In the late XIX century, Frederick Twort and Felix d’Herelle were the first to report antibacterial agents with viral-like properties. Bacteriophages, i.e., viruses, that use bacteria as hosts, undergo two major cycles: (i) Lytic, where progeny virions (single phage particles) are created inside the bacterial cell, and their release results in disruption of the host cell, and (ii) lysogenic, where the phage inserts its genetic material inside the bacterial genome and lies dormant.
Temperate phages in the form of prophage (i.e., embedded in the bacterial genome) can be activated, e.g., via external stimuli, and enter the lytic cycle [
154]. Phages either cause bacterial lysis via proteins called amurins or they form large pores in the cytoplasmic membrane—through the action of small proteins called holins, which permanently affect the integrity of bacterial cell wall [
154,
155]. Only in a few cases, especially filamentous phages, can progeny virions be continuously secreted, causing chronic infections [
156].
D’Herelle sought to exploit these agents’ therapeutic potential by using them on a boy to cure dysentery. Immediately after this, companies such as L’Oréal and Eli Lilly began preparations for the commercialization of phage therapy. Some institutes also started to be devoted entirely to this aspect of microbiology (e.g., Eliava Institute in Tbilisi).
Several phage-based products curing inner ear infections [
158], urinary tract infections [
159], typhoid [
160], and systemic multi-drug-resistant infections [
161] are being tested. The first clinical trial approved by the Food and Drug Administration (FDA) is for intravenous phage therapy in 2019 [
162]. Abedon et al. [
19] and Cisek et al. [
163] gave very compelling reviews on the history of phage therapy, and the current situation is summarized by Altamirano and Barr [
164].
Additionally, phage therapy also plays a significant role in veterinary medicine. Mice and chicken infected with
Salmonella spp. can be treated with
Salmonella phages with a 90–100% success rate [
165]. Other bacteriophage applications include phage-mediated biocontrol, phage bioprocessing for food decontamination, and biodisinfection of objects of veterinary supervision [
166]. Certain phage-based products such as ListShield
TM are now approved to be used in meat and poultry products against
L. monocytogenes [
167].
4.1. Phages Against Bacterial Infections
Despite early success, phage therapies got abandoned with the emergence of antibiotics in the pharmaceutical industry [
168]. However, the spread of drug-resistance superbugs and lack of new antibiotics is causing the renaissance of the use of phages against bacteria [
169]. Bacteriophages are chosen for this purpose due to their bactericidal effect, low toxicity, high specificity, high rate of replication, easy storage, and lack cross-resistance with antibiotic classes [
154]. Since bacteriophages are extremely specific, the patient’s commensal bacteria are not negatively affected during the treatment. Moreover, human cells are not directly affected [
170]. There are particular examples of phage internalization by eukaryotic cells. The structure of the
E. coli receptor is similar to the structure of polysialic acid present on the surface of neuroblastoma cells. Lehti et al. showed that the penetration of eukaryotic cells by
E. coli phage PK1A2 is possible in vitro [
171]. The virus remained in the cells for up to 24 h, but such “infection” did not affect the cells’ viability. Another example showed that the engineered phage could bind and enter the cell, but the replication of M13 was not detected [
172].
However, the human immune system reacts to the appearance of the phages in the body. Phages impact immunity directly as they modulate the innate and adaptive immune response through phagocytosis, cytokine response, and antibody production [
170]. It is difficult to determine precisely which phage components are responsible for the innate immune response modulation. Studies on induced immune reactions usually were conducted using lysates of phages containing leftovers of lysed bacteria (i.e., membrane proteins, LPS, etc.) [
173,
174,
175].
Humoral response to the phages varies by the type of the virus and type of infection [
176]. Immune reaction also depends on the location of bacterial contagion and localization of therapeutic phage injection site [
163]. In some instances, antibodies against phages are formed, but in other cases, the human body tends to be non-responsive without developing antibodies [
176]. Even small differences in phage coats’ protein composition may affect their circulation time and immunogenicity [
177].
