Why are antibiotics becoming problematic?

Unchecked and excessive use of anything is dangerous. The same is true for drugs and antibiotics that create resistance in hosts and pathogens without creating any specific beneficial outcomes. The problem of multidrug and antimicrobial resistance (AMR) is well known to the scientific community and presents broad-scale challenges to the health sector [13]. Although antibiotics are a million lifesavers, problems like AMR cannot now be ignored. The annual mortality rate of 700,000 per year due to AMR- and MDR-linked therapeutic failure is not an ignorable figure [9]. Similarly, the problems are not limited to direct human infections; other environmental entities and living beings, such as dairy farm animals, are also affected [13, 14]. Thus, affected animals create economic losses for farmers and public health hazards for the public in general [14, 15]. Several bacterial species, such as Staphylococcus aureus, are major concerns [3]. Similarly, the illogical use of antibiotics causes direct organ damage, therapeutic failure, economic losses, agricultural problems, agricultural risks, and food manufacturing problems [14].

Antibiotic resistance is primarily acquired by alterations in genes or by the acquisition of resistance-coding genes into the bacterial species [14]. Any further use of antibiotics or drugs could worsen the problem; thus, a solution based on advanced genetics is necessary [16]. Thus, for emerging problems such as AMR and MDR, unconventional strategies are being tried to deal with multidrug-resistant pathogens. Phage therapy is an alternative measure that brings a new era to the treatment of immunosuppressed individuals [17]. Several phage-based medical amenities are thus being introduced, such as phages incorporating medicated dressings, topical solutions, and liposomal entrapments. These medical dressings also involve other antibacterial and drug agents and thus could be easily designed for multi-clinical applications in various diseases [17]. Some high-target bacterial species that are excessively multidrug-resistant are shown in Fig. 1.

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High target bacterial species that are excessively multidrug-resistant [1–7]

Citation: European Journal of Microbiology and Immunology 15, 1; 10.1556/1886.2024.00126

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Bacteriophages: phage therapy

Bacteriophages are viruses that infect bacteria. Their abundance surpasses that of bacteria by 10 times, making them the most abundant organisms on the planet [2]. In addition to phage therapy, they also play an important role in other environmental processes, such as global ecology, animal and plant health effects, and biogeochemical contributions [3]. Phage therapy has been focused on treating bacterial diseases; however, the mutual development of antibiotics in almost the same era limited the efficacy of this technology. After years of work on antibiotic problems, like AMR and MDR, phage therapy became the interest of scientists as an alternative to antibiotics [14–17]. The outstanding number of successful phage therapies for treating patients has encouraged scientists to further work on phage engineering tools and overcome the possible limitations associated with these mainstream applications. It is important to mention here that phage therapy as a classic strategy is not the only benefit associated with phages; rather, they have shown versatile potential for use in agriculture, pest control, disinfectants, drug resistance control, animal farming, and microbiome modulation [18].

Phages result in wide-scale diversity among bacterial species because of their evolutionary developmental dynamics. This dynamic coevolutionary feature of phages has attracted the interest of scientists for their use against MDR problems [17]. These abilities of phages are already being explored in developed countries for their therapeutic and prophylactic purposes, although the problems linked with phage resistance remain a hurdle to their therapeutic success [18]. Similar to AMR and MDR, the problem of phage resistance among bacteria and viruses creates an evolutionary challenge for the health sector. This is because bacterial species simply try to overcome the effects of drugs and viruses by acquiring genes that make their survival possible [18]. The coincidental changes in bacterial virulence and survivability allow them to transfer this resistance through cross-communication to different phages and bacterial species, similar to the effects of MDR. However, the exact mechanisms and development of phage resistance remain to be discussed further [19].

