We are grateful to Leonie de Boer and Bas Zaat (Academic Medical Centre) for providing us with the S. epidermidis O-47 strain. We thank Rico Bongaarts, Francis Smet and Angela Comas (Union Biometrica) for help and advice with the COPAS XL and VAST BioImager analysis. We thank Davy de Witt, Ulrike Nehrdich, and Laura van Hulst for fish caretaking, and other colleagues from Leiden University for helpful discussions. This research forms part of the Project P5.03 IBIZA of the research program of the BioMedical Materials institute, co-funded by the Dutch Ministry of Economic Affairs, and of the Smart Mix Program (NWOA_6QY9BM) of The Netherlands Ministry of Economic Affairs and of The Netherlands Ministry of Education, Culture and Science. Additional support was obtained from the EU project ZF-Health (FP7-Health-2009-242048), and RMJ was supported by Marie Curie fellowship as Experienced Researcher in the EU Initial Training Network FishForPharma (PITN-GA-2011-289209). SJR received funding from the Innovative Medicines Initiative Joint Undertaking under grant agreement n&#176;115337, resources of which are composed of financial contribution from the European Union&#8217;s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies in kind contribution.The authors further acknowledge financial support from the Leiden University Fund (LUF) for robotics and from Cyttron, in the Besluit Subsidies Investeringen Kennisinfrastructuur program, which in turn is financially supported by the Netherlands Organization for Scientific Research for imaging facilities. The COPAS system acquisition was partially supported by the Division for Earth and Life Sciences (ALW) with financial aid from the Netherlands Organization for Scientific Research (NWO, 834.10.004).

