**5. Treatment and control of UPEC**

stage of biofilm formation on a urinary catheter includes deposition of conditioning film of host urinary components, such as proteins, electrolytes, and other organic molecules [59]. These molecules on the surface of the urinary catheter may change its surface and neutralize any antiadhesive properties [60]. Planktonic bacteria are attached to the surface of the urinary catheter through hydrophobic and electrostatic interaction [61]. Development of biofilm on surface of the catheter occurs through the division of binding bacterial cells, appending additional planktonic bacteria and secretion of extracellular matrix. Detachment of single cell or group of bacterial cells from the biofilm may result in the passage of pathogens into the urine [51]. For this reason, biofilm formation on the urinary catheters is critical for initiating and maintaining of CAUTIs and is a reservoir of resistant pathogenic bacteria [62]. Several factors contribute to the formation of biofilm by *E. coli*, e.g. fimbriae, curli, and flagella. Type 1 fimbriae involved in biofilm formation may also support the colonization of urinary catheter surface [15]. The risk of CAUTI depends on the duration of catheterization, the quality of catheter care, and host susceptibility. Prolonged catheterization is the most important risk factor associated with CAUTI [62]. Long-term urinary catheter use (more than 30 days) causes permanent bacterial colonization of the urine in 100% cases [63]. Examination of people in a nursing home showed that long-term catheterization was significantly related with bacteriuria, pyelonephritis, and renal inflammation [58]. Forming of biofilm on the urinary catheters is a public health problem for patients who need these medical devices. It is recommended that patients who are chronically

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

catheterized were treated with 5–10 days of targeted antibiotic therapy [64].

variations during different time periods and in different areas [68, 69].

cline was isolated from people with UTI from different parts of India [68].

Antimicrobial resistance in UPEC is a clinical problem in patients with UTIs, in particular in women with recurrent UTI. The empirical antimicrobial treatment in case of recurrent UTIs exerts significant resistance pressure on the uropathogens and the fecal flora, which serves as resistance reservoirs for potential uropathogens [65–67]. Antimicrobial resistance among *E. coli* causing UTI is increasing in many countries around the world and shows considerable

The level of resistance of UPEC strains from hospitalized patients in Poland and Turkey to ampicillin was 56% [70, 71], while above 85% of UPEC strains from patients in India were resistant to this antibiotic [68]. High percentage (67.3%) of *E. coli* strains resistant to tetracy-

Sanchez et al. [72] suggested that the increase of resistance of UPEC to ciprofloxacin is a result of widespread use of this antibiotic in the treatment of uncomplicated UTIs in the early 2000s. The most recent published data suggested that the level of resistance to trimethoprim-sulfamethoxazole increased and in different countries was over 21–24.2% [71–73]. This trend has continued for decades and the increasing resistance of *E. coli* to trimethoprim-sulfamethoxazole can be explained by frequent use of this antimicrobial agent because it is recommended as the second-line drug in treating acute uncomplicated cystitis in women. Authors reported low resistance of *E. coli* to nitrofurantoin (0.85–1.6%) and no increase in resistance in the last

**4. Antimicrobial resistance of UPEC**

decade was observed [71, 72].

Currently, the antibiotic therapy is an important part of the therapeutic strategy for UTI. The increased antibiotic resistance in recent years suggests that the choice of antibiotic should be guided by the results of sensitivity assay, although in cases of community-acquired UTI, an empirical therapy is often used [23]. The drugs of first-line choice for empirical treatment of uncomplicated UTI in all European countries are fosfomycin trometamol, pivmecillinam, or nitrofurantoin macrocrystals [84]. Trimethoprim-sulfamethoxazole is also used in countries where resistance to this chemotherapeutic is low. Higher rates of side effects in comparison with other drugs limit the use of quinolones as second-line therapy. Moreover, in many countries in Europe, high resistance rates of *E. coli* strains to nalidixic acid were observed [85], and thus aminoglycosides and carbapenem are the drugs of choice. In patients with recurrent infections of the urinary tract, the antibiotics may be recommended prophylactically. It is believed that two recurrences of UTI within 6 months after therapy or three episodes per year could be considered an indication to establish prophylaxis after treatment. The drugs for this purpose are nitrofurantoin, trimethoprin-sulfamethoxazole, fosfomycin trometamol, and cotrimoxazole at lower doses than therapeutic [86]. However, repeated antibiotic treatment of UTI and prophylactic use of antibiotics frequently results in a rise in resistance to antibiotics and adversely affects microbiota of patients which may lead to secondary infections posttreatment, such as gastrointestinal infection and vaginal yeast infection [87, 88].