Phage therapy was first described at the beginning of the last century. The ability of phages to target living bacterial cells was primarily acquired for therapy, along with possible usage as a diagnostic tool for distinguishing bacterial strains [20]. Advances in scientific techniques, such as genetic engineering and gene mapping, have helped to further explore the benefits associated with hybrid bacterial strains and DNA transduction processes across bacteria and host species. Genetic engineering allows the modification of phage particles to exhibit specific proteins of interest on their surfaces via genetic fusion [21]. Such technologies have also helped to create modified versions of page-mediated therapies. These techniques not only enable phages to treat wide-ranging MDR pathogenic bacteria but also increase phage safety and decrease virulence in the host strain [21]. The latest tools in the form of CRISPR-Cas have further improved the bactericidal efficacy of bacteriophages, a perspective that will be further explored in the following sections [22].

Bacteriophages as tools for decreasing antibiotic resistance

In addition to being an alternative to antibiotics, bacteriophages represent another strategy by which antibiotic resistance and bacterial virulence can be reduced in pathogenic species. Bacteria also develop resistance to phages, and the mechanism through which phage resistance is developed is to mutate the phage receptors to overcome or lower their expression [17]. However, it should be noted that such structures are often virulence-causing structures, such as flagella, pili, efflux pumps, and lipopolysaccharides (LPS), which are also involved in the development of antibiotic resistance. Thus, bacteria lose their ability to develop antibiotic resistance, leading to phage resistance [23].

Moreover, other bacterial processes, such as the exchange of genetic materials via conjugation governed by lost structures, such as pilus, are often overturned in therapeutic favor and against the pathogenic nature of bacteria [24]. This act as another mechanism by which antibiotic resistance can be curtailed, making phage therapy an effective alternative to antibiotics. Both of these properties can be used to sensitize antibiotics against bacterial pathogens by evaluating the large-scale performance of phage therapy in clinics, farms, and environmental and waste treatment plants worldwide [24]. In addition, the mechanism of inhibition of quorum sensing (QS), which is an additional feature of phages, may also be used to increase the action of antivirulence compounds. This system regulates gene expression in conjunction with changes in cell population density and the upregulation of virulent genes [25].

Phages modulate microbiomes and prevent other complications

There are several ways in which phage therapy can be used as an alternative to antibiotics. One indirect approach is to treat diseases that otherwise become untreatable through antibiotic dosages because of the MDR effect in diseases caused by bacterial pathogens. Because animals and humans contain a natural microbiome, any irregulation of these microbiomes can lead to certain diseases, such as obesity, autoimmunity, autism, depression, diabetes, dental diseases, and even malignancies of certain kinds [24, 25]. Under such conditions, phages could be a useful alternative for treating dysbiosis and comorbidities by maintaining a balance in the microbial population and withdrawing their disease-causing effects.

An example is the use of phagetherapy against the very common bacterial species Helicobacter pylori, which causes gastric and peptic ulcers in the body [26]. It has gradually developed the ability to protect against common allergies, asthma, and even esophageal cancer [26]. Scientists have attempted to address this issue through the modulation of population size with phacotherapy instead of complete elimination through advanced triple and quadruple therapies, which lead to MDR [25, 26]. However, more work is needed to identify highly lytic phages to control the growing population size of H. pylori. Similarly, phages may also be used against the recurrent diarrhea-causing agent Clostridium difficile in elderly patients because of their ability to kill the pathogens and allow completion of the antibiotic course [26].

Furthermore, the most common skin acne caused by Cutibacterium acnes can also be controlled by phages via direct attack, and vaginosis caused by Gardnerella vaginalis can also be best alternatively treated with phage therapy instead of relying upon antibiotics and hence AR development [17]. Similarly, death-causing phage therapy in periodontal diseases could also be used against the most common Streptococcus mutans (a common cause of dental caries) and Aggregatibacter actinomycetemcomitans (a common cause of progressive periodontitis) [27]. Moreover, the general use of phages in prebiotics and probiotics could also be normalized to promote better growth against harmful bacteria and obesity to overcome bacteria that cause weight gain via fat accumulation [28].

Are bacteriophages able to combat nonbacterial diseases?