1. Bacterial Strains and Growth Conditions
<ol>
	<li>Prepare S. epidermidis&#160;Inoculum
	<ol>
		<li>Take several individual colonies from S. epidermidis strain O-47, containing a pWVW189 derived mCherry expression vector (De Boer L. unpublished) from a Luria Bertani (LB) agar plate supplemented with 10 &#956;g/ml chloramphenicol and culture overnight at 37 &#176;C in 25 ml LB medium supplemented with 10 &#956;g/ml chloramphenicol to midlog stage.</li>
		<li>Centrifuge 1 ml of the culture at 12,000 x&#160;g for 1 min and subsequently wash them 3x with 1 ml sterile phosphate buffered saline (PBS) with 0.3% (v/v) Tween-80.</li>
		<li>Measure the optical density at 600 nm (OD600), and dilute the bacterial suspension to an OD600 of 0.3 in 2% (w/v) polyvinylpyrrolidone40 (pvp40) in PBS. Note: An OD600 of 0.3 corresponds to 1.0 x&#160;108 colony forming units/ml (cfu/ml).</li>
	</ol>
	</li>
	<li>Prepare M. marinum&#160;Inoculum
	<ol>
		<li>Take several individual colonies from M. marinum strain M or E11 containing the pSMT3-mCherry vector stably expressing mCherry10 from a Middlebrook 7H10 agar plate with 10% (v/v) Middlebrook OADC enrichment supplemented with 50 &#956;g/ml hygromycin and culture overnight at 28 &#176;C in 10 ml of Middlebrook 7H9 broth with 10% (v/v) Middlebrook ADC enrichment supplemented with 50 &#956;g/ml hygromycin.</li>
		<li>Centrifuge 1 ml of the culture at 12,000 x&#160;g for 1 min and subsequently wash it 3x with 1 ml sterile PBS with 0.3% (v/v) Tween-80.</li>
		<li>Measure the OD600, and dilute the bacterial suspension to an OD600 of 0.3 in 2% (w/v) pvp40 in PBS. Note: An OD600 of 1 corresponds to 1.0 x&#160;108 cfu/ml.</li>
	</ol>
	</li>
</ol>
2. Prepare Zebrafish Eggs
<ol>
	<li>Place a maximum of 70 male and 50 female wild type zebrafish separately into the large breeding vessel. Note: Place the female fish in the lower part of the large breeding vessel.</li>
	<li>Remove the separator the next day in the morning, to let the zebrafish start breeding.</li>
	<li>Collect the eggs at the bottom of the large breeding vessel through the egg collector in a 50 ml tube filled with egg water (60 &#181;g/ml instant ocean sea salt).</li>
</ol>
3. Injection Needles
<ol>
	<li>Obtain commercially available custom made glass capillaries needles with an inner diameter of 10 &#181;m.</li>
</ol>
4. Experimental Outline of Injection
<ol>
	<li>Boil 100 ml of 1% (w/v) agarose in egg water, and cool until approximately 40 &#186;C. Pour agarose into the automated microinjectors plate and place the 1,024-well stamp into the agarose. Note: The plate is ready to use when cooled down.</li>
	<li>On the automated microinjector operating software click on &#8216;Calibrate stage&#8217;, then click on &#8216;1024&#8217; well grid and place the agarose plate in the microinjector and calibrate the plate by clicking on the screen at the center&#160;position of the well.</li>
	<li>Go to &#8216;needle menu&#8217; and click on &#8216;calibrate needle holder&#8217;.</li>
	<li>Fill the injection needle using a microloader tip with either 10 &#181;l pvp40 containing 100 cfu/nl S. epidermidis or 30 cfu/nl M. marinum, or use pvp40 as mock injection.</li>
	<li>Place the needle in the automated micro-injector and calibrate the x, y position by lowering or moving the needle up and clicking on the screen at the position of the needle. Then calibrate the z position of the needle by clicking on the screen at the position of the tip of the needle.</li>
	<li>Distribute the eggs over the agarose grid using a plastic transfer pipet, and remove excess egg water. Place the agarose grid in the automated microinjector.</li>
	<li>Go to the &#8216;Injection menu&#8217; and adjust the &#8216;Injection pressure&#8217; setting to 200 hPa, &#8216;Injection time&#8217; 0.2 sec and &#8216;Compensation pressure&#8217; 15 hPa, which correlates with 1 nl, at the Femtojet settings menu.</li>
	<li>Click on &#8216;Inject all&#8217; to inject the entire plate.</li>
	<li>Collect the eggs after injection by washing them into a Petri dish (92 x&#160;16 mm), with a maximum of 70 embryos per Petri dish, and incubate at 28 &#176;C.</li>
</ol>
5. Flow-cytometer Analysis
<ol>
	<li>Prepare the large particle flow cytometer according to the manufacturer&#8217;s instructions and fill the sample cup and sheath fluid container with egg water.</li>
	<li>At the operating software, go to the &#8216;PMT&#8217; menu and use the following settings: 650 V for the &#8216;Red&#8217; channel and 0 V for the &#8216;Green&#8217; and &#8216;Yellow&#8217; channels. Then go to &#8216;Thresholds&#8217; menu and use the following settings: &#8216;Optical density&#8217; threshold signal: 975 mV (COPAS XL value: 50) and the &#8216;Time Of Flight&#8217; (TOF) minimum to 320 &#956;sec (COPAS XL value: 800) in order to reduce the influence of debris.</li>
	<li>For analysis without sorting the embryos go to step 5.4, for analysis and sorting the embryos into a Petri dish go to step 5.5 or for analysis and sorting the embryos into a 96-well plate go to step 5.6.</li>
	<li>Place the embryos in the sample cup and click on &#8216;start&#8217; to start the analysis. When all embryos are analyzed stop the analysis by clicking on &#8216;stop&#8217;. Save the data by clicking on &#8216;Store all&#8217;. Note: All data are stored as TXT, LMD, DAT, CSV and BSRT files. Follow the protocol at step 5.7.</li>
	<li>Place the embryos in the sample cup and define the maximum of 70 embryos per plate to be sorted by entering 70 in the &#8216;Sort&#8217; menu. Place an empty Petri dish under the sorter and click on &#8216;Manual Sort&#8217;. When the Petri dish is filled, save the data by clicking on &#8216;Store all&#8217;. Note: All data is stored as TXT, LMD, DAT, CSV and BSRT files. Follow the protocol at step 5.7.</li>
	<li>Place the embryos in the sample cup and define the maximum of 1 embryo per well to be sorted by entering 1 in the &#8216;Sort&#8217; menu. Place an empty 96-well plate into the left plate holder and click on &#8216;Fill plate&#8217;. When the 96-well plate is filled, save the data by clicking on &#8216;Store all&#8217;. Note: All data is stored as TXT, LMD, DAT, CSV and BSRT files. Follow the protocol at step 5.7.</li>
	<li>Get the TXT file to process the raw data, use the following data filter settings: &#8216;Status select&#8217;: 40, and if using the sort module &#8216;Status sort&#8217;: 6. Then use the numbers from the total fluorescence signal from the &#8216;Red&#8217; channel to calculate the average and the standard error of the mean. Plot these data sets into bar or scatter graphs.</li>
</ol>
6. Drug Treatment
<ol>
	<li>Analyze and sort at 3 days post injection (dpi), M. marinum infected embryos in two equal groups using the large particle flow cytometer (step 5.5). Treat one group with a compound of interest in its carrier solvent and other with carrier solvent alone (control). Apply similar treatments to mock injected control to test for antibiotic side effects.</li>
	<li>Repeat at 4 and 5 dpi the analysis (step 5.5) and refresh the egg water or egg water containing the compound.</li>
</ol>
7. High Resolution Imaging
<ol>
	<li>Anesthetize the embryos with 0.02% (w/v) buffered 3-aminobenzoic acid ethyl ester (Tricaine) in egg water 10 min before analysis.</li>
	<li>Prepare the Vertebrate Automated Screening Technology system and the Large Particle Sampler according to the manufacturer&#8217;s instructions.</li>
	<li>Select the reference images corresponding to the age of the embryos from the &#8216;Imaging &#8211; Object &#8211; Detection Setup&#8217; menu.</li>
	<li>Select the amount of pictures and orientation to be made by the Vertebrate Automated Screening Technology system from the &#8216;Imaging &#8211; Auto store images&#8217; menu.</li>
	<li>Place a 96-well plate filled with embryos (from step 5.6) into the left plate holder of the Large Particle Sampler, and click on &#8216;Run plate&#8217;.</li>
	<li>When an embryo is detected and correctly positioned; image the head and the tail separately with the CLSM using a 10X plain dry objective and stitch the images afterwards using image processing software.</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial-associated+infection:+locating+the+finish+line+in+the+race+for+the+surface.">Biomaterial-associated infection: locating the finish line in the race for the surface.</a> Sci Transl Med. 4, (2012).</li><li>Benard, E. 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J.S is owner of Life Science Methods BV that produces instruments used in this article.

