Ana L. Flores-Mireles

Urinary tract infections: epidemiology, mechanisms of infection and treatment options

Urinary tract infections (UTIs) are a severe public health problem and are caused by a range of pathogens, but most commonly by Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis and Staphylococcus saprophyticus. High recurrence rates and increasing antimicrobial resistance among uropathogens threaten to greatly increase the economic burden of these infections. In this Review, we discuss how basic science studies are elucidating the molecular details of the crosstalk that occurs at the host–pathogen interface, as well as the consequences of these interactions for the pathophysiology of UTIs. We also describe current efforts to translate this knowledge into new clinical treatments for UTIs.

EbpA vaccine antibodies block binding of Enterococcus faecalis to fibrinogen to prevent catheter-associated bladder infection in mice

Binding to host fibrinogen is a critical event in Enterococcus faecalis pathogenesis in catheter-associated urinary tract infections in mice. Dinner and a Biofilm Results in Catheter-Associated Infection Enterococci bacteria are a frequent cause of catheter-associated urinary tract infections. Their increased antibiotic resistance has made it essential to understand molecular mechanisms of infection to generate alternative treatments. Flores-Mireles et al. now report that fibrinogen is released from host cells into mouse bladders in which catheters have been inserted. Subsequently, Enterococcus faecalis uses an adhesin (EbpA) at the tip of its pilus to bind to the host fibrinogen. Surprisingly, the bacteria feast on the fibrinogen to form a biofilm on the catheter. By elucidating the E. faecalis EbpA-fibrinogen interaction, these investigators were able to develop an EbpA-based vaccine that prevented catheter-associated infection by E. faecalis in mice. Enterococci bacteria are a frequent cause of catheter-associated urinary tract infections, the most common type of hospital-acquired infection. Treatment has become increasingly challenging because of the emergence of multiantibiotic-resistant enterococcal strains and their ability to form biofilms on catheters. We identified and targeted a critical step in biofilm formation and developed a vaccine that prevents catheter-associated urinary tract infections in mice. In the murine model, formation of catheter-associated biofilms by Enterococcus faecalis depends on EbpA, which is the minor subunit at the tip of a heteropolymeric surface fiber known as the endocarditis- and biofilm-associated pilus (Ebp). We show that EbpA is an adhesin that mediates bacterial attachment to host fibrinogen, which is released and deposited on catheters after introduction of the catheter into the mouse bladder. Fibrinogen-binding activity resides in the amino-terminal domain of EbpA (EbpANTD), and vaccination with EbpA and EbpANTD, but not its carboxyl-terminal domain or other Ebp subunits, inhibited biofilm formation in vivo and protected against catheter-associated urinary tract infection. Analyses in vitro demonstrated that protection was associated with a serum antibody response that blocked EbpA binding to fibrinogen and the formation of a fibrinogen-dependent biofilm on catheters. This approach may provide a new strategy for the prevention of catheter-associated urinary tract infections.

Pilin and Sortase Residues Critical for Endocarditis- and Biofilm-Associated Pilus Biogenesis in Enterococcus faecalis

Enterococci commonly cause hospital-acquired infections, such as infective endocarditis and catheter-associated urinary tract infections. In animal models of these infections, a long hairlike extracellular protein fiber known as the endocarditis- and biofilm-associated (Ebp) pilus is an important virulence factor for Enterococcus faecalis. For Ebp and other sortase-assembled pili, the pilus-associated sortases are essential for fiber formation as they create covalent isopeptide bonds between the sortase recognition motif and the pilin-like motif of the pilus subunits. However, the molecular requirements governing the incorporation of the three pilus subunits (EbpA, EbpB, and EbpC) have not been investigated in E. faecalis. Here, we show that a Lys residue within the pilin-like motif of the EbpC subunit was necessary for EbpC polymerization. However, incorporation of EbpA into the pilus fiber only required its sortase recognition motif (LPXTG), while incorporation of EbpB only required its pilin-like motif. Only the sortase recognition motif would be required for incorporation of the pilus tip subunit, while incorporation of the base subunit would only require the pilin recognition motif. Thus, these data support a model with EbpA at the tip and EbpB at the base of an EbpC polymer. In addition, the housekeeping sortase, SrtA, was found to process EbpB and its predicted catalytic Cys residue was required for efficient cell wall anchoring of mature Ebp pili. Thus, we have defined molecular interactions involved in fiber polymerization, minor subunit organization, and pilus subcellular compartmentalization in the E. faecalis Ebp pilus system. These studies advance our understanding of unique molecular mechanisms of sortase-assembled pilus biogenesis.