For this reason, alternative or additional prophylactic strategies have been investigated. One of them is improving the management of UTI by the development of preventive vaccines. Effective vaccine for UTI will need to generate a strong mucosal immune response in the urinary tract. Designing a UTI vaccine that would be effective against UPEC is difficult due to heterogeneous nature of the UPEC population. UTI vaccine should be designed based on more than one antigen because not all strains express the exact set of virulence genes during infections. A vaccine based on the multiple virulence factors, such as fimbrial adhesins or iron receptors, could be clinically effective against UTI [89]. The vaccine with whole or lysed fractions of inactivated bacteria can be effective to generate protective immunity. Urovac® is one of such vaccines (Solco Basel AG, Birsfelden, Switzerland, and Protein Express, Cincinnati, OH, USA) containing ten heat-killed uropathogens, including six UPEC strains. The UPEC strains in the Urovac® show different virulence factors, such as hemolysin, type 1, P, and S fimbrial adhesins, CNF-1, siderophores, and the *E. coli* CFT073 pathogenicity island marker and many different O and H antigens. Evaluating the efficacy of vaginally administrated Urovac® found that the immunization did not ensure significant long-term protection from UTI or an increase in mean levels of UPEC antibodies in serum, vagina, or urine [90]. However, among the women receiving Urovac, 72% were free from UTIs, while only 30% of women given placebo remained free from UTIs caused by *E. coli*. Moreover, in the Urovac vaccinated group, the number of *E. coli* caused UTIs was significantly lower compared to the control group [91]. Another vaccine which is used in Switzerland since 1988 and sold in other countries worldwide is OM-89/ Uro-Vaxom® (OM Pharma, Myerlin, Switzerland). Uro-Vaxom is an oral capsule containing a lyophilized mix of membrane proteins from 18 UPEC strains. The clinical studies showed that Uro-Vaxom was significantly more effective than placebo in preventing recurrent UTI [92].

with hydrogel containing bacteriophages [98, 99]. Biofilm-associated UTIs are difficult to treat due to the high level of antimicrobial resistance showed by biofilm structures. Many authors recommend macrolides (erythromycin, clarithromycin, and azithromycin) as the treatment of choice in biofilm-associated infections because these antibiotics inhibit the production of primary component of the matrix, alginate [100, 101]. Ciprofloxacin, norfloxacin, gentamicin, or nitrofurazone are often used as components in coating and impregnating the catheters in the aim to inhibit bacterial attachment and development of biofilm [55, 102]. New therapeutic antibiofilm treatments are studied as alternative to antibiotics in order to inhibit biofilm formation and also to avoid the emergence of resistant bacterial populations. The silver showed antimicrobial activity by interacting with bacterial cell membrane and is used to coat catheters. The study showed a statistically significantly lower frequency of bacterial infection in patients treated with a silver alloy-coated catheter compared to those treated with uncoated catheter. Schaeffer et al. [103] reported that bacteriuria was present in 27% of patients with the silver-coated silicone catheter and in 55% of group with uncoated silicone catheter. It was also demonstrated that silver alloy used in hydrogel-coated urinary catheter reduced of up 45% of CAUTI [104]. However, the study conducted by Desai et al. [105] showed that *E. coli* adherence was not significantly lower on silver-impregnated silicone or latex catheters compared

