Bacteriophage Therapy for Urinary Tract Infections Caused by Escherichia coli

Abstract

Urinary tract infections (UTIs) are the most prevalent bacterial diseases affecting 150 million people annually worldwide. Around 85% of UTIs are caused by Escherichia coli from the Enterobacteriaceae family. The pathogenesis of uropathogenic E. coli (UPEC ) involves adherence, colonization, evading host defenses, and damage to host tissue to achieve virulence. The uncontrolled use of antibiotics worldwide during therapy of UTIs has resulted in increased antibiotic resistance and the emergence of multidrug resistance (MDR) and extensive drug resistant (XDR) to UPEC. Bacteriophages have the potential to eliminate and manage resistant biofilm-forming uropathogenic organisms, such as E. coli and control UTIs. The chapter discusses the use of phages as an alternative treatment for UTIs caused by UPEC.

1. Introduction

Urinary tract infections (UTIs) are one of the most frequent bacterial infections and the primary causative agent is Escherichia coli [1]. The other causative agents reported for UTIs include Staphylococcus saprophyticus, Pseudomonas aeruginosa, Klebsiella, Enterobacter, and Proteus species [2]. E. coli, the primary causative agent of UTI, is a Gram-negative bacteria from the Enterobacteriaceae family. The enteropathogenic strains of E. coli are divided into two types: intestinal E. coli, which have enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli, enteroaggregative E. coli, enterotoxigenic E. coli (ETEC). The second category is the uropathogenic E. coli, causing extraintestinal infections (Table 1). The uropathogenic serotypes 01 0K1, 06:K2, 04:K12, 016:K1, or 018:K5 are associated with the majority of UTIs [3]. Uropathogenic E. coli causes both complicated and uncomplicated UTIs. UPEC has fimbria as an important virulence factor. These fimbriae contain subunit protein (pap A) encoded by papA gene [4]. The type I fimbriae are most commonly expressed adhesins that allow the bacteria to attach and colonize the human urogenital tract. The type I fimbriae constitutes of Fim H protein (responsible for binding), Iaminin (part of extracellular matrix), and secretory Ig A. Another fimbriae present in UPEC is P fimbriae (PapG) adhesion of p fimbriae mediates the bacterial binding, thus inducing UTI symptoms [4]. Other virulence factors in UPEC are F1C and S fimbriae enclosing the fac and sfa gene, cytotoxic necrotic zing factor (NF1), iron-binding siderophores, and K1 capsular polysaccharide.

Diarrheagenic E. coli or enteric E. coli	Extraintestinal E. coli (EXPEC)
Pathotypes.; Enterotoigenic E .coli (ETEC). Enteropathogenic E. coli (EPEC). Shiga-toxin-producing E. coli (STEC). Enteroaggregative E. coli (EAEC). Enteroinvasive E. coli (EIEC). Diffusely adherent E. coli (DAEC).	Pathotypes: uropathogenic E. coli,; neonatal meningitis E. coli (NMEC); Serotypes.O1:H4, O1:H6,O1:H7,O1:H, O2:H1, O2:H4, O4:H5,O6:H1, O7:H4,O7:H6, O7:H-, O18ac:H7, O18ac:H-, O22:H, O25:H1, O75:H5 and O75:H7.

Table 1.

Pathogenic E. coli.

Bacteriophages are viruses that attack bacteria. Antimicrobial resistance by bacteria has now become a global threat and could kill 50 million people by the year 2050 as per the World Health Organization estimates [5]. Phages are now known to cure antibiotic-resistant bacterial infections as well as decrease bacterial virulence by overcoming the barriers bacteria used to avoid them. Bacteriophages are now being explored as potential therapeutic tools for the elimination of bacterial pathogens. Bacteriophages can disrupt pathogenic processes associated with biofilm and exopolysaccharide formation by microflora. Bacteriophage therapy is a promising strategy to control bacterial infections as phages are very efficient in killing host bacteria and do not disrupt other flora and have a low cost of production [6]. Antibiotics used alone have a broad spectrum of activity inducing drug resistance in bacteria and are toxic, whereas phages are non-toxic [7]. Using other strategies, such as herbal products, is more costly and less efficient and has a broad spectrum when compared with phages that are safe and efficient even through oral administration [8]. The combination of antibiotics and phage therapy and the use of phage cocktails have great potential in the treatment of drug-resistant bacterial infections, particularly UTIs. This chapter focuses on the use of phages in treating UTIs caused by uropathogenic E. coli.

2. Phages as therapeutics for uropathogenic E. coli (UPEC)

Newer therapeutic options like phages are alternative treatment options for treating UTIs. Phages have been tried as a potential candidate for treating UTIs in a number of studies. Six lytic bacteriophages each at a titer of 10⁶ p.f.u/ml to P. aeruginosa causing UTI showed a decrease in the number of target bacteria [9]. In another study, Pyo bacteriophages to S. aureus, E. coli, Streptococcus species, P. aeruginosa, and Proteus species causing UTI were given twice to patients after transurethral resection of the prostate in a solution form. The patients were asked to retain the phage solution for 30–60 minutes in their bladders. After phage therapy, a decreased bacterial count was seen in 67% of patients with no side effects [10]. In a recent study by Lorenz et al. a pyobacteriophage cocktail solution was given twice daily for 7 days in UTI and was found to be comparable to regular antibiotic therapy [11, 12]. Emerging antimicrobial resistance in UPEC has led to the emergence of multidrug-resistant UPEC and extensively drug-resistant UPEC [10]. Initial studies showed the phage preparations were locally and orally applied to UTIs caused by E. coli, Staphylococcus, and Proteus species. A bacterial reduction of 84% was observed [13].

Broadly bacteriophages are now being used in infecting UPEC strains causing UTIs in four ways (Table 2).

