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 biom....

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.......

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