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 RN4220 strain is mutated in the virulence regulator gene agrA (accessory gene regulator A)34, and therefore may have relatively low virulence in the zebrafish embryo model. Other S. aureus strains or other bacterial species for BAI can be used.
<ol>
	<li>Take 4 to 5 colonies of S. aureus RN4220 bacteria from tryptic soya agar culture plates supplemented with 10 &#181;g/mL chloramphenicol and culture the bacteria to mid-logarithmic growth phase in 10 mL of tryptic soy broth supplemented with 10 &#181;g/mL chloramphenicol at 37 &#176;C under shaking.
	<ol>
		<li>During culture, dilute 100 &#181;L of the bacterial suspension in 900 &#181;L of sterile phosphate buffered saline (PBS) in a cuvette (width of 1 cm) for an optical density (OD) measurement at 620 nm (OD620). Culture the bacteria until the OD620 reaches 0.4&#8211;0.8. 
		NOTE: An OD620 of 0.1 generally corresponds to 3.0 x 107 CFU/mL S. aureus. The OD620 of an inoculum of bacteria in mid logarithmic growth phase is between 0.4-0.8. Different time periods for culturing may be needed for other species and strains of bacteria.</li>
	</ol>
	</li>
	<li>Centrifuge bacteria at 3,500 x g for 10 min and re-suspend the pelleted bacteria in 1 mL of sterile PBS. Subsequently wash the bacteria with sterile PBS 2 times, and finally re-suspend the bacteria in 1.1 mL of 4% (w/v) polyvinylpyrrolidone40 (PVP40) solution in PBS.</li>
	<li>Vortex this bacterial suspension and dilute 100 &#181;L of the suspension in 900 &#181;L of sterile PBS in a cuvette for the OD620 measurement. Adjust the concentration of the bacterial suspension with PVP40 solution. This is the &#34;Bacteria-only&#34; suspension.</li>
	<li>Biomaterials can be freely chosen to mix with bacterial suspension. In the present study, commercial polystyrene (PS) microspheres (blue fluorescent, 10 &#181;m) are used. To generate a Bacteria-Microspheres suspension, centrifuge the microspheres for 1 min at 1,000 x g, room temperature, discard the supernatant and re-suspend the microspheres in the&#160; &#8220;Bacteria-only&#8221; suspension (in PVP40 solution).</li>
	<li>In order to inject approximately equal doses of bacteria in the presence and in the absence of microspheres, the concentration of Bacteria-Microspheres suspension needs to be two thirds of the concentration of the &#8220;Bacteria-only&#8221; suspension (without microspheres). This is achieved by diluting the Bacteria-Microspheres suspension with 0.5 volume of PVP40 solution. Mix the suspensions by vortexing. Of note, this ratio (two thirds) has been assessed for S. aureus. It is also appropriate for S. epidermidis, but it is advised to check whether this ratio also applies to other bacterial species and other sizes (than 10 &#181;m) or shapes of biomaterials (than microspheres) to be injected.</li>
	<li>Check the concentration of the &#8220;Bacteria-only&#8221; and Bacteria-Microspheres suspension by quantitative culture of 10-fold serial dilutions as below: transfer 100 &#181;L of the suspensions to a 96 well-plate and serially dilute by transferring 10 &#181;L aliquots of the suspension into 90 &#181;L of sterile PBS. Plate duplicate 10 &#181;L aliquots of the undiluted and diluted suspensions on agarose plates, incubate the plates at 37 &#176;C overnight, count the colonies and calculate the numbers of bacteria (colony-forming units. CFU).</li>
</ol>
2. Breeding, Harvesting, and Maintenance of Zebrafish Embryos
<ol>
