The invention and usage of antibiotics by humans did not cause the naturally occurring phenomena of antibiotic resistance. Microbes and higher eukaryotes create a wide range of physiologically active compounds with antibacterial capabilities that have been reclaimed as contemporary antibiotics in various conditions [18]. However, the levels of these substances in these natural settings are frequently lower than clinically meaningful thresholds, indicating that resilience does not just develop to counteract their harmful effect. It has been suggested that rather than acting as rigid antimicrobial agents, antibiotics and their interactions with antibiotic resistance pathways function in nature as a means of communication amongst the individuals that make up a microbial community [19]. There is evidence that these chemicals alter community composition and elicit adaptive genotypic and phenotypic responses [19]. When looking at antibiotics from a biological standpoint, it is not surprising that the diversity of ancient bacteria contains genes that provide resistance to modern drugs, like uncontaminated arctic permafrost, indicating that antibiotic resistance persists even without human intervention [20–22].

Antimicrobial resistance is significantly made worse by human activities, notably clinical and industrial abuse of antibiotics. Antibiotics are employed in the production of crops and livestock to cure crop and fish infections, as well as, of course, to combat infectious diseases in people and animals [23]. In reality, others are usually blamed for misusing antibiotics, it has been stated that other nations’ agricultural usage of antibiotics is considerably higher, citing estimates that the United States produces meat with up to 180 mg of a powerful antibiotic drug per kilogram [24]. Unexpectedly, billions of metric tonnes of antibiotics have leaked into wastewater streams and natural reserves as a result of antibiotic usage in agriculture. Farms located closer to cities, pharmaceutical waste being present, and inadequate water purification contribute to this issue by increasing the quantity of antibiotic exposure and its environmental persistence [25]. Continuous exposure of ambient microbial communities and pathogens to various antibiotics has accelerated the emergence and increased number of antibiotic resistance genes as well as their spread [25]. Gene flow, which is the interchange of genetic material among organisms by transformation, conjugation, or transduction, can be involved in development and spreading of antibiotic resistance. This process, in addition to spontaneous chromosomal mutations, strengthened due to the selective pressure of the drug, can result in resistance [26]. One of the primary sources of multidrug resistance for horizontal transmission is the gastrointestinal tract microbiota of antibiotic-treated animals and humans, where antibiotic-resistant bacteria have been preselected [27]. Furthermore, through mechanisms including biofilm formation, swarm adaptability, metabolic dormancy, and longevity, bacterial cells can develop temporary, non-genetically coded resilience [28].

A multitude of resistance mechanisms can be involved in interfering with each stage of antibiotic passage through the bacterial cell: to avoid the penetration of the drug, bacteria can change the structure of the outer membrane or cell wall, or they can increase the expression of the efflux pump to push the harmful chemicals-antibiotic out; they can also produce enzymes such as beta-lactamases that open the ring of the beta-lactam molecule and neutralize its lethal effect. Accordingly, antibiotic-induced synthesis of enzymes that alter the target of the antibiotic, hide it, or quantitatively change it [26, 29]. The bacterial mechanisms to provide antibiotic resistance have been shown in Figure 1.

The “golden age” of antibiotic development spanned from the 1940s through the 1980s, when more than 40 antibiotics were being developed and released for use in clinical settings. During this period, there was the discovery of a large number of new antibiotics, their subsequent use/overuse, and the modern emergence of resistance, but resistance to a particular antibiotic was rarely a cause for concern because newer compounds, often exhibiting stronger pharmacokinetic and pharmacodynamic attributes, were rapidly developed [23, 30]. The implications of this counterproductive cycle, however, became increasingly apparent starting in the 1990s due to a continuous drop in the development and release of novel antibiotics. A situation known as a “dry pipeline” in the study and development of antibiotics refers to the fact that most newly produced antibiotics are modified or coupled variants of previously found chemicals. Innovative medications must prove not just their effectiveness but also their tolerability, an appropriate drug release profile, and cost-effectiveness, which is a difficult undertaking. About 5 of the 5,000 to 10,000 proposed antimicrobial compounds are thought to enter phase I research, of which only one is likely to receive regulatory approval for human use [30]. Pharmaceutical companies are reluctant to invest into antibiotic development when the chances/odds are against them because the drug development process is both costly and time-consuming. Additionally, the short-term use of antibiotics, the need for rigorous management programs, and their susceptibility to the development of resistance can significantly reduce a company’s earnings [30]. The rapid emergence of antibiotic-resistant bacteria is even predicted to surpass recently launched medications, underscoring the need for innovative therapies [31]. Antibiotic resistance is an issue, and the dry pipeline makes it worse because it has slowed down our arsenal of treatment alternatives.

