**2. Virulence factors of UPEC**

### **2.1. Adhesins**

Adhesive proteins as the most important determinants of pathogenicity of UPEC strains are arguable [13], but based on many observations of ABU strains, it was found that these strains are nonadherent and nonhemolytic [14]. UPEC adhesins activate host signaling pathways that promote bacterial invasion [15]. Bacterial adherence to urothelium is important in the development of UTI because it allows the bacteria to persist in the urinary tract against flushing by urine flow. Function of type 1 fimbriae as virulence factors in human pathology remains unclear because they are expressed in both commensal and pathogenic *E. coli* strains [16, 17]. The type 1 fimbriae are heteropolymeric surface organelles that consist of several subunits. These fimbriae bind *E. coli* cells to the urothelial mannosylated glycoproteins uroplakin by subunit FimH, which is located at the distal tip of the type 1 fimbriae. UPEC commonly expresses FimH that efficiently bind monomannose- and trimannose-containing glycoprotein receptors. Whereas, commensal *E. coli* strains usually bind to trimannose residues [18]. Binding of FimH to uroplakins that are expressed in the differentiated urothelium of the bladder and urethers causes adhesion and cellular invasion of *E. coli* and promotes formation of intracellular bacterial communities which leads to the acute stage of infection [19, 20]. FimH adhesin enables UPEC to escape before the immune response by internalization within urothelial cells. Inside infected urothelial cells, *E. coli* is harbored within vesicles [21, 22]. Blocking of FimH adhesin with antibodies or inactivity of the *fimH* gene has a negative effect on the ability of UPEC to colonize the bladder epithelium [5]. *fimH* gene is the most commonly identified virulence gene in the isolates causing UTI [17].

gastroenteritis but sometimes are responsible for diseases outside the intestinal tract [2]. Three pathotypes of the ExPEC are able to exist in the gut but do not cause diseases in this place. Whereas, colonization by the ExPEC strains of other host niches including the central nervous system, blood, and the urinary tract leads to illness in human [3]. Among ExPEC, uropathogenic *E. coli* (UPEC) is the most frequently associated with human diseases. UPEC strains cause 80–90% of community-acquired UTIs and more than 30% of hospital-acquired UTIs [4]. Development of UTIs depends on anatomical factors of host, defense mechanisms, and virulence factors of infecting microorganism. Bacterial infections of the urinary tract are important problem, because about 60% of women in the United States will have at least one UTI during their life. About 8 million physician visits per year are related to these often chronic infections, making UTIs a problem of economic and medical significance [5]. UPEC can colonize the bladder and cause cystitis or may ascend into the kidneys, causing pyelonephritis [3]. *E. coli* may also spread from the urinary tract to the bloodstream causing bacteremia in above 30% of cases and the potential sepsis [6]. The presence of high numbers of *E. coli* in the urine without the clinical symptoms is referred as asymptomatic bacteriuria (ABU) and such infection in healthy, nonpregnant women is generally not treated [7]. Infections of the urinary tract occur when *E. coli* enter through the urethra and effectively colonize the bladder. *E. coli* is the most common pathogen causing cystitis, pyelonephritis with the possibility of causing kidney damage and death. This microorganism can induce acute renal failure and in case of complications after renal transplantation, *E. coli* is the most common clinical isolate [8]. It is considered that human intestinal tract is a primary reservoir of UPEC strains, although in some cases, clonal group of UPEC strains can be transmitted by contaminated food [9]. Host inflammatory responses on the breach of the sterile urinary tract by UPEC consist of the production of cytokines and chemokines, neutrophil influx, the exfoliation of infected bladder epithelial cells, and the generation of reactive nitrogen and oxygen species [5]. Genomic differences among UPEC and other *E. coli* show evolutionary adaptations, which enable UPEC to colonize environmental niches within the urinary tract such as epithelia lining the lumenal walls of the urethra, bladder, renal pelvis, and collecting ducts of the kidneys [10]. UPEC strains have different virulence factors that enable the bacteria to adhere and colonize the uroepithelial cells and to establish the UTIs. UPEC harbor more genes encoding adhesins, iron acquisition systems, and toxins than K12 strains and commensal *E. coli* isolates. These virulence genes are often encoded on mobile genetic elements called pathogenicity islands [11, 12].

