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NOTE: All work described below is to be carried out in a CL2 laboratory within a class 2 microbiological safety cabinet (MSC) following local health and safety guidelines.
1. Preparation&#160;of M. bovis&#160;BCG&#160;lux&#160;for Infection
<ol>
	<li>Defrost a frozen 1.2 mL glycerol (15%) stock of&#160;Mycobacterium bovis&#160;Bacillus Calmette-Gu&#233;rin (BCG)&#160;lux, the Montreal vaccine strain transformed with the shuttle plasmid pSMT1 carrying the&#160;luxAB&#160;genes from&#160;Vibrio harveyi&#160;encoding the luciferase enzyme.</li>
	<li>Inoculate 15 mL of Middlebrook 7H9 broth containing 0.2% glycerol, 10% albumin, dextrose, catalase (ADC) enrichment, and 50 &#181;g/mL hygromycin with a defrosted 1.2 mL aliquot of BCG&#160;lux, in a labeled 250 mL Erlenmeyer flask.</li>
	<li>Place the flask in a sealed biosafety container and incubate at 37 &#176;C in an orbital shaker incubator at 220 rpm for 72 h (or until the culture reaches the mid-log phase of growth).</li>
	<li>Check the growth of BCG&#160;lux&#160;culture by preparing 1:10 dilutions of the culture in luminometer tubes using phosphate-buffered saline (PBS, pH 7.4, 0.01 M phosphate buffer, 0.0027 M potassium chloride, and 0.137 M sodium chloride) in duplicate. Vortex, and load the luminometer tubes into the luminometer and measure the bioluminescence (relative light unit [RLU]/mL) using n-decyl aldehyde as the substrate (1% v/v in absolute ethanol).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE:&#160;The ratio of RLU/colony forming units (CFU) was previously determined to be 3:1 when BCG&#160;lux&#160;was grown in vitro in Middlebrook 7H9 broth.</li>
	<li>Centrifuge the culture at 2,175 x&#160;g&#160;for 10 min at room temperature to pellet the cells and discard the supernatant into an appropriate disinfectant with known mycobactericidal activity. Dispose of all culture waste with disinfectants appropriate for mycobacteria following local guidelines.</li>
	<li>Wash the cell pellet twice in PBS containing 0.05% polysorbate 80 (PBS-T) to prevent bacterial clumping.</li>
	<li>Following the final wash, decant waste supernatant, resuspend the mycobacterial cell pellet in PBS-T, and dilute the mycobacterial suspension to the desired cell density using RLU measurements.</li>
	<li>Prepare ten-fold serial dilutions of the inoculum in 24-well plates using PBS-T. Plate out 10 &#181;L onto Middlebrook 7H11 agar plates (0.5% glycerol, 50 &#181;g/mL hygromycin, 10% oleic acid, albumin, dextrose, catalase [OADC]) in duplicate to enumerate inoculum CFU counts.</li>
</ol>
2. Preparation of&#160;G. mellonella&#160;Larvae
<ol>
	<li>Purchase last instar larvae from appropriate sources and maintain the larvae in the dark at 18 &#176;C upon arrival and use within 1 week of purchase. Alternatively, larvae can be self-reared following the protocol of Joj&#257;o et al. For purchased larvae, discard any dead, discolored, or pupating larvae prior to storage.&#160;&#160;&#160;&#160;&#160; 
	NOTE:&#160;Pupating larvae are morphologically distinguishable from the last instar larvae.</li>
	<li>Identify and select healthy larvae for experimentation, based on uniform cream color with little to no discoloration (melanization), size (2&#8722;3 cm in length), weight (approximately 250 mg), displaying a high level of motility, and possessing the ability to right themselves when turned over.</li>
	<li>Carefully count the healthy larvae (minimum of 20&#8722;30&#160;per group) into a Petri dish (94/15 mm) lined with a layer of filter paper (94/15 mm) using blunt-end tweezers to minimize contamination and store at room temperature in the dark until use.</li>
</ol>
3. Infecting&#160;G. mellonella&#160;with BCG&#160;lux
<ol>
	<li>Minimize the clutter within the MSC to reduce the risk of contamination and needle stick injury.</li>
	<li>Prepare the injection platform by taping a circular filter paper (94 mm) to a flat raised surface. Soft or hard surfaces can be used and are entirely dependent on user preference, e.g., pipette box lids or a nylon scouring sponge.</li>
	<li>Sterilize a 25 &#181;L microsyringe (25 G) by aspirating 3 volumes of 70% ethanol and further rinse with 3 volumes of sterile PBS-T.</li>
	<li>Aspirate 10 &#181;L of BCG&#160;lux&#160;inoculum (prepared in section 1) or PBS-T into the sterilized 25 &#181;L microsyringe. Use a separate syringe for PBS-T negative control. 
	NOTE:&#160;Resuspend the BCG&#160;lux&#160;inoculum following 10 injections to ensure uniform cell suspension.</li>
	<li>Use tweezers to pick up one larva and place it onto the injection platform.</li>
	<li>On the platform, flip the larva onto its back and immobilize it by securing the head and tail with tweezers. Locate the last left proleg counting down from the head of the larva and carefully insert the tip of the needle (5&#8722;6 mm) at a 10&#8722;20&#176; angle to the horizontal plane.&#160;&#160;&#160;&#160;&#160; 
	NOTE:&#160;Short and narrow tweezers allow for easy immobilization with minimum larval stress. Pay attention not to over penetrate which may puncture the gut and cause non-BCG&#160;lux&#160;specific melanization or death.</li>
