Uropathogenic Escherichia coli superinfection enhances the severity of mouse bladder infection

Abstract

Urinary tract infections (UTIs) afflict over 9 million women in America every year, often necessitating long-term prophylactic antibiotics. One risk factor for UTI is frequent sexual intercourse, which dramatically increases the risk of UTI. The mechanism behind this increased risk is unknown; however, bacteriuria increases immediately after sexual intercourse episodes, suggesting that physical manipulation introduces periurethral flora into the urinary tract. In this paper, we investigated whether superinfection (repeat introduction of bacteria) resulted in increased risk of severe UTI, manifesting as persistent bacteriuria, high titer bladder bacterial burdens and chronic inflammation, an outcome referred to as chronic cystitis. Chronic cystitis represents unchecked luminal bacterial replication and is defined histologically by urothelial hyperplasia and submucosal lymphoid aggregates, a histological pattern similar to that seen in humans suffering chronic UTI. C57BL/6J mice are resistant to chronic cystitis after a single infection; however, they developed persistent bacteriuria and chronic cystitis when superinfected 24 hours apart. Elevated levels of interleukin-6 (IL-6), keratinocyte cytokine (KC/CXCL1), and granulocyte colony-stimulating factor (G-CSF) in the serum of C57BL/6J mice prior to the second infection predicted the development of chronic cystitis. These same cytokines have been found to precede chronic cystitis in singly infected C3H/HeN mice. Furthermore, inoculating C3H/HeN mice twice within a six-hour period doubled the proportion of mice that developed chronic cystitis. Intracellular bacterial replication, regulated hemolysin (HlyA) expression, and caspase 1/11 activation were essential for this increase. Microarrays conducted at four weeks post inoculation in both mouse strains revealed upregulation of IL-1 and antimicrobial peptides during chronic cystitis. These data suggest a mechanism by which caspase-1/11 activation and IL-1 secretion could predispose certain women to recurrent UTI after frequent intercourse, a predisposition predictable by several serum biomarkers in two murine models.

Figure 1. Superinfections of C3H/HeN mice.

A) Mice were infected with 10⁷ CFU UTI89, 2×10⁷ CFU UTI89, or PBS and re-infected with UTI89 at the indicated time points. One week total bladder titers are shown. Percentage of mice likely to develop chronic cystitis is displayed at the top of the column based on a CFU cutoff of 10⁶. Asterisks indicate p<0.05 from the PBS control and singly infected mice. B–D) Mice were infected with the indicated strain or PBS and re-infected one hour later. Urine titers were determined over time and four-week bladder titers are displayed. The fraction of mice with chronic cystitis is displayed at the top of each column. Red data points indicate resolved infection. Horizontal bars indicate median values. The dashed line at 20 CFU represents the LOD, and the dashed line at 10⁶ (A) or 10⁴ CFU (B–D) represents the chronic cystitis cutoff for urine and bladder titers. Panel A reflects 2–4 experiments with 5–9 mice per group. Panel B is 2–7 experiments with 4–5 mice per group. Panel C–D are 3 experiments with 4–5 mice per group. Statistical comparisons were determined using Fisher's exact test based on the fraction of mice experiencing chronic cystitis.

Figure 2. Role of caspase 1/11 and exfoliation in C3H/HeN superinfections.

A) Inoculation protocol shown for caspase inhibition studies of panel B. B) Four-week total bladder titer based on inhibitor or vehicle. C) Mice were inoculated with PBS, UTI89 or the indicated dose of protamine sulfate in 50 µL PBS and inoculated one hour later with UTI89. Urine was collected weekly and overall bladder titers are shown at four weeks. Panel B represents 5 experiments with n = 5–10 mice per group. Panel C represents 2 experiments with n = 5–10 mice per group. B–C) Observations in red indicate resolved infection. Percent of mice with persistent bacteriuria and chronic cystitis is shown at the top of each column. For Panel B and C, a Fisher's Exact Test was used to determine significance between proportions of mice experiencing chronic cystitis. Mann-Whitney U Test was used to compare median CFU values in Panel B.

Figure 3. C57BL/6J mice are susceptible to chronic cystitis when superinfected 24 hpi.

A–B) Mice were transurethrally infected with PBS or UTI89 and re-infected at the indicated timepoints with UTI89. Urine was tracked weekly and four-week total bladder (A) and kidney pair (B) titer is displayed. N = 2–4 experiments with 4–5 mice per group. Statistical differences determined by Fisher's Exact test.

Figure 4. UTI89 Superinfection of C57BL/6J mice increases bladder and kidney infection.

A–B) mice were infected with PBS or UTI89 Kan^r and re-infected 24 hrs later with PBS or UTI89 Spect^r. Urine was tracked over four weeks and total bladder (A) and kidney pair titer (B) is displayed. Number above columns indicates number of mice with persistent bacteriuria with bladder (A) or kidney (B) titer>10⁴ CFU. Data represents 3–8 experiments with n = 4–29 mice per group. Panels also include data reproduced from Fig. 3. Statistical differences determined by Fisher's Exact test.

Figure 5. Serum cytokine signature of C57BL/6J mice with persistent bacteriuria.

Serum was obtained 24 hrs after initial inoculation prior to superinfection. Levels of 23 cytokines were determined and cytokines showing significant differences between resolved and chronic superinfected mice are shown. Levels of KC (A), IL-6 (B), and G-CSF (C) shown in pg/mL. Data represent 4–6 experiments with n = 4–29 mice per group. Statistical differences determined by One-Way ANOVA overall and Mann-Whitney U test for pairwise comparisons.

Figure 6. Microarray gene changes for C3H/HeN and C57BL/6J bladders.

A) C3H/HeN heatmap analysis for mice that resolved infection, experienced chronic cystitis, or were mock infected with PBS. B) C57BL/6J heatmap analysis for mice mice that resolved infection, experienced chronic cystitis, or were mock infected with PBS. Depicted is a representative analysis of two biological and three technical replicates.