Published: January 7th, 2019
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.
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.
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 BAI2,3,4,5,6. Implanted biom....
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.......
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 <.......
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 .......
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).....
|Tryptic soya agar
|Media preparation unit at AMC
|Tryptic soya broth
|10 µm diameter polystyrene microspheres (blue fluorescent)
|Glass microcapilary (1 mm O.D. x 0.78 mm I.D.)
|Micropipette puller instrument
|Sutter Instrument Inc
|Light microscope LM 20
|3-aminobenzoic acid (Tricaine)
|Stereo fluorescent microscope LM80
|Microloader pipette tips
|Micromanipulator M3301 with M10 stand
|World Precision Instruments
|FemtoJet express micro-injector
|Microtrube 2ml pp
|MSA-2 plates (Mannitol Salt Agar-2)
|Chapmon 2 medium
|Methyl cellulose 4000cp
|Gyrotory shaker (for bacterial growth)
|New Brunswick Scientific
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