2.1. Pathogenic E. coli
E. coli is a gram-negative, rod-shaped bacterium that is usually found within the lower gut of warm-blooded organisms (endotherms). According to Vogt and Dippold (2005), the majority of E. coli strains are not harmful, but some can lead to severe food poisoning, septic shock, meningitis, or urinary tract infections in humans [144]. The pathogenic strains of E. coli, in contrast to the normal flora, create toxins and other virulence factors that allow them to live in areas of the body where E. coli would not ordinarily reside and cause harm to host cells. Only pathogens have virulence genes that encode these harmful features [101].
Oral and fecal pathways are common routes for the spread of pathogenic E. coli. Unsanitary food preparation, agricultural contamination from manure fertilizer, crop irrigation using contaminated gray water or raw sewage, feral pigs on cropland, and direct ingestion of sewage-contaminated water are common modes of transmission [73]. The main sources of E. coli O157:H7 are dairy and beef cattle, which can harbor the infection asymptomatically and excrete it in their feces [50]. Cucumber, raw ground beef, raw spinach or seed sprouts, raw milk, unpasteurized juice, unpasteurized cheese, and foods contaminated by infected food workers through the fecal–oral pathway are food products linked to E. coli outbreaks.
According to Eckburg et al. (2005), E. coli and similar bacteria make up approximately 0.1% of the gut flora [48]. The main route by which pathogenic strains of the bacterium cause illness is fecal-oral. Because they can only exist outside of the body for a brief period of time, cells are perfect indicator organisms to check environmental samples for the presence of feces [135]. The bacterium has been the subject of extensive research for more than 60 years and is also easily and affordably produced in a laboratory setting. Vibrant strains of E. coli can cause a variety of illnesses in both humans and domestic animals. Neonatal meningitis, urinary tract infections, and gastroenteritis are among these illnesses. Rarely, virulent strains can also cause extraintestinal diseases such as gram-negative pneumonia, peritonitis, mastitis, septicemia, and hemolytic-uremic syndrome [136].
2.1.1. Diarrheagenic Gastroenteritis
Normally, E. coli stays within the intestinal lumen without causing any harm. However, in individuals who are immunocompromised or have weakened gastrointestinal barriers, even nonpathogenic strains of E. coli can lead to infections. Moreover, a number of highly adapted E. coli clones that have evolved to cause a wide range of diseases in humans and animals can infect even the healthiest people. Pathogenic E. coli infections can spread throughout the body or just affect mucosal surfaces. The three primary clinical syndromes resulting from infections produced by intrinsically harmful strains of E. coli include urinary tract infections, sepsis/meningitis, and enteric/diarrheal disorders.
In children and dairy calves, diarrheagenic E. coli (DEC) is a major cause of acute gastroenteritis. According to Liu et al. (2016), acute gastroenteritis ranks fourth in the world for children under the age of five years in terms of mortality and is a frequent cause of morbidity in both developing and developed nations during childhood [89]. E. coli pathotypes that are diarrheagenic (DEC) are distinguished from nonpathogenic and extraintestinal pathogenic (ExPEC) based on virulence factors found in their genomes and phenotypic traits. According to Kaper et al. (2004), the three types of ExPEC are neonatal meningitis-associated E. coli (NMEC), sepsis-inflicting E. coli (SEPEC), and uropathogenic E. coli (UPEC) [77].
The DEC group has been reexamined as seven distinct pathotypes by pathogenomics and phenotypic classification (Table 1). These pathotypes are defined by their essential virulence genes and differential features, which include enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (Non-O157/STEC), enterohemorrhagic E. coli (EHEC/O157), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and E. coli that adheres diffusely (DAEC) [37].
i) Enteropathogenic E. coli (EPEC)
EPEC was the first pathotype of E. coli to be described. For many years, O:H serotyping was the only way to identify this pathotype and the mechanisms underlying EPEC-induced diarrhea remained a mystery. However, since 1979, numerous advances in understanding the pathogenesis of EPEC diarrhea have been made, such that EPEC is now among the best understood pathogenic E. coli.
EPEC infections are linked to a distinct intestinal histopathology. Known as "attaching and effacing" (A/E) infections, these bacteria form close attachments to intestinal epithelial cells and induce dramatic cytoskeletal alterations, such as the accumulation of polymerized actin directly beneath the adherent bacteria. Microvilli effacement and close adherence between the bacteria and the epithelial cell membrane characterize this remarkable phenotype. A whole family of enteric pathogens that cause A/E lesions on epithelial cells has its origins in EPEC strains.
ii) E. coli that is enterohemorrhagic (EHEC/O157)
The E. coli strain EHEC is responsible for producing Shiga toxin. The intestinal wall lining is harmed by the toxin. EHEC was identified as the source of bloody diarrhea in 1982, which occurred when a person consumed raw or undercooked hamburger meat tainted with the bacteria. Since then, unpasteurized milk, unsalted apple juice or cider, salami, spinach, lettuce, sprouts, well water, and surface water areas that animals frequently visit have all been connected to EHEC outbreaks. According to Gomes et al. (2016), outbreaks have also been linked to animals at petting zoos and daycare facilities [56].
