Visualization of Proteus mirabilis within the matrix of urease-induced bladder stones during experimental urinary tract infection

Abstract

The virulence of a urease-negative mutant of uropathogenic Proteus mirabilis and its wild-type parent strain was assessed by using a CBA mouse model of catheterized urinary tract infection. Overall, catheterized mice were significantly more susceptible than uncatheterized mice to infection by wild-type P. mirabilis. At a high inoculum, the urease-negative mutant successfully colonized bladders of catheterized mice but did not cause urolithiasis and was still severely attenuated in its ability to ascend to kidneys. Using confocal laser scanning microscopy and scanning electron microscopy, we demonstrated the presence of P. mirabilis within the urease-induced stone matrix. Alizarin red S staining was used to detect calcium-containing deposits in bladder and kidney tissues of P. mirabilis-infected mice.

FIG. 1.

Confocal images of GFP-expressing P. mirabilis in a bladder stone. GFP-expressing P. mirabilis in a mouse bladder stone were revealed by confocal laser scanning microscopy. Two representative images are shown. Bar, 25 μm.

FIG. 2.

Scanning electron micrographs of P. mirabilis urease-induced bladder stone. (A) One-quarter of the bladder viewed at a low magnification (bar, 500 μm). The orientation of the bladder is indicated by an arrow pointing to the inferior end of the bladder (the end leading to the urethra). (B) Higher magnification (bar, 100 μm) of the area enclosed in a box in panel A. (C) Higher magnification (bar, 5 μm) of the area enclosed in a box in panel B. (D and E) Representative views of the bladder stone (bars, 2 μm).

FIG. 3.

Sections of P. mirabilis-infected mouse bladders stained with hematoxylin and eosin stain and alizarin red S stain. (A and B) Consecutive sections of a mouse bladder that developed a macroscopically visible stone after infection by P. mirabilis, stained with hematoxylin and eosin stain and alizarin red S stain, respectively. (C and D) Consecutive sections of a mouse bladder that developed mild urolithiasis due to P. mirabilis infection, stained with hematoxylin and eosin stain and alizarin red S stain, respectively. (E and F) Magnified views of areas in panel B, showing mineral deposits on the bladder epithelium stained with alizarin red S stain. Bars, 400 μm for panels A to D and 100 μm for panels E and F.

FIG. 4.

Sections of P. mirabilis-infected mouse kidney stained with hematoxylin and eosin stain (A) and alizarin red S stain (B). A severely necrotic area in the kidney, indicated by the heavy infiltration of host immune cells shown in panel A, was stained with alizarin red S stain, as shown in panel B. Bars, 100 μm.