This project was supported by grants from the Biotechnology and Biological Science&#160;Research Council&#160;(BBSRC), awarded to PRL and YL (BB/P001262/1), and the National Center for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs)&#160;awarded to PRL, SMN, BDR, and YL (NC/R001596/1).

NOTE: All work described below are to be carried out in a CL2 laboratory within a class 2 microbiological safety cabinet (MSC) following local health and safety guidelines.
1. Preparation of M. bovis BCG lux for Infection
<ol>
	<li>Defrost a frozen 1.2 mL glycerol (15%) stock of M. bovis BCG lux, the Montreal vaccine strain transformed with the shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi encoding the luciferase enzyme19.</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 lux, in a labelled 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 lux 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)20. 
	NOTE: The ratio of RLU/colony forming units (CFU) was previously determined to be 3:1 when BCG lux was grown in vitro in Middlebrook 7H9 broth20.</li>
	<li>Centrifuge the culture at 2,175 x g for 10 min at room temperature to pellet the cells and discard the supernatant into an appropriate disinfectant with known mycobactericidal activity. Dispose 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 G. mellonella 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.21. For purchased larvae, discard any dead, discolored or pupating larvae prior to storage. 
	NOTE: Pupating larvae are morphologically distinguishable to 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 G. mellonella with BCG 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 is 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 lux 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: Resuspend the BCG lux inoculum following 10 injections to ensure uniform cell suspension.</li>
	<li>Use tweezers to pick up one larva and place onto the injection platform.</li>
	<li>On the platform, flip the larva on to its back and immobilize by securing the head and tail with the 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. 
	NOTE: 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 lux 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 G. mellonella Following Infection
<ol>
	<li>Over the time course, monitor the survival of the larvae every 24 h. Larvae are considered dead when they fail to move in response to touch.</li>
</ol>
5. Measuring the In Vivo Burden of BCG lux in 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. 
	NOTE: 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 g for 5 s to remove homogenate from the lids, and carefully decant the homogenate into sterile luminometer tubes individually. 
	NOTE: 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 lux following in vivo infection. 
	NOTE: Piperacillin eliminates native G. mellonella microbiota with minimal growth inhibition of BCG lux16.</li>
</ol>
6. Statistical Analysis
<ol>
	<li>Plot the Kaplan-Meier survival curve using data collected and carry out the Mantel-Cox (Log-rank) test to determine the significance of the result, where *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001, and ****p &#60; 0.0001.</li>
</ol>

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The authors have nothing to disclose.

The use of G. mellonella as an infection model has been established for a number of bacterial and fungal pathogens for the study of virulence, host-pathogen interaction, and as a screen for novel therapeutics10,22. The following discussion is based on the experimental procedure for&#160;the use of G. mellonella as an infection model for the MTBC. 
The health of the na&#239;ve larvae prior to experimentation c...