The high throughput methodology described in this paper provides a fast and cost effective pipeline to screen high number of fish embryos and larvae with different types of infections. Using the large breeding vessel instead of traditional single or family breeding tanks facilitated control of the spawning process and generation of larger number of synchronous eggs. With an improved version of the automated micro-injection system7, it is possible to inject up to 2,500 eggs almost all in the same cell stage within 1 hr. With these updates and improved software it is feasible to inject more eggs than previously was possible which can be used to perform large drug screens with bacterial proliferation as a read out. However this method is still limited to yolk injection, other injection routes for example described by Benard et al. (2012)6, will hopefully be incorporated in the automated micro-injection system in the near future.
Although these methods are benchmarked for screening zebrafish, it would be useful for applications with other fish species as well. For instance, the common carp has been indicated to have advantages for drug screens. Like zebrafish, eggs and early stage embryos from common carp are transparent but with the main advantage of its large spawn size of hundred thousands of eggs and the availability of inbred lines that offer a more constant genetic background16.
The analysis of large amounts of infected embryos is done with the high throughput large particle flow cytometer. This device can sort analyzed embryos into multi well plates or a Petri dish making it especially suitable for testing large numbers of compounds. If a higher imaging resolution is needed, than the setup is adapted in a way that the large particle flow cytometer technology can be used for pre-screening and subsequently analyze the samples at a medium throughput at a higher resolution. This can be done using the Vertebrate Automated Screening Technology17,18. This device can automatically collect live or fixed embryos between 2 and 7 days post fertilization from a multi well plate or bulk container, image 360&#176; through a capillary using CLSM or stereo microscopy and dispose again in 2 bulk containers allowing manual sorting of the embryos based on the microscopic images. Future improvements will allow the sorting of the embryo after imaging into the multi well plate, therefore making it possible to screen automatically large number of individual embryos over time with CLSM. Assuming that in future applications the Vertebrate Automated Screening Technology system can also be connected to the large particle flow cytometer technology without the need of prior dispensing larvae into multi well plates, will lead to a more advanced sorting.
This paper describes the establishment and optimization of a high throughput setup to study S. epidermidis and M. marinum infection as a model for drug discovery. It demonstrates that the outcome of these bacteria injected into the yolk depends on the developmental stage of the eggs at the time of injection. Injecting M. marinum E11 at 16-128 cell stage or the M strain at 16-64 cell stage leads to the same infection pattern as caudal vein injection2,6. However this setup is not limited to the proliferation of bacterial pathogens only. It was shown before that it is possible to robotically inject solutions containing DNA, RNA or morpholinos for transgenesis, over-expression and gene knock-down studies, respectively13. Furthermore, it was shown that this setup is also useful for the study of cancer cell proliferation and migration. Therefore this pipeline presents a versatile method for high throughput screens of a variety of signal mechanisms in the context of innate immunity, applied to infectious disease and the development of cancer. These screens can be combined with others for medicine discovery but also with analysis of possible toxic effects of identified applicable drugs.

