Characterization of a novel murine model of Staphylococcus saprophyticus urinary tract infection reveals roles for Ssp and SdrI in virulence

Abstract

Staphylococcus saprophyticus, an obligate human pathogen, is the most common Gram-positive causative agent of urinary tract infection (UTI) in young, healthy women. Despite the clinical importance of S. saprophyticus, little is known about how it causes disease in the urinary tract or how the host responds to the infection. Here we established an in vivo model to study both host and bacterial factors contributing to S. saprophyticus UTI. Using this model, we show that S. saprophyticus preferentially infects C3H/HeN murine kidneys instead of the bladder, a trait observed for multiple clinical isolates. Bacterial persistence in the kidneys was observed in C3H/HeN mice but not in C57BL/6 mice, indicating that host factors strongly contribute to the ability of S. saprophyticus to cause UTI. Using C3H/HeN mice as a model, histologic and immunofluorescence analyses of infected tissues revealed that S. saprophyticus induced epithelial cell shedding in the bladder and an inflammatory response characterized by macrophage and neutrophil infiltration in the bladder and kidneys. The inflammatory response correlated with increased production of proinflammatory cytokines and chemokines in both the bladder and the kidneys. Finally, we observed that the putative S. saprophyticus virulence factors Ssp and SdrI were important for persistence, but not for initial colonization, in the murine urinary tract. Thus, we characterized both host and bacterial factors involved in progression of S. saprophyticus UTI, and we describe a useful model system for studying factors involved in the pathogenesis of this Gram-positive uropathogen.

FIG. 1.

S. saprophyticus strain 7108 is rapidly cleared from the bladder but persists in the kidneys of C3H/HeN mice. S. saprophyticus was instilled into the bladders of C3H/HeN mice (12 mice/time point). Bladders (A) and kidneys (B) were removed and homogenized, and 10-fold serial dilutions were plated to determine the number of CFU/organ. Each circle indicates the data for one mouse, and the horizontal lines indicate the geometric means. The data are composite data for two experiments. An asterisk indicates that the kidney titer was significantly higher than the bladder titer at a time point (P < 0.005, Mann-Whitney U test).

FIG. 2.

S. saprophyticus strains preferentially colonize the kidney. Clinical isolates (indicated on the x axis) (Table 1) were inoculated into the bladders of C3H/HeN mice (10 mice/group) using 50 μl containing 10⁷ CFU. Bladders and kidneys were removed and homogenized, and 10-fold dilutions were plated to determine the number of CFU/organ at 48 h postinfection. Each circle indicates the data for one mouse, and the horizontal lines indicate the geometric means. The data are composite data for two experiments. An asterisk indicates that the kidney titer was significantly higher than the bladder titer at a time point (P < 0.005, Mann-Whitney U test).

FIG. 3.

S. saprophyticus is rapidly cleared from the C57BL/6 murine urinary tract. Female C3H/HeN or C57BL/6 mice (5 mice/time point) were infected with 10⁷ CFU of S. saprophyticus strain 7108. Bladders and kidneys were homogenized at 1, 3, 6, 25, and 48 h postinfection, and serial dilutions of organ homogenates were plated to determine the number of CFU/organ. Each circle indicates the data for one mouse; the filled circles indicate the data for C3H/HeN mice, and the open circles indicate the data for C57BL/6 mice. The horizontal lines indicate the geometric means. The data are representative of the data obtained in two separate experiments performed with 5 mice/experiment/mouse strain/time point.

FIG. 4.