The persistent development of antibiotic-resistance traits leads to the production of pathogens that are extensively drug-resistant, pan-drug-resistant (PDR), and multidrug-resistant (MDR) [32]. Researchers, doctors, and public health authorities have been interested in a particular bacterial species since they are mostly responsible for MDR infections, which are most commonly associated/linked to healthcare settings and are also the most severe. The acronym ESKAPE refers to the pathogens in this category, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., describes how its members might use a range of resistance mechanisms to prevent antibiotics’ bactericidal activity [33]. Therefore, the World Health Organization (WHO) has classified the last four of these infections as a significant priority for introducing novel medicines, notably the carbapenem- and cephalosporin-resistant species [34].

MDR infections are more likely to be acquired by patients with underlying medical disorders, immunocompromised, and hospitalized patients (particularly in critical care units, surgical wards, or burns units). But more and more publications are warning that “common” community-acquired illnesses might become resistant to antibiotic therapy in healthy people [35–37]. In the post-antibiotics era, even ordinary diseases and small wounds can be fatal [38]. Even a compilation of these metrics, which include rates of illness and mortality and monetary expenses, cannot fully capture the scope of the burden of antimicrobial resistance. Compared to patients with antibiotic-sensitive infections, people with MDR infections are more likely to experience treatment failure, have worse prognoses, have high death rates, have longer hospital stays, and increased complications risk or long-term consequences [23, 38, 39]. According to a recent analysis, by 2050, resistant strains would result in the yearly loss of 10 million lives, costing the global economy USD 100 trillion [40]. Regardless of age, social class, or place of residence, it poses a danger to world health that might impact everybody [41].

Antimicrobial resistance must be addressed by multifaceted, interdisciplinary, and global strategies. Establishing regulations for antibiotic use in both people and animals is a crucial element that must be considered [42]. Attention is needed since, in many nations, especially in underdeveloped nations, self-medication and easy availability of antibiotics without a prescription are dreadfully prevalent. The extensive use of antibiotics in livestock which has to be reduced is even more concerning. Medical professionals occasionally prescribe antibiotics for conditions that are not appropriate for them. Examples include treating viral or fungal infections with antibiotics, needlessly extending antibiotic courses, or using antimicrobials with a wider spectrum than is typically necessary for a given infection. By supplying the industry with administrative and financial stimulus, the problem of the dry pipeline might be reduced [43] or by “reviving” outdated antibiotics. This is the case with chloramphenicol and polymyxins, which were discontinued because of safety concerns (nephrotoxicity/neurotoxicity and uncommon yet potentially lethal hematological adverse effects, respectively), but are currently being reinstated [44, 45]. The most important thing is to promote research into novel therapeutic options and to reinvigorate interest in underutilized ones, like phage therapy. The detailed comparison of phage and antibiotic therapy has been portrayed in Table 1.

Based on their modes of replication inside the bacterial host, there are two types of phages (i.e., the temperate phages and the lytic phages). Temperate phages bind with specific receptors on the bacterial cell, inject their genetic material into the bacteria, and then integrate it into the host’s genome. Another type of phage is the lytic phages that upon entry subdue the bacterial biosynthetic machinery for synthesis of the genome and viral proteins including endolysins which degrade the bacterial cell wall, allowing the release of newly assembled phages [59, 70, 73].

The biologics behind the phage therapy are also based on the bacteriophages’ replication mechanism. In the case of the lytic phage, the cycle starts with attachment to specific receptor proteins on the bacterial cell. In both gram-negative and gram-positive bacteria, these receptor proteins can be present on cell wall (such as the teichoic acid, peptidoglycan in the case of gram-positive bacteria), or can also be located on the capsules or even the pili and flagella [59, 74]. Once interconnection is established between the bacteriophage and the bacterial receptor, the virus can infect the host by injection of viral genetic material into the bacterial host. When the viral genetic material enters the bacterial cell, it subdues the host’s self-producing machinery. The next step is the production/multiplication of novel phages, followed by their release thanks to activity of lytic proteins-endolysins. The result is bacterial death and newly released phages are ready to infect other bacteria carrying the same receptors as the previous host. The lytic phages, which mostly infect human pathogens, belong to the orders of Microviridae (“the tail-less phages”) and Caudovirales (“the tailed phages”), and these have single-stranded DNA or double-stranded DNA as the genomic material, respectively. Other than the lytic phages, which have great therapeutic significance in treating bacterial infection, the temperate phages cycle begins with attachment to the cellular receptors of bacteria. The subsequent steps differ because, in the case of temperate phages, the incorporation of phage genomic material takes place into the bacterial genome. So the temperate phage replicates with the bacteria itself, and under stress conditions, it may also cause the lysis of bacterial cells [59, 70, 73].

