Enterococcal Biofilm Formation and Virulence in an Optimized Murine Model of Foreign Body-Associated Urinary Tract Infections

Implant loss from murine bladders correlates with less severe infection of the bladder and kidney. Graphs represent bacterial titers in log scale recovered from the bladders (A) and kidneys (B) of animals that have lost the implants (L) (○) or retained the implants (R) (•) at the indicated time of sacrifice. The experiment was done at least three times with five mice in each experiment. The horizontal broken line depicts the limit of detection. The horizontal bar represents the median value for each group of mice. Values that are significantly different by the Mann-Whitney U test are indicated as follows: *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Values that are not significantly different (P > 0.05) (ns) by the Mann-Whitney U test are indicated.

Implantation-mediated histological changes are associated with enterococcal colonization of the murine bladder. (A to F) H&E staining of bladder sections derived from animals without implants inoculated with PBS (A and D), animals with implants inoculated with PBS (B and E), or animals with implants infected with E. faecalis OG1RF (C and F) 24 hpi observed with a light microscope at magnifications of ×40 (A to C) and ×63 (D to F). Areas in black boxes on panels A, B, and C are magnified in panels D, E, and F, respectively. Severe edema, hyperplasia, urothelial sloughing, and immune cell infiltration characterize bladders of implanted animals with or without bacterial challenge. The white arrow in panel E indicates damage to the uroepithelium. Abbreviations: U, uroepithelium; LP, lamina propria; L, lumen; M, muscularis. (G to I) Representative laser scanning confocal microscopic (CLSM) images of bladder sections from PBS-treated nonimplanted, PBS-treated implanted, and OG1RF-infected implanted mice 24 hpi immunolabeled with goat anti-mouse uroplakin III (green), rabbit anti-Lancefield group D antigen (red), and counterstained with TOPRO-3 nuclear dye (blue) reveal alterations to the uroepithelium of implanted bladders and the presence of bacteria in the bladder lumen and on the uroepithelial surface. The white broken line separates the lumen (L) from the uroepithelium surface (U). Bars = 10 μm.

Implantation and E. faecalis infection elicit the production of specific cytokines in the bladder. Graphs represent cytokine expression in the whole bladder of animals with implants inoculated with PBS or E. faecalis OG1RF at 24 hpi. Cytokines reported are expressed with at least twofold difference relative to mock-infected animals with no implants. The presence of implants induces the specific expression of IL-6, G-CSF, and KC and causes the downregulation of GM-CSF and MIP-1α. In contrast, E. faecalis elicits the specific expression of IL-1β, IL-12(p40), and MIP-1α at 24 hpi. Data represent a representative experiment performed four times with five mice for each condition in the experiment. Values are shown as the means plus standard errors of the means (error bars). Values that are significantly different (P < 0.05 by the Mann-Whitney U test) are indicated (*).

E. faecalis produces biofilms on the surfaces of silicone implants in vivo. Representative scanning electron micrographs of silicone implants retrieved from murine bladders infected with E. faecalis OG1RF at 72 hpi showing bacterial biofilms on the outer surface (A and B) and filling the lumen (C and F) of the silicone implant. Bacteria (white arrowhead) are often associated with host cells, indicated by the black arrowhead in panel C. Both bacteria and host immune cells can also be found embedded in a thick extracellular material, indicated by the black arrow, in the lumen of the silicone implant 72 hpi (E and F). Areas in black boxes on panels A, C, and E are magnified in panels B, D, and F, respectively. Bars, 20 μm (A and C), 5 μm (B, D, and F), and 100 μm (E).

Autolytic factors are not required for E. faecalis virulence in the urinary tract. (A to F) Graphs show the numbers of CFU of E. faecalis OG1RF and isogenic in-frame gelE- and atn-deficient mutants recovered from retrieved implants (A and D), bladders (B and E), and kidneys (C and F) from animals 24 hpi following infection with either strain. Horizontal dashed lines represent the limits of detection for viable bacteria. Each symbol represents the value for an individual mouse, and each experiment was done twice with four or five mice for each strain. Only data from animals with implants at the time of sacrifice are represented. The horizontal bars indicate the median values for groups. Values that are statistically significantly different (P < 0.05) by the Mann-Whitney U test are indicated (*). Values that are not statistically significantly different (P > 0.05) by the Mann-Whitney U test are indicated (ns).