Hemorrhagic colitis, hemolytic-uremic syndrome (HUS), diarrhea, and hemorrhagic diarrhea are all caused by the enterohemorrhagic bacterial strain E. coli O157: H7, which is also a significant food source. Three to eight days following infection are when EHEC symptoms start to appear. They include diarrhea that can turn into bloody diarrhea (hemorrhagic colitis) and abdominal pain. Fever and vomiting are possible side effects. The primary means of transmission for EHEC pathogens is eating contaminated food. Raw or undercooked meat, raw (unpasteurized) dairy products, and occasionally raw vegetable products are the main food products affected, as the digestive tract of cattle serves as the primary natural reservoir of EHEC. Such animals may also become contaminated when they are milked or killed. Another possible source of contamination is ruminant feces in the ground, in manure, or in water (ponds and streams). Although uncommon, EHEC can also spread from person to person. Most often, it is seen in a community or family setting [99].
iii) E. coli that is enterotoxigenic (ETEC)
The bacterium enterotoxigenic E. coli (ETEC) is a pathogenic version or pathotype of E. coli that produces heat-labile (LT) and heat-stable (ST) enterotoxins that cause diarrhea. Nearly fifty years have passed since these bacteria were first linked to cholera-like watery diarrhea [120]. Despite this, the bacteria continue to pose a serious threat to global health, especially to young children living in low-resource areas of the world. Here, it is estimated that more than a billion cases of diarrheal illness occur in children under five each year, with hundreds of millions of episodes of diarrhea linked to ETEC alone [78]. Watery diarrhea is caused by ETEC and can vary in severity from a mild self-limiting illness to a severe purging illness. Symptoms of an ETEC infection can include headaches, cramping in the stomach, vomiting, and, in rare instances, a low-grade fever. According to some research, ETEC infection may have some side effects, including an increased risk of childhood stunting from malnourishment and immunological deficiencies, an increased risk of developing other infectious diseases, and even an impact on cognitive development [138]. Moreover, there is a connection between postinfectious irritable bowel syndrome and traveler's diarrhea. Food or water tainted with human or animal excrement is how it spreads. Hand washing with soap on a regular basis and avoiding or properly preparing foods and beverages that may be contaminated with bacteria are two ways to prevent infection.
IV. Enter invasive E. coli (EIEC)
EIEC, which shares a close relationship with Shigella, is believed to induce watery diarrhea by invading the colon epithelial cells. They attach to and penetrate intestinal cells using adhesin proteins, making them extremely invasive. Although they do not produce any toxins, they mechanically destroy intestinal wall cells, causing severe damage. A few minor biochemical tests separate EIEC from Shigella, but these pathotypes share important factors that contribute to their virulence [140]. It is believed that EIEC infection is an example of inflammatory colitis, even though many patients appear to have small bowel syndrome with secretory symptoms. Abdominal cramps, malaise, tenesmus, and occasionally fever are among the symptoms. Dysentery or bloody diarrhea is an unusual consequence [124].
v) E. coli that is enteroaggregative
A pathotype of E. coli known as enteroaggregative E. coli (EAEC) causes both acute and chronic diarrhea in both developed and developing nations. Additionally, they might result in UTIs. According to Jensen et al. (2014), EAECs are identified by their "stacked-brick" pattern of adhesion to the HEp-2 human laryngeal epithelial cell line [69]. It is now accepted that EAEC is a newly discovered enteric pathogen. Specifically, EAEC is known to be a common cause of diarrhea in pediatric populations and the second most common cause of traveler's diarrhea, behind enterotoxigenic E. coli. Additionally, it has been linked to long-term infections in the latter group as well as in immunocompromised hosts, including those with HIV [66]. Intestinal infections are brought on by EAEC; these infections can cause fever, diarrhea, and stomach pain. The majority of severe cases may result in kidney failure, dehydration, or bloody diarrhea [69].
iv) Shiga toxin-generating E. coli (Non-O157 STEC)
The most well-known serotype of Shiga toxin-producing E. coli (STEC) is probably E. coli O157:H7 (EHEC), but non-O157 STEC refers to at least 150 other serotypes of STEC that can infect humans and animals. For a long time, E. coli O157:H7 was linked to the majority of STEC outbreaks that were known to occur. This was mostly due to the ease with which E. coli O157 could be found in stool cultures ordered by medical professionals and carried out in clinical laboratories. Although the virulence of non-O157 STEC is highly variable, some strains can undoubtedly be just as dangerous as O157, even having the capacity to cause hemolytic uremic syndrome (HUS) and even death. All non-O157 STEC pathogenic strains have the potential to result in bloody diarrhea and hospitalization. However, only strains carrying Stx2 (as opposed to only strains carrying Stx1) usually cause HUS. The sources and risk factors for non-O157 STEC outbreaks are often comparable to those of E. coli O157:H7. The main means of transmission are foodborne, although it can also spread through contact with animals, water, and other people.
Vii) E. coli that is diffusely adherent (DAEC)
One group of E. coli that has been linked to diarrhea is diffusely adherent E. coli (DEC). Their diffuse adherence pattern on HeLa or HEp-2 cultured epithelial cells is what distinguishes them [125]. This adherence phenotype is caused by adhesins from the Afa/Dr families, which are present in 75% of DAEC. Much attention has been given to DAEC strains possessing Afa/Dr adhesions, but only those that were positive for the daaC probe, which recognizes a conserved region from Afa/Dr adhesin operons, were found at a higher frequency in diarrheic patients than in asymptomatic controls [93].