It must be noted that lytic phages are highly preferred over lysogenic phages because the latter can transfer virulent genes from one bacterium to another. However, in some cases, the lysogenic phages were considered for the therapy, such as for treating infection caused by Clostridium difficile for which no strictly lytic phage has been yet obtained. Also, temperate phages can be justified in emergencies with time constraints and when lytic phages are unavailable [59, 70, 73]. The mechanism of phage therapy has been sequentially illustrated in Figure 2.

The pharmacodynamics of phage therapy is linked with its pharmacokinetics. Phages must be applied at the side where there is a bacterial infection. When phages are given intravenously, very few of them can reach the site of infection, and they also clear from the blood very quickly. The success of phage therapy depends upon the initial dose and the timing of the administration of phages. Therefore, it is better to use more virulent phages in therapy. A virulent phage has a big burst size which is very beneficial in suppressing the growth of bacteria [72]. There are two categories of phage therapy. One is using a single phage specific for the bacteria causing the infection, and this type is also referred to as “mono-phage therapy”. The other type of phage therapy is called “polyphage therapy” as it employs a combination of phages (i.e., a phage cocktail). Bacteria have developed mechanisms to escape from the phages that kill them, such as using outer membrane vesicles as decoys, prevention of phage adsorption, cleavage of phage genetic material, preventing the phage assembly, blocking the entry of phage DNA, bacterial suicide, and many other newly discovered mechanisms. To combat the resistance, the use of polyphage therapy is recommended. To optimize the phage cocktail, many procedures have been introduced that allow the selection of more lytic phages [75, 76].

There are some advantages of using phage therapy to combat bacterial infection, which is given as follows:

a.

Phages are capable of increasing their numbers when the host is present. They estimate their dose themselves and this phenomenon is called auto-dosing. As a result of this phenomenon, there is no need for the repeated inoculation of phages at the site of infection [77].

Phage therapy can be standardized by strictly using the lytic phages, confirming the antimicrobial activity of phage against the bacteria, identifying the bacterial receptors that allow the elimination of resistance, and using combination therapies to combat the resistance [59]. Another approach is to use phage lytic proteins instead of whole viruses or a combination of phages with antimicrobials to combat the resistance [71, 78].

Clinical phage therapy uses bacteriophages to treat and prevent infections in humans. It is also practiced as a means of modifying the microbiome. In a few countries, clinical phage therapy is permitted for routine use. However, the corresponding data from these efforts remain limited [83]. Only a handful of clinical trials have been approved in this area, and few are currently completed. The PhagoBurn trial, conducted in 2013 under both good manufacturing practice (GMP) and good clinical practice (GCP), is the largest clinical trial on phage therapy in Europe. The results of a trial involving patients with Pseudomonas aeruginosa wound infections were compared to those of routine care to evaluate the effectiveness and acceptability of phage therapy and traditional methods [84]. Each treatment was topically administered for seven days. One group received phages, and the other received a managed treatment. The phage remedy organization had a quicker recovery than the control institution (well-known treatment). No adverse consequences have been observed in the phage-handled institution. The confined efficacy of the phage cocktail became because of a significant drop in phages after GMP manufacturing, leading participants to hold a much lower awareness of phages than predicted at the beginning. Extra importantly, susceptibility checking out was no longer completed before treatment, and bacteria became immune to low doses of phages after treatment failed [84].

Phages have been used to carry vaccines containing antigens and other substances. Antigens expressed on their surfaces can be delivered directly through phage particles [85]. Phages that induce both humoral and cell-mediated immunity can act as effective adjuvants or immune-stimulating agents. However, DNA vaccines are created by incorporating the sequence vital for vaccine antigen synthesis into the phage genome, which then acts as a vehicle for delivering a DNA vaccine. Phage display vaccines do not need adjuvants because bacteriophages are intrinsically immunogenic and can act as natural adjuvants, which makes them more effective than other types of vaccines [77].

The chemicals used to control the microorganisms are toxic to the environment and dangerous to living organisms. Phages are considered one of the most plentiful biological beings on the planet. Phages can be used to control the spread of disease-causing agents without any harmful effects on other living organisms. Bacteriophages are a form of biological defense used in the agriculture and food industries to protect them from contamination with pathogenic and spoilage bacteria. Since bacteriophages are specific with narrow spectra, they can be used to eliminate specific bacteria or even certain types/strains of bacteria [86].

