Establishment of a persistent Escherichia coli reservoir during the acute phase of a bladder infection

Abstract

The vast majority of urinary tract infections are caused by strains of uropathogenic Escherichia coli that encode filamentous adhesive organelles called type 1 pili. These structures mediate both bacterial attachment to and invasion of bladder epithelial cells. However, the mechanism by which type 1 pilus-mediated bacterial invasion contributes to the pathogenesis of a urinary tract infection is unknown. Here we show that type 1-piliated uropathogens can invade the superficial epithelial cells that line the lumenal surface of the bladder and subsequently replicate, forming massive foci of intracellular E. coli termed bacterial factories. In response to infection, superficial bladder cells exfoliate and are removed with the flow of urine. To avoid clearance by exfoliation, intracellular uropathogens can reemerge and eventually establish a persistent, quiescent bacterial reservoir within the bladder mucosa that may serve as a source for recurrent acute infections. These observations suggest that urinary tract infections are more chronic and invasive than generally assumed.

FIG. 1

Kinetics of bacterial clearance from the bladder following infection with type 1-piliated UPEC. (A) The majority of bacteria were cleared within the first 48 h after infection of C57BL/6 mice with UTI89. (B) Significant numbers of bacteria, however, persist within the bladder tissue up to 6 weeks after infection. In addition, the bacteriologic status of urine samples collected from each mouse at the time of death is also indicated in panel B. ○, urine titer of >10³ CFU/ml; ●, urine titer of <10³ CFU/ml; ◑, no urine collected. Horizontal lines indicate the median titer at each time (n = 5 or 6).

FIG. 2

Replication of UPEC within superficial bladder cells. (A and B).Two hours after infection of C57BL/6 mice with UTI89, bacteria were detected in hematoxylin-and-eosin-stained sections entering or already within the superficial epithelial cells lining the lumenal surface of the bladder (arrowheads). (C to E) By 6 h after inoculation, large foci of intracellular E. coli were apparent within many of the superficial bladder cells. (E) Bacteria (red) were stained using an anti-E. coli primary antibody and Cy3-labeled secondary antibody. Host cell nuclei were visualized using Hoechst dye. Bars, 10 μm.

FIG. 3

Efflux of UPEC from superficial bladder epithelial cells. (A and B) Six hours after infection with type 1-piliated UPEC, the superficial bladder cell layer was in the process of undergoing exfoliation. The remaining superficial cells often appeared swollen with evidence of membrane blebbing. (B) Bacteria frequently appeared to be spilling out from within the superficial cells. (C to G) Elongated forms of bacteria, along with their normal-sized counterparts, were observed seemingly emerging from within superficial cells and spilling onto underlying and surrounding epithelial cells. (E) Filamentous bacteria were sometimes seen bridging ss host cell sutures and interacting with two adjoining superficial cells simultaneously. (Inset) The elongated bacteria contained at least partial septa at variable distances along their lengths. (G) Examination of hematoxylin-and-eosin-stained bladder sections indicated that the filamentous forms of bacteria could extend a significant distance through the interior of the superficial bladder cells. Bars, 5 μm (A to F) and 10 μm (G).

FIG. 4

In vitro intracellular persistence and reemergence of UPEC. 5637 bladder epithelial cells were infected with uropathogenic isolates (UTI 89 or NU14) or with a laboratory K-12 strain (AAEC185/pSH2 or MG1655) that expresses type 1 pili, and intracellular growth assays were performed in the presence of gentamicin. (A) Intracellular levels of UTI89 and NU14 remained constant for 48 h in the presence of gentamicin alone. In contrast, intracellular titers of the K-12 strains decreased significantly during the same time interval. Inhibition of bacterial replication using the bacteriostatic antibiotics TMP-SMZ greatly reduced the ability of the clinical isolates to survive intracellularly. TMP-SMZ had no effect on the persistence profile of the K-12 strains. (B) Bacterial fluxing assays indicate that intracellular UPEC isolates can exit host 5637 bladder cells. Following a 24-hour incubation of infected host cells in the presence of gentamicin, the cell culture medium was replaced with fresh medium with and without gentamicin. Within 7 h after removal of gentamicin (to allow extracellular bacterial growth), the titers of total extra- and intracellular UTI89 cells greatly increased. In contrast, the titers of MG1655 remained nearly unchanged regardless of the absence or continued presence of gentamicin.

FIG. 5

In vitro formation of intracellular bacterial inclusions. (A) One day after infection of 5637 cells with UTI89, large foci of intracellular bacteria were detected within 5637 cells by transmission electron microscopy. (B and C) Similar inclusions of intracellular bacteria were visualized by confocal microscopic examination of 5637 cells 72 h after infection with NU14 constitutively expressing green fluorescent protein. Host cells were counterstained with propidium iodide. Gentamicin prevented extracellular bacterial growth during these assays.