-invaziv Vivo içinde Bioluminescence sistemi tarafından fare konak Enterohemorrhagic Escherichia Coli kolonizasyon algılama

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

JoVE Journal

İmmünoloji ve Enfeksiyon

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Bir fare modeli için değerlendirmek doğuştan gelen bağışıklık yanıtı Staphylococcus aureus enfeksiyon

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, due to increasing microbial resistance, the efficacy of the most used antibiotics has declined. To find alternative treatment options, there is a great need for a better understanding of the UTI pathogenesis and the mechanisms that determine UTI susceptibility. In order to investigate this in an animal model, a reproducible, non-invasive assay to study the course of UTI is indispensable.
For years, the gold standard for the enumeration of bacterial load has been the determination of Colony Forming Units (CFU) for a particular sample volume. This technique requires post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. As an alternative, bioluminescence imaging (BLI) is gaining popularity to determine the bacterial load. Labeling pathogens with a lux operon allow for the sensitive detection and quantification in a non-invasive manner, thereby enabling longitudinal follow-up. So far, the adoption of BLI in UTI research remains limited.
This manuscript describes the practical implementation of BLI in a mouse urinary tract infection model. Here, a step-by-step guide for culturing bacteria, intravesical instillation and imaging is provided. The in vivo correlation with CFU is examined and a proof-of-concept is provided by comparing the bacterial load of untreated infected animals with antibiotic-treated animals. Furthermore, the advantages, limitations, and considerations specific to the implementation of BLI in an in vivo UTI model are discussed. The implementation of BLI in the UTI research field will greatly facilitate research on the pathogenesis of UTI and the discovery of new ways to prevent and treat UTI.

This manuscript describes the intravesical administration of uropathogenic bacteria with a lux operon to induce a urinary tract infection in mice and subsequent longitudinal in vivo analysis of the bacterial load using bioluminescence imaging.

Biyolüminesans Görüntüleme Kullanan Farelerde İdrar Yolu Enfeksiyonlarının Boyuna Takibi ve Tedavileri

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, ...

Reporter Enzyme Fluorescence Imaging for In Vivo Tracking of Bacterial Infection

TRANSKRIPT

Reporter Enzyme Fluorescence Imaging for In Vivo Tracking of Bacterial Infection