Table 1
Major Pathotypes of E. coli that cause diarrhea [30].
| No | Strains | Diarrhea pattern | Antecedent condition |
| 1 | Enteropathogenic E. coli (EPEC) | Watery | Can cause diarrhea outbreaks in newborn nurseries |
| 2 | Enterotoxigenic E. coli (ETEC) | Watery | Produces a toxin that acts on the intestinal lining, and is the most common cause of traveler's diarrhea |
| 3 | Enterohemorrhagic E. coli (O157/EHEC) | Bloody/nonbloody | A type of EHEC on which Bloody diarrhea and hemolytic uremic syndrome (anemia and kidney failure) can be brought on by E. coli O157:H7. |
| 4 | Entero invasive E. coli (EIEC) | Bloody/nonbloody | Invades (passes into) the intestinal wall to produce severe diarrhea. |
| 5 | Enteroaggregative E. coli (EAEC) | Watery | Can cause acute and chronic (long-lasting) diarrhea in children. |
| 6 | Shiga toxin producing (non-O157 STEC) | Bloody/nonbloody | Causes of acute diarrhea, dysentery, and HUS. |
| 7 | Diffusely adherent E. coli (DAEC) | Watery diarrhea | Leads to diarrhea, stomach pain and cramps and low-grade fever |
2.1.2. Nondiarrheal pathogenic E. coli
Extraintestinal pathogenic E. coli encompass several well-described pathogens, i.e., uropathogenic E. coli (UPEC), which causes sepsis and urinary tract infections, and neonatal meningitis E. coli (NMEC), which causes sepsis and brain infections. These subspecies are important pathogens and are implicated in the global spread of antibiotic resistance genes.
i) Uropathogenic E. coli (UPEC)
Approximately 90% of urinary tract infections (UTIs) in people with normal anatomy are caused by uropathogenic E. coli (UPEC) [136]. Fecal bacteria colonize the urethra and travel up the urinary tract to the bladder, kidneys (causing pyelonephritis), or, in the case of males, the prostate in ascending infections. Women are 14 times more likely than men to experience an ascending UTI due to their shorter urethras [105]. P fimbriae, or pyelonephritis-associated pili, are used by uropathogenic E. coli to bind urinary tract urothelial cells and colonize the bladder. This receptor is absent in approximately 1% of the human population, and its presence determines whether a person is susceptible to urinary tract infections caused by E. coli. Alpha- and beta-hemolysins are produced by uropathogenic E. coli that cause lysis of urinary tract cells [136].
The Dr family of adhesins, which is especially linked to cystitis and pregnancy-associated pyelonephritis, is another virulence factor frequently found in UPEC. The Dr blood group antigen (Dra), which is found on the decay accelerating factor (DAF) on erythrocytes and other cell types, is bound by Dr adhesins. According to Justice et al. (2006), the Dr adhesins cause the development of lengthy cellular extensions that encircle bacteria, activating multiple signal transduction cascades along the way, including PI-3 kinase. By infiltrating superficial umbrella cells, UPEC can circumvent the body's innate immune defenses, such as the complement system, and create intracellular bacterial communities (IBCs) [74]. Additionally, they are capable of forming capsular polysaccharides, which aid in the formation of biofilms and the K antigen. Biofilm-generating E. coli is frequently the cause of persistent urinary tract infections because it is resistant to immune factors and antibiotic treatment. K antigen-synthesizing upper urinary tract infections caused by E. coli are frequent [49].
ii) Meningitis/sepsis-associated E. coli (MNEC)
Gram-negative neonatal meningitis is most frequently caused by this E. coli pathotype, which has a 15–40% case fatality rate and causes severe neurological defects in many survivors1. While infections by gram-positive organisms seem to be declining, the incidence of infants with early onset sepsis caused by E. coli infections appears to be increasing. Meningitis-causing E. coli strains are primarily composed of K1 capsule strains, accounting for 80% of the strains, and represented by only a small number of O serogroups, similar to E. coli pathotypes with well-established genetic bases for virulence. An intriguing distinction between MNEC and E. coli strains that cause urinary tract or intestinal infections is that, while the latter strains are easily spread through urine or feces, infection of the central nervous system does not seem to provide a clear advantage for the selection and spread of highly pathogenic MNEC strains.
These strains, found in the mother's vagina, colonize the newborn's intestines and cause bacteremia, which eventually results in meningitis. Additionally, the lack of maternal IgM antibodies (which only transfer IgG across the placenta because FcRn only mediates the transfer of IgG) combined with the body's recognition of the K1 antigen as self-due to its similarity to cerebral glycopeptides causes severe meningitis in newborns. These strains, found in the mother's vagina, colonize the newborn's intestines and cause bacteremia, which eventually results in meningitis. Additionally, the lack of maternal IgM antibodies (which only transfer IgG across the placenta because FcRn only mediates the transfer of IgG) combined with the body's recognition of the K1 antigen as self-due to its similarity to cerebral glycopeptides causes severe meningitis in newborns [36].