The frequency and high level of animal interaction with various types of phages in nature are proved by anti-phage antibodies found in the human sera [
163]. Research by Łusiak-Szelachowska et al. has shown that sera of patients with bacterial infections treated with phage cocktails (locally or locally/orally) had high anti-phage activity after fifteen days. Sera of healthy volunteers treated with the same dose of phage therapy showed a low phage inactivation rate [
178]. Interestingly, sera of patients who received phages orally did not exhibit high anti-phage activity [
178]. On the contrary, Żaczek et al. showed that the majority of 20 patients who received MS1 phage cocktail (orally and/or locally) did not show an increased level of anti-phage antibodies at all. Those studies are of great importance as they rant on human phage therapy effectiveness in humans.
Anti-phage immunoglobulins are one of the most significant factors that may potentially limit the therapeutic effectiveness of phage therapy [
179]. Neutralizing antibodies bind viral epitopes within those parts relevant to infecting the bacteria [
163]. This limits the potential of using phages as drugs. On the other hand, phages can support inflammatory response against bacteria via lysis of the bacterial cell wall, enabling them to release pathogen-associated molecular patterns (PAMPs) and activate the immune system [
180]. Therefore, phage therapies might be very effective since, in addition to direct damaging bacteria cells, phages also activate the human immune system.
Bacteriophages also show antibiofilm activity through depolymerase production. Examples of such phages include the ones isolated from Belgrade wastewaters (ISTD) [
181]. Smaller phages specifically encoding (enzyme polymeric substances) EPS-degrading enzymes can penetrate through the biofilms and cause a disturbance [
182]. The same was demonstrated in the case of
P. aeruginosa biofilms in mouse models against cystic fibrosis [
182]. Another approach to overcome multi-drug resistance is the use of phages with a combination of antibiotics. A better clearance of bacterial cells with a reduced evolution of phages or antibiotic resistance was observed [
183]. The main challenge is to determine the robustness of such an approach, explore the role of immune responses that determine therapeutic outcomes, and establish the phage and antibiotic levels necessary for the therapeutic effect [
184].
Supplementation of antibiotic treatment with phages is best suited for cases when the antibiotics do not adequately reach the target area or when the antibiotic resistance is very high. Phage-antibiotic synergy is an evolutionary trade-off wherein bacterial resistance towards phages increases antibiotic susceptibility, resulting in bacterial growth reductions and complete biofilm suppression [
185]. Carmen et al. analyzed this relationship by combining a real-time microtiter plate readout with a matrix-like heat map of treatment potencies that measures the synergy between phages and antibiotics, so-called synography. This synography is performed against a drug-resistant group of pathogenic
E. coli with antibiotic levels ranging from MIC across seven logs of viral load. The results suggest that phages act as adjuvants by lowering the MIC for such strains. Therefore, it is established that lytic phages can rejuvenate an ineffective antibiotic for resistant bacteria [
186]. Several new aspects have surfaced with success in antibiotic/phage combination therapy, requiring further study and experimentation. The following questions require answers and guidelines: Is there an optimal ratio of phage particles to the antibiotic molecules, which triggers high clearance levels? Are there still unknown interactions with the host immune system after application of phage/antibiotic treatment that can further our understanding and aid better therapy design? It is also essential to explore the rate at which bacteria develop or lose “immunity” towards phages. Another concern is the accurate identification of the pathogen causing the infection, as phages are extremely specific in their action [
187]. Further complications in the application of this therapy, e.g., combination with antibiotics, and in-patient care, also require consideration [
188].
Despite the effectiveness of the phage-antibiotic combination, this approach may not be recommended in all cases. For instance, aminoglycoside antibiotics inhibit DNA replication in mycobacteriophage and hence cause interference with pathogen elimination by phages [
189].
Another powerful tool against multi-drug resistance is the combination of phages with nanoparticles. Metallic nanoparticles can be functionalized onto phages to support the antibacterial action. While bacteriophages provide the specificity for delivery, the metallic gold particles act as bactericidal agents. Such a combination of phages and nanoparticles is easy to engineer, and the properties of the constituents complement each other in eliminating resistant bacterial infections [
96]. Bacteriophages can also be conjugated to gold nanorods. These systems can then be used to kill specific bacterial cells using photothermal ablation, i.e., local generation of heat causing bacterial cell death followed by excitation with near-infrared light [
190].
Phages can also be employed to produce novel bio/nanomaterials due to their rapid multiplication with uniform copies. They can either be used as building blocks of such materials or mere templates. Such phage-based nanomaterials have a wide range of applications, their use depends on the properties of individual phages and additional components of the composites [
191]. Phages can themselves be used as natural nanoparticles. They can also be engineered to display peptides that have bactericidal effects [
192].