	<li>Count the infected larvae into a Petri dish lined with a layer of filter paper, a single 90 mm Petri dish can accommodate up to 30 larvae.</li>
	<li>Store the Petri dish containing the larvae in a vented or non-sealed dark box inside an incubator at 37 &#176;C with 5% CO2.</li>
</ol>
4. Monitoring the Survival of&#160;G. mellonella&#160;Following Infection
<ol>
	<li>Over the time course, monitor the survival of the larvae every 24 hours. Larvae are considered dead when they fail to move in response to touch.</li>
</ol>
5. Measuring the In Vivo Burden of BCG&#160;lux&#160;in&#160;G. mellonella
<ol>
	<li>At each time point, randomly select five infected larvae previously prepared in section 3 and gently sterilize the larval surfaces using&#160;cotton bud swabs soaked in 70% ethanol.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE:&#160;This step is important when plating larval homogenate for mycobacterial CFU enumeration as non-sterilized larvae can lead to contamination.</li>
	<li>Place the larvae individually into 2 mL lysing matrix tubes containing 800 &#181;L of sterile PBS.</li>
	<li>Homogenize the larvae using a homogenizer for 60 s at 6.0 m/s.</li>
	<li>Centrifuge the lysing tubes at 3,500 x&#160;g&#160;for 5 s to remove homogenate from the lids, and carefully decant the homogenate into sterile luminometer tubes individually. 
	NOTE:&#160;For CFU enumeration (step 5.7), ensure to reserve 100 &#181;L of homogenate in a sterile 1.5 mL reaction tube.</li>
	<li>Recover any remaining homogenate by washing the lysing matrix tubes with 1 mL of PBS-T and pipette into the corresponding luminometer tubes.</li>
	<li>Vortex the luminometer tubes and measure the bioluminescence of the homogenates previously described in step 1.4.</li>
	<li>Prepare ten-fold serial dilutions of the homogenate in 24-well culture plates using PBS-T. Plate out 10 &#181;L of the dilution onto Middlebrook 7H11 agar plates containing 0.5% glycerol, 50 &#181;g/mL hygromycin, 10% OADC, and 20 &#181;g/mL piperacillin, to determine the RLU/CFU ratio of BCG&#160;lux&#160;following in vivo infection. 
	NOTE:&#160;Piperacillin eliminates native&#160;G. mellonella&#160;microbiota with minimal growth inhibition of BCG&#160;lux.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5ml reaction tube (Eppendorf)</td>
			<td>Eppendorf</td>
			<td>22431021</td>
			<td></td>
		</tr>
		<tr>
			<td>20, 200 and 1000 &#181;l pipette and filtered tips</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well culture plate</td>
			<td>Greiner</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>25 ml pipettes and pipette boy</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>3 compartment Petri dish (94/15mm)</td>
			<td>Greiner</td>
			<td>637102</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II safety cabinet</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask with vented cap (250 ml)</td>
			<td>Corning</td>
			<td>CLS40183</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (&#62;99.7%)</td>
			<td>VWR</td>
			<td>208221.321</td>
			<td></td>
		</tr>
		<tr>
			<td>Galleria mellonella&#160;(250 per pk)</td>
			<td>Livefood Direct UK</td>
			<td>W250</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5150</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer (FastPrep-24 5G )</td>
			<td>MP Biomedicals</td>
			<td>116005500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Corning</td>
			<td>30-240CR</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer (Autolumat LB 953)</td>
			<td>Berthold</td>
			<td>34622</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer tubes</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing matrix (S, 2.0ml)</td>
			<td>MP Biomedicals</td>
			<td>116925500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro syringe (25 &#181;l, 25 ga)</td>
			<td>SGE</td>
			<td>3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H11 agar</td>
			<td>BD Bioscience</td>
			<td>283810</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H9 broth</td>
			<td>BD Bioscience</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook ADC enrichment</td>
			<td>BD Bioscience</td>
			<td>212352</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook OADC enrichment</td>
			<td>BD Bioscience</td>
			<td>212240</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium bovis&#160;BCG&#160;lux</td>
			<td>Various</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>n-decyl aldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>D7384-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaking incubator</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 80 (Tween-80)</td>
			<td>Sigma-Aldrich</td>
			<td>P8074-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Small box</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td>Dark vented or non-sealed box recommended</td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td>Short and narrow tipped/Blunt long tweezers</td>
		</tr>
		<tr>
			<td>Petri dish (94/15mm)</td>
			<td>Greiner</td>
			<td>633181</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper (94mm)</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td>Cut to fit</td>
		</tr>
	</tbody>
</table>

