The ubiquitous nature of microorganisms extends to their presence in the gastrointestinal tract (GIT) ^[1][2][1,2], where they play a host of unique physiological characteristics and functions which contribute to the overall well-being and health [2], including, but not limited to, their help in digestive and metabolic processes [3], gut barrier protection [4], essential vitamin production [5], and the immune system [6].

The gut microbiota is influenced by a potential compounding combination of different factors determining the microbial makeup [7]. These include diet or nutrient type (e.g., plant and fiber-based diets promote the selection/presence of beneficial microorganisms, unlike chemically processed and sugary diets, and breast milk has significant influence on the infant microbiota in the first few years) [7], external chemicals (e.g., antibiotics) ^[8][9][8,9], genetic/hereditary profile (although largely unclear) [10], age [9], environmental pollutants/chemicals (e.g., agrochemicals), medical conditions (e.g., celiac and IBDs), and lifestyle [9].

Pathogenic/unhealthy gut microorganisms can hardly ever be eradicated and can even be potentially beneficial; however, maintenance of the equilibrium between the healthy and harmful gut microorganisms is essential and, through several mechanisms, is related to well-being and overall health and the prevention, development, and management of health/medical conditions [11]. Specifically, IBDs often result from the immune response to the dysbiosis of the gut microbiota, increasing the potentially pathogenic microorganisms (e.g., Pseudomonadota and Enterobacteriaceae) and decreasing the beneficial microorganisms (e.g., Lactobacillaceae and Bifidobacteria) [11]. Accumulative evidence has shown that disruption of the metabolites and the gut microbiota can influence insulin sensitivity [12], optimal brain and central nervous system (CNS) (which, of course, controls most of the GIT physiology) functions, and emotional behaviors ^[13][14][13,14]. High blood pressure, a top risk factor for heart disorders, can be initiated by disruption of the gut microbiota [15]. Also, specific microbial genes are associated with the generation and accumulation of Trimethylamine N-oxide (TMAO), a risk factor for cardiovascular diseases, especially at high concentrations ^[16][17][16,17]. The gut microbiota can be described as the “other organ”, contributing significantly to nutrient and energy acquisition and regulation. Thus, dysbiosis can negatively affect the above functions, triggering excessive tissue deposition and obesity [18].

One prominent and easily the most abundant life forms are the bacteriophages [8]. Bacteriophages have found applications in diverse fields and have peculiar characteristics in clinical practice against pathogens, including their respective specificity against limited bacterial genera and species ^[19][20][19,20]. They are also minimally allergenic with “little or no” side effects [21]. They form a major part of the gut microbiota and constantly interact with host bacterial strains [4]. Specifically, they naturally aid in the regulation/modulation of the gut bacterial population, acting as natural predators, selectively infecting and killing specific bacterial strains and, hence, aiding in maintaining the gut microbiota and its overall composition [22]. Also, specific interactions, which release cytokines and other immune molecules, contribute to the modulation of the immune system [4]. Furthermore, they significantly contribute to human metabolic processes and the general well-being in human health [4].

Many pathogenic and opportunistic bacteria are inherent in the gut system and have led to gut disease initiation, development, and advancement. Phage therapies are, however, developed and targeted at these bacteria. Potential applications of phages for preventive and curative purposes have produced single phages, cocktails, genetically modified phages, and even a combination with some other therapeutics, including antibiotics and probiotics ^[23][108]. The extensive literature review revealed that studies are developing therapies against the most prevalent bacteria associated with gut system infections, including Vibrio spp., Escherichia coli, Clostridioides difficile, Salmonella spp., Fusobacterium nucleatum, Shigella spp., Klebsiella pneumoniae, Listeria monocytogenes, Ruminococcus gnavus, and Campylobacter spp. Though most are still in the in vitro investigation, preclinical, and clinical trial stages, diverse potential applications have been found for many bacteriophage species, and they serve as potent therapeutic alternatives to managing multiple-drug-resistant strains ^[24][109]. These studies have enabled the identification and profiling of phages individually and in combinations using different dosage forms and delivery systems.

