Fibrinolytic-deficiencies predispose hosts to septicemia from a catheter-associated UTI

Abstract

Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat partly due to development of multidrug-resistance from CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, here we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, here we found that Enterococcus faecalis, a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI.

Introduction

Urinary catheterization is a common procedure to drain urine from patients’ bladders due to chronic conditions or while in healthcare facilities, intensive care units, during surgical procedures and recovery^1,2,3. Despite its benefits, catheter placement increases the risk of developing a catheter-associated urinary tract infection (CAUTI)^4,5,6,7. CAUTIs are one of the most common nosocomial infections and often lead to septicemia with a 30% mortality^3,8. In fact, ~25% of sepsis cases come from complicated UTI, including CAUTIs⁹. Current CAUTI management focuses on catheter removal, replacement, and an antibiotic regimen⁶. However, the consistent colonization of catheters by external and fecal microflora, along with polymicrobial infections, pose challenges to management and treatment due to the increasing prevalence of antibiotic-resistant CAUTI pathogens^6,10,11. Leading the CDC and WHO to classify CAUTI as a serious threat^12,13.

Currently, there is no consensus on best practices for CAUTI treatment. Patients are treated with the same protocols as non-catheter associated (uncomplicated) urinary tract infections (uUTI). However, CAUTIs exhibit unique clinical manifestations, causative organisms, and pathologic mechanisms, making these infections distinct from uUTI⁶. For example, uUTIs are more prevalent in women than men (4:1 ratio) while in CAUTI there is no gender bias⁶. Also, E. coli accounts for >95% of uUTI, whereas CAUTI-pathogens are more diverse, including gram-negative, gram-positive, and fungal pathogens⁶.

In both humans and mice, urinary catheterization provokes local tissue damage to the bladder, activating coagulation, and converting the blood coagulation protein, fibrinogen (Fg), into fibrin. Fibrin clots and extravascular fibrin deposits promote blood clotting and wound healing¹⁴. Then, in fibrinolysis, plasminogen (Pg) is activated into serine protease plasmin to degrade fibrin clots and restore tissue homeostasis^14,15. However, constant catheter-induced bladder inflammation induces Fg/fibrin accumulation on the catheter with increasing catheterization time in human and mice^16,17. Fg/fibrin deposition compromises the urothelium, thereby introducing a platform for biofilm formation by CAUTI pathogens^{16,17,18,19,20,21,22,23}.

Here, we identified that mice with genetic or acquired fibrinolytic-deficiencies are susceptible to severe and persistent CAUTI and systemic dissemination by diverse groups of prevalent uropathogens. Moreover, we found that E. faecalis, a prevalent CAUTI pathogen known to bind to Fg, secretes a protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation and persistence during CAUTI. Furthermore, antifibrinolytic agent usage in catheterized mice results in Fg/fibrin accumulation in the bladders, resulting in persistent CAUTI and systemic dissemination in mono- and poly-microbial infection by E. faecalis, E. coli, and C. albicans. Identification of patient populations with higher susceptibility to CAUTI and its sequelae will allow clinicians to improve patient outcomes by implementing efficient patient monitoring to mitigate infection incidences, morbidities, and mortality.

Results

Prolonged urinary catheterization promotes enterococcal burden and persistence in the bladder and systemic dissemination

Clinical studies showed that CAUTI-burdened patients have an increased risk of septicemia and mortality^{10,11,24,25,26}. To understand E. faecalis bladder and catheter colonization progression and systemic dissemination, we performed a temporal study examining acute infection (1, 3, 6, 9, 12 hr post-catheterization and infection (hpi) and 1 day post-infection (dpi)) and prolonged infection (3, 7, 14 dpi). Female C57BL/6 mice were catheterized and infected with of E. faecalis (~2 ×10⁷ CFU) or mock-infected (PBS). At specified timepoints, mice were sacrificed to harvest the bladders, catheters, kidneys, spleens, and hearts. Bladders at 1 hpi have an initial colonization of ~10⁴ bacterial CFU that increased significantly overtime, showing maximum colonization at ~10⁷ CFU by 12 hpi in the bladder and persisting through 14 dpi (Fig. 1a). Similarly, catheter colonization significantly increased with catheterization time and persisted (Fig. 1b). Importantly, robust bladder and catheter colonization allowed E. faecalis to disseminate to the kidneys, spleen, and heart (Fig. 1c–e). Thus, our data recapitulates what is clinically observed^{11,12,25,26,27}.

Progression of bladder inflammation correlates with microbial burden

Catheter-induced inflammation is a CAUTI hallmark^21,27,28. To understand how urinary catheterization changes the bladder environment during acute and prolonged catheterization and in the presence or absence of infection; we examined mouse bladder edema and inflammation by bladder weight and histological analysis. Bladders exhibited a progressive weight increase with the catheter dwell time (Fig. 1f). Bladder histological analysis corroborates the gradual edema progression and increase of the bladder size associated with urinary catheterization (Fig. 1g). Furthermore, there is a significant positive correlation of bladder weight with bacterial burden in the bladder (r = 0.7991; P = 0.0098) or catheter (r = 0.9005; P = 0.0008) (Supplementary Fig. 1). Importantly, E. faecalis infection further increased bladder weight after 12 hpi (Fig. 1f). This indicates that catheterization alone increases bladder edema overtime and is exacerbated by an E. faecalis infection.