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

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

33

The new antibiofilm strategy is phage therapy using engineered bacteriophages that have biofilmdegrading enzymatic activity. It was demonstrated that engineered phages that express biofilmdegrading enzymes are more efficient in removing bacterial biofilms than nonenzymatic phage alone. Lu and Collins [106] generated bacteriophage which expressed biofilm-degrading enzyme (DspB) during infection. The DspB showed simultaneous action against both bacterial cells in the biofilm and the biofilm matrix. The engineered enzymatic phage reduced bacterial biofilm cell in 99.9%. One of the new ways of eliminating biofilm is the use of nanoparticles. Water-based syn-

was described. The minimal inhibitory concentration was observed at 0.01 mg/mL. In addition, YF3 nanoparticles-coated catheters were able to significantly reduce bacterial colonization compared to the uncoated surface, which provides the potential to develop the concept of utilizing

The alternative strategies to decrease UPEC infection include the use of plant-derived antibacterial agents containing different functional groups in their structure and development of resistance in bacteria to these antimicrobials is less frequent [108]. Borges et al. [109] showed that phenolic acids such as gallic and ferulic acid have prevented biofilm formation and show potential to reduce the mass of biofilms formed by the Gram-negative bacteria including *E. coli*. The antibiofilm effect of trans-cinnamaldehyde (TC) on UPEC was reported by Amalaradjou et al. [110]. These authors showed that TC was effective against UPEC biofilm on polystyrene or latex, and the expression of *E. coli* genes encoding attachment and invasion of bladder cells was significantly decreased by TC [111]. Phytochemicals as alternative antimicrobials in preventing and inactivating *E. coli* biofilm on urinary catheters were also assessed. It was demonstrated that TC at the concentration of 0.5% was highly effective for preventing *E. coli* biofilm formation in the lumen of urinary catheter and after 1 day of completely inhibited biofilm formation. Whereas, completely inactivated biofilm after 1 day was observed at 1.25%

yttrium fluoride nanoparticles as novel antimicrobial and antibiofilm agents [107].

) nanoparticles that showed antibacterial properties against *E. coli*

to adherence of *E. coli* on catheters without silver.

thesis of yttrium fluoride (YF<sup>3</sup>

Other prophylaxis method is use of different *Lactobacillus* species in the form of probiotics which reduced the risk of UTI and vaginal infections. Use of *Lactobacillus* species maintains low pH and produces hydrogen peroxide that inhibits growth of *E. coli* in urinary tract but also activates Toll-like receptor-2 and therefore leads to reduced inflammatory reaction [93]. Beerepoot et al. [94] conducted study in which postmenopausal women with recurrent UTI prophylactically received trimethoprim-sulfamethoxazole or oral capsules containing *L. rhamnosus* GR-1 and *L. reuteri* RC-14. After 12 months of treatment, the reduction in recurrence was more than 50% in both groups. However, in group that received trimethoprim-sulfamethoxazole, the twofold increase in resistance was observed.

Research on dietary supplementation showed that cranberry juice and its extracts reduced UTI recurrences. The active metabolite of cranberry, proanthocyanidin A prevents bacterial adhesion to the urothelial layer by inhibiting P fimbriae expression [95]. The minimum daily dose of proanthocyanidin A, which is able to reduce significantly the number of urinary *E. coli* to be 36 mg [96]. The study conducted by Wojnicz et al. [97] showed that cranberry extract Żuravit S.O.S.® reduced motility and adhesion to epithelial cells in *E. coli* strains isolated from urine of patients with pyelonephritis and also limited the ability of these strains to form biofilm.