To date the zebrafish (Danio rerio) has been successfully established as an efficient model to study a variety of infectious diseases1. The zebrafish embryo offers unique in vivo imaging possibilities due to their transparency and the large number of existing transgenic reporter lines expressing fluorescent proteins. This powerful combination makes it possible to track different immune cell types in time while interacting with pathogens such as Mycobacterium marinum, the closest relative of M. tuberculosis2, or Staphylococcus epidermidis, the main causative of biomaterial associated infection3-5. Different routes of infection can be used in zebrafish embryos depending on the purposes of the study6.
One of these infection routes is yolk injection of the bacteria. The main advantage of this method compared to the others is that yolk infection can be performed automatically via robotic injection, significantly reducing the injection time and allowing high reproducibility of the infection7,8.
Previous work, using the zebrafish as a high throughput in vivo model system for the study of S. epidermidis and M. marinum infection showed to be successful7,8. This system is able to screen for disease progression via robotic yolk injection of early embryos and using fluorescence readout as a measure for the bacterial load. In agreement with this notion, this setup has been optimized and established a highly efficient high throughput pipeline with the potential to generate large numbers of homogenously infected embryos and track the progression of the infection during the time after treatment with a number of compounds. With the established setup it is possible to generate up to 8,000 synchronous embryos to screen for disease progression, processing in this way up to 2,500 embryos per hour. Embryos are sorted based on their bacterial load using an automated system, ensuring homogenous groups of infected larvae. Furthermore, to validate the setup, effects of reference known to prevent tuberculosis progression in mammals have been tested on embryos infected with M. marinum E11 strain or the more virulent M strain9.
This study describes in detail the high throughput pipeline that has been established to be able to generate large numbers of infected embryos and the subsequent analysis of the bacterial progression during development and after compound treatment.

