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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 induces hypervirulence in M. abscessus. (A) Simplified diagram of the experimental workflow used to induce lipid-loaded M. abscessus by nitrogen deprivation prior to infection of zebrafish embryos. Zebrafish embryos were infected via caudal vein injection at 30 hpf with approximately 200 CFU of M. abscessus S or R cultured for 48 h in either 7H9 or MSM NL Gly 1%. (B) Nitrogen limitation results in significantly earlier mortality in M. abscessus R-infected zebrafish. Zebrafish embryo survival was monitored daily over a 12-day period following infection. Each group consisted of approximately 20 embryos, with each curve reproduced in triplicate. Statistical analysis was completed using the Mantel-Cox log-rank test. (C) Representative images displaying significantly increased pathology phenotypes at 6 dpi in zebrafish embryos infected with the M. abscessus R morphotype following nitrogen limitation. Transgenic reporter line zebrafish embryos harbouring fluorescent macrophages (mpeg:mCherry) (red overlay) were infected with M. abscessus harbouring pTEC15::mWasabi (green overlay) and the merge was observed in yellow. The open arrow (top) displays an intact abscess, while the closed arrow (bottom) displays a ruptured abscess. Scale bars represent 500 µm. **p-value ≤ 0.01.