Owing to the rapid emergence of antibiotic-resistant bacteria, combating infectious diseases is becoming increasingly difficult. A renewed interest in bacteriophage therapy and trends in the development of CRISPR-Cas antimicrobials have provided new treatments for antimicrobial resistance. Further research on phage therapy may improve delivery of the CRISPR-Cas system using phage-based vectors, and additional RCTs on phage therapy and new studies on phage-delivered CRISPR-Cas antimicrobials

With regard to narrow host range issues, phage adsorption requires recognition and binding to specific structures on the bacterial host surface or, in other words, high species or strain specificity. The primary solutions to these issues include modifying the

The use of phage-based vectors as phage therapy to deliver the CRISPR-Cas system into target bacteria currently has several limitations, including a narrow host range, bacterial resistance, safety issues and phage clearance [8].

The global emergence of MDR bacteria represents an increasing public-health concern. Since the pace of emerging antibiotic resistance exceeds that of new antibiotic development, alternative strategies to combat MDR pathogens have been developed, including phage therapy and the CRISPR-Cas9 system [34].

The CRISPR-Cas system can be delivered to target cells in different contents and in several ways (Fig. 1). Specific target genes in bacteria can be knocked out using genome editing using the

CRISPR, an adaptive immune system that protects microbes against infection, was originally discovered in an archaeal microbe, Haloferax mediterranei, by Francisco Mojica in 2003 [16], [17]. The CRISPR-Cas system has since been adopted as a gene editing and diagnostic tool [18]. This system is separated into two classes (class 1 and class 2), with three major types in each class (class 1 including types I, III and IV, and class 2 including types II, V and VI) according to phylogeny, sequence,

Recently, several RCTs on phage therapy for bacterial infections in humans have been conducted and published (Table 1), including phase II RCTs on the topical administration of phage therapy that have gained significant attention [10], [11]. Wright et al. used a topical (ear drop) phage preparation for chronic otitis due to antibiotic-resistant Pseudomonas aeruginosa infection and their results have revealed improved outcomes compared with those following placebo administration [10]. Jault

In addition to conventional phage therapy, ‘clustered regularly interspaced short palindromic repeats’ and ‘CRISPR-associated protein’, abbreviated as the CRISPR-Cas system, which is an adaptive immune system of prokaryotes [6], has been adopted as a genomic editing tool and a novel treatment for multidrug-resistant (MDR) bacteria [7]. However, the design and execution of the delivery of the CRISPR-Cas system for targeting micro-organisms remains a significant challenge. One effective way to deliver the CRISPR-Cas system is via engineered phage-based vectors, which can also be considered a phage-derived antimicrobial therapy [8]. In this article, we review recent advances in phage therapy and the CRISPR-Cas system as antimicrobial treatments as well as RCTs of bacteriophage therapy. In addition, we display mechanisms of the CRISPR-Cas system antimicrobials in a schematic diagram and summarise current data on phage-delivered CRISPR-Cas systems.

Bacteriophages (phages) are a category of viruses capable of infecting bacteria. Phages were first documented in 1915 and were named bacteriophages in 1917 [1], [2]. Attempts to use phages to treat infectious diseases have been made but were generally abandoned after the 1940s owing to their difficulty in use, poor efficacy and the increasing use of antibiotics [3]. However, with the emergence of antimicrobial resistance, phage therapy has become a promising therapeutic option for combating these ‘superbugs’. Numerous successful cases and clinical trials on phage therapy have been published in the last few years, including: studies on conventional monophage therapy; phage-derived enzymes; synergistic effects of phages, antibiotics and the immune response; and bioengineered phages [4]. However, a lack of randomised controlled trials (RCTs) demonstrating the safety and efficacy of phage therapy, as well as several regulatory issues such as production and marketing authorisation, pose obstacles to its practical use [5].

An increasing number of reports have been published on phage therapy and the successful application of antibacterials derived using this method. Additionally, the CRISPR-Cas system has been used to develop antimicrobials with bactericidal effects in vivo. The CRISPR-Cas system can be delivered into target bacteria in various ways, with phage-based vectors being reported as an effective method. In this review, we briefly summarise the results of randomised control trials on bacteriophage therapy. Moreover, we integrated mechanisms of the CRISPR-Cas system antimicrobials in a schematic diagram and consolidated the research on phage-delivered CRISPR-Cas system antimicrobials.