<table><tbody><tr><td>Middlebrook 7H10 agar</td><td>BD Bioscience, Franklin Lakes, New Jersey, USA</td><td>262710</td><td></td></tr><tr><td>Middlebrook 7H9 broth</td><td>BD Bioscience, Franklin Lakes, New Jersey, USA</td><td>271310</td><td></td></tr><tr><td>BBL Middlebrook oleic acid albumin dextrose catalase (OADC) enrichment</td><td>BD Bioscience, Franklin Lakes, New Jersey, USA</td><td>211886</td><td></td></tr><tr><td>BBL Middlebrook albumin dextrose catalase (ADC) enrichment</td><td>BD Bioscience, Franklin Lakes, New Jersey, USA</td><td>211887</td><td></td></tr><tr><td>LB agar</td><td>Sigma-Aldrich, Saint Louis, Missouri, USA</td><td>L5542</td><td>Multiple suppliers</td></tr><tr><td>LB broth</td><td>Sigma-Aldrich, Saint Louis, Missouri, USA</td><td>L3022</td><td>Multiple suppliers</td></tr><tr><td>Chloramphenicol</td><td>Bio-connect, Huissen, the Netherlands</td><td>16785.03</td><td>Multiple suppliers</td></tr><tr><td>Hygromycin</td><td>Bio-connect, Huissen, the Netherlands</td><td>25966.01</td><td>Multiple suppliers</td></tr><tr><td>Tween-80</td><td>Sigma-Aldrich, Saint Louis, Missouri, USA</td><td>P1754</td><td>Multiple suppliers</td></tr><tr><td>Polyvinylpyrrolidone40</td><td>Calbiochem, San Diego, California, USA</td><td>529504</td><td>Multiple suppliers</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td><td>Sigma-Aldrich, Saint Louis, Missouri, USA</td><td>A5040</td><td></td></tr><tr><td>Agarose</td><td>Sphaero-Q, Gorinchem, the Netherlands</td><td>S103</td><td>Multiple suppliers</td></tr><tr><td>Instant ocean sea salt</td><td>Sera Marin, Heinsberg, Germany</td><td>5460</td><td></td></tr><tr><td>iSPAWN</td><td>Techniplast, Buguggiate, Italy</td><td>iSPAWN</td><td></td></tr><tr><td>Automated microinjection system</td><td>Life Science Methods BV, Leiden, the Netherlands</td><td>Automated microinjection system</td><td></td></tr><tr><td>Complex Object Particle Analyzer and Sorter XL (COPAS XL)</td><td>Union BioMetrica Inc., Holliston, Massachusetts, USA</td><td>COPAS XL</td><td></td></tr><tr><td>Vertebrate Automated Screening Technology BioImager (VAST BioImager)</td><td>Union BioMetrica Inc., Holliston, Massachusetts, USA</td><td>VAST BioImager</td><td></td></tr><tr><td>LP Sampler</td><td>Union BioMetrica Inc., Holliston, Massachusetts, USA</td><td>LP Sampler</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica Microsystems, Wetzlar, Germany</td><td>TCS SL</td><td></td></tr><tr><td>Injection needle 10 &amp;micro;m inner diameter</td><td>Qvotek, Mississauga, Canada</td><td></td><td></td></tr></tbody></table>

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.