Apart from being an alternative to antibiotics for treating bacterial diseases, it is pertinent to share that there is a vast ground where phage therapy can be used for treating certain other types of diseases. For instance, they can be successfully applied to viral infections where phages work immunomodulatory and act as genetic vehicles for gene delivery mechanisms [8]. Similar properties could also be used for the discovery of new diseases, their diagnosis, and possible treatment via antivirals and vaccine development [1–4]. They create antiviral responses that can be used to treat viral infections such as those triggered by SARS-COV-2 and Epstein– Barr virus (EBV), apart from having the ability to prevent secondary bacterial infections in such viral diseases [29]. One example is a phage infecting A. baumannii, which downregulates the levels of tumor necrosis factor (TNF) in cellular pathways. Because upregulation of the anti-inflammatory cytokine TNF has previously been observed in patients with COVID-19, causing more viremia and possible secondary infection, the aforementioned phages could be a possible implication for combating such situations [29].

In addition, phages may directly regulate the entry and infection of COVID-19 through surface-based integrin motifs that compete with coronaviruses for entry into host cells [29, 30]. The same motifs may also interfere with the adsorption and internalization of EBV, thereby preventing direct infection. Furthermore, the gene delivery capabilities of phages can be used to deliver genes of interest to specific organisms. A recent experiment by Przystal et al. used the hybrid phage vector M13 with a single-stranded adeno-linked transgene cassette inserted within the intergenomic region of the phage genome to treat glioblastoma multiforme (GBM) in mice models [31]. Upon binding and internalization of the M13 genome, the hybrid vector delivers the recombinant genome to the host cell and helps in the expression of the therapeutic host gene that induces apoptosis. The entire experiment was successful; however, the limitations and further work are a definitive requirement for future work [31].

The same mechanism has been observed for the aforementioned CRISPR-Cas9 approach, in which engineered phages deliver the CRISPR-Cas system as therapeutic machinery to eukaryotic cells to initiate genome editing in either virus or host [22]. This approach could be implemented for viruses that integrate themselves into the host genome, such as HIV, HPV, and HSV. Taking the example of HPV further, we found that its dsDNA integration into the host genome could be edited via CRISPR-Cas9 [32]. The two main target oncogenes for this purpose are E6 and E7, which majorly block the cell cycle by degrading tumor-suppressive protein (p53) and retinoblastoma (Rb) [32]. Thus, CRISPR-Cas via RNA-guided endonuclease and two pairs of single guide RNAs specific for E6 and E7 in HPV-18 and HPV-16, respectively, can induce viral genomic cleavage and eventual cell death [32, 33]. These experiments were mostly conducted on lentiviruses but could be updated with phage-based vector experiments to improve the efficiency of the overall infection model.

Similarly, in addition to viral diseases, certain degenerative diseases, such as Parkinson's disease and Alzheimer's disease, can also be treated through phage therapy [8]. For example, these diseases can be characterized by the accumulation of misfolded proteins against which current therapy works by specifically targeting such aggregates. In a recent study, phage M13 capsid protein motifs were observed to interact and reverse the canonical amyloid folds in the brain [8, 34]. Thus, a new scientific paradigm can be further explored where cell-specific targeting, specific cell tropism, large gene packing vectors, and relatively simpler genetic and gene transduction abilities can be used for disease management [34]. Nonetheless, further work on phage-immune reactions should be well explored along with the development of inexpensive engineered phages in the future.

Pest control and phage therapy: an indirect alternative to antibiotics

As mentioned earlier, the direct use of phages as an alternative to antibiotics is becoming popular; however, besides direct attacks on commercially deteriorating bacterial species, there is an indirect use of phages via the killing of insect pests that work only under microbiome conditions (endosymbiosis) to exert harmful impacts on plants [8, 35]. One such obligate endosymbiotic bacterial genera are Buchnera, which interacts with aphids and produces tryptophan to develop their hosts. Similarly, some symbiotic bacteria interact with other pests, such as mosquitoes, roaches, flies, and termites [35]. Thus, eliminating the required microbiomic species provides an indirect way to overcome the use of antibiotics to treat health conditions.

