Name: a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated staphylococcus aureus infection
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection at JoVE.com

This study was financially supported by the IBIZA project of the BioMedical Material (BMM) program and co-funded by the Dutch Ministry of Economic Affairs. The authors would like to thank Prof. Dr. Graham Lieschke from Monash University, Australia for providing the zebrafish transgenic line (mpeg1:Gal4/UAS:Kaede).

In this protocol, maintenance of adult zebrafish is in compliance with the local animal welfare regulations as approved by the local animal welfare committee. Experiments with embryos were performed according to the 2010/63/EU Directive.
1. Preparation of &#34;Bacteria-only&#34; and Bacteria-microspheres Suspensions
NOTE: The S. aureus RN4220 strain expressing mCherry fluorescent protein (S. aureus-mCherry) is used. The S. aureus...

Biomaterial-associated infection (BAI) is a serious clinical complication. A better understanding of the pathogenesis of BAI in vivo would provide new insights to improve the prevention and treatment of BAI. However, current experimental BAI animal models such as murine models are costly, labor-intensive, and require specialized personnel trained in complex surgical techniques. Therefore, these models are not suitable for high throughput analysis. Since requirements for zebrafish embryo models are less complex and costs ...

A variety of medical devices (referred to as &#34;biomaterials&#34;) are increasingly used in modern medicine to restore or replace human body parts1. However, the implantation of biomaterials predisposes a patient to infection, called a biomaterial-associated infection (BAI), which is a major complication of implants in surgery. Staphylococcus aureus and Staphylococcus epidermidis are two most prevalent bacterial species responsible for BAI2,3,4,5,6. Implanted biomaterials form a surface susceptible to bacterial biofilm formation. Moreover, local immune response may be deranged by the implanted biomaterials, causing reduced effectiveness of bacterial clearance. The initial clearance of infecting bacteria is performed mainly by infiltrating neutrophils, which have strongly reduced bactericidal capacity in the presence of an inserted or implanted biomaterial7. Moreover, macrophages infiltrating the tissue after the initial influx of neutrophils will phagocytose the remaining bacteria but cannot effectively kill them intracellularly, due to deranged immune signaling that is a consequence of the combined presence of the biomaterial and bacteria8. Thus, the presence of biomaterials can facilitate intracellular survival of bacteria9,10,11,12,13 and biofilm formation on the implanted biomaterials4,14. Consequently, BAI may lead to the failure and need for replacement of implanted biomaterials, causing increased morbidity and mortality and prolonged hospitalization with additional costs2,15.
An increasing number of anti-BAI strategies are being developed2,16,17. In vivo evaluation of the efficacy of these strategies in relevant animal models is essential. However, traditional experimental BAI animal models (e.g., mouse models) are usually costly, time-consuming, and therefore not suitable for high throughput testing of multiple strategies18. Recent development of bio-optical imaging techniques based on bioluminescent/fluorescent labeling of host cells and bacteria may allow for the continuous monitoring of BAI progression and host-pathogen/host-material interactions in single small animals such as mice18,19,20,21. However, this technique is relatively complex and still in its infancy, and several issues must be addressed for quantitative analysis of BAI18. For instance, a high challenge dose is required to visualize bacterial colonization. In addition, light scattering and adsorption of bioluminescence/fluorescence signals in tissues of mammalian test animals must also be addressed18,19,21. Therefore, novel, cost-effective animal models allowing for intravital visualization and quantitative analysis over time are valuable complementary systems for studying BAI in vivo.
Zebrafish (embryos) have been used as a versatile in vivo tool for dissecting host-pathogen interactions and infection pathogenesis of several bacterial species such as mycobacteria22, Pseudomonas aeruginosa23, Escherichia coli24, Enterococcus faecalis25, and staphylococci26,27. Zebrafish embryos have many advantages such as optical transparency, a relatively low maintenance cost, and possession of an immune system highly similar to that in mammals28,29. This makes zebrafish embryos a highly economic, living model organism for intravital visualization and analysis of infection progression and associated host responses28,29. To allow visualization of cell behavior in vivo, transgenic zebrafish lines with different types of immune cells (e.g., macrophages and neutrophils) and even with fluorescently tagged subcellular structures have been developed28,29. In addition, the high reproduction rate of zebrafish provides the possibility of developing high throughput test systems featuring automated robotic injection, automated fluorescence quantification, and RNA sequence analysis27,30.
In the present study, we aimed to develop a zebrafish embryo model for biomaterial-associated infection using fluorescence imaging techniques. To this end, we developed a procedure to inject bacteria (S. aureus) in the presence of biomaterial microspheres into the muscle tissue of zebrafish embryos. We used S. aureus RN4220 expressing mCherry fluorescent protein (S. aureus-mCherry), which was constructed as described elsewhere for another S. aureus strain10,31. The transgenic zebrafish line (mpeg1: UAS/Kaede) expressing Kaede green fluorescent protein in the macrophages32 and blue fluorescent polystyrene microspheres were used. In a previous study, we have shown that intramuscular injection of microspheres into zebrafish embryos to mimic biomaterial implantation is feasible33. To quantitatively analyze the progression of BAI and associated cell infiltration in single embryos over time, we used the &#34;Zebrafish-Immunotest&#34; project file which is operated within &#34;ObjectJ&#34; (a plug-in for ImageJ) to quantify the fluorescence intensity of bacteria residing and macrophages infiltrating in the vicinity of the injection site of microspheres33. In addition, we determined the numbers of colony-forming units (CFU) of bacteria in the presence and absence of microspheres in the embryos to study potential effects of biomaterials on infection. Our present study demonstrates that with the methods developed here, the zebrafish embryo&#160;is a promising, novel&#160;vertebrate animal model for studying biomaterial-associated infections in vivo.