The present results show that the high throughput pipeline to study S. epidermidis and M. marinum infection has been successfully established and that may be extended to other infection models. Firstly, the use of the large breeding vessel (Figure 1A), based on the published system by Adatto et al. (2011)11, enables to generate large numbers of synchronous eggs in single events affording a high control of the spawning process. Next to be able to inject large number of embryos in a short period of time, an improved version of the previously developed automated micro-injection system7 was used (Figure 1A). To asses which is the best developmental stage for yolk infection, injections with S. epidermidis and M. marinum were performed at all the different stages between 1 and 512 cell stage, according to the description made by Kimmel et al. (1995)12.
Injections with 100 cfu S. epidermidis between the 16 and 128 cell stage provided the best infection pattern (Figure 2). The bacteria proliferated inside the yolk for 3 days and spread into the body from 3 dpi onwards. Performing injections before the 16 cell stage led to high mortality from 4 dpi, and injection after the 256 cell stage showed mainly bacterial growth inside the yolk with hardly any bacteria spreading inside the body of the embryo. Quantification of bacterial burden was performed by fluorescence intensity analysis using the large particle flow cytometer as described by Veneman et al. (2013)8 (Figure 3).
Observations showed that the optimal developmental stage for injection of 30 cfu M. marinum injection is between 16 to 128 cell stage for the E11 strain (Figure 4A) and between 16-64 cell stage with the more virulent M strain (Figure 4B). Embryos injected at these stages showed bacterial growth inside the yolk and spreading of the bacteria through the embryo (Figure 7). Infection with both strains at earlier stages presented nonspecific generalized bacterial growth leading the embryos to die after 4 dpi. On the other hand, in embryos injected at later stages bacterial burden was restricted to the yolk.
Next, presorting with large particle flow cytometer (Figure 1B) generated large homogenous groups of infected fish excluding non- or highly infected embryos (Figures 5A and 6A). After presorting, M. marinum infected embryos were treated with Rifampicin, a first line antituberculosis drug. Previous studies demonstrated that treatment with Rifampicin at a dose of 200 &#956;M efficiently reduces M. marinum infection in zebrafish7,13. Taking advantage of the large number of homogenously infected embryos generated with the high throughput setup, treatment with different doses was performed. Embryos infected with M. marinum M strain and treated for 48 hr&#160;with 12, 24, and 200 &#956;M Rifampicin showed to reduce efficiently mycobacterial infection in a dose dependent manner (Figure 5B). In view of the efficient reduction of the infection using Rifampicin at a dose of 200 &#956;M this concentration was used for the future experiments. In line with the previous result, studying bacterial burden progression using M. marinum E11 strain a significant reduction 24 hr&#160;and onwards after treatment with 200 &#956;M Rifampicin was observed (Figure 6B). &#160;
Furthermore, if high magnification imaging is required of these embryos, they can be displayed automatically in 96-well plates (Figure 1C), from where the samples can be analyzed using the Vertebrate Automated Screening Technology system with the Large Particle Sampler mounted onto a CLSM.
The Vertebrate Automated Screening Technology system with the Large Particle Sampler is a system that can either be mounted onto a CLSM or stereo microscope. This device allows the loading of live or fixed embryos from a 96-well plate or bulk container automatically through a glass capillary, and orientates it in front of the camera at the desired angle (e.g., dorsal or lateral). Images of the embryo in all orientations can be made with the on board camera or with an external CLSM (Figure 7). Embryos will subsequently be transferred in the collection or waste container.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig1highres.jpg" src="/files/ftp_upload/51649/51649fig1.jpg" /> 
Figure 1. Mainstream experimental outline. A) Adult fish are put together to mate, eggs are collected, aligned into an agarose plate and injected. B) The injected eggs are incubated at 28 &#176;C and will be pre-sorted for possible drug treatment. C) Subsequent analysis by large particle flow cytometer and/or Large Particle Sampler/Vertebrate Automated Screening Technology with CLSM. <a href="https://www.jove.com/files/ftp_upload/51649/51649fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig2highres.jpg" src="/files/ftp_upload/51649/51649fig2.jpg" /> 
Figure 2. Establishment of best cell stage for S. epidermidis yolk injection. Zebrafish embryos were injected in the yolk at different developmental stages from 1 to 512 cell stage with 100 cfu of S. epidermidis. Embryos injected between 1 and 8 cell stage showed bacterial growth in the yolk and high mortality from 4 dpi. Embryos injected between 16 and 128 cell stage showed bacterial growth in the yolk and inside the body starting at 3 dpi. Embryos injected between 256 and 512 cell stage showed many bacterial growth inside the yolk. <a href="https://www.jove.com/files/ftp_upload/51649/51649fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig3highres.jpg" src="/files/ftp_upload/51649/51649fig3.jpg" /> 
