Establishment and Optimization of a High Throughput Setup to Study <em>Staphylococcus epidermidis</em> and <em>Mycobacterium marinum</em> Infection as a Model for Drug Discovery

Abstract

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.

Tuberculosis causes highly conserved metabolic changes in human patients, mycobacteria-infected mice and zebrafish larvae

“…Zebrafish embryos at 2 to 4 hours post fertilization (hpf) were used for M.marinum injection. For mass spectrometry analysis we used M.marinum strain E11 labelled with a mCherry-expressing pSMT3 vector 26 . For the NMR experiments we used M.marinum strain M labelled with mWasabi plasmid pTEC15 vector 22 .…”

“…For the NMR experiments we used M.marinum strain M labelled with mWasabi plasmid pTEC15 vector 22 . M.marinum preparation and injection were followed by the protocol of a previous study 26 . Infection levels were checked with fluorescence microscopy (Fig.…”

“…Zebrafish has been shown to be an excellent model for the study of TB [21][22][23][24][25] . Because zebrafish have large amounts of offspring at one time and the availability of high throughput automated infection methods 26 , many zebrafish larvae can be tested for TB in a short time. The test system is independent of food intake and adaptive immune responses that only start later during development.…”

Outside‐In Systems Pharmacology Combines Innovative Computational Methods With High‐Throughput Whole Vertebrate Studies

“…9 Integrating such devices with automated fluorescence microscopy could lead to a closed experimental setup delivering systems pharmacology data in an automated high-throughput fashion. 10 The availability of these methodologies welcomes experimentalists and modelers to combine forces aimed to tailor experimental data to a systems pharmacology model, and vice versa, continuing the learn-and-confirm iterative approach. Indeed, as the first outside-in data analysis without prior knowledge on the system or target pathways will result in information on important features of the system and identify promising candidates, more detailed experiments, and subsequent analyses can be designed and executed to elucidate underlying physiological mechanisms.…”

Three-dimensional reconstruction and measurements of zebrafish larvae from high-throughput axial-view in vivo imaging

“…The space and time features of these images could, de facto, be used efficiently. In order to scale experiments to true high-throughput, other solutions need be probed [1]. Moreover, spatial acuity is required to enable the 3D image analysis.…”