26 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

This paper describes key virulence factors of UPEC, the role of biofilm formation by UPEC in development of UTIs and in catheter-associated UTIs. The resistance to antibiotics and new

Adhesive proteins as the most important determinants of pathogenicity of UPEC strains are arguable [13], but based on many observations of ABU strains, it was found that these strains are nonadherent and nonhemolytic [14]. UPEC adhesins activate host signaling

therapeutic approaches of treatment and control of UPEC will be also discussed.

**2. Virulence factors of UPEC**

**2.1. Adhesins**

About 80% of UPECs express P fimbriae that are frequently associated with acute pyelonephritis [23]. P fimbriae are encoded by *pap*A-K gene operon which can be localized on one or more pathogenic-associated islands [24]. The P-fimbrial–tip adhesins (PapG adhesins) bind to Gal *α* (1–4) Gal in glycosphingolipids of the membrane of urothelial cells localized in the kidney. The PapG adhesins are encoded by four classes of *papG* genes but only two of them are associated with uropathogenicity. Class II adhesin genes are predominant among the isolates from pyelonephritis and from renal transplant patients, while class III genes are found more frequently among cystitis isolates [25–27]. Attachment of P fimbriae to receptors leads to activation of the immune cell response and to the development of inflammation- and painassociated with UTIs. P fimbriae improve bacterial colonization of the tubular epithelium that can adversely affect renal filtration leading to total obstruction of the nephron and consequently contributes to the full pathophysiology of pyelonephritis [14].

S fimbriae of *E. coli* bind to sialyl galactosides occurring in the receptors of erythrocytes and renal tubular epithelium cells, and are also involved in UTIs development. S fimbriae show binding to epithelial cells of lower urinary tract and kidney and may facilitate bacterial dissemination within host tissues [15, 28].

*E. coli* strains harboring operons coding fimbrial Dr and afimbrial Afa adhesins are also associated with UTIs. Dr adhesins bind to decay-accelerating factor (DAF) which is widely distributed along the urinary tract and plays an important role in colonization of urinary tract by Dr adhesin-producing *E. coli* [29]. UPECs with Afa adhesins have a tropism to renal tissue and have the ability to induce chronic or recurrent infection [30]. The research recently conducted by Muenzner et al. [31] showed that uropathogenic *E. coli* strains, which express the Dra/AfaE adhesins, bind to CEACAMs (carcinoembryonic antigen-related cell adhesin molecules) present on epithelial cells. The interaction of CEACAMs with Dra/AfaE adhesins causes increase of integrin activity, promote matrix adhesion, and suppress epithelial exfoliation, which promotes host infection.

Curli are highly adhesive extracellular amyloid fibers produced by UPEC and other *Enterobacteriaceae* [32]. The major subunit of curli is the CsgA [33]. Curli promote adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, and also cause induction of the proinflammatory cytokine IL-8. They exhibit exclusive role in promoting UPEC biofilms and represent one of the major biofilm components [34]. Curli are produced at limitation of nutrients and salts, at reduced oxygen tension and at temperature below 30°C. However, it is believed that many pathogenic bacteria and commensal strains can also express curli at 37°C during infection in humans [35]. Curli fimbriae interact with serum proteins and this might promote bacterial dissemination in host. UPEC strains-producing curli are more likely to cause urosepticaemia than strains which do not produce curli [36].

**2.3. Iron acquisition systems**

virulence factors of UPEC [48].

**3. Biofilm formation by UPEC**

duced matrix and attached to an abiotic or living surface [49].

Limiting iron availability in the urinary tract is an important host defense against bacterial pathogens. For growth and metabolic activity, bacteria require a cytoplasmic iron concentration of about 10−6 M, while free iron concentrations in the mammalian host are extremely low (10−25 M in the blood and lower at other sites of organism) [47]. Consequently, pathogenic bacteria have to be equipped with systems for acquisition of iron from the host. Bacteria produce siderophores, low-molecular-weight molecules that bind and transport iron (Fe3+) through the bacterial membrane into cytosol where the iron is released. Iron bound siderophores are transported through (with) specific receptors at the outer membrane that facilitate carrying of siderophore-iron complexes through the bacterial membrane. Common siderophore system is enterobactin and its receptor FebA, which is expressed by both pathogenic and K12 *E. coli* strains, although in the context of infection and also other siderophore systems (salmochelin and IroN, aerobactin and IutA, and yersiniabactin and FyuA) have been observed in UPEC [3]. The occurrence of these systems in UPEC strains difficult to identify certain systems as