	<li>Follow the general procedures described earlier35,36 for breeding, harvesting, and maintenance of zebrafish embryos, with modifications described below. Cross a family of wild type Tupfel long fin (TL) zebrafish or zebrafish of the selected transgenic line (here, Mpeg1: Kaede) in a tank with a net added to induce the adult females to produce eggs after the light turns on, then separate adults from the produced eggs.</li>
	<li>Collect the embryos the next day and discard the non-transparent ones which are not viable. Keep approximately 60 embryos per Petri dish (100 mm in diameter) in E3 medium37 and incubate at 28 &#176;C. Remove dead and abnormal embryos, and refresh the E3 medium daily.</li>
</ol>
3. Preparation of Injection Needles
<ol>
	<li>Prepare the glass microcapillary needles for injection using a micropipette puller instrument. Use the following settings: heat: 772, pull: 100, vel: 200, time: 40, gas: 75.</li>
	<li>Break the needle tip with forceps at the position where the needle has an outer diameter of approximately 20 &#181;m (for 10 &#181;m microspheres), using a light microscope with a scale bar in the ocular. Avoid needles with a very large opening size, as they will compromise survival of the embryos. 
	NOTE: The opening may be chosen to be smaller or larger, depending on the size and shape of biomaterials to be injected. In the literature, injections using needles with an opening of approximately 50 &#181;m have been reported to cause a significant decrease in embryo survival38.</li>
</ol>
4. Injection of &#34;Bacteria-Only&#34; or Bacteria-microspheres Suspension into Zebrafish Embryos
<ol>
	<li>Heat agarose solution (1&#8211;1.5% (wt) in demi-water) using a microwave oven and pour into a 100-mm Petri dish. Place a plastic mold template on top of the agarose solution in the Petri dish to create indentations in the agarose for placing embryos in proper positions, facilitating injections. Incubate at room temperature and remove the mold when the agarose solution has solidified.</li>
	<li>At 3 d post-fertilization, place the embryos in a 100 mm Petri dish containing 0.02% (w/v) 3-aminobenzoic acid (Tricaine) to anaesthetize them. After 5 min, transfer the embryos to the agarose plate overlaid with E3 medium containing 0.02% (w/v) Tricaine and align them in one orientation for injection. For the Mpeg1: Kaede transgenic line first anaesthetize embryos in E3 medium containing 0.02% (w/v) tricaine. Then&#160;select embryos expressing green fluorescent proteins using a stereo fluorescence microscope.</li>
	<li>Load the needle with approximately 10 &#181;L of the &#34;Bacteria-only&#34; or Bacteria-Microspheres suspension using a microloader pipette tip. Mount the needle onto a micromanipulator connected to the micro-injector. For the injector used here (see Table of Materials), use the following settings for injections of 2&#8211;3 nL: pressure: 300&#8211;350, back pressure: 0, time: 2 ms. 
	NOTE: The settings for the micro-injector depend on the injector used. Injector settings may need to be adjusted for injections of bacteria mixed with biomaterials with other shapes or sizes.
	<ol>
		<li>Use needles with the same opening for the injection of the &#34;Bacteria-only&#34; suspension and the Bacteria-Microspheres suspension. If the needle is broken or clogged, always change for a new needle for further injections.</li>
	</ol>
	</li>
	<li>Insert the needle into the muscle tissue of embryos under a light microscope (Figure 1), at an angle of 45&#8211;60&#176; between the needle and the body of embryos. Adjust the position of the needle in the tissue by gently moving it back and forth. Inject the embryos using a foot pedal connected to the micro-injector.</li>
	<li>After injection of fluorescent bacteria, score the embryos for successful infection under a stereo fluorescence microscope. Discard the embryos scored negative (no visible fluorescent bacteria or no visible fluorescent microspheres). Maintain embryos individually in E3 medium in 48-well plates. Refresh the medium daily.</li>