Antibody-Based Therapy for Enterococcal Catheter-Associated Urinary Tract Infections

ABSTRACT Gram-positive bacteria in the genus Enterococcus are a frequent cause of catheter-associated urinary tract infection (CAUTI), a disease whose treatment is increasingly challenged by multiantibiotic-resistant strains. We have recently shown that E. faecalis uses the Ebp pilus, a heteropolymeric surface fiber, to bind the host protein fibrinogen as a critical step in CAUTI pathogenesis. Fibrinogen is deposited on catheters due to catheter-induced inflammation and is recognized by the N-terminal domain of EbpA (EbpANTD), the Ebp pilus’s adhesin. In a murine model, vaccination with EbpANTD confers significant protection against CAUTI. Here, we explored the mechanism of protection using passive transfer of immune sera to show that antisera blocking EbpANTD-fibrinogen interactions not only is prophylactic but also can act therapeutically to reduce bacterial titers of an existing infection. Analysis of 55 clinical CAUTI, bloodstream, and gastrointestinal isolates, including E. faecalis, E. faecium, and vancomycin-resistant enterococci (VRE), revealed a diversity of levels of EbpA expression and fibrinogen-binding efficiency in vitro. Strikingly, analysis of 10 strains representative of fibrinogen-binding diversity demonstrated that, irrespective of EbpA levels, EbpANTD antibodies were universally protective. The results indicate that, despite diversity in levels of fibrinogen binding, strategies that target the disruption of EbpANTD-fibrinogen interactions have considerable promise for treatment of CAUTI. IMPORTANCE Urinary catheterization is a routine medical procedure, and it has been estimated that 30 million Foley catheters are used annually in the United States. Importantly, placement of a urinary catheter renders the patient susceptible to developing a catheter-associated urinary tract infection, accounting for 1 million cases per year. Additionally, these infections can lead to serious complications, including bloodstream infection and death. Enterococcus strains are a common cause of these infections, and management of enterococcal infections has been more difficult in recent years due to the development of antibiotic resistance and the ability of strains to disseminate, resulting in a major threat in hospital settings. In this study, we developed an antibiotic-sparing treatment that is effective against diverse enterococcal isolates, including vancomycin-resistant enterococci, during catheter-associated urinary tract infections. Urinary catheterization is a routine medical procedure, and it has been estimated that 30 million Foley catheters are used annually in the United States. Importantly, placement of a urinary catheter renders the patient susceptible to developing a catheter-associated urinary tract infection, accounting for 1 million cases per year. Additionally, these infections can lead to serious complications, including bloodstream infection and death. Enterococcus strains are a common cause of these infections, and management of enterococcal infections has been more difficult in recent years due to the development of antibiotic resistance and the ability of strains to disseminate, resulting in a major threat in hospital settings. In this study, we developed an antibiotic-sparing treatment that is effective against diverse enterococcal isolates, including vancomycin-resistant enterococci, during catheter-associated urinary tract infections.

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

Catheterization alters bladder ecology to potentiate Staphylococcus aureus infection of the urinary tract

Significance Staphylococcus aureus is a cause of catheter-associated urinary tract infections (CAUTIs). S. aureus CAUTIs are problematic because they are usually caused by antibiotic-resistant strains, and patients who develop these infections have a high risk of developing serious complications. Catheterization in humans and mice causes damage in the bladder that results in the release of host protein fibrinogen (Fg). This study suggests that S. aureus exploits the presence of Fg via interactions mediated by the Fg-binding protein ClfB to facilitate colonization of the bladder and the catheter to cause a persistent infection in both mice and humans. Insights into S. aureus CAUTI pathogenesis is facilitating the development of more-targeted therapies to better treat these infections. Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging cause of catheter-associated urinary tract infection (CAUTI), which frequently progresses to more serious invasive infections. We adapted a mouse model of CAUTI to investigate how catheterization increases an individual’s susceptibility to MRSA UTI. This analysis revealed that catheterization was required for MRSA to achieve high-level, persistent infection in the bladder. As shown previously, catheter placement induced an inflammatory response resulting in the release of the host protein fibrinogen (Fg), which coated the bladder and implant. Following infection, we showed that MRSA attached to the urothelium and implant in patterns that colocalized with deposited Fg. Furthermore, MRSA exacerbated the host inflammatory response to stimulate the additional release and accumulation of Fg in the urinary tract, which facilitated MRSA colonization. Consistent with this model, analysis of catheters from patients with S. aureus-positive cultures revealed colocalization of Fg, which was deposited on the catheter, with S. aureus. Clumping Factors A and B (ClfA and ClfB) have been shown to contribute to MRSA–Fg interactions in other models of disease. We found that mutants in clfA had significantly greater Fg-binding defects than mutants in clfB in several in vitro assays. Paradoxically, only the ClfB− strain was significantly attenuated in the CAUTI model. Together, these data suggest that catheterization alters the urinary tract environment to promote MRSA CAUTI pathogenesis by inducing the release of Fg, which the pathogen enhances to persist in the urinary tract despite the host’s robust immune response.

Manganese acquisition is essential for virulence of Enterococcus faecalis

Manganese (Mn) is an essential micronutrient that is not readily available to pathogens during infection due to an active host defense mechanism known as nutritional immunity. To overcome this nutrient restriction, bacteria utilize high-affinity transporters that allow them to compete with host metal-binding proteins. Despite the established role of Mn in bacterial pathogenesis, little is known about the relevance of Mn in the pathophysiology of E. faecalis. Here, we identified and characterized the major Mn acquisition systems of E. faecalis. We discovered that the ABC-type permease EfaCBA and two Nramp-type transporters, named MntH1 and MntH2, work collectively to promote cell growth under Mn-restricted conditions. The simultaneous inactivation of EfaCBA, MntH1 and MntH2 (ΔefaΔmntH1ΔmntH2 strain) led to drastic reductions (>95%) in cellular Mn content, severe growth defects in body fluids (serum and urine) ex vivo, significant loss of virulence in Galleria mellonella, and virtually complete loss of virulence in rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI) models. Despite the functional redundancy of EfaCBA, MntH1 and MntH2 under in vitro or ex vivo conditions and in the invertebrate model, dual inactivation of efaCBA and mntH2 (ΔefaΔmntH2 strain) was sufficient to prompt maximal sensitivity to calprotectin, a Mn- and Zn-chelating host antimicrobial protein, and for the loss of virulence in mammalian models. Interestingly, EfaCBA appears to play a prominent role during systemic infection, whereas MntH2 was more important during CAUTI. The different roles of EfaCBA and MntH2 in these sites could be attributed, at least in part, to the differential expression of efaA and mntH2 in cells isolated from hearts or from bladders. Collectively, this study demonstrates that Mn acquisition is essential for the pathogenesis of E. faecalis and validates Mn uptake systems as promising targets for the development of new antimicrobials.

MP23-19 FIBRINOGEN DEPOSITS ON URINARY CATHETERS IN A TIME-DEPENDENT MATTER AND CO-LOCALIZES WITH E. FAECALIS IN PATIENTS WITH POSITIVE E. FAECALIS URINE CULTURES

INTRODUCTION AND OBJECTIVES: There is mounting evidence that fibrinogen deposition on urinary catheters is a key step in the pathogenesis of catheter-associated urinary tract infection (CAUTI). The aim of this study was to investigate whether fibrinogen and Enterococcus faecalis co-localize on catheters acquired from patients with post-operative urine cultures positive for E. faecalis. METHODS: Urinary catheters from a series of 50 patients undergoing elective urologic procedures were collected post-operatively and analyzed via immunofluorescence to detect deposited fibrinogen. Pearson correlation was performed to measure the correlation between fibrinogen deposition and dwell time. Additional catheters and urine cultures were collected at time of catheter removal. Catheters from patients with positive Enterococcus cultures were probed for fibrinogen and Enterococcus via immunofluorescence. RESULTS: A total of 50 adult patients undergoing urinary catheterization as standard of care were prospectively identified at our institution. Fibrinogen concentration quantified as mg/catheter using a standard curve was highly correlated with catheter dwell time (r1⁄40.63; p<0.0001) (Figure 1). E. faecalis was capable of binding to fibrinogen on these catheters ex vivo.Five additional catheters were obtained from patients with E. faecalis-positive post-operative urine cultures. Fibrinogen was present at all time points (18 hours, 1 day, 1 day, 8 days, 9 days) and co-localized with E. faecalis (Figure 2) in vivo. CONCLUSIONS: We have previously shown that fibrinogen deposits on urinary catheters, and that E. faecalis is capable of binding to these catheters ex vivo. In this study, we demonstrate that E. faecalis co-localizes with fibrinogen in catheterized patients with urine cultures positive for E. faecalis. This data strengthens the clinical association of fibrinogen deposition with CAUTI and suggests that targeting the binding of E. faecalis with fibrinogen may help reduce the rate of Enterococcus CAUTI. Source of Funding: 1F32DK104516-01 (ALF-M), and National Institute of Allergy and Infectious Diseases and National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK051406, R01-AI108749-01 and P50DK0645400 (ALF-M, JNW, HLS, JSP, MGC, SJH).