Visualization of Proteus mirabilis morphotypes in the urinary tract: the elongated swarmer cell is rarely observed in ascending urinary tract infection

Abstract

Proteus mirabilis, a common cause of nosocomial and catheter-associated urinary tract infection, colonizes the bladder and ascends the ureters to the proximal tubules of the kidneys, leading to the development of acute pyelonephritis. P. mirabilis is capable of swarming, a form of multicellular behavior in which bacteria differentiate from the short rod typical of members of the family Enterobacteriaceae, termed the swimmer cell, into hyperflagellated elongated bacteria capable of rapid and coordinated population migration across surfaces, called the swarmer cell. There has been considerable debate as to which morphotype predominates during urinary tract infection. P. mirabilis(pBAC001), which expresses green fluorescent protein in both swimming and swarming morphotypes, was constructed to quantify the prevalence of each morphotype in ascending urinary tract infection. Transurethral inoculation of P. mirabilis(pBAC001) resulted in ascending urinary tract infection and kidney pathology in mice examined at both 2 and 4 days postinoculation. Using confocal microscopy, we were able to investigate the morphotypes of the bacteria in the urinary tract. Of 5,087 bacteria measured in bladders, ureters, and kidneys, only 7 (0.14%) were identified as swarmers. MR/P fimbria expression, which correlates with the swimmer phenotype, is prevalent on bacteria in the ureters and bladder. We conclude that, by far, the predominant morphotype present in the urinary tract during ascending infection is the short rod-the swimmer cell.

FIG. 1.

pBAC001 and transformed GFP-expressing swarmer and swimmer cells. (A) Schematic of pBAC001. The148 bases upstream of the start codon for the flagellin gene (flaA) were amplified by PCR, and this fragment was ligated 19 bases upstream of the gene coding for GFP and 57 bases downstream of the lac promoter (Pr lac) in pXL7301. (B) Fluorescent P. mirabilis bull's-eye colony. Five microliters of a culture of P. mirabilis HI4320(pBAC001) grown overnight was spotted onto the center of a swarming plate and incubated for 8 h at 37°C. P. mirabilis(pBAC001) swarmed normally and is fluorescent. (C and D) Swarmer (C) and swimmer (D) morphotypes expressed GFP. Bacteria from several regions of the bull's-eye colony were sampled and viewed by confocal microscopy.

FIG. 2.

GFP-expressing bacteria colonize the urinary tract. Confocal micrographs showing typical fields (seen with a 63× lens objective) of bladder (A) and kidney parenchyma (B and D) sections and pus in kidney (C). Host cells and actin are red, and host cell nuclei are blue. GFP-expressing bacteria are green. e, epithelium; l, lumen; c, capillary; t, tubule; p, polymorphonuclear cell; a, actin. Bars, 10 μm.

FIG. 3.

Rare swarmer cells. Of 5,087 bacteria measured, 7 were swarmer cells. Swarmer cells are indicated in the micrographs by a white asterisk. These elongated bacteria were found in bladder (A) and kidney (B to G) tissue. The swarmer cells are all >12 μm long, and all but one (B) are in close contact with surrounding swimmer cells and have a gentle curve. The swarmer cell in panel E is not expressing MR/P fimbriae, as are most of the surrounding swimmer cells (blue). The host nuclei are stained blue with DAPI in panels B to D.

FIG. 4.

Confocal micrographs of MR/P fimbria-expressing swimmer cells in ureters. (A) Nine Z sections were taken, one every 0.5 μm through the ureter section. All the bacteria (green) are expressing MR/P fimbriae (red). (B) MR/P fimbriae are seen as red colocalizing with the green bacteria to yield yellow in this image in which the above 18 images are stacked together.