S. saprophyticus strain 7108 induces inflammatory infiltration in the kidney. Mice were inoculated with either PBS (A and D), S. saprophyticus 7108 (B, C, and E), or S. saprophyticus Top58 (F). Bladders (A to C) and kidneys (D to F) were harvested and prepared for hematoxylin and eosin staining as described in the text. An inflammatory infiltrate was observed in S. saprophyticus-infected kidneys at 48 h p.i. (panels E and F, arrows). Conversely, no infiltrate was observed in the bladder at the same time point (B and C), but areas of epithelial exfoliation were observed (arrowheads). (A) The bladder epithelium from PBS mock-infected mice was intact, and no inflammatory infiltrate was observed in the bladders or kidneys of mock-infected animals (A and D). The area showing epithelial exfoliation in a box in panel B (magnification, ×10) is shown at a higher magnification (×40) in panel C. The magnification of all other images is ×10. L, bladder lumen; Pe, renal pelvis; Py, pyramid; C, cortex.

FIG. 5.

S. saprophyticus infection induces a soluble proinflammatory innate immune response in the urinary tract of C3H/HeN mice. S. saprophyticus strain 7108 (10⁷ CFU) was instilled into the bladders of C3H/HeN mice (5 mice/time point). Bladders (A) and kidneys (B) were removed at 6 h, 48 h, 7 days, and 14 days postinfection and homogenized, and supernatants were collected. The cytokine levels in organ homogenate supernatants are expressed as fold changes compared to the levels in PBS mock-infected mice (the fold change for mock-infected mice was defined as 1 and is indicated by a dashed line). The error bars indicate the standard deviations. Cytokine levels were measured in three separate experiments (48 h), two separate experiments (7 days), or one experiment (6 h and 14 days). The results of one representative experiment are shown. Cytokines significantly induced by S. saprophyticus in this experiment are indicated by carets, and cytokines significantly induced in multiple experiments are indicated by asterisks. Statistical significance was determined using a one-sample t test (P < 0.1). The lack of significance when induction appears to be high is a result of a small sample size.

FIG. 6.

Immune infiltrates in the murine urinary tract in response to S. saprophyticus strain 7108. C3H/HeN mice were infected with S. saprophyticus, and organs were removed at the time points indicated, fixed, and either dissociated for flow cytometry or sectioned for immunofluorescence microscopy. (A to D) Representative dot plots resulting from cellular infiltration analysis. CD45⁺ cells, which were ∼2 to 4% of the total bladder cells (A and B) or kidney cells (C and D), were gated (A and C), and the percentages of Gr1⁺ neutrophils and F4/80⁺ macrophages were determined (B and D). (E to H) Immune infiltrate as assessed by determining the percentage of CD45⁺ immune cells in each organ which costained with the neutrophil/monocyte marker Gr1 or the monocyte/macrophage marker F4/80. The data are composite data for two separate experiments (2 or 3 mice/time point/experiment). The samples used for PBS mock-infected organs were pooled samples from 6 h, 48 h, and 7 days p.i. The error bars indicate the standard deviations. Statistical significance (indicated by an asterisk) was determined by a paired t test (P < 0.05). (I to L) Kidney sections from mice inoculated with S. saprophyticus for 6 h (I and J) or 48 h (K and L) stained with anti-Aas (red) and anti-CD-45 antibodies (green). Tissue sections were counterstained with Hoescht dye for nuclear labeling (blue). Magnification, ×40. The renal pelvis (Pe), pyramid (Py), and cortex (C) areas are outlined with dashed lines. In negative controls for each preparation, in which the primary anti-Aas and anti-CD-45 antibodies were not included, there was no cellular or bacterial staining (data not shown).

FIG. 7.

Putative S. saprophyticus virulence factors are necessary for infection of C3H/HeN mice. Female C3H/HeN mice were infected with 10⁷ CFU of S. saprophyticus wild-type strain 7108 or isogenic strains with a mutation in ssp (Ssp⁻) or sdrI (SdrI⁻). Bladders and kidneys were homogenized at 6 h, 48 h, and 7 days postinfection, and serial dilutions of organ homogenates were plated for determination of the number of CFU. The data are composite data for two (6 h and 7 days) and four (48 h) experiments (5 to 10 mice/time point/experiment). The horizontal lines indicate the geometric means. Statistical significance was determined by the Mann-Whitney U test.