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. 2017 Oct 18;85(11):e00475-17. doi: 10.1128/IAI.00475-17

FIG 1.

FIG 1

C. albicans and P. aeruginosa demonstrate synergistic virulence in mucosal infection of the swimbladder. (A and B) Wild-type zebrafish larvae at 4 days postfertilization were separated into 4 groups and microinjected into the swimbladder with 5 nl of PVP (control), C. albicans (C. a.) at 2.5 × 107 CFU/ml, P. aeruginosa (P. a.) at 2.5 × 108 CFU/ml, or C. albicans and P. aeruginosa (C. a. + P. a.) at 2.5 × 107 CFU/ml and 2.5 × 108 CFU/ml, respectively. Fish were screened immediately postinjection to select for consistent inocula, and mortality was recorded every 24 h out to 96 h postinjection. Confocal images were acquired at ×10 and ×20 magnification, with scale bars at 100 μm and 200 μm. Data are representative of 4 pooled, independent experiments. Pooled numbers of individual fish are the following: n = 98, 58, 49, and 58 for PVP, C. albicans, P. aeruginosa, and C. albicans plus P. aeruginosa, respectively. A Kaplan-Meier survival analysis and log-rank (Mantel-Cox) test with Bonferroni correction demonstrated a significant reduction in survival. (C) Larvae were injected and monitored as described above with the following groups: PVP control, C. albicans (2.5 × 107 CFU/ml) plus UV-inactivated (UV-x) P. aeruginosa (2.5 × 108 CFU/ml), P. aeruginosa (2.5 × 108 CFU/ml) plus UV-x C. albicans (2.5 × 107 CFU/ml), or C. albicans plus P. aeruginosa at 2.5 × 107 CFU/ml and 2.5 × 108 CFU/ml, respectively. Data are representative of six pooled, independent experiments. Pooled numbers of individual fish are the following: n = 142, 86, 92, 33, and 96 for PVP, C. albicans, C. albicans plus UV-x P. aeruginosa, UV-x C. albicans plus P. aeruginosa, and C. albicans plus P. aeruginosa, respectively. A log-rank (Mantel-Cox) test with Bonferroni correction determined significant differences as indicated. Statistical significance was assigned based on GraphPad Prism conventions (not significant [n.s.], P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; adjusted in panels A and C with Bonferroni correction). The swimbladder is outlined in a dotted magenta line for clarity.