Tuberculosis (TB) is a major threat to global public health with 9 million new cases per year and 1.5 million deaths1. In addition, it is estimated that one quarter of the world&#8217;s population is infected with the causative agent of the disease, Mycobacterium tuberculosis (Mtb). Amongst the infected population, 5&#8722;10% will develop active TB disease over their lifetime. Furthermore, the emergence and spread of multi-drug resistant (MDR) and extensively-drug (XDR) resistant Mtb poses a serious threat to disease control, with 123 countries reporting at least one XDR case1. Treatment of TB requires a cocktail of at least four anti-mycobacterial drugs, of which isoniazid and rifampicin are prescribed for a minimum duration of six months; treatment is often associated with complex side effects and toxicities. Protection from the only licensed vaccine against TB, Mycobacterium bovis Bacillus Calmette-Gu&#233;rin (BCG), is variable2. An incomplete understanding of the pathogenesis of TB significantly hampers the development of new therapeutic and vaccination strategies.
For decades animal infection models have been vital for TB research to understand the basic pathogenesis and host response to infection, and to evaluate novel anti-mycobacterial agents, immuno-therapeutics and new vaccine candidates3,4. However, research using animal infection models of TB is notoriously difficult as the pathogenesis and progression of TB infection are complex, and there is no single animal model that mimics the full spectrum and important features of the disease5,6. Furthermore, animal experiments are expensive, time consuming to undertake and require full ethical justification. Nevertheless, animal infection models of TB have been described in non-human primates (e.g., macaques), guinea pigs, rabbits, cattle, pigs, mice and zebrafish, with each having their limitations3,4. The murine model is the most commonly used model due to cost, availability of inbred lines, reproducibility of infection and abundance of immunological reagents. However, they do not typically form granulomas associated with areas of hypoxia that are characteristic of latent tuberculosis infection (LTBI)6. Guinea pigs are highly susceptible to Mtb infection, with pathology and early granuloma formation similar to those in humans, and are widely used in vaccine testing; yet the lack of immunological reagents hampers their use as an infection model7. Zebrafish are suitable for large-scale screening in early-stage preclinical studies due to their small size, rapid reproduction and advanced genetic tools, but are anatomically and physiologically different to humans and are only susceptible to Mycobacterium marinum infection3. The animal models most closely resembling human Mtb infection are non-human primates (e.g., the macaque), but they are expensive and have significant ethical and practical considerations which considerably limits their use8.
The insect larva of the greater wax moth or honeycomb moth, Galleria mellonella, have become increasingly popular as an infection model for a variety of bacterial and fungal pathogens9, and as a screen for novel antimicrobial drug candidates10. G. mellonella is a successful invertebrate model due to its sophisticated innate immune system (comprised of cellular and humoral defenses) that shares a high degree of structural and functional similarity to that of vertebrates11. This includes immune mechanisms such as the phagocytosis of pathogens by hemocytes (functionally similar to mammalian macrophage and neutrophils)12,13, the production and circulation of anti-microbial peptides (AMPs) and complement-like proteins within the hemolymph (analogous to mammalian blood) of G. mellonella11. Other advantages9,14,15 of G. mellonella larvae as a model include 1) their large size (20&#8722;30 mm) which allows for easy manipulation and infection, as well as the collection of tissue and hemolymph for analyses, 2) easy maintenance at 37 &#176;C, compatible for studying human pathogens, 3) precise infection by injection without the need for anesthesia, 4) efficacy of antimicrobial agents can be assessed utilizing less drug for evaluation, 5) lack of ethical constraints compared to the use of mammals, 6) large group sizes can be used compared to animal models allowing greater reproducibility, and 7) shorter times for infection experiments are required.
In a recent study, we demonstrated that G. mellonella can be used as a novel infection model for studying the pathogenesis of infection by bioluminescent M. bovis BCG lux, a genetically modified version of the vaccine strain and member of the Mtb complex (MTBC)16. While G. mellonella has previously been used as an infection model for non-tuberculous mycobacteria (NTM), mainly M. marinum and Mycobacterium abscessus17,18, studies using MTBC are limited to that of Li et al.16. Bioluminescent non-pathogenic mycobacterial strains, which can be used at containment level (CL) 2 as a surrogate for Mtb, offer the advantages of safety and practicality over pathogenic mycobacteria. Following infection with BCG lux, larvae begin to develop early granuloma-like structures, which could provide valuable insight into the role of innate immunity in the establishment of TB infection16. In addition, this simple invertebrate infection model has the potential to provide a rapid, low-cost, and reliable evaluation of TB pathogenesis incorporating controlled challenge and multiple replicates for reproducibility. Furthermore, the model has the potential to be used to screen novel anti-TB drug and vaccine candidates in early development, reducing the overall number of animals in experimentation. The ability to measure changes in host and pathogen structure, transcriptome and proteome to determine drug targets and assess mechanisms of action of novel drugs and therapeutic vaccines, are also advantageous.
Here we describe the experimental protocols for the preparation of a bioluminescent M. bovis BCG lux inoculum and G. mellonella larvae for mycobacterial infection, as well as the determination of both larval and mycobacterial survival in response to infection.

<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 saftey 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 (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>Homogeniser (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 BCG 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>Winterm (V1.08)</td>
			<td>Berthold</td>
			<td>n/a</td>
			<td>Program LB953.TTB</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>

use of the invertebrate galleria mellonella as an infection model to study the mycobacterium tuberculosis complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Here we present representative data that can be obtained using the G. mellonella &#8212; BCG lux infection model and highlight the benefits of G. mellonella as an infection model for members of the MTBC (Figure 1). Experimental procedures with key technical points are outlined in Figure 2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_up...

Watch this Scientific Journal Video about Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex at JoVE.com

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...

use-invertebrate-galleria-mellonella-as-an-infection-model-to-study

Research

JoVE Journal

Immunology and Infection

11.7K Views.  Imperial College London. Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

Video: Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

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 ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...