measurement of the in vivo burden of bacteria expressing luciferase in an insect larva

Source: Asai, M. et al., <a href="https://app.jove.com/v/59703">Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex.</a>&#160;J. Vis. Exp. (2019)
This video showcases the in vivo bacterial burden measurement using Bacillus Calmette-Gu&#233;rin, BCG, lux-infected insect larvae. The process involves larval homogenization, treatment with a luciferase substrate, and bioluminescence measurement to determine relative light units that correlate with bacterial burden.

Measurement of the In Vivo Burden of Bacteria Expressing Luciferase in an Insect Larva

Source: Asai, M. et al., Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex.&#160;J. Vis. ...

To measure the in vivo bacterial burden, begin with sterilized insect larvae pre-infected with Bacillus Calmette-Gu&#233;rin, or BCG lux. BCG lux is a genetically modified Mycobacterium bovis BCG strain expressing luciferase &#8212; a bioluminescence-producing enzyme.
Transfer an insect larva into a buffer-containing lysing matrix tube with steel balls. Using a homogenizer, homogenize the larva, releasing the intracellular components, including bacteria, into the buffer.
Centrifuge the tube to collect the homogenate and transfer it to a luminometer tube.
Wash the matrix tube with a detergent-containing buffer to solubilize cellular fragments and release residual bacteria.
Combine the fractions in the same luminometer tube and add the luciferase substrate. Substrates enter into bacteria and interact with luciferase enzymes, resulting in bioluminescence. Load the tube into a luminometer and measure the bioluminescence.
The luminometer measures the intensity of emitted light and calculates the relative light unit, or RLU &#8212; indicative of the in vivo burden of BCG in an insect larva.
Spread the diluted homogenate suspension on a piperacillin-containing agar plate and incubate to selectively grow BCG as distinct colonies. The colonies on the plate represent the colony-forming unit, or CFU. The RLU to CFU ratio correlates bioluminescence with bacterial viability.

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Immunology

Immunopathology

Host-Pathogen Interaction Models

72 Views. Source: Asai, M. et al., Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex. J. Vis. Exp. (2019) This video showcases the in vivo bacterial burden measurement using Bacillus Calmette-Guérin, BCG, lux-infected insect larvae. The process involves larval homogenization, treatment with a luciferase substrate, and bioluminescence measurement to determine relative light units that correlate with bacterial burden. 

Video: Measurement of the In Vivo Burden of Bacteria Expressing Luciferase in an Insect Larva - Concept

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

JoVE Journal

Immunology and Infection

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.

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

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...

Measurement of the In Vivo Burden of Bacteria Expressing Luciferase in an Insect Larva

Transcript

Measurement of the In Vivo Burden of Bacteria Expressing Luciferase in an Insect Larva

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

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

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis