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Clinical Microbiology Reviews, January 2008, p. 26-59, Vol. 21, No. 1
0893-8512/08/$08.00+0 doi:10.1128/CMR.00019-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Complicated Catheter-Associated Urinary Tract Infections Due to Escherichia coli and Proteus mirabilis
S. M. Jacobsen,1 D. J. Stickler,2 H. L. T. Mobley,3 and M. E. Shirtliff1,4*Department of Microbiology and Immunology, School of Medicine, University of Maryland—Baltimore, 655 W. Baltimore Street, Baltimore, Maryland 21201,1 Cardiff School of Biosciences, Cardiff University, Cardiff, Wales CF10 3TL, United Kingdom,2 Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109,3 Department of Biomedical Sciences, Dental School, University of Maryland—Baltimore, 650 W. Baltimore Street, Baltimore, Maryland 212014
SUMMARY INTRODUCTION PATHOGENESIS OF CAUTIs CAUTIs DUE TO E. COLI Adhesins Motility Invasion and Biofilm Formation Avoidance of Host Immune Response Damage to the Host and Acquisition of Nutrients CAUTIs DUE TO PROTEUS MIRABILIS Adhesins Motility Biofilm Formation Avoidance of Host Immune Response Damage to the Host and Acquisition of Nutrients PREVENTION OF CAUTIs Limiting Catheter Usage Condom and Suprapubic Catheters and Intermittent Catheterization Closed-System Foley Catheters and Proper Use by Health Care Professionals Prevention of Bacterial Colonization of the Urinary Meatus and Urinary Tract Novel Surfaces Catheters Containing Antimicrobial Agents Probiotics Future Promising Technologies DIAGNOSIS AND TREATMENT OF CAUTIs PROGNOSIS OF CAUTIs REFERENCES
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Catheter-associated urinary tract infections (CAUTIs) represent the most common type of nosocomial infection and are a major health concern due to the complications and frequent recurrence. These infections are often caused by Escherichia coli and Proteus mirabilis. Gram-negative bacterial species that cause CAUTIs express a number of virulence factors associated with adhesion, motility, biofilm formation, immunoavoidance, and nutrient acquisition as well as factors that cause damage to the host. These infections can be reduced by limiting catheter usage and ensuring that health care professionals correctly use closed-system Foley catheters. A number of novel approaches such as condom and suprapubic catheters, intermittent catheterization, new surfaces, catheters with antimicrobial agents, and probiotics have thus far met with limited success. While the diagnosis of symptomatic versus asymptomatic CAUTIs may be a contentious issue, it is generally agreed that once a catheterized patient is believed to have a symptomatic urinary tract infection, the catheter is removed if possible due to the high rate of relapse. Research focusing on the pathogenesis of CAUTIs will lead to a better understanding of the disease process and will subsequently lead to the development of new diagnosis, prevention, and treatment options.
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Indwelling urinary catheters are standard medical devices utilized in both hospital and nursing home settings to relieve urinary retention and urinary incontinence. Of the almost 100 million catheters that are sold annually worldwide, one-quarter of them are sold in the United States (50). The most common urinary catheter in use is the Foley indwelling urethral catheter, a closed sterile system that is comprised of a tube inserted through the urethra and held in place by an inflatable balloon to allow urinary drainage of the bladder. Although these devices were originally designed for short-term use in patients, indwelling catheter use is now commonplace in the long-term setting.
Due to the frequent and sometimes unnecessary use of indwelling catheters during hospitalization (21 to 50% of patients) (153), many patients are placed at risk for complications associated with the use of these devices. A study of 1,540 nursing home residents determined that the risk of hospitalization, length of hospitalization, and length of antibiotic therapy were three times higher in catheterized residents than in noncatheterized residents (205). The most notable complication associated with indwelling urinary catheters is the development of nosocomial urinary tract infections (UTIs), known as catheter-associated UTIs (CAUTIs). Infections of the urinary tract associated with catheter use are significant not only due their high incidence and subsequent economic cost but also because of the severe sequelae that can result.
CAUTIs, the most common type of nosocomial infection, account for over 1 million cases annually (401) or over 40% of all nosocomial infections in hospitals and nursing homes (382, 383, 438) and constitute 80% of all nosocomial UTIs (132). Due to this high incidence, the overall cost for medical intervention of nosocomial UTIs is staggering, with an estimated $424 million to $451 million spent annually in the United States to manage these infections (157). Furthermore, catheter-associated bacteremia is estimated to cost approximately $2,900 per episode (339). Costs for treatment of nosocomial UTIs include antimicrobial therapy, increases in length of stay during hospitalization, physician visits, and morbidity (98). These costs will inevitably rise due to advances in preventive medicine that extend life expectancy, increasing the elderly population. This population today (those 
65 years old) accounts for approximately 12.6% (37,849,672) of the total population of the United States (301,139,947) (422); their care accounts for about one-third (6) of the estimated $1 trillion in U.S. health expenditures (279).
Individuals requiring an indwelling catheter are predisposed to the development of CAUTIs due to the presence of an indwelling catheter device and potentially pathogenic multidrug-resistant organisms in the hospital setting. Despite the imminent threat of infection from these potent opportunistic nosocomial multiresistant strains, most cases of catheter-associated bacteriuria or the presence of bacteria in the urine are asymptomatic.
However, when an episode of CAUTI becomes symptomatic, the resulting sequelae can range from mild (fever, urethritis, and cystitis) to severe (acute pyelonephritis, renal scarring, calculus formation, and bacteremia). Left untreated, these infections can lead to urosepsis and death (284, 438). These complicated infections commonly recur and result in long-term morbidity due to the presence of encrustation and blockage of the catheter by crystalline biofilms that increase resistance to the host immune response and to antibiotics (394). Since the incidence of symptomatic CAUTIs is a major health concern due to the complications and recurrence associated with this type of infection, research directed at understanding the pathogenesis of CAUTIs is warranted and should lead to new and improved diagnosis, prevention, and treatment options.
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Despite innate mechanical safeguards against microbial infection of the intact human urinary tract, specific organisms are capable of colonizing and persisting in this environmental niche. Similar to other mucosal pathogens, uropathogens employ specific strategies to infect the urinary tract, including colonization of a urinary catheter and/or mucosal site (uroepithelial cells), evasion of host defenses, replication, and damage to host cells. The insertion of a foreign body such as an indwelling catheter into the bladder increases the susceptibility of a patient to UTIs, as these devices serve as the initiation site of infection by introducing opportunistic organisms into the urinary tract. The majority of these uropathogens are fecal contaminants or skin residents from the patient's own native or transitory microflora that colonize the periurethral area (56, 66, 217, 288, 462). Transitory microflora that originate from hospital personnel or from contact with other patients may represent antibiotic-resistant nosocomial strains, complicating treatment for these infections. Bacterial entry into the bladder can occur at the time of catheter insertion, through the catheter lumen, or along the catheter-urethral interface (439). The preferred mechanism of bladder entry during CAUTIs is extraluminal (66%), where organisms ascend from the urethral meatus along the catheter urethral interface. Organisms can also enter the bladder intraluminally (34%), where the bacteria migrate into the bladder as a result of manipulation of the catheter system (400, 440).
Indwelling urinary catheters further favor the colonization of uropathogens by providing a surface for the attachment of host cell binding receptors that are recognized by bacterial adhesins, thus enhancing microbial adhesion. Upon insertion, urinary catheters may damage the protective uroepithelial mucosa, which leads to the exposure of new binding sites for bacterial adhesins (108). Lastly, the presence of the indwelling catheter in the urinary tract disrupts normal host mechanical defenses, resulting in an overdistension of the bladder and incomplete voiding that leaves residual urine for microbial growth (133).
Organisms capable of infecting the urinary tract during catheterization use approaches to establish infection that are similar to those used by organisms that cause uncomplicated UTIs. However, due to the introduction of a foreign body, organisms causing CAUTIs require fewer recognized virulence factors to colonize and establish infection than those required by pathogens to infect a fully functional urinary tract.
Bacterial adhesins initiate attachment by recognizing host cell receptors located on surfaces of the host cell or catheter. Adhesins initiate adherence by overcoming the electrostatic repulsion observed between bacterial cell membranes and surfaces to allow intimate interactions to occur (61). These factors are differentially expressed during the course of infection not only for the recognition of different surfaces and cell types that the uropathogen encounters (e.g., in bladder versus the kidney) but also to evade the host immune response. These bacterial cell surface structures recognize specific host cell surface and extracellular matrix components such as proteins, glycoproteins, glycolipids, and carbohydrates. Gram-negative uropathogens produce an assortment of adhesins including those attached to the tip of hair-like projections, known as fimbriae or pili, as well as adhesins anchored directly within bacterial cell membranes, known as nonfimbrial adhesins.
Once firmly attached on the catheter surface or the uroepithelium, bacteria begin to phenotypically change, producing exopolysaccharides that entrap and protect bacteria. These attached bacteria replicate and form microcolonies that eventually mature into biofilms (Fig. 1). During biofilm development, intracellular communication by quorum sensing regulates formation and detachment from biofilms through the collective expression of genes after cellular populations reach a threshold concentration. The rate of genetic material exchange occurring within the biofilm is greater than that between planktonic cells (134, 326), thereby allowing the potential spread of antibiotic resistance genes and other traits. Once established, biofilms inherently protect uropathogens from antibiotics and the host immune response (63). The shedding of daughter cells from actively growing cells and the shearing of biofilm aggregates from the mature biofilm seed other sections of the catheter and bladder.
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FIG. 1. Pathogenesis of biofilm formation on urinary catheters during CAUTIs. The inset (reprinted from reference 393) shows a scanning electron micrograph of a urinary catheter encrusted with P. aeruginosa.
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Once colonized on the catheter and uroepithelium, uropathogens must adapt to the urinary tract environment and acquire nutrients. The production and secretion of degradative enzymes and toxins into the local environment may lead to a breakdown of tissue, releasing nutrients. As iron is a limiting nutrient in the human host (447), uropathogens have developed complex iron acquisition systems such as heme transporters, ferric and ferrous iron transport systems, and siderophore iron uptake systems to circumvent host iron-sequestering mechanisms. Certain uropathogens are capable of using urea, found in high concentrations in human urine (up to 500 mM) (35, 170), as a nitrogen source due to the expression of urease. As a consequence of urease-mediated hydrolysis of urea to ammonia and carbon dioxide, the local environment becomes alkalinized, which leads to the precipitation of polyvalent ions that become enmeshed in the biofilms on catheters and urinary epithelial surfaces (118). These crystalline biofilms must be removed from the host to completely resolve the infection, since antimicrobial agents may be ineffective at eliminating biofilm-associated bacterial populations.
To maintain an infection in the human urinary tract, pathogens must be capable of evading the host immune response. Gram-negative uropathogens enact a number of mechanisms of host immune evasion, including the production of capsules, immunoglobulin A (IgA) proteases, and lipopolysaccharides (LPSs). Capsules, comprised of repeating units of polysaccharides, play a role in evading the immune system by resisting phagocytosis, antimicrobial peptides, and the bactericidal effects of human serum (46, 311, 454). Capsular structures elicit a poor immunogenic response due to their structural similarities to polysialic acid residues found on human cells (415). Additionally, this barrier plays a role in late biofilm development (346) and protects against desiccation and phage attack. It also assists in accelerating the urinary stone crystallization process via electrostatic interactions that accumulate urinary ions at the bacterial surface (55, 87, 408). During the course of UTIs, antibodies that recognize antigenic components of uropathogens are produced. However, proteases targeting immunoglobins and other host defense components such as complement (C1q and C3) and antimicrobial peptides (human beta-defensin 1 [hBD1] and human cathelicidin LL-37) protect uropathogens from the host response (24). LPS, a requisite constituent of gram-negative bacterial outer membranes, is composed of three components: a lipid A molecule that anchors LPS to the membrane, a core consisting of polysaccharides, and a variable O antigen. This macromolecule elicits a potent inflammatory response that initiates the development of septic shock in systemic infections. Various components of LPS have been demonstrated to be important for resistance to antimicrobial peptides (95) and complement-mediated lysis. A summary of virulence factors expressed by gram-negative bacteria is shown in Fig. 2.
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FIG. 2. Virulence factors of the gram-negative uropathogens E. coli and P. mirabilis. IM, inner membrane; OM, outer membrane. (The micrographs are reprinted from references 172, 219, and 346 with permission.)
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E. coli, undoubtedly the most researched microorganism, is a facultative anaerobe that is a member of the family Enterobacteriaceae. While both commensal and uropathogenic E. coli (UPEC) strains colonize the large intestines of humans, only UPEC strains are primarily selected for growth in the urinary tract. Virulence factors that differentiate these avirulent commensals from virulent strains of E. coli were acquired on mobile genetic elements by horizontal gene transfer; examples of such transfer can be found on the E. coli chromosome in the form of pathogenicity islands (125). These virulence factors enable E. coli strains to colonize and persist in the human host despite highly effective host defenses (278). E. coli strains have evolved to cause a variety of human diseases including sepsis, meningitis, diarrhea, and UTIs (276). These organisms are serotypically diverse, spanning over 250 serotypes based on O, H, and K antigens (292). Strains of E. coli associated with infections of the urinary tract are referred to as UPEC strains and are a subset of strains called extraintestinal pathogenic E. coli strains, which cause UTI, sepsis, and meningitis.
UPEC strains are the most commonly isolated organisms in community-acquired UTIs (70 to 90%) and among the most commonly isolated in nosocomially acquired UTIs (50%) including CAUTIs (202). E. coli has been identified as the causative agent in 90% of all case of UTI in ambulatory patients (167). UPEC strains can be classified into four phylogenetic groups, designated A, B1, B2, and D, with strains classified as B2 and D usually causing the most extraintestinal infections including UTIs (287). Since these organisms are capable of colonizing the intestinal and vaginal tracts as well, these sites can serve as potential reservoirs for UTIs and CAUTIs (83, 160).
As with other organisms, UPEC strains possess an arsenal of virulence factors that specifically contribute to their ability to cause disease in the human urinary tract. Genes encoding hemolysin, P fimbriae, S fimbriae, and cytotoxic necrotizing factor 1 (CNF1), for example, have been identified on various pathogenicity islands in different UPEC strains (125). This genetically heterogenous group of organisms varies in its capacity to colonize and persist in the urinary tract (99, 158). DNA microarray analysis of E. coli CFT073, a pyelonephritis strain, compared transcriptional profiles of this strain grown in LB, in human urine, and in the murine bladder cystitis model of infection (126, 376) and verified the in vivo expression of type 1 pili, iron acquisition proteins, and capsule (15, 335, 336, 345). In a prevalence study conducted by Kanamaru et al. (180), who compared 427 E. coli strains (377 UTI isolates and 50 fecal isolates) using PCR assays, the putative virulence factors iroN, iha, kpsMT, ompT, and usp were found 2.0 to 4.3 times more frequently in UTI isolates than in fecal isolates and were strongly associated with a specific anatomical site of infection (i.e., kidney or bladder).
Since UPEC strains are more commonly associated with infections of the intact urinary tract, it is thought that less-virulent organisms are capable of causing complicated UTIs such as CAUTIs. These bacteria may express less and perhaps different virulence factors during this process compared to organisms that are able to infect structurally and functionally normal urinary tracts (261). It has been implied that UPEC strains that infect the catheterized urinary tract have a reduction in the expression of P fimbriae and possibly other factors such as hemolysin, serum resistance, colicin production, and certain H, O, and K serotypes (261). An analysis of 70 clinical urinary strains of E. coli isolated from patients with spinal injuries undergoing long-term bladder catheterization identified that these strains rarely possess a complete arsenal of virulence factors possessed by strains isolated from cases of uncomplicated UTI (26). Among 70 urinary isolates, the prevalences of virulence factors were as follows: mannose-resistant hemagglutinins, 30%; P fimbriae, 17%; hemolysin, 27%; K antigens, 28%; and aerobactin, 33% by bioassay and 39% by gene probe (26). These findings indicate that the presence of a urinary catheter and a neuropathic bladder increases susceptibility to colonization of the urinary tract (26). Despite its prominent role in CAUTIs, limited research specifically addressing UPEC and its ability to cause these types of infections has been performed. Because of this, we will focus on the most recent developments in the research on UPEC and its role in UTIs and, when applicable, any research that is devoted to the field of bacterial virulence during CAUTIs.
UPEC strains and other uropathogens must attach to uroepithelial cells and the catheter surface to colonize and initiate CAUTI and may express a variety of adhesins to assist in this initial attachment. These adhesins also contribute to the direct triggering of host and bacterial signaling pathways, assisting in the delivery of bacterial products to host tissues, and promoting bacterial invasion into host cells (271). A study by Reid et al. (321) suggested that nonspecific adhesins, not specific fimbriae, expressed by UPEC are responsible for attachment to urinary catheter material. It is unknown which specific adhesin molecules are involved in the colonization of UPEC on catheter surfaces. However, potential adhesins associated with UTIs, including type 1, P, S, FC1, and F9 fimbriae and Iha and Dr adhesins, could possibly play a role during CAUTIs. The most extensively studied adherence factors of UPEC are type 1 and P fimbriae (271); an in-depth description of these structures has been reported previously (96, 271).
Type 1 fimbriae, the most frequently expressed virulence factor of UPEC (80 to 100% of strains), are composite helical cell surface structures consisting of repeating major pilin FimA subunits, tip fibrillum (FimF and FimG), and tip adhesin FimH assembled via the chaperone (FimC)-usher (FimD) pathway (344). These pili undergo phase variation and are regulated by the recombinases FimB and FimE.
In a study by Mobley et al. (261) examining urine cultures of 51 long-term catheterization patients over a year, type 1 fimbriae were expressed by a significantly higher number of UPEC isolates causing the most persistent infections than by strains causing transient infections. These pili are thought to be critical for the interaction of UPEC with uroepithelial cells during colonization of the bladder (59, 211, 272, 403). FimH of type 1 pili is thought to be involved in the adherence of these organisms to the bladder epithelium through the recognition and binding of the mannosylated integral membrane glycoproteins uroplakin Ia (467) and uroplakin Ib located on superficial epithelial cells. This tip adhesin also recognizes extracellular matrix proteins including collagen (types I and IV), fibronectin, and laminin as well as Tamm-Horsfall protein. Therefore, these bacterial adhesive structures are able to recognize epithelia (bladder and kidney), immune cells (macrophages, neutrophils, and mast cells), erythrocytes, and extracellular matrix proteins. This tip adhesin may also mediate bacterial autoaggregation and biofilm formation (313, 347, 348).
In addition, type 1 pili are believed to induce an inflammatory response associated with UPEC attachment and invasion (202) through the binding of FimH to specific mast cell receptors that initiate this response by the secretion of inflammatory mediators (1). Lastly, Snyder et al. (377) demonstrated that the expression of type 1 fimbriae coordinately affects the expression of P fimbriae in an inverse manner that may coordinate sequential events during colonization during UTIs in vitro and in vivo as examined by CFT073-specific DNA microarray analysis and mutagenesis studies using a mouse model. This adhesin has been associated with the invasion process as will be discussed later in the review. As these pili have been suggested to be expressed for the initial interactions between UPEC and various surfaces, it is speculated that type 1 pili could be involved in the initial interactions with the catheter surface or in interactions with uroepithelial cells during CAUTIs associated with UPEC.
P fimbriae or pyelonephritis-associated pili (pap) are the second most common virulence factors associated with UPEC uropathogenesis. The genetic determinants responsible for the production of these fimbriae are encoded on the UPEC chromosome by the papABCDEFGHIJK operon. P fimbriae are composed of heteropolymeric fibers consisting of different protein subunits (148), including proteins involved in the structure of the pilus (major pilin PapA, pilus anchor PapH, tip fibrillum PapKEF, and tip adhesin PapG), pilus assembly (periplasmic chaperone PapD and outer membrane usher PapC), and pilus regulation (PapB and PapI). Some studies have shown that P pili are needed by UPEC strains during UTIs; others have failed to show this requirement. UPEC strains expressing P fimbriae attach to globoside residues present on human kidney epithelial cells, which is suggested to play a role in the virulence associated with pyelonephritis (present in 80% of pyelonephritic E. coli strains) as well as ascending UTI (79, 310). Attachment to uroepithelial cell digalactoside receptors mediated by these fimbriae has been shown to induce cytokine secretion (interleukin-6 [IL-6] and IL-8) by this cell type in vitro (137). Studies have proposed that P pili may be important in establishing a bacterial reservoir in the intestinal mucosa (113, 233). However, experimental evidence suggests that these adhesins appear to have a less important role in colonizing abnormal or obstructed urinary tracts (156, 416). Based on these findings, it is thought that P pili may have either no role or a limited role during CAUTIs caused by UPEC.
UPEC is capable of expressing other surface adhesins including S pili (271), F1C pili, F9 fimbriae, IrgA adhesin, and Dr adhesins (271). S pili, consisting of the major subunit SfaA and the minor subunits SfaG, SfaH, and SfaS, recognize and bind sialyl galactosides on human kidney epithelial cells (191) and have been shown to play a role in UTIs caused by UPEC in rats (238). F1C pili, encoded by 14% of UPEC isolates, recognize and attach to kidney epithelial (distal tubules and collecting ducts) and endothelial (bladder and kidney) cells (183). Recently, Ulett et al. described a novel fimbria for UPEC strain CFT073 known as F9 fimbriae (420). These fimbriae were suggested to play a role during biofilm formation and are found in other UPEC and other pathogenic E. coli strains. The precise role of these surface structures during infection is currently unknown. UPEC expresses an iron-regulated gene homologue adhesin IrgA, designated Iha, during UTIs. This outer membrane protein is prevalent among clinical UPEC strains (38 to 74%) (16, 166) compared to fecal E. coli isolates (14 to 22%). Recombinant Iha from the pyelonephritogenic E. coli isolate CFT073 conferred adherence to cultured T-24 human uroepithelial cells to nonadherent E. coli strain ORN172 (163). In addition, a mutant in iha was more attenuated in a mouse model of ascending UTI than wild-type strain CFT073 and UPEC76 (CFT073 pap mutant) (163).
The Dr adhesin family of UPEC includes the uropathogen-associated fimbrial adhesin Dr and nonfimbrial adhesins (AFA-I, AFA-II, AFA-IV, Nfa-I, and Dr-II) (271) and has been associated with cystitis (30 to 50%) in children (114). The Dr operon consists of six genes encoding the main structural subunit DraA, the chaperone DraB (308), the usher DraC (308), the potential invasin DraD (106), DraP, and the adhesin DraE (465). The structural adhesin DraE determines the receptor-binding specificity of Dr adhesins (285). This adhesin is believed to be important in bacterial persistence in the urinary tract through the invasion of bladder and kidney epithelia via the interaction of these fimbriae with decay-accelerating factor (CD55) (286), a regulatory protein that protects tissues from autologous complement-mediated damage. The Dr adhesin also binds type IV collagen (286) and integrins, thus promoting recognition and the adherence of these organisms to interstitial compartments of the kidney, neutrophils, and erythrocytes. Strains of UPEC expressing Dr adhesin were capable of causing chronic experimental pyelonephritis in C3H/HeJ mice (114). They were also capable of long-term survival in human epithelial cells and persisting in the kidneys of experimental animals for months (215). It is unknown if this family of adhesins is expressed during CAUTIs caused by UPEC.
In summary, UPEC is known to express a number of adherence factors that assist in its ability to persist in the urinary tract. However, there is limited research on how this organism adheres to catheter surfaces. It can be speculated that some of the known adhesins that UPEC uses during UTIs could be expressed during CAUTIs caused by this organism as host cell components attach to the catheter surface to provide binding sites. However, more extensive research on the adherence of UPEC during CAUTIs is warranted and necessary to better understand the pathogenesis of this infection.
It is postulated that once UPEC is established on the catheter surface, flagellum-mediated motility is important for the ascent of this uropathogen from the catheter to the bladder and subsequently to the upper urinary tract (ureter and kidney). The synthesis of the flagellar structure is coordinated in a complex regulon consisting of several operons arranged in a hierarchical system (discussed in detail in a review by Fernández and Berenguer [96]). The filament of flagella consists of flagellin, the major filament subunit encoded by the fliC gene, that extends into the extracellular milieu from the outer membrane. The filament is connected to the flagellar hook FlgE through its attachment to the junction proteins FlgK and FlgL and the filament scaffolding protein FlgD (96). Two recent mutagenesis studies by Lane et al. (208) and Wright et al. (459) demonstrated that flagella, while not absolutely required for virulence during UTIs, greatly enhanced the persistence and fitness of UPEC during this type of infection. Therefore, flagellum-mediated motility should likely be considered to be important for the movement of UPEC on the catheter surface and from the catheter surface to the uroepithelium. This, however, has not been directly demonstrated.
Once initial attachment and permanent adherence commence on either the surface of catheters or uroepithelial cells, the establishment of UPEC infection occurs through the colonization of the bladder by the invasion of host cells and the subsequent formation of biofilms. As demonstrated in murine models, UPEC has developed mechanisms to invade host cells, and several reviews discussed this phenomenon in detail (32, 271). UPEC strains have been observed in vitro and in vivo to be internalized by bladder epithelial cells (103, 241, 251) and renal epithelial cells (82, 294, 380, 442). Several adhesins and toxins have been implicated to be involved in the process of invasion, including type 1 fimbriae, the Afa/Dr adhesin family (Dr, Dr-II, F1845, Afa-1, and Afa-3), S pili, P pili, and CNF1. Type 1 fimbria-mediated invasion is dependent upon FimH expression (241). E. coli strains that express Dr adhesins have been observed to invade epithelial cells including Caco-2 intestinal cells (115, 286). Dr adhesin-mediated invasion of uroepithelial cells is dependent upon the presence of the decay-accelerating factor receptor on host cells (117) and may contribute to persistence within the upper urinary tract (271). Research into the roles of S and P pili (241) during bacterial invasion of epithelial cells has not been studied in depth. However, it has been proposed that these pili, in conjunction with toxins, may facilitate the invasion of host tissues (115, 116).
CNF1, discussed later in the review, has been implicated in the invasion of UPEC into uroepithelial cells. This secreted toxin enters the host via the low-pH-mediated endocytotic pathway (54) and then constitutively activates key Rho GTPases that signal the reorganization of the actin cytoskeleton in the host cell. CNF1 has been shown to induce apoptosis in bladder epithelial cells via terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. Thus, this toxin may play a role in bladder cell shedding in vivo and exposing the underlying tissue for bacterial invasion (257).
The ability of UPEC strains to persist in the urinary tract has been demonstrated by Justice et al. to reside within the superficial umbrella cells of C3H and BALB/c murine bladders due to the formation of biofilm structures known as intracellular bacterial communities (IBCs) (175). These IBCs are formed in a sequential manner. First, during murine cystitis, UPEC cells are attached to the cell surface by type 1 fimbriae and then invade the uroepithelium (240, 241) 1 to 3 h after the initial inoculation. Localized actin rearrangements occur and engulf the bound organism via zipper-like phagocytosis (240). After being internalized in the murine superficial bladder cell, UPEC replicates rapidly, forming clusters known as early IBCs (175). Recently, type 1 fimbriae have been shown to have an additional intracellular role during this stage of IBC formation (460). As IBCs mature, around 6 to 8 h after inoculation of the murine bladder (175), they more closely resemble classical biofilm structures (88), where the bacterial doubling time is increased (from approximately 30 to 60 min) and the bacterial cell length is shortened (0.7 µm versus 3 µm). At this stage, pod-like protrusions are observed on the surface of murine bladder epithelial cells (8). Around 12 h postinoculation, bacterial detachment is observed (175) as either a whole community or individual highly motile cells that burst out of the murine bladder lumen in a process referred to as fluxing (7). It is postulated that IBCs and biofilms contribute to the persistence of these organisms due to the increase in resistance to antibiotics and the host immune response. IBC formation has not yet been substantiated in humans.
There are several factors that are known to contribute to the formation of biofilms by E. coli. These include fimbriae, curli, flagella, antigen 43, and extracellular matrix molecules including cellulose, colanic acid, and poly-β-1,6-N-acetyl-D-glucosamine (67, 68, 74, 81, 437, 469). Specifically, biofilm formation mediated by type 1 fimbriae may assist in the colonization of urinary catheter surfaces (271).
There have been recent studies examining biofilm formation on catheters and UPEC. Ferrières et al. (97) showed that certain catheter materials such as silicone and silicone-latex actually select for and promote biofilm formation for the most virulent UPEC strains, whereas asymptomatic bacteriuria strains form better biofilms on polystyrene and glass (Fig. 3). Koseoglu et al. (194) revealed that UPEC type O4 had formed mature biofilms after 12 to 24 h and developed biofilms completely in almost all latex/silicone balloon catheter samples after 4 to 7 days as examined by scanning electron microscopy.
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FIG. 3. Cross section of a silicone catheter removed from a patient after blockage. Crystalline material can be seen completely occluding the catheter lumen. (Reprinted from reference 387a) with permission of the publisher.)
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Once established, bacteria must express factors to avoid the host immune response in order to persist in the urinary tract; these factors include fimbriae that are subject to phase variation, capsules, and LPS. As mentioned above, type 1 fimbriae are subject to phase variation to evade the host immune response. UPEC strains, as with other uropathogens, have been shown to produce an exopolysaccharide capsule as a means of avoiding the immune response and thereby contributing to serum resistance. There are over 80 types of these capsular polysaccharides (CPSs) (K antigens), with K1 capsules most frequently observed among urinary and clinical isolates (160, 291). These organisms produce group II or group III capsules, compared to commensals that express group I polysaccharide capsules (161). These capsules are thin, highly anionic structures that tend to aggregate spontaneously (174). The gene locus encoding the group II capsule has been described for UPEC strain NU149 and is organized into three regions. The genes involved in the assembly and transport of capsules are located at conserved regions designated regions 1 (kpsFEDUCS) and 3 (kpsMT). In contrast, region 2 (kfiABCD) is unique to each serotype and is involved in capsular biosynthesis (451). Schwan et al. (355) described the down-regulation of the kpsFEDUCS operon, possessing the genes of capsule assembly region 1 responsible for K-antigen expression (451), in UPEC upon type 1 fimbria attachment to mannose-coated Sepharose beads as a possible initiating event of UTIs. These acidic polysaccharide capsules assist in the avoidance of phagocytosis and complement activation (161). There are no studies that have investigated the role of these structures during CAUTIs caused by UPEC.
Upon entry into the bladder, UPEC contacts uroepithelial cells and initiates a robust innate immune response through the activation of various signaling pathways. Cell activation is accomplished by interactions between bacterial surface molecules, such as fimbriae and LPS, and uroepithelial cell receptors. LPS, a critical constituent of the gram-negative cell wall, was shown to be important for resistance to antimicrobial peptides (95) and complement-mediated lysis. However, the compound activates cells of innate immunity via its interaction with Toll-like receptor 4 (TLR4) and CD14 located on the host cell membrane (349, 350). These TLR molecules (TLR1 to TLR10) are pattern recognition receptors expressed on the cell surfaces of leukocytes and epithelial cells that recognize preserved molecular motifs located on various pathogens (202). TLR4 is important in these events since mice deficient in this surface receptor are incapable of efficiently clearing UPEC from the bladder (127). These initial interactions activate signal pathways that subsequently lead to the release of cytokines (tumor necrosis factors, gamma interferon, IL-8, and IL-1) that guide neutrophil infiltration, stimulate complement and coagulation (130, 351), and trigger the shedding of superficial bladder facet cells (273). Prior to expulsion from the host, intracellular UPEC emerges from these exfoliated bladder epithelial cells to invade the exposed underlying tissues to persist in the host. Type 1 fimbriae, as described by Blomgran et al. (30), interact with neutrophils in a mannose- and LPS-dependent manner, which leads to subsequent neutrophil apoptosis. It is suggested that LPS plays an important role in the persistence of UPEC during UTIs and CAUTIs. However, there is currently no experimental evidence that substantiates these claims.
Once established on catheter and uroepithelial surfaces, uropathogens such as UPEC strains must adapt to the urinary tract environment and acquire nutrients. This is accomplished in part by the production of degradative enzymes, toxins, and nutrient acquisition systems. These factors include toxins such as hemolysin, CNF1, and autotransporters such as secreted autotransporter toxin (Sat), Pic (139, 295), and Tsh (139) to break down host peptides and iron acquisition proteins to acquire iron sequestered by the host.
UPEC strains expressing toxins such as hemolysins, CNF, and Sat have been isolated (121, 144, 202). Of the 91 UTI isolates examined by Caprioli et al. (47), 37% produced both the hemolysin HlyA and CNF1, whereas only 1 of 114 fecal isolates produced CNF1. The two common forms of hemolysin expressed by UPEC are the alpha-hemolysin HlyA (384), and cell-associated beta-hemolysin. The presence of hlyA was found to be 31 to 48% for UTI isolates, compared to only 15% for fecal isolates (239). The genes encoding these heat-labile secreted pore-forming proteins are found on either plasmids or the chromosome (252). Hemolysin has been shown to lyse human kidney proximal tubule cells (263). The effect of these toxins is the induction of cell lysis due to an increase in intracellular osmotic pressure caused by the dissipation of calcium ion gradients. A proposed function of these toxins is to cause the release of host cell iron into the extracellular environment that can then be captured by the bacterium.
The toxins classified as CNF (CNF1 or CNF2) are known to constitutively activate the Rho GTPases RhoA, Rac1, and Cdc42, which regulate the actin cytoskeleton (75). Cells exposed to this toxin form enlarged multinucleated cells, as exhibited by membrane ruffling, the formation of focal adhesions and actin stress fibers, and DNA replication in the absence of cell division. The existence of the CNF1 gene was found in 27 to 41% of UTI isolates but in only 9% of fecal isolates (239). CNF1 has been shown to modulate polymorphonuclear leukocyte function through the down-regulation of phagocytosis and altering the distribution of the complement receptor CR3 (CD11b/CD18). A study by Falzano et al. (94) supplied evidence that CNF1 of UPEC is capable of blocking the cell cycle G2/M transition in the T24 uroepithelial cell line.
UPEC has been shown to secrete a number of autotransporters during UTIs. The autotransporter toxin known as Sat (121) is a vacuolating cytotoxin that damages kidney epithelial cells during acute pyelonephritis in the mouse model (122). UPEC strains have also been found to express two other autotransporters, Pic and Tsh, that possess serine protease activity (139). These proteins are expressed in vivo during pyelonephritis in the mouse model (139). Since these proteins have been shown to cause host damage during UTIs in an animal model, it is plausible that these autotransporters could be expressed by UPEC during CAUTIs. However, research to address this hypothesis has not been undertaken.
Although nutrient acquisition systems are important for the survival of UPEC during growth in the urinary tract, this review focuses on the nutrient acquisition systems for iron, as these are the best-studied systems. Iron acquisition systems are important virulence factors produced during UTIs and CAUTIs, as this nutrient is limiting in urine (363). Since iron acquisition is important for the viability of UPEC during infection, these organisms produce multiple systems including siderophore-siderophore receptor and heme uptake systems (334). As stated above, E. coli strains likely use alpha-hemolysin as another means of retrieving iron from the host (290). The known siderophore systems of UPEC include the catechol enterobactin and the hydroxamate aerobactin along with their corresponding receptors, such as the IreA, IroN, and Iha receptors (161, 378). Most E. coli strains produce the siderophore enterobactin. However, the genes encoding aerobactin are found significantly more often in strains isolated from the urinary tract (69.4% of 124 isolates [P = 0.001]) than in fecal E. coli samples (41.2% of 51 isolates) (77). UPEC strains have been shown by hydrolysis fluorescence detection to produce novel siderophores including salmochelins, C-glucosylated enterobactins that are dependent upon the biosynthesis of enterobactin and the iroBCDEN operon (468), and yersiniabactin (423).
Iron acquisition systems have been shown to be important during UTIs caused by UPEC. Snyder et al. demonstrated that five iron acquisitions were upregulated in UPEC strain CFT073 during infection in the CBA mouse model of ascending UTI compared to static in vitro growth in LB (376). Recently, Reigstad et al. (322) revealed that heme- and siderophore-associated iron acquisition systems play key roles in IBC development in female C3H/HeJ mice. Further research is needed to determine if these acquisition systems are important during CAUTIs caused by these organisms.
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Proteus species, members of the family Enterobacteriaceae (301), are distinguishable from most other genera by their ability to swarm across an agar surface. These organisms are widely distributed in the environment, including polluted water, soil, and manure, where Proteus plays a role in the decomposition of organic matter from animals, and in the intestinal tract of mammals. Proteus species are the causative agent of a variety of opportunistic nosocomial infections including those of the respiratory tract, eye, ear, nose, skin, burns, throat, and wounds; it also may cause gastroenteritis (302, 331). Antibodies specific for Proteus have been isolated from patients with active rheumatoid arthritis (76, 90), suggesting some association with this malady. Proteus bacilli are more commonly associated with UTIs in those individuals with structural or functional abnormalities, especially ascending infections in patients undergoing urinary catheterization (441, 444). Colonization of the intestinal tract allows Proteus to establish reservoirs for transmission into the urinary tract by intermittent colonization of the periurethral region. This intermittent colonization can lead to the subsequent contamination of the catheter, thus allowing nosocomial infections to develop (53).
Proteus-associated UTIs may be difficult to treat, and the bacterium persists due to complications associated with this type of infection, including bladder and kidney stone formation (urolithiasis) that can lead to the obstruction of catheters and the urinary tract (200, 356, 444). The three species of Proteus associated with UTIs are Proteus mirabilis, Proteus vulgaris, and Proteus penneri. While UTIs caused by P. vulgaris (361) and P. penneri (199, 200) have been identified, P. mirabilis is the third most common cause of complicated UTI (12%) and the second most common cause of catheter-associated bacteriuria in patients catheterized long term (15%) (439).
P. mirabilis is a common cause of CAUTIs. It was observed by Roberts et al. (327) that P. mirabilis has the greatest ability to attach to catheters out of all gram-negative organisms. As monitored by a low-light optical imaging system, catheter sections colonized with bioluminescent P. mirabilis were inserted into murine bladder lumen. These animals developed severe cystitis that persisted significantly longer than that in mice challenged with bacterial suspensions alone and required prolonged antibiotic treatment to reduce the infection (178). To establish and maintain infections of the urinary tract and colonization of catheters, Proteus species must adapt to the catheterized urinary tract and produce an arsenal of strictly regulated virulence factors.
Indwelling urinary catheters serve as the initiation site of CAUTIs by introducing uropathogens such as Proteus spp. into the urinary tract and providing a surface for coating by host cell debris and protein that may be recognized by bacterial adhesins. P. mirabilis strains tend to attach to catheters with a greater propensity than other gram-negative bacteria (327). Studies have demonstrated that P. mirabilis strains are capable of attaching to a number of catheter polymers including ethylene, propylene, polystyrene, sulfonated polystyrene, silicone, and red rubber (135, 327).
To facilitate binding to these different surfaces, P. mirabilis must be capable of producing a variety of adherence factors, such as fimbriae and hemagglutinins, that are thought to play an important role in the establishment of CAUTIs. Proteus species have been shown to produce various fimbriae and hemagglutinins involved in the colonization of the urinary tract and possibly catheter surfaces, including MR/P fimbriae (289), MR/K hemagglutinin (289), uroepithelial cell adhesin (UCA)/nonagglutinating fimbriae (NAF) (456), P. mirabilis fimbriae (PMF) (10), and ambient-temperature fimbriae (ATF) (243). Others have also been predicted for the genome sequence of strain HI4320 (M. Pearson, J. Parkhill, and H. L. Mobley, unpublished data).
MR/P fimbriae are perhaps the best-understood fimbriae expressed by P. mirabilis strains during UTIs. These fimbriae are thick channeled (7 to 8 nm) and are classified as mannose-resistant fimbriae (36, 371). These fimbriae assemble through the chaperone-usher pathway (379). The genes required for the expression of MR/P fimbriae on the cell surface are encoded on the Proteus chromosome on two divergent transcripts, mrpABCDEFGHJ (designated the mrp operon) and mrpI (14). Some of the proteins encoded by the mrp operon include the fimbrial structural subunit MrpA (13); the terminator for fimbrial assembly, MrpB (222); the minor fimbrial subunit MrpG (224); the tip adhesin MrpH (219); and the repressor of flagellin synthesis, MrpJ (223).
Expression of MR/P fimbriae is subject to phase variation (13, 14). The mrpI gene encodes a site-specific recombinase that reverses the orientation of the 251-bp invertible element that precedes the mrp operon. Expression of MR/P fimbriae correlates with the orientation of this invertible element (466). MrpI orients the invertible element in either an "on" position, allowing the expression of the MR/P fimbria, or an "off" position, in which the promoter is in the opposite orientation and is thus unable to drive transcription.
Many studies have suggested that MR/P fimbriae play a role in the virulence observed during UTIs caused by uropathogenic P. mirabilis strains. In the CBA model of ascending UTI, infection with P. mirabilis elicited a strong immune response to MrpA, the major structural subunit of MR/P fimbria, indicating that MR/P fimbriae were expressed in vivo (11). Isogenic mutants incapable of expressing MR/P fimbriae were attenuated when examined in this mouse model (12, 219, 221, 224). A mutant constitutively expressing MR/P fimbriae outcompeted the wild-type strain in the murine bladder but not the kidneys in a cochallenge experiment, thereby establishing MR/P fimbriae as being an important bladder colonization factor for P. mirabilis (221). Tissue binding studies by Sareneva et al. (343) revealed the propensity of this fimbrial type to adhere specifically to the human renal tubular epithelial cells and to the exfoliated uroepithelial cells of urinary sediment.
Experiments conducted by Jansen et al. (155) suggest that MR/P fimbriae dictate the localization of bacteria in the bladder and contribute to biofilm formation, a process essential for the establishment of CAUTIs. A P. mirabilis HI4320 construct with an invertible element locked in the "on" position colonized the luminal surfaces of murine bladder umbrella cells and formed significantly more biofilms after 2 days of growth in urine (P = 0.05) compared to a construct with the invertible element in the "off" position. The off-position mutant colonized the lamina propria underlying exfoliated uroepithelium. Although studies have associated the expression of MR/P fimbriae with virulence during UTIs caused by P. mirabilis, there is no direct evidence substantiating a role of these fimbriae in CAUTIs.
On the other hand, MR/K fimbriae have been linked with the attachment of organisms to catheter surfaces and with the persistence of catheter-associated bacteriuria (262, 331, 461). These fimbriae have also been detected during Providencia stuartii infections in catheterized elderly patients (262). Expression of these thin (4- to 5-nm) nonchanneled type 1 mannose-resistant fimbriae (36, 371) enables P. mirabilis to attach tightly to the Bowman's capsule of the host kidney glomeruli and to the tubular basement membranes (343). Although more associated with P. penneri strains (461), it is speculated that MR/K fimbriae play a possible role in the initial adherence to catheter biomaterials during P. mirabilis CAUTIs.
Besides MR/P and MR/K fimbriae, the other fimbriae produced by P. mirabilis during UTIs may contribute to attachment to the catheter surface. Surface adhesins determined not to be involved in the hemagglutination caused by MR/P and MR/K fimbriae have been identified in P. mirabilis, including UCA/NAF (10, 29, 60, 406, 456), PMF (244), and ATF (242). Wray et al. (456) characterized UCA, a nonagglutinating fimbria from P. mirabilis HU1069 that was demonstrated to weakly attach to exfoliated human desquamated uroepithelial cells. The 540-bp ucaA gene, which encodes the major fimbrial subunit of UCA, has nucleic acid homology to the F17A gene of E. coli F17 pilin (58%) (60) and was identified in all 26 P. mirabilis strains tested (29). Due to its homology to the F17 pilin of E. coli (60), it has been suggested that these fimbriae might be involved in the colonization of the intestines by these organisms (58). Based on studies conducted by Bahrani et al. (10, 11, 13), there was some ambiguity as to which fimbrial types were identified as UCA (456) since thin (4-nm) and thick (6-nm) fimbrial filaments were observed by electron microscopy, and multiple bands were isolated on sodium dodecyl sulfate-polyacrylamide gels (406). These fimbrial subunits were isolated and characterized from P. mirabilis strain 7570 from a patient with struvite urolithiasis and renamed NAF by Tolson et al. (406). The N-terminal sequence of this fimbrial subunit was confirmed to be identical to the N-terminal sequence from P. mirabilis strain HU1069 of the study reported by Wray et al. and not homologous to the N termini of MR/P, ATF, or PMF fimbrial subunits (406). Bacteria expressing NAF adhered strongly to a number of cell lines in vitro, including uroepithelial cells (407) and MDCK (Madin-Darby canine kidney) (5, 216) and EJ/28 urinary tract tumor (214) cell lines. Purified NAF from P. mirabilis binds to a number of glycolipids such as asialo-GM1, asialo-GM2, and lactosyl ceramide, as demonstrated by thin-layer chromatography overlay assays and solid-phase binding assays (216). Because of its homology to fimbriae that assist in intestinal tract colonization, it is possible that these fimbriae may play a role in the initiation of CAUTIs by allowing P. mirabilis to attach and establish in the intestines and thus form a reservoir of organisms that can potentially cause CAUTIs. However, there have been no definitive studies examining this possibility.
PMF are encoded by genes located in the pmf gene cluster and consist of five polypeptides: PmfA, the 18.9-kDa major subunit of PMF (10); PmfC (93.1 kDa); PmfD (28.2 kDa); PmfE (38.9 kDa); and PmfF (19.7 kDa). The pmf gene cluster has >25% amino acid sequence identity with the pap, mrp, and sfa fimbrial gene clusters. However, pmfE has been identified as being unique to this gene cluster. Thus far, no regulatory elements for the production of these fimbriae have been identified (245). There are conflicting results as to the function these fimbriae during UTI. In a study by Massad et al. (244), PMF were demonstrated to play a role during the colonization of the bladder since an isogenic mutant in the pmfA gene of P. mirabilis HI4320 was 83-fold more attenuated than the wild-type strain during independent challenge in the CBA mouse model of ascending UTI (244). However, in this same study, PMF could not be shown to be involved with attachment to human uroepithelial cells since attachment to this cell type is similar in both the wild type and the pmfA mutant. Its role in the colonization of the kidney is also questionable since no significant difference between numbers of the wild type and pmfA was observed in kidney tissue (244). Contrary to this, a study by Zunino et al. (472) showed that there was significant attenuation observed in the kidney and bladder by the isogenic pmfA mutant compared to the parent strain Pr2921 during cochallenge in a model of ascending UTI in female CD-1 mice. Furthermore, attachment of this isogenic pmfA mutant to T24/83 human-derived bladder carcinoma cells and human uroepithelial cells was significantly less than that of the wild type. These conflicting results require resolution.
ATF were classified as a new fimbrial type, as examined by electron microscopy and immunogold labeling (243), and were identified in all eight P. mirabilis strains analyzed. The genes responsible for the production of ATF are organized in the atf gene cluster and encode a 19-kDa major-subunit AtfA (243), the chaperonin-like protein AtfB, and the outer membrane usher AtfC (243). AtfA has significant amino acid sequence identity to type 1 major fimbrial subunits of several enteric species (38% to 41%) (243). An allelic-replacement atf mutant colonized the murine urinary tract at a gene comparable to that of the wild type in independent challenge and outcompeted the wild type in cochallenge experiments in the murine model of ascending UTI (243). As nonclinical strains of Proteus were shown to express AtfA, as observed by Western blot analysis (470), it is suggested that these fimbriae are involved in the colonization of P. mirabilis in the environment and are most likely not involved in CAUTIs.
Currently, only MR/K fimbriae are known to be associated with the process of attachment during CAUTIs. Clearly, additional studies must ascertain whether known factors or currently uncharacterized factors are involved in adherence, as this process is essential for these types of infections. The identification of novel adherence factors as well as other virulence factors will be facilitated by the recent annotation of the P. mirabilis HI4320 genome by the Sanger Centre in conjunction with the Mobley Laboratory.
In general, flagella on the surface of bacterial pathogens are thought to assist in host colonization and dissemination, initial attachment, and sensing of the extracellular environment (25). For Proteus species, these surface structures are important in the process known as swarming, a distinct characteristic of these organisms. Therefore, it is speculated that flagellar motility and, potentially, swarming are important during CAUTIs, as the ability of P. mirabilis to disseminate from the initial site of colonization on the catheter surface to the uroepithelial cells of the urinary tract is critical for the establishment of these types of infections.
Swarming is a surface-induced multicellular differentiation process that allows organisms to move in a coordinated manner and expand the population to new locations over solid surfaces (258, 318, 453). During growth in liquid medium, Proteus species assume the form of an infectious single-cell, motile, 1.0- to 2.0-µm-long bacillus that displays a distinct phenotype including the presence of peritrichous flagella on its cell surface and swimming behavior. However, when transferred onto solid medium, these swimmer cells differentiate into hyperflagellated, multinucleated, nonseptated elongated swarmer forms measuring 20 to 80 µm in length. These differentiated swarmer cells migrate out from the original inoculation site in a rapid and highly coordinated manner that is dependent upon multicellular interactions and cell-to-cell signaling (18).
Swarmer cells align themselves in multicellular rafts and are enveloped in the extracellular slime material of the colony migration factor Cmf that is required for and facilitates translocation through a reduction in surface friction (124, 172, 381). The swarming process continues until the cell number is reduced by cell loss or when the bacterial mass changes the direction of motion (18). The cessation of movement, known as consolidation, is accompanied by the dedifferentiation and replication of swarmer cells into vegetative swimmer cells. This periodicity distinguishes P. mirabilis swarming from other swarming processes. For a more in-depth description on the process of Proteus swarming, refer to reviews by Rather (318) and Rozalski et al. (331).
Since swarming is such a dominant characteristic of this genus, any factors that affect or regulate this phenomenon would likely affect the fitness of the organism. The swarming phenomenon is a metabolically complicated and demanding process that must genetically coordinate the expression of over 50 genes (21), including those involved in the production, assembly, and operation of flagella and virulence factors such as flagellin, urease, hemolysin, and the ZapA metalloprotease (4, 24).
To identify potential genes that may be involved in the process of swarming, transposon mutagenesis studies of P. mirabilis using Tn5 transposons were performed (20, 21, 23, 41). Those studies identified over 50 genes that were involved in the swarming process and included genes that encoded proteins involved in flagellar biosynthesis (20), flagellar rotation (20), surface elongation (20), control and coordination of multicellular motility (20), production of LPS and peptidoglycan (23), and cell division (23). Burall et al. (41) identified a mini-Tn5 transposon mutant in dsbA of P. mirabilis HI4320 that was defective in the colonization of the murine urinary tract and in swarming. This gene encodes an oxidoreductase that forms disulfide bonds in periplasm proteins. However, no definitive role of this gene during swarming has been defined. The utilization of signature-tagged mutagenesis has greatly assisted in the identification of genes not previously associated with the swarming process, and any of these genes could play an essential part in the pathogenesis of CAUTIs caused by P. mirabilis.
The definitive roles of flagella and swarmer cell differentiation in the virulence of P. mirabilis during UTIs remain controversial, but some of these suggested roles include dissemination of P. mirabilis from the initial site of infection to other sections of the catheter or to the urinary tract and avoidance of the host immune response. Flagella are believed to contribute to the virulence of swimmer cells by allowing motility from the catheter to the bladder epithelium and onward, ascending into the ureters and kidneys. An isogenic, nonpolar, nonmotile, flagellum-negative mutant in flaD of P. mirabilis WPM111 was attenuated 100-fold compared to the wild type in the CBA model of ascending UTIs, indicating the importance of flagella in murine UTIs (259).
The biosynthesis of flagella is a key process in both motility and swarming and involves numerous genes on the Proteus chromosome (22). Flagellin is encoded by flaC, and the flaD gene encodes the flagellar filament capping protein (19). Studies suggest that the major flagellin protein for P. mirabilis is subject to antigenic variation through homologous recombination as three copies of flagellin-determinant gene (flaA, flaB, and flaC) that reside on the P. mirabilis genome with only one copy that is actively expressed (19, 275). It was proposed that flagellin gene rearrangement is a mechanism for host immune system evasion by P. mirabilis and is extremely relevant for Proteus infections since flagella are highly immunogenic. As a result, any antigenic change could increase the survival of Proteus species in the urinary tract through the evasion of secretory IgA directed toward flagella during colonization in the bladder (19). Due to its relevance during UTIs, it is probable that antigenic variation via flagellin gene rearrangement is a method of host immune response evasion by P. mirabilis during CAUTIs.