</ol>
5. Crushing of Embryos, Microscopic Scoring, and Quantitative Culture of Bacteria
<ol>
	<li>Score all embryos microscopically for the presence of fluorescent bacteria using a stereo fluorescence microscope, starting immediately after the injections, and on each subsequent day until the embryos are randomly selected for quantitative culture.</li>
	<li>Randomly select a few live infected embryos (5 to 6 in the present study) shortly after injection and transfer them individually to separate 2 mL microtubes using sterile tips. Remove the medium, wash the embryos gently with sterile PBS once and add 100 &#181;L of sterile PBS.</li>
	<li>Add 2&#8211;3 sterile zirconia beads (2 mm in diameter) to each vial and crush the embryos using the homogenizer (see Table of Materials) at 3,500 rpm for 30 s. Culture the homogenate quantitatively as described in step 1.3. 
	NOTE: Other homogenizers may require different settings.</li>
	<li>Randomly select multiple embryos on subsequent days after injection for quantitative culture according to step 5.3.</li>
</ol>
6. Fluorescence Microscopy of Infection Progression and Provoked Cell Infiltration in Zebrafish Embryos
<ol>
	<li>Anaesthetize the embryos as described in step 4.2. Pipette 500 &#181;L of a PBS-2% (wt) methyl cellulose solution into a Petri dish containing E3 medium with 0.02% (w/v) Tricaine. Place the embryos in the methyl cellulose solution and keep them straight and horizontal. 
	NOTE: Methyl cellulose solution is used as a &#8220;glue&#8221;, which due to its viscosity, can temporarily immobilize embryos in best orientation for imaging.</li>
	<li>Use a stereo fluorescence microscope equipped with bright field, mCherry, green fluorescent protein (GFP), and UV filters to image individual embryos under identical optimized settings (e.g., intensity, gain, and exposure time) at 160x magnification. Set the focal plane such that the tissue damage caused by the injection is in focus, using the bright field filter. Set the Z-stack depth at 10 &#181;m and step size at 5 &#181;m, which allows for recording of 3 consecutive images.</li>
	<li>Image individual embryos once daily from 5 h post-injection until 2 days&#160;post-injection. Exclude dead embryos (no heartbeat or embryo partly degraded) for further imaging and analysis. Maintain embryos individually in E3 medium in 48-well plates. Refresh the medium daily.</li>
</ol>
7. Quantitative Analysis of Fluorescence Intensity of Infection Progression and Provoked Cell Infiltration Using Object J Project File &#8220;Zebrafish-Immunotest&#8221;
<ol>
	<li>Download &#8220;Zebrafish-Immunotest&#8221; and the detailed manual from the link: &#60;https://sils.fnwi.uva.nl/bcb/objectj/examples/zebrafish/MD/zebrafish-immunotest.htmL&#62;, as described in a previous study33. In brief, open images for analysis with &#8220;Zebrafish-Immunotest&#8221;, operating under the freeware program Image J. In each loaded image, manually mark the injection site based on the observed tissue damage of embryos. 
	NOTE: The marked site is used by &#8220;Zebrafish-Immunotest&#8221; as the center point to detect the fluorescence peak within a distance of 50 &#181;m. The detected fluorescence peak is then used as the center of a standardized area (diameter of 100 &#181;m) for fluorescence measurement.</li>
	<li>Click &#8220;calculation&#8221; in the Object J menu to run the fluorescent measurement for multiple channels, if applicable, automatically for all images. Export the data and analyze by appropriate statistic test methods.</li>
</ol>