Virulence Factors and Innovative Strategies for the Treatment and Control of Uropathogenic *Escherichia coli*

http://dx.doi.org/10.5772/67778

29

Currently biofilm is defined as a structured bacterial community embedded in a self-pro-

The biofilm matrix is composed with exopolysaccharides, which form a hydrated viscous layer and protects enclosed bacterial cells against dehydration, toxic molecules such as antibiotics, and from immune system of host [50]. Bacteria within the biofilm differ in gene expression resulting in a phenotype different from the planktonic bacteria. The slow growth of pathogens in biofilms is the major factor conferring resistance to antibiotics [51]. The ability of bacteria to form biofilm is associated with pathogenesis of numerous diseases. Biofilm formation results in chronic, persistent infections that are difficult to eradicate with antimicrobial treatment. It is believed that biofilms occur in up to 60% of human infections [52]. UPEC can persist within the bladder tissue in underlying epithelial cells or create biofilm-like pods in the recurrent cystitis [53]. Biofilm of *E. coli* may form on the urothelium and is involved in infections associated with biomaterials such as catheters or prostheses. UPEC strains are frequently isolated from biofilms formed in the lumen of catheters and showing resistance to antibiotic treatment [54]. Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection, and approximately 80% of UTIs acquired in the hospital are associated with catheterization [55]. The insertion of indwelling catheter into the bladder increases the susceptibility of patients to UTIs, because these devices are the initiation site of infection by introducing opportunistic organisms into the urinary tract [56]. UPEC strains are capable of colonizing the intestinal and vaginal tracts, and these sites are potential reservoirs of microorganisms for UTIs and CAUTIs [57]. The urinary catheter connects the colonized perineum with the sterile bladder providing a route for bacterial entry along the catheter lumen or the external surface of the catheter [58]. CAUTI is related to the susceptibility of catheter material to microbial colonization. The initial

## **2.2. Toxins**

Production of toxins by UPEC is an important virulence factor because they may induce an inflammatory response and lead to symptoms of urinary tract infections. The most important virulence factor of UPEC is α-hemolysin (HlyA). This toxin is strongly proinflammatory and leads to secretion of IL-6, IL-8, and chemotaxins that increase clinical severity in UTI patients [27, 37]. HlyA belongs to the family of RTX (repeats in toxin) [38]. HlyA is a lipoprotein of 110 kDa that forms pores in host cells, leading at high concentrations of HlyA to cell lysis, that enable UPEC to defeat mucosal barriers, damage effector immune cells, and gain access to nutrients and iron [39]. At sublytic concentrations, HlyA implicates the inhibition of chemotaxis and bacterial killing by phagocytes and induces apoptosis of neutrophils and renal cells, and also promotes the exfoliation of bladder epithelial cells [40]. Hilbert et al. [41] found that cytotoxicity, cytokine suppression, and HlyA production were tightly linked in clinical strains, and that *E. coli* utilizes HlyA to inhibit epithelial cytokine production *in vitro*. HlyA is responsible for about 50% of UTIs cases which leads to renal complications [27].

Cytotoxic necrotizing factor 1 (CNF1) is produced by approximately one third of UPEC [14]. The toxicity of this protein is linked with its ability to constitutive activation of the Rho GTPases that affect numerous cellular functions such as the formation of actin stress fibers and membrane ruffle formation. The result is the entry of *E. coli* into urothelial cells [42]. CNF1 promote apoptosis of bladder epithelial cells, probably stimulating their exfoliation and increasing bacterial entry to underlying tissue [43]. Besides, CNF1 inhibits activities of neutrophils, reducing phagocytosis and antimicrobial activity [44].

Secreted autotransporter toxin (Sat) is referred to as serine protease autotransporter and is associated with pyelonephritic *E. coli* strains. Sat is considered a virulence factor because it has toxic activity against cell lines of bladder or kidney origin. Sat induces elongation of cells and loosening of cellular junctions in cell lines of kidney. Furthermore, Sat triggers vacuolation within the cytoplasm of both human bladder and kidney cell lines [45]. Another secreted toxin called Vat (vacuolating autotransporter toxin), often expressed by UPEC strains, shows the ability to induce a variety of cytopathic effects in target host cells, including swelling and vacuolation. However, the role of Vat in UTI pathogenesis has not been thoroughly studied [46].