4.3. Bacterial Resistance Mechanisms
Temperate bacteriophages form prophage by inserting their genome into bacterial cells. Some DNA fragments are left behind that help surviving bacteria acquire immunity (from bacteriophages) during subsequent infections. These fragments might also be horizontally transferred to other bacterial cells, providing them with similar immunity. Such DNA sequences family has come to be known as clustered regularly short palindromic repeats (CRISPR).
CRISPR has an associated protein, an endonuclease responsible for creating cuts in a double-stranded DNA, thereby helping modify the genome. This protein is called Cas9, and its association with a guide RNA, responsible for matching the target gene, is called the CRISPR-Cas9 system. Cas protein is directed by RNA-spacers (flanked by repeats) to target DNA and cleave it [
146]. These spacers are fragments of DNA congregated from phages that have attacked the bacterial cell in the cell. Insertion of spacers into CRISPR loci on the host genome ultimately leads to the prevention against phage infection [
208]. A popular application of CRISPR-Cas9 is in the treatment of infectious diseases such as HIV [
145]. Other applications include the target and cleavage of DNA responsible for antibiotic resistance [
146].
It is speculated that the CRISPR-Cas immune system of certain bacteria such as
P. aeruginosa is controlled by quorum sensing [
209]. The CRISPR-Cas9 system protects bacteria from horizontally transferred mobile elements. MDR bacteria, lacking this system, acquire new genes easily and rapidly adapt to new antibiotics [
9]. On the other hand, CRISPR-Cas9 prevents the completion of the phage life cycle.
In the context of the ever-evolving arms race between bacteria and phages, it was now crucial for the latter to develop a system of their own against CRISPR. Such a system, called anti CRISPR, was soon discovered within bacteriophages. Anti-CRISPR (Acr) proteins that can easily block different CRISPR-Cas systems were found in the genomes of viruses, bacteria, and archaea. Acr genes were first discovered in
P. aeruginosa where they encode a range of small proteins preventing the functioning of the CRISPR-Cas9 system against the bacterial genome [
210]. Acr-phages overcome CRISPR with the first phage blocking the host CRISPR-Cas immune system to allow a subsequent Acr-phage to attack [
211]. Acr-phages work as a community to replicate and escape extinction successfully [
212].
Acr proteins specifically inhibit the CRISPR-Cas system, and therefore have an enormous potential for application as modulators of genome editing tools. Several approaches were employed to discover Acr families, two primary ones being: (i) Guilt by association and (ii) self-targeting. Guilt by association functions by searching for helix-turn-helix (HTH)-containing proteins encoded downstream of Acr proteins. Such proteins are referred to as Aca (anti-CRISPR associated) and are more conserved than Acr themselves. Self-targeting is referred to CRISPR-Cas systems that enclose spacers targeting regions of the same genome. Organisms with such self-targeting genomes can only survive with the presence of Acrs to prevent CRISPR-Cas from functioning [
213].
Anti-CRISPR proteins have also found application in precise and efficient gene editing. One such example is the anti-CRISPR protein, AcrIIA4, fused with the N-terminal region of human Cdt1 that is degraded in S and G
2 phases of the cell cycle. The expression of AcrIIA4-Cdt1 can increase the frequency of homology-directed repair (HDR) in these phases. This efficiency can also be enhanced by tuning the delivery timing of SpyCas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes. This combination of SpyCas9 and AcrIIA4-Cdt1 is the cell cycle-dependent Cas9 activation system for successful genome editing [
214].
Acr genes are found adjacent to genes encoding the HTH DNA-binding motif. These HTH encoding genes are used as markers to identify anti-CRISPR families. Interestingly, another system, popularly known as the anti-CRISPR system, comprises these HTH encoding genes. Their function is to act as the repressor of Acr promoter, thereby attenuating CRISPR transcription. Not much is known about such systems yet, but they have given a new direction to CRISPR-based genome-editing tools [
215].
To summarize, bacteria counteract phage attack by preventing phage adsorption through biofilms, inhibiting DNA injection via inactivation of proteins involved in the cell wall synthesis, targeting bacteriophage nucleic acids with the help of nucleases, and employing CRISPR systems against the attacking bacteriophage. Another ingenious method, known as abortive infection, is an altruistic action wherein the release of functional phage virions is prevented by the host cell’s programmed cell death [
216].