The authors have nothing to disclose.

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 in general are lower than for murine models, the present study assessed whether zebrafish embryos can be used as a novel vertebrate animal model for investigating BAI in vivo, complementary to the mammalian models.
In order to set up the zebrafish embryo BAI model, we developed a protocol of co-injection of bacteria and microspheres based on a method for intramuscular injection of microspheres into embryos described before33. In this way, most of the bacteria are present in the vicinity of the microspheres in embryos, mimicking the bacteria-biomaterial interaction that takes place during or shortly after the implantation of biomaterials in humans/mammals.
To be able to compare infection progression in the presence and in the absence of microspheres in zebrafish embryos, embryos should be challenged with similar initial numbers of bacteria with and without microspheres. However, according to our experience, co-injection of Bacteria-Microspheres suspension results in more bacteria being injected than injection of &#34;Bacteria-only&#34; suspension. To address this issue, the ratio between the concentration of bacteria in &#34;Bacteria-only&#34; suspensions (without microspheres) and in Bacteria-microspheres suspensions should be tailored. In the present study, the concentration of S. aureus in the &#34;Bacteria-only&#34; suspension needed to be approximately 1.5 times higher than the concentration in the Bacteria-Microspheres suspension. Whether this ratio also applies to other bacterial species and other biomaterials remains to be tested. Of note, to inject similar numbers of bacteria with and without microspheres, the opening of the needles used for injections of either suspension should be as close to identical as possible.
Several properties of a biomaterial such as shape and size play an important role in determining its injectability and co-injection with bacteria. In the present study, we used 10 &#181;m polystyrene microspheres (PS10) as model biomaterials. According to our experience, microspheres of a size of up to 15 &#181;m are injectable for 3 days old embryos following the protocol developed in the present study33. For microspheres of a relatively large size (e.g., &#8805; 10 &#181;m), we discourage the use of non-monodispersed or irregularly shaped microspheres/microparticles which tend to cause clogging of the needle. If biomaterials in other shapes than round and in different sizes are chosen to be used, their injectability needs to be tested. For (co-)injection of microspheres and bacteria, we used a 4% polyvinylpyrrolidone (PVP) solution to facilitate dispersion. This may be helpful for injection of biomaterials with and without bacteria into embryos in general.
As was the case for injections of microspheres only33,40, the numbers of microspheres injected together with bacteria into embryos varied per injection, mostly ranging from 1 to 4 microspheres per embryo. However, since such variations did not result in differences in levels of the provoked immune cell response in embryos33, they were also not expected to influence the potential effects of microspheres on infection progression. Nevertheless, novel approaches allowing injections of a single microsphere (alone or together with bacteria) would be desired.
We assessed whether microscopic scoring of presence of fluorescent bacteria can be used as a &#34;yes or no&#34; scoring system to analyze infection progression in live embryos. The criterion of a positive microscopic score was defined as the presence of visible fluorescent bacteria, regardless of the fluorescence intensity. Compared to quantitative culture of bacteria, our representative results suggested that microscopic scoring can distinguish embryos with 20 or more CFU of S. aureus bacteria from embryos with lower numbers of CFU or no infection. Thus, since only low numbers of bacteria are required for a positive score, this method is almost as sensitive as quantitative culture. Since the embryos do not need to be sacrificed and no high-end microscopes or sorting systems for fluorescence are required to perform analyses using this method, we consider microscopic scoring a simple but reliable method to monitor the infection progression of bacteria in live embryos. For quantitative analysis of the level of infection (rather than the &#34;yes or no&#34; scoring system as described above) and immune cell response in single live