The population of mosquitoes (an important biological vector) requires larval microbiota and water habitat, and any alterations in their habitat or microbiota via phages could directly interfere with the life history traits of this widespread common vector [36, 37]. Recent work on anopheles mosquitoes showed interesting outcomes in which larval development was reduced through phage addition to the larval habitat, which targeted specific species of bacteria and hence deregulated the mosquito population [37]. Thus, further studies are required to explore other direct and indirect ways to control healthcare concerns and avoid drugs and antibiotics.

Crispr-Cas system

CRISPR-Cas9 is considered an alternative unconventional molecular tool to combat antibiotics [33]. CRISPR-Cas9 uses small guide RNAs (crRNAs) derived from the adaptive immune system of prokaryotes, thus exhibiting a unique adaptive nature and therapeutic potential [45]. The molecular tool has two main parts: CRISPR and Cas. The CRISPR genomic regions contain short repeats with spacer sequences acquired from genetic entities such as bacteria, plasmids, and transposons [45]. The CRISPR region comprises the adenine– thymine repeat region, which often separates the Cas genes encoding Cas proteins. The CRISPR-Cas system is primarily divided into two major classes with multiple types and subtypes. Class 1, with subtypes I, III, and IV, contains multi-Cas protein complexes, whereas Class 2, containing types II, V, and VI, contains only a single effector protein region [45–47].

CRISPR-Case system for combating antibiotic resistance

The CRISPR-Cas system is also being progressively explored as an alternative to antibiotics because it disables MDR genes and bacterial virulence factors while sensitizing drug-resistant bacteria via the specific cleavage of crucial genomic domains in bacterial species [45–47]. The entire molecular machinery uses bacteriophages as direct predators of bacteria for delivering CRISPR-Cas9 into the bacterial species [46]. Phage vectors are packed with CRISPR to target bacterial species containing antibiotic resistance plasmids or DNA sequences. Upon entry, Cas programs in such a way that it recognizes targeted sequences by guiding RNAs to cause cleavage of the double-stranded targeted bacterial DNA, thereby causing loss of the resistant plasmid genes and consequent cell death [47]. Because this system is only being evaluated for its efficacy in early clinical trials, further studies are needed to determine the efficacy of CRISPR-based therapy.

The CRISPR-Cas system expresses specific antibacterial products that not only discriminate between commensal and pathogenic bacteria but also directly kill antibiotic-resistant pathogens from targeted environments of complex bacterial populations [33, 48]. As discussed earlier, the targeting function is performed with the help of DNA endonuclease Cas9, which uses gRNA to target the specific DNA of invader organisms. The futuristic natural infection of phages by bacteria renders the phage-mediated delivery method most appropriate for acute to chronic infections [49]. However, there is a need to ensure phage susceptibility of the targeted bacteria to facilitate the process.

Thus, properly designing a broad-spectrum phage and upgrading its delivery system will help manage the complex microbiota. Apart from being a common alternative to antibiotics, the CRISPR-Cas9 system has several other sticking abilities and is considered the best tool for future experiments [49]. For example, the specific marketability of phages for pathogenic bacteria reduces the side effects compared with antibiotics. Moreover, the time and cost involved in the overall process from selection to isolation, engineering and development, self-replication, and the ability to pass the blood– brain barrier and biofilms make phages far less expensive than antibiotics [50].

Phagetherapy for secondary bacterial infection of COVID-19

Studies on COVID-19 pathogenesis have revealed that it creates uncontrolled inflammatory immune responses that cause damage to the lungs, where a nutrient-rich environment is created by the damaged alveolar cells. This nutrient-rich environment attracts various opportunistic pathogens to create secondary infections, which promote immunostimulatory effects, and an excessive fluidic environment, which continues to add more severity to the pathogenic condition [56]. In such cases, clinicians prescribe more antibiotics to support the overall treatment and limit secondary immune infections. However, this has led to excessive global antibiotic use during the COVID era. Thousands and millions of excess antibiotics are administered, resulting in up to 51% of unjustified antibiotic prescriptions [57]. These prescriptions were made without prior blood culture, antibiotic sensitivity assessment, procalcitonin examination, or other relevant tests [56–58]. The overuse of antibiotics during this period might have increased AMR resistance in organisms spread throughout the world in water bodies and wastewater treatment plants, a perspective that is slowly coming to the surface and needs to be dealt with an iron fist [57].