Considering the already established potential of phage therapy in gastrointestinal (GI) disorders, Jaiswal et al. ^[24][109] evaluated the in vitro and in vivo (using rabbits) therapeutic efficacy of a pure lytic vibriophage cocktail against the V. cholerae strain MAK 757 (ATCC 51352) that is implicated in cholera. The results of the lytic effects of individual vibriophages, B1, B2, B3, B4, and B5, applied singly and in combination as a cocktail showed synergistic effects, with the cocktail outperforming each of the individual phages. A comparison of the therapeutic management of orally induced V. cholerae infection in mice using a phage cocktail, conventional antibiotics (ciprofloxacin), and an oral rehydration system by Jaiswal et al. ^[25][110] showed that although the antibiotics had a significantly better anti-V. cholerae effect, the phage cocktail presented a significantly better safety and specificity profile and, thus, was more reliable in managing the infection. Jun et al. ^[26][111] showed a 74% (20 out of 27) lysis of multi-resistant Vibrio parahaemolyticus, a marine bacterium implicated in gastroenteritis and transmitted via raw oyster consumption, with optimal phage protein achieved following immediate phage therapy after infection. Yen et al. ^[27][112] utilized the potential prophylaxis of a cocktail of three virulent V. cholerae-specific phages, ICP1, ICP2, and ICP3, with specific effects against V. cholerae domiciled in the small intestine. From the in vivo results, the 24-h oral administration of the phages before cholera infection reduced intestinal tract colonization, thereby preventing cholera-like diarrhea. In addition to successfully demonstrating prophylaxis in V. cholerae-induced diarrhea, phage resistance was also not observed in the V. cholerae colonies.