2.3. Antibiotic therapy and resistance
Bacterial infections are typically handled with antibiotics. However, there are significant differences in the antibiotic sensitivity of various E. coli strains. Since E. coli are gram-negative bacteria, they are immune to many antibiotics that work well against gram-positive bacteria. Amoxicillin and other semisynthetic penicillins, numerous cephalosporins, carbapenems, aztreonam, trimethoprim-sulfamethoxazole, ciprofloxacin, nitrofurantoin, and aminoglycosides are among the antibiotics that can be used to treat an E. coli infection. The issue of antibiotic resistance is becoming worse. A portion of this can be attributed to human antibiotic overuse, but a portion is most likely caused by the use of antibiotics in animal feed as growth promoters [71]. "On the order of 10 − 5 per genome per generation, which is 1,000 times as high as previous estimates," according to a study, is the rate of adaptive mutations in E. coli. This finding may be important for the management and study of bacterial antibiotic resistance [111].
resistantance to antibiotics Moreover, E. coli may use a process known as horizontal gene transfer to transfer antibiotic resistance genes to other bacterial species, including Staphylococcus aureus. E. coli frequently carries several drug resistance plasmids, which can easily spread to other species when under stress. Plasmids from and to other bacteria can be accepted and transferred by E. coli due to species mixing in the intestines. Consequently, E. coli and other enterobacteria are significant sources of antibiotic resistance that can be transferred [121]. Since the prevalence of bacterial strains that produce extended-spectrum beta-lactamases has increased in recent decades, resistance to beta-lactam antibiotics has become a particular issue. These beta-lactamases make many, if not all, of the penicillin’s and cephalosporins ineffective as therapies. Extended-spectrum beta–lactamase–producing E. coli (ESBL E. coli) are highly resistant to an array of antibiotics, and infections by these strains are difficult to treat. In many instances, only two oral antibiotics and a very limited group of intravenous antibiotics remain effective [109].
2.4. Phage therapy
2.4.1. Fundamentals of phage biology
Phages are nonliving biological entities that are simple yet highly diverse. They are made of protein capsids containing either DNA or RNA (Fig. 1). Phages are naturally occurring bacterial parasites that are dependent on their bacterial host for survival because they are unable to reproduce on their own and are therefore nonliving. Generally, phages attach themselves to particular receptors on the surface of the bacterial cell, inject their genetic material into the host cell, and either integrate this material into the bacterial genome (temperate phages reproduce vertically from mother to daughter cell) or use the bacterial replication machinery to produce the next generation of phage progeny (lytic phages), which lyse the destination cell. When the number of phage progeny reaches a critical mass, which varies based on the environment and can range from a few to over 1000 viral particles, the lytic proteins activate and hydrolyze the peptidoglycan cell wall, releasing new phage to restart the lytic cycle [145].
The majority of phages exhibit infectious properties solely to bacteria harboring their corresponding receptor, thereby effectively defining the host range of lytic phages [118]. Phages differ in their host specificity; some are strain specific, while others have shown the ability to infect a variety of bacterial strains and even genera [103]. Bacteria have developed a multitude of defense mechanisms against lytic phage infection, and phages possess an equally remarkable array of defense mechanisms against this resistance. The integration of phage DNA into the clustered regularly interspaced palindromic repeat/CRISPR-associated system and the alteration or loss of receptors in bacteria is examples of this [83]. For phages, this can include the recognition of new or altered receptors and anti-CRISPR genes. The two orders of lytic phages that are most frequently linked to human pathogens and the gut microbiota are microviridin, which are tailless single-stranded DNA viruses, and Caudovirales, also referred to as "tailed phages" because they have double-stranded DNA genomes [100].
Lysogenic phages incorporate their genetic material into the bacterial chromosome as an endogenous prophage, as opposed to lytic phages (Fig. 2). The bacterial lysogen then multiplies the prophage with each cell division. The lytic cycle and the release of phage progeny into the environment can be initiated by environmental stressors acting on the bacterial host, which can also induce the lysogenic phage from the latent prophage form. Prophage-encoded genes become accessible for transcription by the host upon integration of their genetic material into the bacterial genome [39]. Conventional phage therapy employs only lytic phages, which are inherently fatal to their bacterial host. Lithic phages are used in "phage cocktails," which are preparations made up of several phages that have been shown to be effective in vitro against the pathogen of interest.
Lytic bacteriophages undergo the lytic cycle, in which the host is lysed and offspring bacteriophages are released into the surroundings. Bacteriophages specifically attach to the bacterial host on a receptor present on the surface of the bacteria and inject their genetic material into the cell. The host cell supplies the necessary molecular building blocks and enzymes to replicate the bacteriophage's genetic material and produce offspring bacteriophages. During the release of new viruses, bacteriophage enzymes participate in disrupting the structures of host cell-cell lysis [85]. Bacteriophage-encoded proteins such as endolysin and holin lyse the host cell internally. Holins are small proteins that accumulate in the cytoplasmic membrane of the host and allow endolysin to degrade peptidoglycan, enabling offspring bacteriophages to escape. Subsequently, in the external environment, lytic bacteriophages can infect and destroy all nearby bacteria. The production of large numbers of offspring by lytic bacteriophages is an advantage when they are used in bacteriophage therapy.
