FIG 6.

Fluorescence micrographs showing the formation of neutrophilic extracellular traps (NETs) in vivo in loop fluid from rabbits infected with E22. (A) Loop fluid from rabbit ileum infected in vivo for 20 h with EPEC strain E22 was subjected to low-speed centrifugation (21 × g for 5 min) onto glass microscope slides in a cytological “cytospin” centrifuge using Shandon centrifuge funnels. After fixation in alcohol-acetone, the glass slides were dried on a warmer plate and then stained for DNA with 10 μg/ml propidium iodide. An example of a DNA NET is indicated by the black arrow. E22 bacteria grow as short coccobacilli when in contact with host tissues and stain red or pink, often adhering to the DNA NETs. Host cells, mostly heterophils, the rabbit equivalent of neutrophils, also stain with propidium iodide (red arrow). The size bar is at the bottom right. The photograph was taken at a ×600 magnification under oil immersion. (B) Loop fluid from rabbit ileum was mixed with a fluorescently labeled, green fluorescent protein (GFP)-expressing laboratory strain of E. coli, DH5α-(pGFP), and allowed to incubate for 20 min at 37°C. The mixture was again applied to the funnels of a cytological centrifuge and spun onto glass slides as described above for panel A. After fixation and drying, the slides were again stained with propidium iodide. Panel B shows that the laboratory strain DH5α-(pGFP) becomes enmeshed in the DNA NETs along with the pathogenic strain E22. E22 bacteria, stained red by propidium iodide, are short, about 2 μm in length, and are indicated by the black arrow. The DH5α-(pGFP) bacteria fluoresce bright green and are about 4 μm in length. Diffuse pink background staining represents extracellular DNA. The main photograph in panel B is again at a ×600 magnification under oil immersion. The inset photographs, taken at a ×1,000 magnification, show red E22 bacteria (red arrows) in close contact with green-fluorescing DH5α bacteria (green arrows), and the size bar in the top left inset, 8 μm, applies to the top right inset photograph as well. The proximity of the two different bacterial strains, trapped together in DNA NETs, could facilitate the horizontal transfer of antibiotic resistance genes via conjugation or transformation.