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. 2019 Jun 17;9:8667. doi: 10.1038/s41598-019-45164-5

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Nitrogen limitation results in increased bacterial burden and granuloma abundance. Transgenic zebrafish embryos harbouring red fluorescent macrophages (mpeg1:mCherry) were infected with approximately 200 CFU of M. abscessus S or R (producing mWasabi fluorescent protein) cultured in either 7H9 or in MSM NL Gly 1% and were imaged at 2, 4 and 6 dpi to quantify bacterial burden and granuloma number. (A,B) Bacterial burden was quantified using ImageJ ‘Analyse Particles’ function to determine fluorescent pixel counts. Error bars represent standard deviation, with each data point representing a single embryo. Data shown represent a pool of three individual experiments with approximately 20 embryos per group. Statistical analysis was completed using a Student’s t-test. (C,D) Granuloma number was quantified manually following colocalization (yellow) of bacteria (green) and macrophage aggregates (red) using ImageJ. Error bars represent standard deviation. Data shown is the average of three individual experiments with approximately 20 embryos per group. Statistical analysis was done using a Student’s t-test. (E,F) Representative images of granuloma quantification following bacterial and macrophage colocalization in the cranial and trunk region at 6 dpi. White arrows highlight granuloma presence. Scale bars represent 200 µm. **p-value ≤ 0.01, ***p-value ≤ 0.001.