Features such as physiological heterogeneity, controlled growth rates, hypoxia, quorum sensing, and genetic transfer may make secondary bacterial infection more common in COVID-19 patients; however, phages help maintain coevolutionary features with bacteria [58, 59]. They exhibit host specificity, easy isolation, accessibility, excessive availability, and the ability to degrade antibiotic-resistant biofilms because of the presence of degrading enzymes such as endolysins and depolymerizes [8, 15, 58]. CRISPR-Cas9 further limits the bacterial ability to protect other taxa against phage resistance, making phage therapy a good alternative to antibiotics [55].

Another advantage of phage therapy against secondary infections in patients with COVID-19 is its anti-inflammatory properties, an aspect truly required in COVID infections, which are dominated by excessive immunostimulatory inflammation. Experiments using T4 phages reduced inflammatory responses associated with cytokines and C-reactive proteins [58]. Scientists predict that two possible mechanisms dictate this lower rate of anti-inflammatory responses in phage therapy, compromising either the indirect effect by reduced bacterial load, by lysing and clearing from the body, or a direct effect by interacting with the host immune system [41, 58]. The rapid clearance of bacterial load and immune interactions help reduce cytokine influx, thereby reducing inflammatory responses [41]. Such characteristics help bacteriophages maintain community dynamics by killing common bacterial pathogens [59]. As explained earlier in detail, phages outperform antibiotics and can be used alone or in combination to treat bacterial infections [60].

Are bacteriophages a noteworthy alternative to antibiotics?

Here, the question arises whether bacteriophages are noteworthy to be used as alternatives to antibiotics, and the answer lies in various factors that make it implicit to imply and check the workability of phage therapy. First, bacteriophage viruses are specialized to kill bacteria, a property that is similar to antibiotics [5, 6]. Second, they can even infect pathogenic species that are highly antibiotic- and multidrug-resistant [16, 17]. Additionally, it is estimated to have an unbelievable number of phages in the world, much more than that of bacteria, which gives it an advantage in infecting and killing bacterial populations [8]. Moreover, phages are a highly diverse group of organisms, as detected by transmission electron microscopy. They have a simple structure containing a head/capsid with packaged genomic material and a tail used to infect bacterial hosts. They may also have other morphologically beneficial features, such as tailless and filamentous forms, which make them suitable for phage therapy [1–7].

The phage life cycle is also similar to that of animal viruses, which begins with the adsorption of the virus by the host bacterium. After attachment, the phage ejects its genome into the cell wall of the bacterial cytoplasm, activating it in the host and expressing various desired genes for phages [38]. Phages control the bacterial cell machinery to produce thousands of copies via a carefully programmed chain of events. Then, the phages perform bacterial cell lysis via phage-specific proteins such as lysins and Hollins, thereby releasing the phages into the outer environment and finally killing the lytic bacteria [39]. The particles are now ready to infect other bacteria and repeat the lytic life cycle with ultimate killing of their host until a favorable environment is found. They could also wait for a long inactive period until new hosts are available and the surrounding conditions work in favor of them [38, 39].

Thus, a continuous race between phages and bacteria persists in nature, which makes phages a favorable entity to work against bacteria as a natural tool instead of producing antibiotics and facing problems such as AMR. However, the mutual competition between phages and bacteria makes both species more competitive in a way that bacteria try to defend themselves by incorporating surface changes or varying defense mechanisms to destroy the phage nucleic acid [40]. This process often leads to phage resistance in bacteria; however, unlike MDR or AMR, phage resistance in bacteria is not a serious problem because the loss of cell surface characteristics or internal defense mechanisms of bacteria also causes loss of virulence, which is linked with certain surface entities, as explained previously [40]. Furthermore, the rate of phage resistance in bacteria is slow and manageable. This is because of the low occurrence of spontaneous mutation events in bacteria. If a few bacteria become phage-resistant, their large population size and fast life cycle quickly eliminate 99.9% of pathogens. The remaining few species can be easily handled by the host immune system, except in immune-compromised individuals [41]. The need is to carefully regulate phage therapy to select the phages that could infect the mutant from the available collection of phages.