### **2.3. Iron acquisition systems**

Curli are highly adhesive extracellular amyloid fibers produced by UPEC and other *Enterobacteriaceae* [32]. The major subunit of curli is the CsgA [33]. Curli promote adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, and also cause induction of the proinflammatory cytokine IL-8. They exhibit exclusive role in promoting UPEC biofilms and represent one of the major biofilm components [34]. Curli are produced at limitation of nutrients and salts, at reduced oxygen tension and at temperature below 30°C. However, it is believed that many pathogenic bacteria and commensal strains can also express curli at 37°C during infection in humans [35]. Curli fimbriae interact with serum proteins and this might promote bacterial dissemination in host. UPEC strains-producing curli are more

Production of toxins by UPEC is an important virulence factor because they may induce an inflammatory response and lead to symptoms of urinary tract infections. The most important virulence factor of UPEC is α-hemolysin (HlyA). This toxin is strongly proinflammatory and leads to secretion of IL-6, IL-8, and chemotaxins that increase clinical severity in UTI patients [27, 37]. HlyA belongs to the family of RTX (repeats in toxin) [38]. HlyA is a lipoprotein of 110 kDa that forms pores in host cells, leading at high concentrations of HlyA to cell lysis, that enable UPEC to defeat mucosal barriers, damage effector immune cells, and gain access to nutrients and iron [39]. At sublytic concentrations, HlyA implicates the inhibition of chemotaxis and bacterial killing by phagocytes and induces apoptosis of neutrophils and renal cells, and also promotes the exfoliation of bladder epithelial cells [40]. Hilbert et al. [41] found that cytotoxicity, cytokine suppression, and HlyA production were tightly linked in clinical strains, and that *E. coli* utilizes HlyA to inhibit epithelial cytokine production *in vitro*. HlyA is responsible for about 50% of UTIs cases which leads to renal

Cytotoxic necrotizing factor 1 (CNF1) is produced by approximately one third of UPEC [14]. The toxicity of this protein is linked with its ability to constitutive activation of the Rho GTPases that affect numerous cellular functions such as the formation of actin stress fibers and membrane ruffle formation. The result is the entry of *E. coli* into urothelial cells [42]. CNF1 promote apoptosis of bladder epithelial cells, probably stimulating their exfoliation and increasing bacterial entry to underlying tissue [43]. Besides, CNF1 inhibits activities of

Secreted autotransporter toxin (Sat) is referred to as serine protease autotransporter and is associated with pyelonephritic *E. coli* strains. Sat is considered a virulence factor because it has toxic activity against cell lines of bladder or kidney origin. Sat induces elongation of cells and loosening of cellular junctions in cell lines of kidney. Furthermore, Sat triggers vacuolation within the cytoplasm of both human bladder and kidney cell lines [45]. Another secreted toxin called Vat (vacuolating autotransporter toxin), often expressed by UPEC strains, shows the ability to induce a variety of cytopathic effects in target host cells, including swelling and vacuolation. However, the role of Vat in UTI pathogenesis has not

neutrophils, reducing phagocytosis and antimicrobial activity [44].

likely to cause urosepticaemia than strains which do not produce curli [36].

28 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

**2.2. Toxins**

complications [27].

been thoroughly studied [46].

Limiting iron availability in the urinary tract is an important host defense against bacterial pathogens. For growth and metabolic activity, bacteria require a cytoplasmic iron concentration of about 10−6 M, while free iron concentrations in the mammalian host are extremely low (10−25 M in the blood and lower at other sites of organism) [47]. Consequently, pathogenic bacteria have to be equipped with systems for acquisition of iron from the host. Bacteria produce siderophores, low-molecular-weight molecules that bind and transport iron (Fe3+) through the bacterial membrane into cytosol where the iron is released. Iron bound siderophores are transported through (with) specific receptors at the outer membrane that facilitate carrying of siderophore-iron complexes through the bacterial membrane. Common siderophore system is enterobactin and its receptor FebA, which is expressed by both pathogenic and K12 *E. coli* strains, although in the context of infection and also other siderophore systems (salmochelin and IroN, aerobactin and IutA, and yersiniabactin and FyuA) have been observed in UPEC [3]. The occurrence of these systems in UPEC strains difficult to identify certain systems as virulence factors of UPEC [48].
