**2.2 Genetically engineered phages against UPEC**

Genetically modified or engineered phages have been reported for use in UTIs particularly multidrug-resistant uropathogens. These genetically engineered phages having desirable properties are made using genetic engineering methods, such as homologous recombination, phage recombination of electroporated DNA, *in vivo* recombination, and CRISPR-CAS- mediated genome engineering [33]. An enzymatic engineered phage T7DspB, which expresses exopolysaccharide (EPS)-degrading enzyme dispersin B (DspB), hydrolyses an adhesin required by *E. coli* K12 and clinical *E. coli* isolates for biofilm formation. This genetically modified phage T7DspB had more efficiency in reducing biofilm as compared to natural lytic phage T7 [18]. A

*Bacteriophage Therapy for Urinary Tract Infections Caused by* Escherichia coli *DOI: http://dx.doi.org/10.5772/intechopen.105940*

phage specific for UPEC (*E. coli* K1) has been genetically modified using the CRISPR-CAS mechanism. The phage called K1F-GFP was very effective in killing host bacteria *E. coli* EV 36-RFP present in T24 epithelial cells of the human urinary bladder [19]. A recent study shows ε 2 phages having mosaic intercrossing of 2–3 ancestor phages and devoid of genes conferring lysogeny, antibiotic resistance, or virulence were more virulent and effective for 47 *E. coli* strains found in UTI [34]. Genetically engineered phages can be especially beneficial in the treatment of UTIs caused by multidrugresistant bacteria, however, the cost factor, narrow host range, and host immune responses are the limitation. The above studies show that engineered phages can be used in killing biofilm-forming *E. coli* causing UTI as future therapy in humans.

#### **2.3 Phage lytic proteins for UPEC**

With the advancement of genomics phage, lytic proteins or enzymes are being developed. They have high antibacterial activity against biofilm-forming multidrugresistant clinical isolates. Phages produce cell wall lytic proteins, such as endolysins and virion-associated peptidoglycan hydrolases (PGH). Endolysins or lysins are produced by the phages in the later stages of the lytic cycle. They lyse the host bacteria "from within" when the phage lytic cycle ends [35]. Endolysin integrated with outer membrane permeabilizers (omps) against UPEC and other Gram-negative bacteria, which lead to the lysis of the bacterial cell wall. This endolysin showed high antibacterial activity against the multidrug clinical isolates of Gram-negative bacteria [36]. A study used *E. coli*-specific phage lyase lysep3 fused with the N-terminal region of Bacillus amyloliquefaciens found to be highly efficient in lysing clinical isolates of *E. coli*, *P. aeruginosa*, *A. baumanni*, and Streptococcus strains [20].

Virion-associated peptidoglycan hydrolases (PGH) produced by phages are enzymes that cause "lysis of cell wall from without" thereby killing the host bacteria [37]. Early studies showed the use of phage lytic proteins in combination with a chelating agent like ethylene diamine tetra acetic acid disodium dehydrate (EDTA) to disrupt the Gram-negative bacterial cell outer membrane barrier [21]. Protein engineering techniques are now being used to increase the efficiency of endolysin penetration in UPEC. These endolysins engineered to fuse with OMPs can distort the LPS of the Gram-negative bacteria and are called "artilysins." The first study on artilysin used modular endolysin OBPgp279 of *P. fluorescens* phage and PVP-SE1gp146 of *Salmonella enterica serovar enteridis* phage PVP-SE1 in integration with seven outer membrane peptides. These resulting artilysins had a high antibacterial effect against isolates of *E. coli* [22]. Another artilysin Art-175 was made and tested on colistin-resistant *E. coli* isolates. High bactericidal activity was observed against colistin-resistant *E. coli* isolates [23]. The c-terminal of *E. coli* phage lysin Lysep3 was genetically engineered. It showed increased antibacterial activity against *E. coli* [24]. Endolysins have recently been engineered as "Innolysins," which combine the binding capacity of phage receptor binding proteins (RBPs). Twelve innolysins were made by fusing phage T5 endolysin and RBPb5 in different configurations. Innolysin Ec6 was highly effective against *E. coli*, innolysin Ec21 displayed bactericidal activity to *E. coli* resistant to third-generation cephalosporins [25].

#### **2.4 Phages in combination with antibiotics**

Phage-antibiotic combinations are based on phage-antibiotic synergy (PAS) that is antibiotics are more effective in treating biofilm infections in sub-lethal

concentrations combined with phages than phages applied alone. The PAS also significantly reduces the development of bacterial resistance as compared to phages used singly [26]. The first study using phage-antibiotic combination to control *E. coli* biofilm *in vitro* was when T4 phage and cefotaxime resulted in effective destruction of T4 host E. coli ATCC 11303 biofilms as compared when antibiotic was given alone [27]. With other antibiotics, such as beta-lactam, quinolone, and mitomycin C, there was a similar effect and an increase in T4 phage plaque size. PAS has been studied in other pathogens like biofilm-forming Pseudomonas aeruginosa. When *P. aeruginosa* biofilms were treated in combination with phages and different antibiotics like ceftazidime, ciprofloxacin, colistin, gentamicin, and tobramycin showed high bactericidal activity to *P. aeruginosa* in biofilms grown on human epithelial cell culture [38]. Another study has shown the synergism effect of Cpl-711 endolysin of *S. pneumoniae* and amoxicillin or cefixime on multidrug-resistant isolates of *S. pneumoniae* using mouse and zebrafish models for experimental *in vivo* infection [39]. In a recent study, phage cocktail and antibiotics were used together to combat drug-resistant uropathogens (UPEC). Synergistic effects of the phage cocktail with antibiotics showed phage antibiotic synergism at a lower MIC value of antibiotics [28]. The PAS is quite complex and influenced by many factors like phage and class of antibiotics used, at what concentration the phage lowers the MIC value of antibiotics, and the combination is effective on drug-resistant uropathogens besides host factors like urine and serum. Thus, more studies are needed in PAS to make it a successful therapy for uropathogens mainly UPEC. The use of phages and limiting bacteria to nutrients like iron, which have an important role in biofilm development has also been reported. By adding divalent metal ions, such as Co(II) and Zn(II) to the culture medium a reduction in biofilm development by UPEC was seen [40].