Nasr-Eldin et al. ^[28][113] isolated and characterized highly stable Siphoviridae and Podoviridae phages for their lytic potential against Escherichia coli-causing gastrointestinal diseases using phage and E. coli incubation in a high saline environment. The characterizations, including the host range and synergistic profile, suggested that these bacteriophages were ideal candidates for therapeutic use. Similarly, Abdulamir et al. ^[29][114] targeted E. coli strains implicated in human gastroenteritis using a cocktail of 140 specific lytic phages administered to mice via their drinking water, oral injection, or vegetable capsules. The results revealed that the group receiving phage therapy via vegetable capsules obtained the least positive fecal cultures. While the peak reduction in E. coli was seen between 5–10 days post phage feeding, the second best-performing group following the phages’ administration was the group treated via the drinking water, with the study providing insight into the possible use of phage feed as a biocontrol for eliminating E. coli from animal intestines. Also, Abdelaziz et al. ^[30][115] isolated, characterized, and reported the broad-coverage lytic efficacy of phage phPE42 against E. coli clinical isolates implicated in gastrointestinal tract infections in an in vivo experiment. Bourdin et al. ^[31][116] acknowledged the downside of phage species and strain specificity and underlined the need for page therapies with broad host ranges. Also, the need to develop better-targeted phage therapies for disease conditions is needed. In treating childhood diarrhea-associated E. coli infection, they obtained and tested the lytic effects of 89 T4-like phages against four (4) batches of E. coli isolates. The result revealed that specific phage therapies for the tested pathogens were difficult and complex owing to the geographical, epidemiological, and time differences, thus recommending the need to identify flexible and specific therapeutic phages. With over 80% of traveler (TD) and childhood diarrhea cases caused by a variety of enteropathogens, and with multi-resistant E. coli usually responsible for about 30–40% ^[32][117], Aleshkin et al. ^[32][117] developed and assayed a phage cocktail for prophylaxis against TD caused by E. coli, Shigella flexneri, Shigella sonnei, Salmonella enterica, Listeria monocytogenes, and Staphylococcus aureus, and obtained specific prevention effects against TD caused by E. coli. In a similar experiment, Vahedi et al. ^[33][118] assayed the potential of combining a specific bacteriophage and antibiotics targeted against enteropathogenic E. coli (EPEC), using both in vitro and in vivo models. The in vitro study showed that 10⁶ PFU/ml of the phage eliminated EPEC from infected HEp-2 cells. In vivo, administration of the phage:antibiotic combination presented a total reduction in EPEC after 24 h and is attributed to the potentiation effect of both the antibacterial agent and the phage. However, slight weight loss was observed in the mice, possibly due to the adverse impact of antibiotics on the microbiota. However, Sarker et al. ^[34][119], in their work against acute bacterial diarrhea in children, used safe, orally administered species-specific T4-like coliphages in human subjects and showed no improvement in diarrhea symptoms, which was attributed to insufficient phage coverage and low E. coli titers. However, they assume that higher oral phage doses might be necessary to obtain the desired outcome, which triggers the need for more knowledge using in vivo phage–bacterium interaction strategy to understand E. coli propagation in childhood diarrhea. Galtier et al. ^[35][120] utilized three virulent bacteriophages in therapy against the colonization of adherent invasive E. coli (AIEC) strain LF82 implicated in inflammatory bowel diseases, Crohn’s disease, and ulcerative colitis and showed sufficient phage replication in the ileum, cecum, and colon following murine gut analysis. A single day of treatment with the bacteriophages administered to LF82-colonized AIEC strain CEABAC10 transgenic mice, which express the human carcinoembryonic antigen-related cell adhesion molecule 6CEACAM6 glycoprotein receptor for AIEC, revealed a notable decrease in the AIEC count. Over two weeks of continuous treatment resulted in the absence of colitis symptoms in mice colonized with the bacterial strain. Cieplak et al. ^[36][121] demonstrated the safety of phages in relation to their ability to induce dysbiosis following a