2.4.2. Phage classifications
Bacteriophages are classified on the basis of their morphological structure and genetic materials. The majority of phages are tailed phages with dsDNA and are members of the Caudovirales order. The DNA translocase molecular motors that pack the chromosomes of tailed phages into the procapsid are identical, but the DNA replication technique and the resulting genome end are different [32]. The type and unique features of the receptor on the surface of the host cell determine how the phages are absorbed. Phages are primarily divided into virulent and temperate phages based on their life cycles. The lytic life cycle is followed by virulent phages. However, under certain circumstances, temperate phages can occasionally switch from the lysogenic to the lytic cycle. The two main proteins employed by lytic phages to kill their host cells are holin and lysine. The International Committee on Taxonomy of Viruses (ICTV) categorizes phages based on their appearance and nucleic acids (Table 2). The Caudovirales order, which contains the Myoviridae family with a contractile tail, the Podoviridae family with a short tail, and the Siphoviridae family with a noncontractile long tail, makes up approximately 96% of the documented bacteriophages. The same order includes filamentous, cubic, and polymorphic phages, which are divided into 10 distinct families and account for approximately 3.6% of all known bacteriophages [76].
Table 2
Bacteriophage classification [54]
| Family | Morphology | Nucleic acid | Examples |
| Myoviridae | Contractile tail, Non enveloped | Linear dsDNA | T4 virus, P1, P2, FO1, Jilinvirus, Vequintavirus |
| Siphoviridae | Long non contractile tail, Non enveloped | Linear dsDNA | Lambda, T5, N15, Kagunavirus, Dhillonvirus |
| Podoviridae | Short non contractile tail, Non enveloped | Linear dsDNA | T7 virus, P22, T3, SP6 |
| Tectiviridae | Isometric, Non enveloped | Linear dsDNA | PRD1 |
| Corticoviridae | Isometric, Non enveloped | Circular dsDNA | PM2 |
| Lipothrixviridae | Rod-shaped, Enveloped | Linear dsDNA | Acidianus filamentous virus |
| Plasmaviridae | Pleomorphic, Enveloped | Circular dsDNA | Acholeplasma laidlawii virus L2 |
| Rudiviriade | Isometric, Non enveloped | Linear dsDNA | SIRV1 |
| Fuselloviridae | Lemon-shaped, None enveloped | Circular dsDNA | SSV-1 |
| Inoviridae | Filamentous, Non enveloped | Circular ssDNA | M13 |
| Microviridae | Isometric, Non enveloped | Circular ssDNA | ΦX174 |
| Leviviridae | Isometric, Non enveloped | Linear ssDNA | |
| Cystoviridae | Spherical, Enveloped | Segmented dsDNA | Φ6 |
2.4.3. Therapeutic application of phages
i) History of phage therapy
Bacteriophages were separately discovered in 1915 by Frederick William Twort and in 1917 by Felix d'Hérelle. Twort reported on a possible "ultramicroscopic virus" that he recovered from vaccinia virus cultures using "white micrococcus" cultures. It appears that the lytic phages that were identified were bacteriophages that targeted Staphylococcus species that were present in a vaccinia virus culture.
On the other hand, Felix d'Hérelle isolated bacteriophage active against Shigella bacillus from the stools of patients recuperating from bacillary dysentery. According to Abedon et al. (2011), there were indications of bacteriophage presence even before their discovery, a time frame known as bacteriophage prehistory [3]. Felix d'Hérelle used bacteriophages in medicine for the first time in 1919 [133].
Worldwide, the use of bacteriophages to treat infectious disorders increased between the early 1920s and the late 1930s [43]. This phase of inflated expectations was succeeded by a period of waning excitement for phage therapy throughout much of the western world, which was followed by antibiotics replacing its usage following World War II and a shift in emphasis toward the use of phages as model genetic systems. Phage therapy was difficult to administer since, at the time of its discovery, relatively little was understood about phages. In fact, until they were seen in the 1940s with the development of electron microscopy, their very existence was a matter of debate. Although phage research did not cease in the former USSR, with the establishment of the Eliava Institute in Tbilissi, Georgia, and other nations such as Poland (including its well-known Hirsfeld Institute in Wroclaw), phage therapy for animals was rediscovered in the English literature in the 1980s [128]. Phages have been used therapeutically for a very long time in Eastern Europe and the former Soviet Union [131]. It was proposed that bacteriophages could be used to prevent and/or treat bacterial infections before the discovery and widespread use of antibiotics [132]. After antibiotics were discovered, phage therapy was widely abandoned due to several logistical and technical challenges. The English literature rediscovered phage therapy in animals in the 1980s, although phage research was never abandoned in the former USSR. This was due to the establishment of the Eliava Institute in Tbilissi, Georgia, as well as other countries such as Poland, which included the well-known Hirsfeld Institute in Wroclaw.
Eastern Europe and the former Soviet Union have long employed phages as medicinal agents [131]. It was proposed that bacteriophages had been used to prevent and/or treat bacterial infections before the discovery and widespread use of antibiotics [132]. Phage therapy was largely discontinued after antibiotics were discovered because of numerous logistical and technological difficulties. However, in the world before antibiotics, when the standard of care for treating bacterial illnesses was incredibly ineffective, phages, with their innate antibacterial qualities, might give much-needed hope. Poor use documentation and inconsistent results were major contributing factors to phage therapy [87]. However, there is still much data supporting their clinical application at present, and several historical innovations have been linked to significant phage therapy-influencing events (Fig. 3).
ii) Phage therapy principles
The keys to antibacterial therapy success are building a library of different bacteriophages. According to Cui et al. (2017), bacteriophages exhibit stringent specificity and can range in host range from very narrow to broad [38]. A range of virulent bacteriophages that can kill the same strain as well as different lytic bacteriophages that can kill different species should be kept in the library. The establishment of stringent enrollment criteria for bacteriophages intended for clinical use is crucial.