Furthermore, the property of pages; to maintain their infectivity is also a positive point to consider them as the best alternatives to antibiotics. The pages respond to the bacterial defense mechanism by exhibiting changes to retain their functional attacks, which makes them a natural encounter with evolving bacterial resistance in nature [42]. Note that phages do not completely kill the entire population of bacteria because they depend on their hosts for survival and progeny. They try to maintain a bacterial concentration near 10 mL⁻¹ g⁻¹, which is often called the critical phage proliferation threshold [39]. A natural flow between the bacterial and phage populations and an increase in the bacterial population causes a subsequent delay in the phage population, which in turn delays the subsequent delay in the bacterial population and the cycle repeats [39, 40]. This cycle does not cause any organisms to be eliminated to keep the cycle flowing. This natural threshold implies that phages will not destroy bacteria; thus, a functional immune system of the host is required to eradicate pathogens from the host body [39, 41]. Scientists believe that a combination of antibiotics and phages could resolve this problem, creating a synergistic effect between phages and antibiotics to work efficiently in the host against infection [40].

Then another question arises regarding the threat of phages to beneficial bacteria. The answer lies in the natural process in which an arms race occurs between bacteria and phages. The phage population outgrows in several subtypes that attack specific subtypes of the bacteria; some phages-sensitive and some are resistant [42]. This phenomenon has resulted in a horizon in which a single bacterial species only has a few strains sensitive to specific phages, resulting in a narrow host range for these phages [27]. For successful phage therapy, specific phage isolates from laboratory specimens must be tested individually for each patient. These phages should target and eliminate only the pathogenic bacteria that are attracted to them. It is crucial that the identified bacteria are also sensitive to specific phages within the host cell. One thing to notice is that the beneficial microbiome will hardly be affected by phage therapy [42, 43].

Bacteria coexist with other organisms, making them impossible to avoid. However, bacteria are not always harmful; they may also benefit hosts by developing symbiotic relationships or may do nothing at all. Although pathogenic bacteria depend on living hosts, as in the human body, for nutrients, they acquire specific virulence and pathogenicity against the host defense system [5, 8, 42]. They can cause infection, sometimes as a single bacterial species, and sometimes in millions. Their diverse lifecycle and ability to cause several infections have led to the need for diverse treatment actions in response, such as improving hygiene, living standards, waste management, and proper cleaning, apart from the therapeutic journey that comes as a secondary precaution [39. 43]. The vaccination also becomes successful against less virulent bacterial species that show limited antigenic variability across bacterial isolates, such as vaccines against diphtheria, pneumococcus, tetanus, influenza, plague, tuberculosis, typhoid, anthrax, cholera, and pertussis [44]. However, vaccination is not always successful against pathogenic bacteria because of their large number, antigenic variations, and fast disease-spreading nature [44].

Because all bacteria are not disease-causing, some have beneficial health effects. Such bacteria create a normal microbiota in the body and help prevent infection from disease-causing pathogens [42]. Similarly, fecal microbial transplantation is performed to normalize the gut of superinfected individuals. Similarly, vaginal infections are often treated with marginal microbial transplantation. Moreover, during transplantation, fecal microbiota transplantation (FMT) is performed, and bacteriophages can play a vital role in maintaining a healthy gut [42–44]. The general benefits of phage therapy as an alternative to antibiotic therapy are summarized in Fig. 2.