<table>
	<tbody>
		<tr>
			<td>Tryptic soya agar</td>
			<td>BD Difco</td>
			<td>236950</td>
			<td>Media preparation unit at AMC</td>
		</tr>
		<tr>
			<td>Tryptic soya broth</td>
			<td>BD Difco</td>
			<td>211825</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone40</td>
			<td>Applichem</td>
			<td>A2259.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;m diameter polystyrene microspheres (blue fluorescent)</td>
			<td>Life technology/ThemoFisher</td>
			<td>F8829</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microcapilary (1 mm O.D. x 0.78 mm I.D.)</td>
			<td>Harvard Apparatus</td>
			<td>30-0038</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller instrument</td>
			<td>Sutter Instrument Inc</td>
			<td>Flaming p-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope LM 20</td>
			<td>Leica</td>
			<td>MDG33 10450123</td>
			<td></td>
		</tr>
		<tr>
			<td>3-aminobenzoic acid (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose MP</td>
			<td>Roche</td>
			<td>11388991001</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo fluorescent microscope LM80</td>
			<td>Leica</td>
			<td>MDG3610450126</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader pipette tips</td>
			<td>Eppendorf</td>
			<td>5242956.003</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator M3301 with M10 stand</td>
			<td>World Precision Instruments</td>
			<td>00-42-101-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>FemtoJet express micro-injector</td>
			<td>Eppendorf</td>
			<td>5248ZO100329</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtrube 2ml pp</td>
			<td>Sarstedt</td>
			<td>72.693.005</td>
			<td></td>
		</tr>
		<tr>
			<td>Zirconia beads</td>
			<td>Bio-connect</td>
			<td>11079124ZX</td>
			<td></td>
		</tr>
		<tr>
			<td>MagNA lyser</td>
			<td>Roche</td>
			<td>41416401</td>
			<td></td>
		</tr>
		<tr>
			<td>MSA-2 plates (Mannitol Salt Agar-2)</td>
			<td>Biomerieux</td>
			<td>43671</td>
			<td>Chapmon 2 medium</td>
		</tr>
		<tr>
			<td>Methyl cellulose 4000cp</td>
			<td>Sigma-Aldrich</td>
			<td>MO512-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378</td>
			<td></td>
		</tr>
		<tr>
			<td>Gyrotory shaker (for bacterial growth)</td>
			<td>New Brunswick Scientific</td>
			<td>G10</td>
			<td></td>
		</tr>
		<tr>
			<td>Zebrafish incubator</td>
			<td>VWR</td>
			<td>Incu-line</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvettes</td>
			<td>BRAND</td>
			<td>759015</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hettich-Zentrifugen</td>
			<td>ROTANTA 460R</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Pharmacia biotech</td>
			<td>Ultrospec&#174;2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F6521-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>48 well-plates</td>
			<td>Greiner bio-one</td>
			<td>677180</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well-plates</td>
			<td>Greiner bio-one</td>
			<td>655161</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri-dish</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri-dish</td>
			<td>Biomerieux</td>
			<td>NL-132</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>Not applicable</td>
			<td>Not applicable</td>
			<td>link: https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr>
			<td>GraphPad 7.0</td>
			<td>Prism</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
	</tbody>
</table>

a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated staphylococcus aureus infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

The present study assessed the applicability of zebrafish embryos as a novel vertebrate animal model for investigating biomaterial-associated infection. Microinjection technique has been commonly used to inject different bacterial species into zebrafish embryos to cause infection22,26,27,30,36. Using the procedure depicted in <...

Watch this Scientific Journal Video about A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection at JoVE.com

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

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