Figure 3. Quantification of bacterial burden using large particle flow cytometer. 100 cfu of S. epidermidis were injected into the yolk of zebrafish embryos. A) Up to 5 dpi, each day, groups of 10 embryos were homogenized and plated directly, showing the average exponential growth based on two biological replicas (error bars = SEM). B) Large particle flow cytometer analysis shows the average fluorescence signal from non-injected and S. epidermidis injected embryos. 30-160 embryos per condition were analyzed (error bars = SEM), different letters indicate statistical significant differences by one way ANOVA followed by Tukey&#39;s post-hoc test (P &#60; 0.001), ns: not significant differences. C) Correlation between cfu and average fluorescence signal of groups of 10 S. epidermidis infected embryos (error bars = SEM). This figure has been modified from Veneman et al. (2013)8. <a href="https://www.jove.com/files/ftp_upload/51649/51649fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig4highres.jpg" src="/files/ftp_upload/51649/51649fig4.jpg" /> 
Figure 4. Establishment of the best cell stage for M. marinum yolk injection. Zebrafish embryos were injected at all the different developmental stages from 1 to 512 cell stage with 30 cfu of M. marinum E11 and M strain. A, B) Embryos injected from 1-8 cell stage showed similar spreading and mortality with both strains. A) Embryos injected between 16-128 cell stage with E11 strain showed formation of granulomas and systemic infection while those injected from 256 to 512 cell stage kept bacterial burden into the yolk. B) Embryos injected between 16-64 cell stage with M strain showed formation of granuloma like structures and systemic infection while those injected from 128 to 512 cell stage kept bacterial burden into the yolk. <a href="https://www.jove.com/files/ftp_upload/51649/51649fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig5highres.jpg" src="/files/ftp_upload/51649/51649fig5.jpg" /> 
Figure 5. Treatment of M. marinum acute infection with a first-line anti-tuberculosis drug. Embryos injected between 16-64 cell stage with 30 cfu of M. marinum M strain were run through the large particle flow cytometer at 3 dpi to be sorted in two groups after discarding the non- and/or highly infected embryos. A) Fluorescence of individual embryos in both groups. B) Embryos treated with Rifampicin (RIF) for 48 hr at doses of 12, 24, and 200 &#956;M were analyzed at 4 dpi; their bacterial load is significantly reduced. C) Representative COPAS profiles of embryos treated with DMSO and Rifampicin at doses of 12, 24, and 200 &#956;M for 24 hr. Bacterial load and distribution is indicated by the red peaks. Blue line represents the profile of the element sorted (4 dpf zebrafish embryo) by the COPAS. 60-90 embryos per condition were analyzed. Each data point represents an individual embryo. Values are indicated as mean &#177; SEM. ns: not significant differences. Analysis of statistical significance of differences was performed by one way ANOVA followed by Tukey&#39;s posthoc test. <a href="https://www.jove.com/files/ftp_upload/51649/51649fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig6highres.jpg" src="/files/ftp_upload/51649/51649fig6.jpg" /> 
Figure 6. Treatment of M. marinum chronic infection with a first line antituberculosis drug. Embryos injected between 16-64 cell stage with 30 cfu of M. marinum E11 strain were run through the large particle flow cytometer at 3 dpi to be sorted in two groups after discarding the non- and/or highly infected embryos. A) Fluorescence of individual embryos in both groups. B) Embryos treated with Rifampicin (RIF) at 200 &#956;M during 4 days were analyzed showing a significant reduction of the bacterial load after 1 day of treatment. 90 embryos per condition were analyzed. Values are indicated as mean &#177; SEM. Different letters indicate significant differences between time points of the same treatment. * indicates significant differences with control group. ns: not significant differences. Analysis of statistical significance of differences was performed by one way ANOVA followed by Tukey&#39;s posthoc test. (P &#60; 0.05). Figure B) has been modified from Spaink et al. (2013)13. <a href="https://www.jove.com/files/ftp_upload/51649/51649fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig7highres.jpg" src="/files/ftp_upload/51649/51649fig7.jpg" /> 
Figure 7. Result of M. marinum E11 yolk injection imaged using Vertebrate Automated Screening Technology and CLSM. Confocal Z stack (stitched 3 images) of a 5 dpi fli1-egfp14 embryo. A) Live embryo showing proliferation of M. marinum E11 bacteria (red) throughout the body. B) Fixed 5 dpi fli-egfp embryo showing M. marinum E11 bacteria (red) throughout the body co-localizing with leucocytes (light blue) detected by L-plastin immunostaining15.