comparison with antibiotics in the in vitro decolonization of E. coli populations in the small intestine. From the study, unlike the synthetic drug, the phage preparation had a targeted lytic effect on the E. coli populations. It impacted no other commensal bacteria used in the study, thus supporting its application in personal medicine, as characterized by its targeting of the bacteria of interest and evasion of dysbiosis induction. The overall efficacy of an ascertained safe commercial phage cocktail, PreforPro^®, on the gut microbiota and markers of intestinal and systemic inflammation in a healthy human population was studied by Gindin et al. ^[37][122]. Twenty-eight (28) days of phage consumption did not alter the normal gut microbiota of most individuals but significantly reduced the target E. coli population and the pro-inflammatory cytokine interleukin 4 (Il-4) responsible for inflammatory reactions in the gastrointestinal tract. Shiga-toxin-producing E. coli is implicated in severe, difficult-to-treat infections. The safety and tolerability of PreforPro^® were reported by Grubb et al. ^[38][123]. They demonstrated the possible enhancement of the combined impacts of a probiotic microorganism and phage (Bifidobacterium lactis BL04 + PreforPro^®) on gastrointestinal discomfort and stool consistency in a healthy adult population. They presented significant improvement in gastrointestinal symptoms over four weeks of therapy administration without disruption of the gut microbiota. There was also an associated increase in the relative abundance of some microorganisms, including Lactobacillaceae. Thus, the study suggests a strong connection between phage and the use of probiotics in improving the microbiota of the intestinal environment. The study by Alomari et al. ^[39][124] also further supported the simultaneous administration of bacteriophages and probiotics. They administered Lactobacillus spp. and phages of pathogenic E. coli combinations in suppositories in treating diarrheal calves. They reported a reduction in the diarrheal symptoms following therapy, complete elimination 24–48 h post-therapy, and a significant increase in the body weight of the treated calves compared with the control. Hsu et al. ^[40][95] proposed the use of genetics-based anti-virulence mechanisms in neutralizing the expression of the bacterial toxin and minimizing resistance as a better opinion to the conventional antibacterial approach. Unlike the conventional mechanism of bacterial lysis, temperate phages can be genetically engineered and integrated into the bacterial chromosome, and they are capable of neutralizing targeted gut bacterial toxins, impeding the virulence factors by modifying bacterial function at the genetic level; thus, they are good candidates for this therapy ^[41][125]. Hsu et al. ^[40][95] and Hsu et al. ^[41][125] utilized temperate phages capable of self-integration into the bacterial genome in in vivo and in situ studies, respectively, and both reported significant repression of the Shiga toxin secretion from E. coli in the mammalian gut. Cepko et al. ^[42][126], using a mouse model, isolated a strictly lytic phage that kills strains of enteroaggregative E. coli associated with both acute and chronic diarrhea ^[43][127]. A single dose of the phage one day post-infection-administration significantly reduced the bacterial count without altering microbiota diversity. Green et al. ^[44][128] explored a location-targeting mechanism aimed at enhancing the phage specificity and lytic cycle and treating gut infection, having observed that invasive pathobionts could reside deep within the mucosal epithelium of the gastrointestinal tract. With the ability to bind to heparan-sulfated glycans on the epithelial surface, the bacteriophage can position itself close to the target host. Here, phage HP3 showed lytic activities toward E. coli ST131 in vitro in a murine sepsis model. However, it proved ineffective in similar activities in the murine intestinal tract. The absence of lytic abilities in the latter was attributed to the intestinal mucins. Under a simulated intestinal environment, a podovirus phage isolated from wastewater, while not altering the intestinal microbiota compared with antibiotics, proved to be more effective due to its inherent ability to bind to heparan-sulfated proteoglycans on the surface of intestinal epithelial cells, thus strongly suggesting this feature to be responsible for the targeted lytic activity against the host bacterium.