Swarming cell differentiation is thought by some to be important for the virulence of P. mirabilis during UTIs since several virulence factors, including flagellin, urease, the hemolysin HmpA, and the IgA metalloprotease ZapA, are upregulated in the differentiated swarmer cell compared to swimmer cells (4, 101). Mutants in FlhA synthesis, proteins required for flagellar synthesis, are nonmotile due to the loss of fliC transcription but also have reduced transcription of hpmA hemolysin (123). Therefore, it has been suggested, based in part on evidence of the coordinate expression of virulence factors during swarming cell differentiation, that factors involved in the swarming process are critical for pathogenesis and that a similar signal must be regulating both swarming and virulence (3, 4, 52, 258). Interestingly, however, swarmer cells are rarely observed in the murine model of ascending UTI, bringing into question the relevance of this morphotype in the absence of a catheter (154).
Swarming may play a role in the migration of Proteus strains on catheter materials; however, swarmer cells of Proteus species are capable of migrating across 1-cm-long sections of Foley catheters consisting of either hydrogel-coated latex, hydrogel/silver-coated latex, silicone-coated latex, and all silicone in vitro (337). Swarmer cells of P. mirabilis have been observed to migrate through populations of E. coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis and then continue to migrate with little or no reduction over hydrogel-coated latex catheter sections (337). Nonswarming mutants were shown to have lost the ability to migrate over these catheter sections (172). However, upon their introduction into models of the catheterized bladder, these mutants were just as capable of encrusting and blocking catheters as the wild type (171). It seems that while swarming might have a role in the initiation of infection, facilitating the passage of the cells from the urethral meatus to the bladder, it is not required for the rapid formation of crystalline biofilm once bacteria have colonized the residual urine in the bladder.
There is conflicting experimental evidence about the significant of swarming during the ascension of P. mirabilis into the urinary tract and during pyelonephritis. Swarming-defective mutants and motile, nonswarming mutants of P. mirabilis were significantly attenuated in the colonization of the kidney compared to the wild type upon intravesical (bladder) inoculation of mice (2). Furthermore, the swarming-defective mutant was still able to colonize the murine bladder albeit at a lower rate than that of the wild-type strain. In contrast, the nonswarming mutant was incapable of colonization of the murine kidney. These findings suggest that swarming is important for P. mirabilis-associated ascending UTIs and pyelonephritis. Histological analysis of murine renal tissue has supported these results, as swarmer cells were found to be the predominant cell type (2). Other studies suggested that flagella and/or swarming is not involved in the process of ascension into the upper urinary tract by P. mirabilis. A clinical strain of P. mirabilis that lacks flagella has been isolated (471). In addition, confocal microscopy studies by Jansen et al. (154) demonstrated that the predominant P. mirabilis morphotype was the short swimmer cell, not the swarmer cell, in the mouse model of ascending UTI. However, the differences observed between these experimental results are most likely due to differences in experimental parameters for each study (318). More conclusive studies are required to resolve these conflicts.
After the initial colonization of the catheter surface, Proteus species, as with other uropathogens, form distinctive crystalline biofilm structures during CAUTIs. These structures assist in the persistence of P. mirabilis in the urinary tract by protecting these organisms from antibiotics and the host immune response and obviously contribute to adhesion to surfaces (146). Urinary stone formation during Proteus-mediated UTI is characteristic of this type of infection and is critical for the development of crystalline biofilms. Bacterially derived stones account for up to 30% of all urinary tract stones worldwide and account for approximately 75% of the urinary stones classified as staghorn calculi (141). Upper urinary tract stones are classified as staghorn calculi if stone formation occurs in the renal pelvis and extends out into at least two calyces (253). Crystalline biofilms are especially problematic during CAUTIs since catheters become blocked due to encrustration caused by the formation of these structures.
It should be recognized that there are powerful physical and chemical factors involved in the initiation and development of the crystalline biofilms that block catheters. Experiments in parallel-plate flow cells showed that when urine cultures flow over polymer surfaces, the pH of the urine can be a major factor in determining bacterial adhesion. For example, some polymers with strongly-electron-donating surfaces will resist colonization by cells until the pH of the urine rises above the pH at which calcium and magnesium phosphates precipitate out of solution. In alkaline urine, macroscopic aggregates of cells and crystals form in the urine, settle on the polymer surface, and initiate crystalline biofilm formation (391). These observations indicate that to stop biofilm formation on devices in the urinary tracts of patients infected with P. mirabilis, it is essential to prevent the rise in urinary pH and the crystallization of apatite and struvite (Fig. 4).
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FIG. 4. Crystalline material that blocked a patient's catheter after just 4 days. The large coffin-shaped crystals were shown by X-ray microanalysis to be a form of magnesium ammonium phosphate (struvite), and the microcrystalline aggregates were shown to be calcium phosphate (apatite). A four-membered bacterial community was isolated from this crystalline biofilm composed of E. coli, P. aeruginosa, E. faecalis, and P. mirabilis. (Modified from reference 390a with permission from Elsevier.)
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Bacterial urease and capsule polysaccharides are two major factors known to be involved in urinary crystal formation and, hence, crystalline biofilm formation in P. mirabilis (277). Since urea is present in concentrations of up to 500 mM in human urine (35, 170), it is not surprising that bacterial ureases play a pivotal role in Proteus-associated UTI. Urease contributes to the development of urinary stones due to urease-mediated hydrolysis of urea to ammonia and carbon dioxide that alkalinizes the local environment. This increase in urinary pH causes the local supersaturation and precipitation of calcium phosphate and magnesium-ammonium phosphate from urine to form crystals of carbonate apatite [Ca10(PO4)6CO3] and struvite (MgNH4PO4·6H2O), respectively (118). These crystals accumulate in the biofilms of catheters and urinary epithelial surfaces and eventually obstruct the flow of urine through the catheter and from the bladder or kidney. Incontinence can develop due to urine leakage around the catheter or retention of urine in the bladder that can seriously complicate the care of patients undergoing long-term bladder catheterization (392). Urease is produced by Proteus species known to cause clinical infections (P. mirabilis, P. vulgaris, and P. penneri) (266), and the urease produced by P. mirabilis is the best characterized one.
The P. mirabilis urease is a 250-kDa multimeric nickel metalloenzyme that is produced in the cytoplasm (266). As this enzyme is inducible in urea, it is assumed to be constitutively expressed during growth in urine (58). This urease is homologous to the urease of Klebsiella aerogenes (266), and the urease operon of P. mirabilis has homology to the urease operon of Providencia stuartii (169, 265, 435) and likely all urease genes. This operon possesses seven genes, ureDABCEFG, that encode proteins involved in the production of urease. The urease apoenzyme is composed of a trimer of trimers consisting of the polypeptides UreA, UreB, and UreC [(UreABC)3] (264) and is activated upon the insertion of the divalent nickel metallocenter into each of the UreC subunits. The insertion and assembly of the nickel ions into the metallocenter are accomplished by the urease accessory proteins UreD chaperone (258), UreE (nickel ion donator), UreF, and UreG (264). The exact mechanism for the assembly of urease is not fully understood (331).
The urease gene cluster is regulated by UreR and the histone-like nucleoid structuring protein (H-NS). The 33-kDa polypeptide UreR, a member of the AraC/XylS family of transcriptional activators (281), initiates transcription of the genes encoding the urease subunits and accessory proteins and of its own gene in a urea-inducible manner through binding to the intergenic region between ureR and ureD (84, 150, 405). UreR binds the promoters of the ureR and ureD genes in the absence of urea, albeit with less affinity than in the presence of urea, suggesting that the organism is prepared for the rapid induction of urease (84, 150, 405). The transcriptional repressor H-NS recognizes and binds to the poly(A)