Bacteriophages are highly specific and very effective in lysing bacteria. The use of lytic phages that are able to pass through the extracellular matrix against *E. coli* biofilm causes a reduction of bacteria number in biofilm and also prevents biofilm formation on catheter coated with hydrogel containing bacteriophages [98, 99]. Biofilm-associated UTIs are difficult to treat due to the high level of antimicrobial resistance showed by biofilm structures. Many authors recommend macrolides (erythromycin, clarithromycin, and azithromycin) as the treatment of choice in biofilm-associated infections because these antibiotics inhibit the production of primary component of the matrix, alginate [100, 101]. Ciprofloxacin, norfloxacin, gentamicin, or nitrofurazone are often used as components in coating and impregnating the catheters in the aim to inhibit bacterial attachment and development of biofilm [55, 102]. New therapeutic antibiofilm treatments are studied as alternative to antibiotics in order to inhibit biofilm formation and also to avoid the emergence of resistant bacterial populations. The silver showed antimicrobial activity by interacting with bacterial cell membrane and is used to coat catheters. The study showed a statistically significantly lower frequency of bacterial infection in patients treated with a silver alloy-coated catheter compared to those treated with uncoated catheter. Schaeffer et al. [103] reported that bacteriuria was present in 27% of patients with the silver-coated silicone catheter and in 55% of group with uncoated silicone catheter. It was also demonstrated that silver alloy used in hydrogel-coated urinary catheter reduced of up 45% of CAUTI [104]. However, the study conducted by Desai et al. [105] showed that *E. coli* adherence was not significantly lower on silver-impregnated silicone or latex catheters compared to adherence of *E. coli* on catheters without silver.

For this reason, alternative or additional prophylactic strategies have been investigated. One of them is improving the management of UTI by the development of preventive vaccines. Effective vaccine for UTI will need to generate a strong mucosal immune response in the urinary tract. Designing a UTI vaccine that would be effective against UPEC is difficult due to heterogeneous nature of the UPEC population. UTI vaccine should be designed based on more than one antigen because not all strains express the exact set of virulence genes during infections. A vaccine based on the multiple virulence factors, such as fimbrial adhesins or iron receptors, could be clinically effective against UTI [89]. The vaccine with whole or lysed fractions of inactivated bacteria can be effective to generate protective immunity. Urovac® is one of such vaccines (Solco Basel AG, Birsfelden, Switzerland, and Protein Express, Cincinnati, OH, USA) containing ten heat-killed uropathogens, including six UPEC strains. The UPEC strains in the Urovac® show different virulence factors, such as hemolysin, type 1, P, and S fimbrial adhesins, CNF-1, siderophores, and the *E. coli* CFT073 pathogenicity island marker and many different O and H antigens. Evaluating the efficacy of vaginally administrated Urovac® found that the immunization did not ensure significant long-term protection from UTI or an increase in mean levels of UPEC antibodies in serum, vagina, or urine [90]. However, among the women receiving Urovac, 72% were free from UTIs, while only 30% of women given placebo remained free from UTIs caused by *E. coli*. Moreover, in the Urovac vaccinated group, the number of *E. coli* caused UTIs was significantly lower compared to the control group [91]. Another vaccine which is used in Switzerland since 1988 and sold in other countries worldwide is OM-89/ Uro-Vaxom® (OM Pharma, Myerlin, Switzerland). Uro-Vaxom is an oral capsule containing a lyophilized mix of membrane proteins from 18 UPEC strains. The clinical studies showed that Uro-Vaxom was significantly more effective than placebo in preventing recurrent UTI [92].

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

Other prophylaxis method is use of different *Lactobacillus* species in the form of probiotics which reduced the risk of UTI and vaginal infections. Use of *Lactobacillus* species maintains low pH and produces hydrogen peroxide that inhibits growth of *E. coli* in urinary tract but also activates Toll-like receptor-2 and therefore leads to reduced inflammatory reaction [93]. Beerepoot et al. [94] conducted study in which postmenopausal women with recurrent UTI prophylactically received trimethoprim-sulfamethoxazole or oral capsules containing *L. rhamnosus* GR-1 and *L. reuteri* RC-14. After 12 months of treatment, the reduction in recurrence was more than 50% in both groups. However, in group that received trimethoprim-sulfamethoxazole, the two-

Research on dietary supplementation showed that cranberry juice and its extracts reduced UTI recurrences. The active metabolite of cranberry, proanthocyanidin A prevents bacterial adhesion to the urothelial layer by inhibiting P fimbriae expression [95]. The minimum daily dose of proanthocyanidin A, which is able to reduce significantly the number of urinary *E. coli* to be 36 mg [96]. The study conducted by Wojnicz et al. [97] showed that cranberry extract Żuravit S.O.S.® reduced motility and adhesion to epithelial cells in *E. coli* strains isolated from urine of patients with pyelonephritis and also limited the ability of these strains to form biofilm.