Watch this Scientific Journal Video about Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discover

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.

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 ...

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Research

JoVE Journal

Immunology and Infection

13.2K Views.  Leiden University. 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.

Video: Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

Bioengineering

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

High-Throughput Screening Assay for Mycobacterium abscessus Using Dual Reporters

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

Author Spotlight: Scalable Drug Screening Protocol for Efficient Discovery of M. abscessus Treatments

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

Optimized In Vivo Models for Intracellular S. aureus Infection Studies

Development and Assessment of Intracellular Infection Models for Staphylococcus aureus

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular persistence contributes to chronic and recurrent infections, complicating treatment and prolonging the disease. Consequently, there is a critical need for an intracellular infection model to better understand, prevent, and treat infections caused by S. aureus. This study indicated that antibiotics effectively eliminated extracellular bacteria but could not eradicate those that had entered the cells. Thus, a stable intracellular infection in vitro was established by RAW264.7 infected with S. aureus and co-culturing them with antibiotics. Subsequently, an intracellular infection model in mice was established by injecting peritoneal macrophages containing the intracellular infection. Vancomycin effectively cleared bacterial loads in mice challenged with planktonic S. aureus; however, it was ineffective against mice infected with equal or lower levels of intracellular bacteria within the peritoneal macrophages. This indicates that the intracellular infection model of S. aureus was successfully established, offering potential insights for the prevention and treatment of intracellular infections.

Staphylococcus aureus (S. aureus) has the capability to disseminate throughout the body, causing persistent and recurrent infections. To better understand these processes, this study establishes an intracellular infection model for S. aureus. This model will provide a crucial foundation for investigating the mechanisms behind intracellular infections.

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular ...

Visualizing Antibiotic Persistence in Bacterial Pathogens

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment that kills the other genetically identical cells. Bacterial persisters can resume replication once antibiotic treatment ends and are commonly thought to&#160;underlie clinical treatment failure.&#160;Recent work harnessing the power of time-lapse fluorescence microscopy, in which bacteria are labeled with fluorescent transcriptional reporters, translational reporters, and/or dyes for a variety of cellular features, has advanced our understanding of Escherichia coli persisters beyond what could be learned from population-level antibiotic survival assays. Such single-cell approaches, rather than bulk population assays, are essential for delineating the mechanisms of persister formation, damage response, and survival. However, methods for studying persisters in other important pathogenic species at this level of detail remain limited.
This study provides an adaptable approach for time-lapse imaging of Pseudomonas aeruginosa (a gram-negative rod) and Staphylococcus aureus (a gram-positive coccus)&#160;during antibiotic treatment and recovery. We discuss molecular genetic approaches to introduce fluorescent reporters into these bacteria. Using these reporters, as well as dyes, we can track the phenotypic changes, morphological features, and fates of individual cells in response to antibiotic treatment. Additionally, we are able to observe the phenotypes of individual persisters as they resuscitate following treatment. In all, this work serves as a resource for those interested in tracking the survival and gene expression of individual antibiotic-treated cells, including persisters, both during and after treatment, in clinically important pathogens.

In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize&#160;phenotypic variants, such as persisters, by time-lapse epifluorescence&#160;microscopy.

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment ...

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

Summary

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