For bacteriophage therapy to be used in clinical settings, standard operating procedures for bacteriophage preparations, storage, and transportation must be developed. Several crucial steps for their application were included in the documented clinical trials of bacteriophage therapy: bacteriophage isolation, characterization, susceptibility testing, endotoxin removal, and production of relevant products. Monitoring of bacteria resistant to bacteriophages and assessment of bacteriophage pharmacokinetics during therapy were also aspects of a documented successful case of bacteriophage therapy. Nevertheless, bacteriophage pharmacokinetics, endotoxin removal, and monitoring of bacteriophage-resistant bacteria were not part of a double-blind phase 1/2 trial. In the event of a bacterial infection in the gastrointestinal tract, it is important to prevent the phage from being neutralized by stomach acid when administering it orally. Additionally, other methods of treatment that have the potential to deactivate phages, such as antiseptic agents, should not be utilized in conjunction with phage preparations [41]
Before lytic phages can be used therapeutically in the West, more study is needed to gather reliable pharmacological data about them, including thorough toxicological studies. Therapeutic phages are believed to kill their target bacteria by multiplying inside and lysing the host cell through a lytic cycle as part of their bactericidal function. However, later research showed that lytic and lysogenic phages have significantly different replication cycles and that not all phages replicate in the same way (Fig. 2).
Lytic phage lysis of host bacteria is a complex process involving a cascade of events involving several structural and regulatory genes, as demonstrated by the recent delineation of the full sequence of the T4 phage and years of elegant studies of the mechanism of T4 phage replication. Since the T4 phage is a typical lytic phage, it is conceivable that many therapeutic phages work in a manner similar to this; however, it is also conceivable that some therapeutic phages possess particular, as yet undiscovered genes or mechanisms, which enable them to successfully lyse their target bacteria [87].
Phages have been given to humans orally, in tablet or liquid formulations (105 to 1011 PFU/dose); rectally; locally (skin, eye, ear, nasal mucosa, etc.), in tampons, rinses, and creams; compared to the first four methods, there have been almost no reports of serious complications related to the use of aerosols or intrapleural injections, and intravenously [53]
Two different phage therapy methods were created at the time of the early 2000s phage therapy renaissance [113]. These are one size fits all strategies and personalized phage therapy approaches. Broad-spectrum-defined phage cocktails, which were intended to target the majority of bacteria thought to be responsible for several infectious disorders, were used in what might be considered the one-size-fits-all method [6]. These predetermined broad-spectrum phage mixtures were created, manufactured, and evaluated using the pharmacoeconomic models that are now in use and were created to support "static" medications such as antibiotics [18]. However, true broad-spectrum phage cocktails that were effective against the majority of gram-positive and/or gram-negative bacteria frequently found in infectious disorders needed a significant number of phages and proved to be extremely challenging to create. It was possible to create phage cocktails with a narrower spectrum that were only effective against one or a small number of bacterial species, to be utilized in specific situations and with the knowledge of the bacterial species that would be infected beforehand. Phages with very extensive host ranges have been isolated and characterized for various bacterial species, including E. coli [142].
In the case of personalized phage medicine, one or more phages were chosen for phage therapy concepts from phage banks or the environment, and they may have been modified (in vitro selection of phage mutants exhibiting increased infectivity) to more effectively infect the bacteria isolated from the patient's infection site. Large therapeutic phage banks were set up and maintained by several phage therapy facilities. These banks were frequently updated with new phages, expanding and adapting the bank's host range to the constantly shifting bacterial populations. As only the infecting bacterium is targeted, there is less selection pressure toward the development of bacterial phage resistance, making personalized phage therapy approaches potentially more sustainable [52]. They shipped bacterial strains and corresponding phages all over the world, which made them more intricate and logistically challenging than one-size-fits-all methods.
2.4.4. Phage therapy against E. coli
Phage treatment for E. coli involves identifying and isolating specific phages that can infect and kill the target E. coli strain. These phages are then purified and prepared for treatment. E. coli phages are commonly isolated from marine environments including fresh and salt water, sewage, hospital waste, human and animal faces, various food sources (such as vegetables, fruits, dairy, and fish), soil, plants, and other environmental sources [81]. Additionally, phages are frequently found in human skin, vagina, mouth, and other parts of the gastrointestinal tract, where their population is thought to be approximately 1 × 1015 [40].
