A. Quantification of E. faecalis uptake by zebrafish phagocytes. Embryos were infected with 1,600 CFUs of E. faecalis cells constitutively producing mCherry and fixed in 4% paraformaldehyde 1.5 h post infection. Phagocytes were immunolabelled using rabbit anti L-plastin antibodies and detected with goat anti-rabbit antibodies conjugated to Alexafluor 488. The infected and immunolabelled embryos were imaged using a scanning confocal microscope. The ratio of mCherry fluorescence signal area associated with phagocytosed and free bacteria was measured using the Fish Analysis Fiji plugin. The uptake of mutant OG1RF Δ11714 (ΔepaX) was significantly higher when compared to the wild-type (OG1RF; ***P = 0.0006) and complemented strain (OG1RF Δ11714 + pTetH-OG1RF_11714; **P = 0.0049). No difference in phagocytosis was observed between the wild-type and complemented strains (ns, P>0.05). Representative images showing E. faecalis uptake in zebrafish embryos are shown. Each picture corresponds to the quantification result indicated with a red dot in A, following infection with OG1RF (B), OG1RF Δ11714 (ΔepaX) (C) and the complemented strain (D). Phagocytes labeled with L-plastin appear in green, mCherry labelled bacteria in red. Scale bar is 25 μm.