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Benefits of phage therapy as an alternative to antibiotics

Citation: European Journal of Microbiology and Immunology 15, 1; 10.1556/1886.2024.00126

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Evolutionary trade-offs between phage and antibiotic resistance

Phages and bacteria co-evolve to develop resistance against each other and positively or negatively compromise the resistance of antibiotics and certain drugs [1–7]. Thus, patients may experience both phage resistance and susceptibility to antibiotics. Similarly, bacteria undergoing phage resistance may exhibit changes in growth pattern, virulence, antibiotic resistance, nutrient usage, and biofilm-forming abilities [51]. However, the bacterial population may face enhanced antibiotic susceptibility, which provides an advantage of phage resistance, making combined therapy the best option for treating bacterial diseases [52].

The first mechanism by which bacteria can acquire resistance to phages is through surface receptor binding changes [51, 52]. Bacteria use cell surface characteristics for various cellular processes, such as mobility, biofilm formation, cell envelope formation, metabolism, nutrient and genetic material transport, and reproduction, which dictate bacterial fitness [52]. Upon phage-mediated resistance changes in bacterial structural conformation, virulence may be lost along with increased antibiotic susceptibility, which imposes fitness losses and ultimately low growth rate as a target of phage therapy. Thus, the loss of bacterial surface characteristics promotes phage resistance and phenotypic conversion with a possible trade-off between bacterial survival, virulence, and antibiotic resistance [53].

Similarly, the surface efflux pumps of bacterial surfaces function variably to control virulence and transportation, adhere to surface or secretion, and uptake antimicrobials, metabolites, and other quorum-sensing signals to the surrounding materials [53, 54]. These efflux proteins include inner membrane transporters, outer membrane proteins, and lipoproteins, which also play major roles in antibiotic resistance and bacterial pathogenicity against host, environment, and other wild pathogens. Thus, bacteria that develop phage resistance become deficient in their efflux pumps and slowly rely on surface binding receptors for efflux actions [55]. The compromised efflux pumps can be used for phage engineering and antibiotic sensitization.

The trade-offs and interactions between phages and bacteria represent a double-edged sword. On the one hand, phage resistance develops, whereas the loss of receptors and efflux pumps from bacterial surfaces decreases antibiotic resistance. The overall reduction in antibiotic resistance diminishes the survivability of bacteria. It results in decreased virulence, growth, motility, reproductive capacity, attachment to host colonization, immune evasion, and compromised fitness [51–55]. In such cases, clinicians often prescribe additional antibiotics to support the overall treatment and prevent secondary immune infections. However, this has led to an excessive global use of antibiotics during the COVID era. Thousands and millions of excessive antibiotic doses were administered, reaching up to 51% of unjustified antibiotic prescriptions. These prescriptions were made without prior blood culture, antibiotic sensitivity assessment, procalcitonin examination, or other relevant tests [55]. However, the case is different for CRISPR-Cas in which the traditional trade-off does not occur between phage resistance and virulence [45–47]. Therefore, the therapeutic profile of phage therapy needs to be drawn alongside the experimental CRISPR-Cas system to consider both the antagonistic and synergistic pleiotropic and control AMR and MDR in bacteria.

Future perspectives and limitations of phage therapy

Phage therapy offers a wide spectrum of benefits and advantages over antibiotic therapy, but it does not come without limitations. There is still much to be done in the future to improve phage therapy in practice. First and foremost, proper permissions from medical and laboratory authorities throughout the world should be obtained for clinical practice [61]. Second, wide-scale knowledge sharing via proper databases and data sharing among physicians and researchers would be an additional recommendation for the practice of phage therapy globally [62]. Third, the curriculum for medical and biology students should include phage therapy. Fourth, there is a need to properly channel reliable data from clinical trials and regulatory guidelines for phage therapy across the world [61–63]. Additionally, there is a need for a better understanding of the pharmacokinetics and pharmacodynamic parameters linked to bacteriophages to address the disparity in phage therapy-based efficacy among patients [61, 62].