Pathogenic E. coli bacteria are divided into two groups mainly according to the location of the disease: extraintestinal pathogenic E. coli (ExPEC) and enteric pathogenic E. coli (InPEC). Enteric pathogenic E. coli is generally divided into those causing diarrhea by expressing heat-labile or heat-stable toxin or Shiga toxin. Diarrheal diseases, and/or the enteric pathogens are one of the leading causes of death in children under the age of five. One of the main worldwide causative groups of these infections is E. coli. Enteropathogenic E. coli (EPEC) and enterotoxigenic (ETEC) E. coli pathogens are endemic primarily in developing nations, and ETEC strains are the primary cause of diarrhea in visitors to these regions. Conversely, enterohemohergic E. coli (EHEC) is the origin of major epidemics in the world, mainly affecting industrialized countries, responsible not only for diarrhea but also for serious clinical complications such as hemorrhagic colitis and hemolytic-uremic syndrome. Overall, the emergence of antibiotic-resistant strains, the annual increase in healthcare costs, the high incidence of travelers' diarrhea, and the increase in the number of episodes of the hemolytic-uremic syndrome have increased the need for effective treatments. Bacteriophage may be an alternative to antibiotics and have potential therapeutic capacities in treating the disease E. coli (Table 3).
A mouse study by Chibani-Chennoufi et al. (2004) showed that broad host range T4-like coliphages for diarrhea-associated E. coli serotypes were isolated from stool samples from infants with diarrhea and from ambient water samples. All of these isolated phages showed very efficient passage through the digestive tract of adult mice when added to drinking water. Viable phages were recovered from the feces in a dose-dependent manner. Just 103 PFU of phage per milliliter of drinking water was the lowest oral dose needed for sustained fecal recovery. In conventional mice, orally administered phage remained confined to the gut lumen and, as expected for noninvasive phage, no histopathological changes were observed in the gut mucosa of phage-exposed animals. E. coli strains introduced into the gut of conventional mice and monitored for ampicillin-resistant colonies were successfully lysed in vivo by phage added to the drinking water [33].
According to Dissanayake et al. (2019), phage treatment reduced viable E. coli O157:H7 in infected mice with efficacy comparable to ampicillin therapy [45]. However, compared to ampicillin, the bacteriophage preparation had less of an impact on the gut microbiota. With no negative effects on the normal, and frequently beneficial, gut flora, lytic bacteriophage preparations can be used prophylactically or therapeutically to prevent or treat bacterial infections of the gastrointestinal tract, including those brought on by eating food contaminated with important foodborne bacterial pathogens such as L. monocytogenes, Salmonella spp., and enterohemorrhagic E. coli (e.g., E. coli O157:H7) [82].
The reports from Dalmasso et al. (2016) demonstrate that three human intestinal phages showed promise as potential phage therapies. According to these authors, the three-phage cocktail completely inhibited the growth of E. coli. The phage cocktail also reduced biofilm formation and prevented the emergence of phage-resistant mutants that appeared in a single phage. Phage combined with ciprofloxacin alone or in cocktails inhibited the growth of E. coli and interrupted the emergence of resistant mutants. These new phage isolates are promising agents for the biological control of E. coli infections. The human gut is a natural reservoir of many phages with promising antibacterial properties [40].
Bruttin and Brussow. (2005) used E. coli T4 phage to treat acute infectious bacterial diarrhea in adults and children. Fifteen healthy adult volunteers received a lower dose of E. coli T4 phage (103 PFU/ml), a higher dose of phage (105 PFU/ml), and a placebo by drinking water. Fecal coliphage was detected in a dose-dependent manner in volunteers who were orally exposed to the phage. All volunteers receiving the highest dose of phage showed fecal phage 1 day after the challenge; this rate was only ± 50% in subjects receiving the lowest dose of phage. One week after a 2-day oral phage application, no fecal phage was detectable. Oral administration of the phage did not cause a reduction in the total number of E. coli in the stool. In addition, in the commensal population of E. coli. No side effects associated with the use of phage have been reported. They found that while the E. coli T4 phage is safe, its therapeutic efficacy is still controversial [27].
However, a report by Sarker et al. (2017) indicated that oral administration of phage to hospitalized children with acute diarrhea did not improve diarrhea scores, possibly due to phage's insufficient ability to fight a broad spectrum of diarrhea or genetic variability of E. coli [122]. In addition, it was not clear whether E. coli was actually responsible for diarrhea since the fecal samples were largely dominated by streptococci. The reduced efficiency of the phage titers after passing through gastric acid was identified as another possible reason for the failure of the assay. In addition, possible differences between the fecal and intestinal physiological status of E. coli and a low titer of the fecal pathogen could have prevented E. coli phage replication. These results confirm that much more knowledge of phage-bacteria interactions in vivo is needed if we are to develop effective phage therapy assays. On a positive note, the coliphages administered during the study passed the gut safely, which helped demonstrate the safety aspects of phage therapy [122].