Bacteriophages are highly specific and very effective in lysing bacteria. The use of lytic phages that are able to pass through the extracellular matrix against *E. coli* biofilm causes a reduction of bacteria number in biofilm and also prevents biofilm formation on catheter coated

fold increase in resistance was observed.

The new antibiofilm strategy is phage therapy using engineered bacteriophages that have biofilmdegrading enzymatic activity. It was demonstrated that engineered phages that express biofilmdegrading enzymes are more efficient in removing bacterial biofilms than nonenzymatic phage alone. Lu and Collins [106] generated bacteriophage which expressed biofilm-degrading enzyme (DspB) during infection. The DspB showed simultaneous action against both bacterial cells in the biofilm and the biofilm matrix. The engineered enzymatic phage reduced bacterial biofilm cell in 99.9%. One of the new ways of eliminating biofilm is the use of nanoparticles. Water-based synthesis of yttrium fluoride (YF<sup>3</sup> ) nanoparticles that showed antibacterial properties against *E. coli* was described. The minimal inhibitory concentration was observed at 0.01 mg/mL. In addition, YF3 nanoparticles-coated catheters were able to significantly reduce bacterial colonization compared to the uncoated surface, which provides the potential to develop the concept of utilizing yttrium fluoride nanoparticles as novel antimicrobial and antibiofilm agents [107].

The alternative strategies to decrease UPEC infection include the use of plant-derived antibacterial agents containing different functional groups in their structure and development of resistance in bacteria to these antimicrobials is less frequent [108]. Borges et al. [109] showed that phenolic acids such as gallic and ferulic acid have prevented biofilm formation and show potential to reduce the mass of biofilms formed by the Gram-negative bacteria including *E. coli*. The antibiofilm effect of trans-cinnamaldehyde (TC) on UPEC was reported by Amalaradjou et al. [110]. These authors showed that TC was effective against UPEC biofilm on polystyrene or latex, and the expression of *E. coli* genes encoding attachment and invasion of bladder cells was significantly decreased by TC [111]. Phytochemicals as alternative antimicrobials in preventing and inactivating *E. coli* biofilm on urinary catheters were also assessed. It was demonstrated that TC at the concentration of 0.5% was highly effective for preventing *E. coli* biofilm formation in the lumen of urinary catheter and after 1 day of completely inhibited biofilm formation. Whereas, completely inactivated biofilm after 1 day was observed at 1.25% and 1.5% TC solution. *p*-Coumaric and ferulic acids have preventive action on *E. coli* biofilm formation on urinary catheter but complete inactivation of the biofilm formed at presence of these phytochemicals was not observed [112]. Recently showed that two alkaloids, piperine from black pepper and reserpine from Indian snakeroot, decreased swarming and swimming motilities of the uropathogenic *E. coli* CFT073. Additionally, piperine increased penetration of ciprofloxacin and azithromycin in to biofilm of *E. coli* CFT073. Authors suggest that these substances can affect on bacterial colonization by inhibition bacterial motility and also may help in treatment of infection by strengthening the penetration of antibiotic in biofilms [113].

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One of the other strategies to prevent colonization, invasion, and biofilm formation by UPEC is inhibition of the assembly of pili by family of bicyclic 2-pyridones, termed pilicides.

The activity of pilicides was evaluated in two different pilus biogenesis systems in UPEC. Hemagglutination mediated by either type 1 or P pili, adherence to bladder cells, and biofilm formation mediated by type 1 pili were all reduced by 90% in laboratory and clinical *E. coli* strains [114]. Pilicide ec240 was found to disrupt type 1 pili, P pili, S pili, and flagellar motility [115]. Other pilicides also inhibit the production of Dr pili that are important in pyelonephritis [116]. Mannosides are FimH receptor analogues and bind to this pilus with high affinity, which results in blocking FimH binding to mannosylated receptors. The use of mannosides is considered a new strategy in treating and preventing UTIs because they prevent bladder colonization and invasion and are effective against multidrug-resistant UPEC and against established UTIs [117].
