Virulence of ST131 B2 group Escherichia coli strains in zebrafish embryos compared to other non-ST131 B2 group strains.

A. Survival probability of zebrafish embryos following infection with three ST131 strains and two non-ST131 strains. Thirty hour post fertilization (hpf) embryos were microinjected with an average inoculum size of 1.9×10³.CFU. The data represent the average of 5 independent experiments (n = 20 for each strain per experiment). NEC20 and NECS81153 were analysed twice. A Mantel-Cox test was performed (between strains p<0.001). NECS81153 and NEC20 did not differ significantly (p = 0.08) from each other in virulence. The non-ST131 group B2 strains NEC20 and NECS81153 induced a significantly higher rate of embryo mortality than the two ST131 strains MECB5 (p<0.001) and FV7563 (p = 0.002). MECB5 was significantly less virulent than S250 (p<0.001), but did not differ from FV7563 (p = 0.73). Although S250 and NECS81153 did not differ in virulence (p = 0.82), S250 was less virulent than NEC20 (p = 0.009). In this model, NEC20 was significantly (p<0.05) more virulent than all the other group B2 strains except for NECS81153(p = 0.08). B. Survival probability of zebrafish embryos following injection of ST131 strain MECB5 compared with group A strain NEC3, and non-ST131 group B2 control strains CFT073, 536 and ED1a. Thirty hour post fertilization (hpf) embryos were microinjected with an average inoculum size of 1.4×10³.CFU. The data represent the average of 2 independent experiments (n = 20 for each strain per experiment) in this context, but each strain was analysed at least 5 times. A Mantel-Cox test was performed. MECB5 was significantly less virulent than the strains CFT073, 536 and NEC3 (p<0.0001). ED1a was significantly less virulent than MECB5 (p = 0.047). Within the group of fast killing strains, strain 536 was less virulent than CFT073 (p = 0.011) and NEC3 (p = 0.020), whereas NEC3 and CFT073 were not significantly different (p = 0.079). C. Infection kinetics after microinjection with the following inocula: FV7563 (300 CFU), S250 (520 CFU), MECB5 (1760 CFU), NECS81153 (2200 CFU) and NEC20 (880 CFU). Bacterial counts were performed in 5 individual embryos per time point per strain (geometric mean ± SEM, n = 5), however, dead embryos were not included in the counts as explained in the text. Shown is a typical experiment. The experiment was repeated 5 times. D. Bright field and fluorescence overlay of a NEC20 infected embryo, 48 hpi, showing a close up of bacteria in a phagocytic cell. Inset shows an enlargement of the phagocytic cell (arrow). n = notochord, arrowhead marks an erythrocyte. E. Fluorescent image of an embryo, 24 hpi, infected with NEC20-DSRed showing bacteria have multiplied in the vasculature. A non-infected control embryo does not show any fluorescence with similar camera settings (not shown). Inset: corresponding bright field image. F. Embryo, 24 hpi, microinjected with MECB5-DSRed. Fluorescence and bright field overlay image of central nervous system infection.