E. faecalis presence modulates levels of inflammatory cytokines in the catheterized bladder

Previous studies show that acute urinary catheterization induces many inflammatory cytokines in the bladder²⁷, of which IL-1β, IL-6, 12(p40), IL-17, CSF 3, and CXCL1 are further induced during E. faecalis infection²⁸. Since E. faecalis significantly exacerbates bladder inflammation during catheterization (Fig. 1f, g), we profiled cytokine level changes of E. faecalis- or mock-infected catheterized bladder, relative to naïve bladders. We found that E. faecalis infection altered cytokine expression patterns differently than in mock-infected bladders. IL-6, CSF 3, CXCL1, and IL-17 levels were significantly higher during infection (Fig. 1h, i, and Supplementary Fig. 2). Conversely, infection significantly reduced levels of IL-1α, IL-2, IL-9, IL-10, IL-12 (p70), IL-13, IFNγ, IL-3, CCL4 (MIP-1β), CSF 2 (GM-CSF), CCL5 (RANTES), and TNFα (Fig. 1h, Supplementary Figs. 2 and 3a, c). IL-1β, IL-12 (p40), IL-4, and CCL3 levels were modulated, showing significantly lower induction during early infection (1–9 h) followed by a significant increased after 12 h (Fig. 1h, Supplementary Figs. 2 and 3b). IL-5 and CCL2 levels increased regardless of the infection (Fig. 1h and Supplementary Fig. 2). This indicates that there is a differential inflammatory cytokine profile on infected catheterized bladders.

During catheter-induced bladder inflammation in mice and humans^16,17, Fg, a liver-produced proinflammatory protein, is recruited into the bladder and deposited on urinary catheters, serving as a platform for microbial CAUTI^{16,18,21,22,23,24,29}. IL-1 (α and β), IL-6, and TNFα are known to stimulate Fg production by migrating from damaged tissues, through the bloodstream and signaling to Fg-producing hepatocytes in the liver^30,31. To understand their contribution to CAUTI-associated inflammation, we evaluated levels of these proinflammatory mediators in the bladder. High levels of IL-6 during catheterization were observed, which significantly increased during infection (Fig. 1h, i). We also assessed the levels of all four cytokines in the bloodstream and livers. Expectedly, IL-6 levels were elevated in the bloodstream and liver (Fig. 1i and Supplementary Fig. 3). To understand the role of IL-1α, IL-1β, IL-6 and TNFα in human urinary catheterization, we collected urine from patients that had a urinary catheter for ~24 h and from healthy donors to compare and analyze their levels. We found that IL-1α, IL-1β, IL-6 and TNFα levels significantly increased in urine from catheterized patients when compared with urine from non-catheterized healthy donors (Fig. 1j). Furthermore, IL-6 was significantly elevated compared with IL-1α, IL-1β, and TNFα in the catheterized patients (Fig. 1j). A limitation of this study is that urine samples were not collected from catheterized patients before catheterization as a further pair-wise comparison. Without this data, any comorbidity prior to catheterization could also contribute to the cytokines present in patient urine post-catheterization. However, based on this data provided as is, we can speculate IL-6 could be an important cytokine during human and mouse CAUTIs and, in mice, IL-6 may communicate with the liver’s Fg expression and release, resulting in its accumulation in the catheterized bladder. Further work should be done to fully assess this.

Fg deposition increases on urinary catheters during E. faecalis infection

Previously, clinical and animal studies from our and other groups have shown that Fg is a major component of proteins deposited on urinary catheters^{16,17,19,20,21,23,32,33}. Importantly, our previous immunofluorescence analyses on catheters showed increased Fg deposition during E. faecalis infection^16,17,18, suggesting that infection may promote Fg recruitment. To test this, we performed a temporal quantitative proteomic analysis to compare Fg abundance on urinary catheters by analyzing the three Fg chains, α, β, γ, between catheterized (mock-infected) and catheterized and infected mice. Five catheters were harvested at the indicated timepoints, pooled, processed, and trypsin digested. Total protein analysis showed 230 proteins associated with mouse catheters (Fig. 2a, c, and Supplementary Data 1). Fg is among the most abundant deposited proteins on the catheter, corroborating our previous proteomic study at 24 hpi¹⁹ and its abundance significantly increased in acute and prolonged infection starting 1 dpi (Fig. 2b, Supplementary Fig. 4, and Supplementary Data 1). Higher Fg -α, -β, -γ total peptide counts in infected bladders suggest that E. faecalis may have a mechanism to promote Fg accumulation….