As an alternative to the use of antibiotics in treating GIT dysbiosis due to C. difficile infection (CDI), Nale et al. ^[45][129] assayed individual phages and a phage cocktail containing different phage combinations for their synergistic potential in the adequate clearance of C. difficile. Results obtained after 36 h post-infection supported the potential application of phage combinations for the targeted eradication of CDI and also concluded that specific phage combinations caused the complete lysis of C. difficile in vitro and prevented the appearance of resistant strains. Selle et al. ^[46][130] attempted to repurpose the endogenous type I-B CRISPR-Cas system in C. difficile as an antimicrobial agent through the use of bacteriophage capable of expressing a self-targeting CRISPR that redirected endogenous CRISPR-Cas3 activity against the bacterial chromosome and demonstrated that a recombinant bacteriophage expressing bacterial-genome-targeting CRISPR RNAs had significant lytic activities against C. difficile in both in vitro and mouse models. The study suggested that phage-delivered programmable CRISPR therapeutics have the potential to increase safety, specificity, and efficacy in complex gut microbial communities and offer a novel mechanism for the treatment of gut pathobionts.

Using animal models, Dallal et al. ^[47][131] analyzed phage SE20 active against Salmonella enteritidis, a Gram-negative bacterium that occurs mainly in human gastroenteritis and is often implicated in salmonellosis, a disease commonly caused by the ingestion of animal-derived products, mainly poultry products (meat and eggs), that are significant carriers of Salmonella spp. ^[48][132]. The in vivo study revealed that a single dose (2 × 10⁹ PFU/mL) of the phage isolate provided a targeted and prophylactic effect against S. enteritidis. Additionally, while there was no bacterial resistance over twelve months of observation, compared with the animal groups receiving phage therapy, the test synthetic antibiotic used caused apparent weight loss in the administered experimental mice. Moye et al., 2019 ^[49][133] demonstrated that the direct ingestion of phages against Salmonella could enhance intrinsic gut resilience and provide protection against Salmonella-induced foodborne diseases. In their study, Simulator of the Human Intestinal Microbial Ecosystem (SHIME), a system aimed at exploring the potential of phage cocktails termed foodborne outbreak pills (FOPs) to eliminate foodborne pathogens and maintain the balance of the host microbiome, effectively depopulated the Salmonella without any distortion of stability of the gut microbiota. Thanki et al. ^[48][132] also reported that an increase in phage dosage resulted in the proportional and effective control of colonization by Salmonella spp. Using poultry and swine assays in vitro and in vivo, Nale et al. ^[50][134] determined the potential of twenty-one myoviruses and one siphovirus in eliminating Salmonella. Individual phages significantly reduced the growth of test isolates within six hours post-infection and the subsequent phage administration. However, bacterial regrowth within an hour following treatment suspension was reported, indicative of bacterial resistance to phage therapy. A novel three-constituent phage cocktail was employed in vitro for its lytic efficacy in an optimized Galleria mellonella larva model infected with Salmonella to remedy the resistance. Comparatively to the individual phages, the cocktail had broader bacterial range coverage, improved lytic efficiency, and prevented the emergence of resistant strains. The study is further supported by Pelyuntha et al. ^[51][135] in their comparison of the lytic profile of individual phages and a phage cocktail against Salmonella colonization, implicated in the broiler gastrointestinal tract, to enhance poultry consumption safety. Pronounced synergistic enhanced lytic activities and evasion of resistance were obtained with the cocktail compared with the individual agents. The study also affirmed that phages, generally considered safe by the FDA and specific in action, remain potential ideal biocontrol agents for bacteria colonization and biofilm formation in various edible products.

Pro-tumoral F. nucleatum is significant in advancing colorectal cancer and potentially influences the therapeutic response ^[52][136]. By incorporating the principles of nanotechnology in a strategic attempt at gut microbiota manipulation, Zheng et al. ^[52][136], in demonstration of phage-guided nanotechnology and the potential to control F. nucleatum colonization in the gut, drastically improved the treatment of colorectal cancer. The oral or intravenous administration of irinotecan-loaded dextran nanoparticles covalently linked to azide-modified phages inhibited the growth of F. nucleatum and thus enhanced the effectiveness of first-line chemotherapy therapies for cancer. Similarly, Dong et al. ^[53][137] formulated F. nucleatum-binding M13-phage-loaded silver nanoparticles (AgNPs) to achieve targeted clearance of F. nucleatum and remodeling of the tumor-immune microenvironment. The in vitro and in vivo studies showed efficient eradication of the bacteria from the gut. Also, significant suppression of the myeloid-derived suppressor cells at the tumor site and the activation of antigen-presenting cells by the M13 phages were observed. These immunomodulatory activities boosted the capacity of the host immune system for colorectal cancer suppression.

Shahin et al. ^[54][138] determined the efficacy and specificity of individual Shigella-specific bacteriophages (vB_SflS-ISF001 and vB_SsoS-ISF002) and a cocktail of both. The phage preparations were investigated against multidrug-resistant Shigella sonnei and Shigella flexneri isolates. The individual bacteriophages showed high lytic activity in about 75% of the isolates. However, the phage cocktail inhibited 85% of the isolates, indicating higher effectiveness and specificity against a wide range of ESBL-positive and -negative isolates of S. sonnei and S. flexneri.

Gut commensals like K. pneumoniae opportunistically worsen gut conditions such as inflammatory bowel diseases (IBDs). Federic et al. ^[55][139] identified multi-resistant K. pneumoniae strains strongly associated with the exacerbation of gastrointestinal disease following enhancement of intestinal inflammation in colitis-prone, germ-free mice challenged with IBD-associated K. pneumoniae strains. Stepwise production of lytic cocktails of five-phage targeting strains enabled the effective control of the bacteria in vivo and further supports the use of phage combination therapy in addressing resistance and generally managing gut disease-contributing pathobionts. The lytic effect of some commercially available bacteriophage preparations on strains of K. pneumoniae isolated from infants with functional gastrointestinal disorders (FGIDs) was assessed by Grigorova et al. ^[56][140] via the drip method and according to clinical recommendations. However, low-level lytic activities and sensitivity to K. pneumoniae correlated with age. Significant levels of lysis were observed in children of three to six months but still reflected the inefficiency of this therapy in eliminating K. pneumoniae from the intestinal microbiota of children with FGID and suggested that more ingenious and radical approaches to ensuring complete eradication of the associated K. pneumoniae are needed.

