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

Summary

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.

Abstract

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 µm polystyrene microspheres (PS₁₀) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file "Zebrafish-Immunotest" 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.

Introduction

A variety of medical devices (referred to as "biomaterials") 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 BAI^2,3,4,5,6. 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 bacteria^{9,10,11,12,13} and biofilm formation on the implanted biomaterials^4,14. Consequently, BAI may lead to the failure and need for replacement of implanted biomaterials, causing increased morbidity and mortality and prolonged hospitalization with additional costs^2,15.

An increasing number of anti-BAI strategies are being developed^2,16,17. 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 mice^18,19,20,21. 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 addressed^18,19,21. 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 staphylococci^26,27. 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 mammals^28,29. This makes zebrafish embryos a highly economic, living model organism for intravital visualization and analysis of infection progression and associated host responses^28,29. 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 developed^28,29. 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 analysis^27,30.

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 strain^10,31. 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 "Zebrafish-Immunotest" project file which is operated within "ObjectJ" (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 is a promising, novel vertebrate animal model for studying biomaterial-associated infections in vivo.

Protocol

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 "Bacteria-only" 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.

1. Take 4 to 5 colonies of S. aureus RN4220 bacteria from tryptic soya agar culture plates supplemented with 10 µg/mL chloramphenicol and culture the bacteria to mid-logarithmic growth phase in 10 mL of tryptic soy broth supplemented with 10 µg/mL chloramphenicol at 37 °C under shaking.
   1. During culture, dilute 100 µL of the bacterial suspension in 900 µL 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 0.4–0.8.
      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.
2. 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.
3. Vortex this bacterial suspension and dilute 100 µL of the suspension in 900 µL of sterile PBS in a cuvette for the OD₆₂₀ measurement. Adjust the concentration of the bacterial suspension with PVP₄₀ solution. This is the "Bacteria-only" suspension.
4. Biomaterials can be freely chosen to mix with bacterial suspension. In the present study, commercial polystyrene (PS) microspheres (blue fluorescent, 10 µm) 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  “Bacteria-only” suspension (in PVP₄₀ solution).
5. 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 “Bacteria-only” 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 µm) or shapes of biomaterials (than microspheres) to be injected.
6. Check the concentration of the “Bacteria-only” and Bacteria-Microspheres suspension by quantitative culture of 10-fold serial dilutions as below: transfer 100 µL of the suspensions to a 96 well-plate and serially dilute by transferring 10 µL aliquots of the suspension into 90 µL of sterile PBS. Plate duplicate 10 µL aliquots of the undiluted and diluted suspensions on agarose plates, incubate the plates at 37 °C overnight, count the colonies and calculate the numbers of bacteria (colony-forming units. CFU).

2. Breeding, Harvesting, and Maintenance of Zebrafish Embryos

1. Follow the general procedures described earlier^35,36 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.
2. 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 °C. Remove dead and abnormal embryos, and refresh the E3 medium daily.

3. Preparation of Injection Needles

1. 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.
2. Break the needle tip with forceps at the position where the needle has an outer diameter of approximately 20 µm (for 10 µm 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 µm have been reported to cause a significant decrease in embryo survival³⁸.

4. Injection of "Bacteria-Only" or Bacteria-microspheres Suspension into Zebrafish Embryos

1. Heat agarose solution (1–1.5% (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.
2. 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 select embryos expressing green fluorescent proteins using a stereo fluorescence microscope.
3. Load the needle with approximately 10 µL of the "Bacteria-only" 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 2–3 nL: pressure: 300–350, 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.
   1. Use needles with the same opening for the injection of the "Bacteria-only" suspension and the Bacteria-Microspheres suspension. If the needle is broken or clogged, always change for a new needle for further injections.
4. Insert the needle into the muscle tissue of embryos under a light microscope (Figure 1), at an angle of 45–60° 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.
5. 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.

5. Crushing of Embryos, Microscopic Scoring, and Quantitative Culture of Bacteria

1. 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.
2. 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 µL of sterile PBS.
3. Add 2–3 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.
4. Randomly select multiple embryos on subsequent days after injection for quantitative culture according to step 5.3.

6. Fluorescence Microscopy of Infection Progression and Provoked Cell Infiltration in Zebrafish Embryos

1. Anaesthetize the embryos as described in step 4.2. Pipette 500 µL 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 “glue”, which due to its viscosity, can temporarily immobilize embryos in best orientation for imaging.
2. 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 µm and step size at 5 µm, which allows for recording of 3 consecutive images.
3. Image individual embryos once daily from 5 h post-injection until 2 days 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.

7. Quantitative Analysis of Fluorescence Intensity of Infection Progression and Provoked Cell Infiltration Using Object J Project File “Zebrafish-Immunotest”

1. Download “Zebrafish-Immunotest” and the detailed manual from the link: <https://sils.fnwi.uva.nl/bcb/objectj/examples/zebrafish/MD/zebrafish-immunotest.htmL>, as described in a previous study³³. In brief, open images for analysis with “Zebrafish-Immunotest”, 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 “Zebrafish-Immunotest” as the center point to detect the fluorescence peak within a distance of 50 µm. The detected fluorescence peak is then used as the center of a standardized area (diameter of 100 µm) for fluorescence measurement.
2. Click “calculation” 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.

Results

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 infection^{22,26,27,30,36}. Using the procedure depicted in <...

Discussion

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 ...

Materials

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