The global spread of antimicrobial resistance poses a considerable threat to human health. Phage therapy is a promising approach to combat MDR bacteria. An increasing number of reports have been published on phage therapy and the successful application of antibacterials derived using this method. Additionally, the CRISPR-Cas system has been used to develop antimicrobials with bactericidal effects in vivo. T

Multidrug-resistant (MDR) bacterial infections in humans are increasing worldwide.

Traditional antibiotic treatments will never be totally replaced by phage therapy. There are great advantages with phage therapy to treat resistant strains but theoretical treatment methods and successful clinical trials are essential before their implementation. Therefore, it is pivotal that scientists conduct the highest standards of rigorous trials to convince the regulatory authorities that phage portray a viable substitute or inclusion to existing therapies. It is vitally important that we learn from past mistakes and explore the possible applications of phages in order to appropriately exploit their very potential in human medicine. Further, more research is required to give new facelift towards phage therapy in future medicine.

Antibiotics are treasures and many times lifesaving agents. They can retain this stature only if they are handled correctly and prescribed appropriately. Emergence of resistant strains is the consequence of wholesale abuse of antibiotic drugs, lack of awareness about antibiotic resistance among the individuals and patient's ignorance on antibiotic treatments. To address antimicrobial drugs resistance issues, collaborations of various disciplines and modernization is required for the development of novel therapeutics and antimicrobial strategies.

All bacteriophages are not good therapeutic bioagents. Significant challenge for phage application is their stability and their competency to reach and lyse the host bacteria [69].

Pharmacokinetics of bacteriophage therapy is much more complex than other therapeutic methods. Several critical parameters in phage therapy need to be fulfilled including rate of phage adsorption, replication cycles, latency period, ideal phage dosing and administration route which are obscured may be due to elimination by some natural barriers (immune system), inter-phage variables, inter-individual differences and differential access to infection sites [67,68].

Narrow host range of bacteriophages could constitute a crucial impediment to presumptive phage therapy [69].

Template bacteriophages potentially transfer harmful genes (antimicrobial resistant genes) to their host bacteria, a serious constraint in phage therapy [29].

Some phages are antigenic and known to elicit anti-phage antibodies seem to be a major constraint in phage therapy [32].

Phage therapy can stimulate adaptive immune system to produce phage neutralizing antibodies that clear the phages from the body. Thus, prevent phage from reaching site of bacterial infection [68].

Replication of phage genome can be occluded either by bacteriophage exclusion (BREX) system and abortive system, anti-phage defense system [67].

Mechanisms developed by host bacteria to digest extrinsic (Phage) DNA: Restriction modification system comprised of enzymes such as endonuclease which recognize and cleave specific sequence in phage DNA and methyltransferase which protect host DNA due to specific methylation or development of adaptive immune response through interfering with CRISPR-cas system.

Phage adsorption blocking one of the major resistance mechanisms is achieved by surface modification of bacterial cell via receptor concealment, downregulation or conformation changes [66].

Extracellular matrix production as a barrier between bacterial receptor and phage is often observed in the biofilm growth or production of competitive inhibitors which specifically bind to phage receptors expressed on bacterial surface and rendering receptors unavailable for phages. Thus, avoid bacteria from phage infection [41].

1)Phage infection resistance strategy: Over millions of years bacteria have coevolved with viruses (Bacteriophages), ergo they have adapted numerous resistance mechanisms.

Phages exploited for vaccines delivery can be in two formats, namely phage display vaccines and phage DNA vaccines. However, phage display technology serves as a huge contribution of phages for phage-based vaccines [5]. Phage display vaccines are produced by expressing cloned antigen on the immunogenic phage particle surface, thereby inducing effective and sturdy immune response. In phage DNA vaccines, eukaryotic gene expression cassette clone the sequence of antigen inserted into the phage genome. Upon administration into the host, the phages are recognized and as a consequence significantly higher and long-last adaptive immune response can be induced [65]. The other mechanism is the use of avirulent bacteria that have acquired phage resistance as attenuated vaccines, since their immunogenicity is retained [60]. With ingenious approaches we can foretell a miraculous therapeutic potential of bioengineered phage.

On the other hand, vaccines rarely induce phenotype resistance as they often aim to elicit immune response of multi-targets and their prophylactic use reduce the probability of spreading resistant mutants. Vaccination has been a solitary intervention capable of eradicating infectious disease and can be considered as the most promising strategy against future global threats [64]. Phages are receiving huge attention as optimal vaccine delivery vehicle, as they are highly stable and simple. In addition, inexpensive mass production and potent adjuvant capacities make them compatible for vaccine design. Phage vaccines present high safety profile and efficient immunostimulatory effects because they have constant relationship with the body during a long-established evolutionary period [65].