1. Phage cocktails

2. Genetically engineered phages

3. Phage lytic proteins

4. Phages in combination with antibiotics

(1) Phage cocktails	phage cocktail of T4 and KEP 10 phage induced in the peritoneal cavity of mouse for treatment of UTI caused by UPEC [14]. Phage cocktail of T1, T4 and phi X 174 like phages evaluated against UPEC were capable of lysing a variety of UPEC strains [15]. Phage cocktail of 9 phages from 99 T4 like coliphages to UPEC was found to be safe and effective in UTI [16]. Dual receptor phages T4, T2 and K1-5 infecting K1 and K5 strains of UPEC were found to be efficient [17].
(2) Genetically engineered phages	Enzymatic engineered phage T7DspB was found to be more efficient in reducing biofilm formed by clinical E. coli isolates as compared to natural lytic phage T7 [18]. Engineered phage K1F-GFP was found to be very effective in killing host bacteria E. coli present in T24 epithelial cells of human urinary bladder [19]. Genetically engineered ε2 phages were found to be more effective for 47 E. coli strains found in UTI.
(3) Phage lytic proteins	E. coli specific phage lyase lysep 3 fused with N-terminal region of Bacillus amyloliquefaciens was found to be highly efficient in lysing clinical isolates of E. coli [20]. Phage lytic proteins in combination with chelating agent like EDTA was used to disrupt Gram-bacterial cell outer membrane barrier [21]. Endolysins called ‘artilysins ‘ which can distort the LPS and has high antibacterial effect against isolates of E. coli [22]. Artilysin Art-175 had high bactericidal activity against colistin resistant E. coli isolates [23]. Phage lysin LySep^3 has high increased antibacterial activity against E. coli [24]. Innolysins [combination of fused phage T5 endolysin and phage receptor binding proteins (RBPs)]. Innolysin Ec6 and Ec21 was found to be highly effective against UPEC [25].
(4) Phages in combination with antibiotics	T4 phage and cefotaxime were highly effective in destruction of T4 host E. coli ATCC11303 biofilms as compared to antibiotic given alone [26]. T4 phage with beta lactam, quinolone and mitomycin C were more effective in destruction of E. coli biofilms [27]. Phage cocktail with antibiotics was found to be effective in combating drug resistant uropathogens [28].

Table 2.

Types of phage therapy for UPEC.

2.1 Phage cocktails against UPEC

Monophage therapy or using a single phage has an important limitation, which is a narrow host range. Phage cocktails use two or more phages for therapy making the host range broad and overcoming host bacterial resistance to phages. The phage cocktail or combination of phages to UPEC can recognize more than one host receptor and hence infect many uropathogenic strains. A phage cocktail of T4 phage and KEP10 phage was introduced in the peritoneal cavity of the mouse as the first therapeutic candidate for the treatment of UTI caused by UPEC [14]. The efficacy of T1, T4, and phiX174-like phages was also evaluated against UPEC. T1 phage was found to be the most effective in killing UPEC as it had a broad lytic spectrum; however, a combination of T1, T4, and phiX174 was capable of infecting a variety of antibiotic-tolerant UPEC strains [15]. A cocktail of nine phages without horizontal gene transfer and undesired genes from 99 T4-like coliphages to UPEC was used to produce a cocktail and given to 15 healthy adults. No side effects were seen, indicating that phage therapy was safe to use in UTIs [16]. In a study, it was seen that phage SP21 uses OmpC of E. coli 0157:H7 as a receptor, when this receptor was deleted, the phage-resistant bacteria emerged after 8 hrs of incubation. On modifying the lipopolysaccharide of the bacteria, the resistant bacteria emerged after 6 hrs of incubation with phage SP22. When a combination of two phages SP21 and SP22 binding to different host receptors of EHEC (E. coli 0157:H7) was used, it resulted in significant delay at the time of emergence of phage-resistant E. coli (upto 30 hrs) as compared to phages used alone [29]. Dual receptor phages to UPEC, which identify more than one receptor, have also been identified. Dual receptor phages to UPEC reported are T4 phages, T2 phages, and phage K1-5 of the family Podaviridae that infects both K1 and K5 strains of E. coli [17, 30, 31]. UPEC causes UTI by adhering to the urothelium producing biofilms successfully evading them from the host immune system and antibiotics. Phage cocktails have been found to be suitable for killing bacteria in biofilms. Biofilms of E. coli on the surface of polyvinyl chloride were susceptible to phage T4D+ [32]. Phage cocktails can be used for treating UTIs caused by E. coli biofilms present on urinary catheters. However, any mutational or conformational change in the host bacterial receptors can make the phages resistant to the bacteria, which is a limitation of using this strategy.

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 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 ε² 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 multidrug-resistant 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 multidrug-resistant 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].

3. Conclusion

The major cause of UTIs worldwide is uropathogenic E. coli. The development of resistance in uropathogenic E. coli is a serious therapeutic problem that requires newer antibiotics and alternative forms of therapy, such as phages. In the treatment of UTIs, studies are being conducted on various forms of bacteriophages, such as phage cocktails, genetically modified phages, phage lytic enzymes and their derivatives, and phage-antibiotic combinations. Clinical trials are being conducted on phage cocktails and phage lytic enzymes for treating UTIs and no randomized control trials. The phage therapy still requires validated clinical research to use different types of phage therapy to eliminate UPEC and the biofilm formed in the urinary tract to control UTIs. More research on phage therapy is still required on drug-resistant uropathogens. Undoubtedly in the future phages can emerge as pharmaceutical compounds, an alternative to conventional antibiotics particularly for treating UTIs caused by drug-resistant uropathogenic E. coli.

Conflict of interest

The author has no conflicts of interest.