Table 3
Some reports of phage therapy in pathogenic E. coli
| Diseases | Phage/s applied | Effectiveness | Reference |
| Urinary tract infection | Single-phage/T4 | Bacterial inoculum rendered untreated mice 100% fatal; however, phage (MOI 60) saved all mice. | [106] |
| Gastroenteritis | Single-phage/unspecified | Dysbiosis-related weight loss and behavioral abnormalities were found in rats given antibiotics alone or in combination, despite the absence of bacterial contamination in these groups. | [140] |
| Urinary tract infection | Single-phage/KEP10 | Bacterial inoculum rendered infected, untreated mice 100% fatal; however, phage (MOI 60) saved 90% of the mice. | [106] |
| Gastroenteritis | Single-phage/T4 | Rats treated with phage outlived untreated rats by 83–0%. | [119] |
| Lung infection | Single phage/536-P1 | The phage saved 100% of the animals from death compared to 25% survival in infected, untreated controls; Reduction of mortality from 80–25% by adapted phages | [47] |
| Systemic infection | Single-phage/K1 phages | Following the lowest treatment dose, K1 capsule-dependent phages produced a 6 log10 reduction (specimen unspecified) in comparison to K1 capsule-independent phages. | [28] |
| Gastroenteritis | Cocktail-phages/ EcD7, V18, SE40, SI3, CH1, Lm1, ST11 | Mice not given any treatment had 104 cfu of bacteria per gram of stool, while mice given the phage had none. | [13] |
| Gastroenteritis | Cocktail/ CLB_P1, CLB_P2, CLB_P3 | Bacterial colonization in ileum-treated mice was 88% lower than in control mice, but by day 7 post treatment, bacterial density had rebounded to levels similar in both groups. | [95] |
| Systemic infection | Single-phage/ EC200PP | 7-hour post infection treatment results in 100% rescue and bacterial elimination in blood; 24-hour post infection treatment results in 50% rescue. | [127] |
| Meningitis | Single-phage/ EC200PP | 100 of the 100 meningitis-induced death rats were saved after receiving treatment with 108 pfu 1 or 7 h | [127] |
2.4.5. Challenges regarding phage therapies
Bacteriophages typically affect specific types of bacteria, and some only affect a few species, and therefore cannot attack all pathogenic strains of the same bacterial species [67]. Although single-bacterium diseases can be effectively treated with bacteriophages, many infections reported in case studies involve multiple pathogenic bacteria. As a result, certain bacteriophages frequently struggle to produce the intended therapeutic outcome. The lysogenic phenomenon is caused by certain lysogenic phages that, once integrated with the host bacterium, are unable to lyse the host bacterium and prevent other phages from acting lytically on the host bacterium. When a virus is lysogenic, it replicates its genome from the host DNA, either before or after joining the bacterial chromosome. In addition, there is a major concern that bacteriophages in the lysogenic state can also transmit toxins and antibiotic-resistance genes to bacteria [31]. The limits of phage therapy, therefore, lie in the stability of the phages in the preparation, the evolutionary resistance of the bacteria, the limited effect of the phages, and the difficulty of the screening methods.
i) The storage stability issue of phage preparation
A promising phage therapy candidate should have a long-life span; it should be stored in a preparation that provides activity without a significant decrease in phage titer during treatment and long-term storage, as such a decrease may adversely affect treatment outcome [68]. However, the stability of phages in different preparations (e.g., liquids, gels, powders) is very variable, especially between different phage types. An alternative strategy to improve the durability of phages is to encapsulate them on various matrices such as liposomes, alginate, cellulose, or other polymers. In vitro and in vivo studies demonstrated the ability of encapsulated phages to persist for a long time at low pH, improving the efficacy of oral administration in animal models [143]. Another issue with phage stability is the occurrence of spontaneous mutations in phage stocks stored for long periods or accumulated during phage production, which can affect viral fitness [26]. Although difficult, it would be useful to predict the evolution of phages during production to establish a production process that minimizes the mutation rate in phage genomes [53].
ii) Bacterial phage resistance developing
One of the main problems with phage therapy is the possible emergence of bacteriophage-insensitive mutants (BIMs), which could impede the success of this therapy. In recent years, several studies have addressed the issue of bacterial phage resistance, showing that the emergence of phage-resistant mutants is common and almost inevitable [108]. In most of these studies, bacterial phage resistance was caused by mutations in genes encoding phage receptors, which include lipopolysaccharides, outer membrane proteins, envelopes, flagella, pili, and others. Several animal models, human pilot studies, and case reports have all seen the emergence of phage-resistant variants in action. Bacterial phage resistance can be circumvented by different approaches [97]. The most typical is the phage combination, which preferentially targets various receptors and has complementary host ranges, into a single preparation, commonly known as a phage cocktail. Such cocktails not only show greater coverage against a specific bacterial species but can also prevent BIM from occurring. Finally, the combination of phage with antibiotics or other antibacterial agents can also be used to avoid the development of bacterial resistance and improve therapeutic efficacy [134].
iii) Problems with phage screening techniques
Because of the increased selectivity of phage action, finding a phage that preys a particular strain often requires screening large collections of phages. The most traditional method for detecting phage activity against a strain is the bilayer agar (DLA) method, in which multiple phages are plated on a bacterial carpet of interest [75]. Depending on the growth rate of the specific target strain, it can take up to 48 h to obtain results; therefore, the DLA method is impractical in a therapeutic setting where rapid diagnosis is essential. High throughput and rapid screening methods are desirable to quickly identify phages capable of successfully infecting target strains. Many methods for the detection and quantification of phages by direct or indirect measurement have been developed, but few appear to be applicable in the clinical setting. Real-time PCR (qPCR) [91], flow cytometry, surface plasmon resonance capacity (SPR), cellular respiration, and optical density kinetics have been developed for rapid and sensitive detection analysis and identification of infections, e.g., by detecting elevated concentrations of phage. If phage therapy is to be widely used as a treatment option in the future, a simple and fast, high-throughput method should be developed and implemented in clinical and banking settings. The strict host effect and non-unique pharmaceutical properties of phages are also major limitations of phage therapies.