embryos, we applied the ObjectJ project file &#34;Zebrafish-Immunotest&#34; to quantify fluorescence intensity of the fluorescent bacteria and fluorescent macrophages recorded over time. We used a standardized area (with a diameter of 100 &#956;m) surrounding the injection site to measure fluorescence, since this area was shown to include a majority of the bacteria and the infiltrating macrophages. A detailed manual of &#34;Zebrafish-Immunotest&#34; was published previously33,40. Of note, &#34;Zebrafish-Immunotest&#34; is an open access plug-in for ImageJ, which can be tailored by the user. For instance, the diameter of the area of analysis and other parameters of &#34;Zebrafish-Immunotest&#34; can be freely changed according to the study setup (see examples in the link provided in step 7 in the Protocol).
In order to show the possible infection-enhancing effects of biomaterials in zebrafish embryos, the challenge dose of bacteria needs to be assessed. In the present study, we found that a challenge dose of 1,000 CFU of S. aureus per embryo or higher is required. Higher levels of S. aureus infection in embryos with PS10 than in those without PS10 were found on the first 2 days after injection, indicating that the presence of biomaterials transiently facilitates outgrowth of S. aureus in the embryos. Whether infection remains at high levels in presence of biomaterials at later time points should be studied using older embryos under ethical approval according to applicable regulations. Since the clearance of S. aureus in zebrafish embryos is mainly dependent on phagocytosis and killing by macrophages and neutrophils23,24, the outgrowth of bacteria might be explained by a reduced effectiveness of the phagocytes to kill the bacteria due to the presence of biomaterials. This is in line with the well-known infection-risk enhancement by biomaterials in patients2,4,15, also commonly observed in more complex animal models such as mouse and other animal models10,11,12,13. In addition to S. aureus infection, infection progression of S. epidermidis or other pathogens in the presence of biomaterials can be investigated following the protocol described in the present study. From the point of biomaterials, several material properties (e.g., chemical composition, hydrophobicity, roughness, and surface charges) may influence the cellular responses and/or bacteria-material interaction41,42. Our previous study has shown that injection of a biomaterial (in the presence of PVP) provoked a stronger macrophage infiltration compared to injection of only PVP in zebrafish embryos. Moreover, zebrafish embryos reacted differently to microspheres made of poly (-caprolactone) and polystyrene33. Therefore, injection of microspheres of different nature may also have differential effects on enhancement of infection, which can be relatively easily assessed by the method described here.
For further development, this zebrafish embryo BAI model may be amended for a high throughput system featuring automated robotic injection27,43, complex object parametric analysis and sorting (COPAS) systems, and high throughput RNA sequence analysis27,44. Such zebrafish embryo-based high throughput BAI models may be used as whole animal systems for in vivo screening and testing of (novel) anti-infective biomaterials as well as testing of the efficacy of antibiotic treatments or other anti-BAI strategies. Moreover, intracellular survival is one major strategy of bacteria to survive in the presence of biomaterials9,10,11,12,13. The usefulness of zebrafish embryos for studying intracellular infection has been shown in several studies36,46. Thus, our embryo model may be used to study the intracellular survival of staphylococci or other intracellular pathogens in the presence of biomaterials, and strategies to treat such intracellular infections. In addition, in situ hybridization techniques45 can be used in the model to study the influence of bacteria or biomaterials or their combination on the expression of particular genes at the injection site, which may be helpful to discover marker genes for BAI.
In summary, the present study shows the potential of zebrafish embryos with the methods developed here to study biomaterial-associated infection in real time in vivo. This zebrafish embryo model may therefore be used to investigate novel infection-resistant biomaterials and promising prevention and treatment strategies of BAI, and to close the gap between in vitro studies and larger animal models.