As human populations grow, so do the numbers of resistant bacteria, which has created a demand for natural biocontrol agents. More resistant bacterial species are constantly rising and the food industry may take a hit. The discovery of some natural biocontrol agents has challenged the food industry, which does not drastically affect human health and food quality [87]. Bacteriophages have been shown to be effective in protecting food from bacterial pathogens. As a result, phages are now being used at all food processing steps. Salmonella and Campylobacter are the two main pathogens that cause diseases in the poultry industry. The researchers show promising results for using phages against these organisms [88].

A biofilm is a community of microbes living on or in another material, such as a multicellular organism or on the surface of a solid object. The cells in biofilms are surrounded by an extracellular matrix (EPS) that is formed by the microorganisms themselves [89]. Bacteria that reside inside biofilms show a high degree of resistance to antibiotics and disinfectants. Bacteria are protected with a matrix made of lipids, nucleic acids, polysaccharides, and proteins [90]. The bacteriophage can disrupt this layer, compromising the bacteria’s protection. Phages secrete a polysaccharide-degrading enzyme termed depolymerases, which break down matrix and open up new entry points for the phage to interact with its receptor on the host cell [91]. Biofilms, the slime-like layer that Clostridium difficile forms around itself to protect itself from antibiotics, can impair the antimicrobial activity of antibiotics and contribute to their virulence. A cocktail of Clostridium difficile phage has been shown to reduce biofilms, preventing colonization when used alone or in a mixture with vancomycin [92].

In recent years, regenerative medicine has gained popularity with the creation of new therapies based on the idea that we can reprogram cells to form new tissues and organs. The motivation is to use several in vitro and in vivo techniques that take advantage of the body’s natural healing processes to treat patients with chronic illnesses like diabetes, osteoarthritis, cardiovascular and central nervous system (CNS) degenerative disorders, and crippling injuries [93].

It is known that bacteriophages may penetrate the blood-brain barrier to reach brain cells, where they can then use their antibacterial properties to provide immunity [94]. Scientists have controlled this characteristic of phages to develop a therapy for nervous system illnesses. A study tested anti-Shiga phage medication and found that it prevents bacterial meningitis in mice [77]. Plaque binding potential of bacteriophages has been used to develop treatments for Alzheimer’s and Parkinson’s illnesses. In general, the plaque-binding potential of bacteriophages has been shown to be directly related to the effectiveness of immunizations in treating Alzheimer’s and Parkinson’s diseases. Bacteriophage preparations can be used as an effective way to lower the potential of neurodegenerative diseases caused by elevated pro-inflammatory cytokines and amyloid-beta protein ingestion [95].

The mechanisms used to distinguish between bacteriophages and their products may recognize a non-self-antigenic molecule or “mask” that results from phage production or degradation. Furthermore, the response induced by bacteriophages is likely beneficial in reducing the potential impact of phage administration [96]. When the phage strains were injected, the immune system response was observed in animal and human experiments despite the prior exposure and course of administration. In animals, research shows that the phagocytic cells operated upon the bacteriophages within a few minutes after their insertion [97].

Phage selection was often underestimated and led to wrong phage choices for treatment. Phages, like other proteins, have distinct shapes that enable them to bind specific targets on their prey. These binding sites can differ from phage to phage and even within a single phage species, making selecting and identifying good candidates difficult [98]. Despite the limited success of phage therapy, there seems to be a lack of studies on this subject. Although phage therapy is still being tested and not yet fully understood, researchers have also pointed out that several parameters need to be controlled. A bacteriophage must be specifically active against a given bacterial strain to be considered a potential therapeutic agent. It can be demonstrated by incubating the bacteriophage with the target bacteria and neutralizing it. For example, researchers found that it is considerably simpler if Pseudomonas aeruginosa instead of Streptococcus aureus is the aim of the bacteriophage for infection control or diagnosis [99].

The topic of phage-resistance emergence is a critical component in comprehending the ecological consequence of phages on the bacterial population when addressing the connections between bacteriophages and their hosts [100]. The development of bacterial resistance to bacteriophages is possibly plausible since bacteria possess or may evolve in many ways to avoid viral infections. It may be possible when bacteria change their recognition molecules, switch off the signaling pathways involved in phage infection, or they can probably modify their metabolism and acquire additional resources to make them resistant to phage attacks [96]. The protein modification of membrane for the alteration or receptor loss has been observed in Vibrio cholerae [101], Streptococcus aureus, Bordetella bronchiseptica, and Escherichia coli [96]. The bacteria provide adaptive immunity against the phages with the help of the CRISPRs and CRISPR-associated genes. However, introducing a cocktail of phages and combined therapy (antibiotics + phages) can be very influential in reducing the development of resistance to phages among bacteria. Given that all the findings indicate that the virus’s capacity to promote bacterial resistance, the quantity required to prevent bacterial resistance development is an essential consideration in selecting a therapeutic bacteriophage [102].