Moreover, it is necessary to carefully regulate the immunogenicity of particular phages to overcome host immune clearance or immunodominance reactions [41]. In addition, the problem associated with phage-mediated bacterial lysis and the release of toxic bacterial compounds may be harmful to the host or may result in complicated diseases in which more than one type of bacteria are concurrently working; hence, phacotherapy needs to be properly regulated for such cases [64]. Additionally, there is a need to develop properly connected phage laboratories in which a diverse range of phage species are made available to overcome the phage's narrow susceptibility to their host and to implicate phage combination therapies [64, 65]. Moreover, the high prevalence of phages in the environment increases the risk of food contamination. This poses a threat to dairy products that rely on bacterial production for nurturing. The contamination of these products with antibiotic resistance genes, which are transferred by phages, can contribute to the spread of antibiotic resistance [65]. Thus, proper regulation of phage therapy is required by improvising temperate phages to lysogenize the industrial strain rather than relying on environmental phages, which reduces the risk of phage infection [65, 66].

Furthermore, data from across the world must be carefully analyzed to improve this technology in laboratories and hospitals. There is an extensive need to further research on bacteriophages and CRISPR-Cas-9 systems to increase our understanding of phages, their bacterial and environmental interactions, and vice versa [63, 54]. This will allow us to identify potentially dangerous phages harboring virulent and resistant genes. Moreover, the practice of combined therapy with phages and antibiotics should be encouraged and experimented upon, since a previous study showed that most patients receiving combined therapy showed a 100% success rate, whereas those only receiving phage therapy had an approximately 88% success rate [60]. This is because combined therapy allows for a decrease in reliance on phage-resistant bacteria with increased sensitivity to antibiotics, making antibiotics an adjuvant against pathogens in immunocompromised patients [60].

Beyond the above-mentioned practical approaches, there is also a need to govern financial, technical, technological, community, and commercial challenges with appropriate measures so that society does not face the same circumstances as MDR and AMR in the future. Only through these considerations and practical measures will the future of phage therapy be able to strengthen, if not completely replace, antibiotic-based treatments to overcome multidrug and antimicrobial resistance in pathogens [61–66]. Therefore, antibiotics are a long-term solution for acute infections until alternative phage therapy gains roots in the medical community. If allowed to progress, phage therapy can target any infectious, sensitive, or resistant bacteria associated with diverse diseases. The chain of events in precision phage therapies follows the isolation of pathogens from patient samples, including MDR, transferred to the phage laboratory to screen, collect, order, or isolate specific phage collection or phage cocktails to be delivered to clinicians within a limited period [60, 66]. The other limitations associated with phage therapy are summarized in Fig. 3.

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Limitations of phagetherpy as an alternative to antibiotics

Citation: European Journal of Microbiology and Immunology 15, 1; 10.1556/1886.2024.00126

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Limitations of CRISPR-Cas9-mediated phage therapy

Similar to phage therapy, the CRISPR-Cas system also encounters certain limitations that need to be addressed. As in nature, microbes residing in living hosts or environments possess diverse ranges of resistant genes; thus, targeting such a diverse range of genes individually via CRISPR technology is a time- and cost-consuming process [67]. Furthermore, to highlight that some bacteria are opportunistic pathogens that work by mimicking the commensal microflora to attain desired benefits, the use of the CRISPR system to remove them completely or by accidentally increasing their growth will cause serious metabolic disadvantages to the host [68]. In addition, the CRISPR-Cas system may also increase antibiotic resistance in bacterial species while addressing the microbial resistance genes in some other bacterial species [47, 67].

Similarly, the CRISPR system seems to easily move between species, causing the reversibility of nucleases by the host repair system and possible host cell death; another problematic limitation of CRISPR [48, 68]. Moreover, designing a CRISPR-Cas system against rarely cultured bacterial species is even more difficult, highlighting the need to redesign the delivery vector against target bacterial species [49, 69]. Moreover, genetic variability and biosafety analyses of phages and CRISPR-Cas regarding possible toxins, virulence factors, and resistance genes are still under observation. Thus, the system requires further work and thorough mutation and genetic analyses to be used as an advanced molecular modality as a therapeutic tool in the future [70, 71].