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 &amp;micro;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 2 mL 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&amp;reg;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 Figure 1, S. aureus alone or together with 10 &#181;m PS microspheres (PS10) were injected into the muscle tissue of zebrafish embryos in the present study. Intramuscular injection of S. aureus caused dose-dependent infection in embryos (Figure 2). The doses injected were close to the aimed challenge doses (Day 0 in Figure 2). Only minor variations within groups were observed. Embryos injected with high challenge doses showed variable infection progression at 1 day and 2 days post-injection (Figure 2, 6000 and 2000 CFU). The injected S. aureus bacteria either were eradicated or had proliferated and subsequently established high levels of infection within the embryos. This is similar to the reported progression of S. aureus infection after intravenous injection into zebrafish embryos26. Embryos injected with low challenge doses of S. aureus cleared the bacteria (Figure 2, 600 and 200 CFU). These results indicated that the challenge dose of S. aureus strongly influences their infection progression in zebrafish embryos.
Co-injection of bacteria and microspheres resulted in higher numbers of bacteria injected than injection of bacteria only (data not shown). By preparing the bacterial inoculum for the &#34;Bacteria-only&#34; injections at a higher concentration (approximately 1.5 times higher) than in the Bacteria-Microspheres suspension, we were able to overcome the difference in numbers of CFU injected. This way, it is possible to inject similar numbers of bacteria into embryos in the presence and in the absence of PS10 (day 0, Figure 3). The numbers of microspheres per injection varied. As an example, in one of our experiments, half of the embryos received 1 to 2 microspheres per injection (12 out of 25 embryos in the S. aureus + PS10 group), some received 3 to 4 (8 out of 25 embryos), and a few received 5 to 7 microspheres (5 out of 25 embryos). However, such variations did not influence the levels of the provoked cellular responses, as reported in a previous study33.
Next, we investigated whether the presence of microspheres has an impact on the infection progression in zebrafish embryos challenged with low doses of S. aureus. To this end, we injected approximately 600 CFU of S. aureus-mCherry with or without PS10. We used two methods to study the infection progression: (i) conventional quantitative culture of the infected embryos after crushing, and (ii) scoring of microscopic detection (&#34;microscopic scoring&#34;) of fluorescent bacteria (S. aureus-mCherry). We compared the results from these two methods. The score was defined as &#34;yes or no&#34; visibility of fluorescent bacteria in the embryos, regardless of the fluorescence intensity. Our results showed that all embryos with more than 20 CFU of S. aureus-mCherry were positive in microscopic scoring (red dots in Figure 3). For 600 CFU of S. aureus per embryo, the presence of PS10 seemed not to significantly influence the infection progression. At all the time points, both the frequency of infected embryos (indicated on top of Figure 3, low challenge dose) and the numbers of CFU after crushing were not different for embryos with or without PS10 (Figure 3, low challenge dose). This suggested that higher challenge doses of S. aureus are required for studying the potential effects of biomaterials on infection progression in zebrafish embryos. Hence, we injected approximately 1,000 CFU of S. aureus in the presence and absence of PS10 into embryos. The microscopic scoring did not show differences in frequency of the infected embryos with and without PS10 at any time point (Figure 3, high challenge dose). The quantitative culture, however, showed higher CFU numbers retrieved from embryos in the S. aureus + PS10 group than those retrieved from the S. aureus-only group at 2 days post-injection (Figure 3, high challenge dose).
To quantify the extent of infection in the presence and in the absence of biomaterials in zebrafish embryos by image analysis, we recorded a series of images showing the infection progression of S. aureus-mCherry (1,000 CFU per embryo) in the presence and absence of PS10 in live embryos over time (Figure 4A). We also recorded the provoked infiltration of Kaede green fluorescent protein-expressing macrophages in the embryos to quantify the immune cell response (Figure 4B). Kaede fluorescent protein may undergo color conversion from green to red under exposure to UV light, depending on the exposure time and light intensity39. However, such color conversion was not observed under the experimental condition used in the present study. The fluorescence intensity of the infecting bacteria as well as of the infiltrating macrophages within a standardized area around the injection site was measured by our ObjectJ project file &#34;Zebrafish-Immunotest&#34;, operating within Image J. &#34;Zebrafish-Immunotest&#34; defines a standardized area with a diameter of 100 &#181;m (yellow circles in Figure 4) around an indicated point of injection. In the embryos, this area included the majority of the fluorescent bacteria residing in the proximity of the injected microspheres, as well as the infiltrating macrophages. The presence of a biomaterial was shown to influence both initial susceptibility to infection and immune cell responses. At 5 h post-injection, macrophage infiltration in response to S. aureus only was significantly higher than in response to S. aureus + PS10 (Figure 5, macrophage infiltration, 5 hpi). At 1 day&#160;post-injection embryos with PS10 showed significantly higher levels of S. aureus infection than embryos without PS10 (Figure 5, infection progression, 1 dpi). Thus, these quantitative results demonstrated that the combination of fluorescence image recording and the ObjectJ project file &#34;Zebrafish-Immunotest&#34; can be used to quantify the infection progression and provoked immune cell response in the presence of a biomaterial in the zebrafish embryo model. The model allows for assessing small differences in immune cell responses and levels of bacterial infection in vivo, and it only requires a stereo fluorescence microscope (with image recording) and the open-access &#34;Zebrafish-Immunotest&#34; for evaluation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58523/58523fig1.jpg" /> 