L. monocytogenes is a facultative anaerobic Gram-positive bacterium prevalently implicated in foodborne and gastroenteritis diseases ^[57][141]. A phage cocktail designated as a foodborne outbreak pill (FOP) and targeted at the implicated L. monocytogenes was evaluated by Jakobsen et al. ^[58][142] in simulated small intestine, large intestine, and Caco-2 models. Extensive inhibition of L. monocytogenes with results comparable to that of a standard drug (ampicillin) was reported. Strikingly, unlike ampicillin, the FOP did not inhibit commensal bacteria in the small intestine, significantly and selectively lysing the L. monocytogenes population while being stable in the gastric environment. Furthermore, the FOP prevented the invasion and adhesion of L. monocytogenes through a Caco-2 monolayer. Generally, the study highlighted the essential health benefits of phage in this regard and their delivery as dietary supplements, enhancing natural defenses of the gastrointestinal tract against specific foodborne pathogens.

The bacteria Ruminococcus gnavus, a Gram-positive anaerobe, is also widely prevalent in the microbiota of humans with inflammatory bowel disease conditions due to Crohn’s disease ^[59][143]. Buttimer et al. ^[60][144] isolated, characterized, and analyzed six phages that infect the R. gnavus JCM 6515T strain. Although no significant decrease in the bacterial count was reported post-phage administration, the study provided insight into two significant mechanisms through which phages interact with R. gnavus in the human gut microbiome.

Campylobacter, a major component of the gut microbiota, especially in birds and livestock, is a major foodborne and diarrheal disease-causing bacterial species ^[61][145]. D’Angelantonio et al. ^[62][146], in their study against Campylobacter jejuni, demonstrated colony reduction in a broiler before slaughter, following a two-step phage administration process involving two double-stranded phages (Φ 16-izsam and Φ 7-izsam) belonging to the Caudovirale order. A 0.1 MOI of Φ 16-izsam was administered to a broiler group on the 38th day of rearing, while Φ 7-izsam at an MOI of 1 was administered on the 39th day to another group; these showed a significant one to two log reduction in C. jejuni counts on the cecal content compared with the control group after sacrificing the birds on the 40th day. The lowest colony count was, however, observed with an MOI of 0.1 of Φ 16-izsam. Also, Nowaczek et al. ^[63][147] isolated 48 strains from 140 broiler chickens (31 Campylobacter jejuni and 17 Campylobacter coli), which exhibited varying and high-level multi-resistance to the selected antibiotics ciprofloxacin, erythromycin, gentamicin, and tetracycline. They further identified and characterized bacteriophages, including bacteriophages φ4, φ44, φ22, φCj1, φ198, and φ287, placed in the Myoviridae and Siphoviridae of Caudovirales order, and demonstrated the susceptibility of a significant number of the Campylobacter spp. to the phage isolates, which had a lytic spectrum of 6, 4, 4, 3, 8, and 7, respectively.

Amidst the substantial efforts at identifying effective phages and their cocktails against bacteria-implicated gastrointestinal disorders, it remains desirable to develop effective delivery systems capable of protecting and stabilizing phage particles and products from degradation, destruction, and inactivation under the gastrointestinal tract’s acidic conditions. A smart biocontrol chitosan-encapsulated bacteriophage cocktail formulation was employed by Rbahimzade et al. ^[64][148]. The formulation was evaluated as a prophylactic and treatment option for gastrointestinal infections, specifically diarrhea. Experimental animals were challenged with S. enterica, Shigella flexneri, and E. coli, after which treatment commenced with the formulation. Findings reveal that phage encapsulation protected therapeutic life forms from enzymatic degradation, with the non-treated experimental animals experiencing weight loss. However, the highest lytic activity was obtained three days post-phage treatment compared with other studies, where lytic activities took seven to ten days to become evident.