Figure 1: Schematic procedure of co-injection of Bacteria-Microspheres suspension into the tail muscle of zebrafish embryos. This figure has been modified from Zhang et al.33 with permission. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58523/58523fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58523/58523fig2.jpg" /> 
Figure 2: Numbers of CFU of S. aureus retrieved from zebrafish embryos injected with inocula, ranging from 6,000 to 200 CFU per embryo and assessed at 0 to 2 days post-injection. The red lines represent the median numbers of CFU. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58523/58523fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58523/58523fig3.jpg" /> 
Figure 3: Numbers of CFU and microscopic scoring of mCherry protein-expressing S. aureus (S. aureus-mCherry) in zebrafish embryos, assessed at different time points. Low challenge dose: 600 CFU per embryo; High challenge dose: 1,000 CFU per embryo; PS10: 10 &#181;m PS microspheres; &#34;+&#34;, embryos injected with S. aureus-mCherry together with PS10; &#34;-&#34;, embryos injected with S. aureus-mCherry only, so without PS10. Embryos were microscopically scored for fluorescent bacteria and randomly selected for quantitative culture of bacteria after crushing of embryos at the day of injection (Day 0) and at Day 1 and Day 2 post-injection. The embryos microscopically scored positive and negative for fluorescent bacteria are shown in red and black dots, respectively. The numbers of microscopically positive-scoring embryos divided by the total numbers of embryos scored (frequency of infected embryos) at each time point are indicated at the top of the graph. Differences in frequencies of infected embryos and in numbers of CFU between the S. aureus + PS10 and S. aureus only groups at each time point were analyzed by the Fisher&#39; exact test and Mann-Whitney test, respectively. *p&#160;&#8804; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58523/58523fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58523/58523fig4.jpg" /> 
Figure 4: Representative images recording the infection progression of mCherry-expressing S. aureus (A, red) in the presence and absence of 10 &#181;m PS microspheres (PS10, blue) and the provoked infiltration of Kaede protein-expressing macrophages (B, green) in embryos, from 5 h post-injection (hpi) to 2 days post-injection (dpi). Non-treated embryos (NT) were used as controls. The yellow circles (100 &#181;m in diameter) indicate the standardized area at the injection site for fluorescence quantification using the ObjectJ project file &#34;Zebrafish-Immunotest&#39;. The quantified fluorescence of bacteria and of macrophages is depicted in Figure 5. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58523/58523fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58523/58523fig5.jpg" /> 
Figure 5: Fluorescence quantification of S. aureus infection progression and the provoked macrophage infiltration in the presence and absence of 10 &#181;m PS microspheres (PS10) in zebrafish embryos from 5 hours post-injection (hpi) to 2 days post-injection (dpi), using the ObjectJ project file &#34;Zebrafish-Immunotest&#34;. A standardized area at the injection site was used for fluorescence quantification (with a diameter of 100 &#181;m, indicated as the yellow circles in Figure 4). Non-treated embryos (NT) were used as controls. Differences between each two groups at each time point were analyzed by the Mann-Whitney test, *p &#8804; 0.05, **p &#60; 0.01, ***p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58523/58523fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Nanyang Technological University

Research

JoVE Journal

Bioengineering

6.9K Views.  Amsterdam UMC. Deze methode kan helpen bij het beantwoorden van belangrijke vragen over hoe biomaterialen de gevoeligheid voor infectie beïnvloeden, hoe ze de progressie van dergelijke infecties beïnvloeden en hoe ze het immuunsysteem gedrag rond dergelijke biomaterialen in vivo beïnvloeden. Het belangrijkste voordeel van deze techniek is dat het een nieuw diermodel biedt dat in vivo visualisatie en intravitale analyse van biomaterieel-geassocieerde infecties mogelijk maakt. Visuele demonstratie van deze...

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

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Developmental Biology

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Immunology and Infection

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

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 ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

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 ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

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

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