Bioengineered phage has also found its application in the field of vaccinology.

The engineered phage potentially reduced bacterial biofilm cell count by > 100 fold compared to that of wild type phage.

Phage engineering has received much attention ever since the emergence of synthetic biology era, evolved with novel and efficient techniques. Bioengineered phages are designed, created or modified form of existing biological entities to accomplish desired task as they would not do naturally [24]. Bioengineering of phages could significantly enhance their therapeutic potential via expanded host range, delivery of exogenous genes, and modification of phage capsids or switching host tropism. Bacteriophage can be bioengineered with various methods to create sequence specific to antimicrobials (CRISPR/Cas 9 sequences to deactivate virulence genes), reverse antibiotic resistance or to enhance antimicrobial activity [25,48].

udies performed in experimental animals have reported that phage therapy did not produce any adverse reactions or toxic effects in animals

Anti-phage antibodies may be produced due to the rapid release of endotoxins which might cause major life-threatening immune reactions like anaphylaxis.

Bacteriophage is known to induce both humoral and cellular immune system

Bacteriophages are generally regarded as safe may be due to their abundance and our constant exposure to them. They are known to exert minimal impact on normal microbiota due to their high host specificity [13], usually exhibit the ability to infect a few strains of bacterial species. Phage resistant bacteria may develop mutated receptors that are not always disadvantageous because the bacteria may completely lose their virulence [60] and become more sensitive to other phages.

Researchers worldwide are trying to apply phage-based techniques to obliterate pathogens in wastewater treatment to ameliorate sludge and effluent emissions into the environment

Large amount of antibiotics along with waste are released into the environment from industries, domestic, medical and agriculture which result in the transmission of drug resistant genes among bacteria in the environment. It has elevated the risk from soil to humans as it enters the food chain, threatens antimicrobial efficacy to combat pathogenic infections

Phage therapy may serve as a potential substitute to antimicrobial and medically superior to antimicrobials in certain cases.

Thus, fatal infections can be treated successfully with phages

Phage cocktails found to be effective than individual phage in the treatment of UTIs caused by UPECs. Phage

Bacteriophages coexist with the bacterial communities in gastrointestinal tract and are known to play a significant role in restoring intestinal eubiosis by eliminating pathogenic strains. They maintain immune homeostasis by potentially exerting immunomodulatory and bactericidal effects.

In 2015, phage therapy to treat wound infections in animal model was demonstrated by the US Navy research group. In their study, cocktails of bacteriophages against A. baumannii demonstrated therapeutic effect in immunosuppressed full-thickened dorsally infected wound mice model. The cocktail reduced the biofilm production in wound, averted the spread of infection and necrosis to surrounding tissues as well as infection–associated morbidity

There was reduction in the bacterial load, cytokines and lactate dehydrogenase in the group treated with high bacteriophage dose compared to non-treated group

The phage not only effectively reduced the biofilm formation but also inhibited growth of phage resistant bacterial mutants [47]. To overcome the limitation of endolysins as antibacterial elements against Gram-negative pathogens, “Innolysins” were designed by combining the enzymatic activity of phage T5-endolysin and its receptor binding protein (RBP)-Pb5 in different configurations.

Studies have shown promising results with phage isolated from hospital sewage against Enteropathogenic E. coli (EPEC) in mice [44] and coliphages effective against planktonic and biofilm related infections [45]. Cocktail of varied phages are used to vanquish phage resistant pathogenic bacteria.

Bacteriophages are used as an effective therapeutic tool against MDR and biofilm forming pathogens. T

Uncontrolled microbial diseases in aquaculture industries often endure economic loss, threaten their sustainability and development.

Several reports have demonstrated phage applications to evade bacterial infections from various crops such as onions [34] and citrus [35]. US FDA has also approved phage cocktails in food processing industries. Phage preparations are accepted as antibacterial food additives for poultry products and ready-to-eat meat

Studies have shown promising results with phage isolated from hospital sewage against Enteropathogenic E. coli (EPEC) in mice [44] and coliphages effective against planktonic and biofilm related infections

Bacteriophages are used as an effective therapeutic tool against MDR and biofilm forming pathogens.

Currently bacteriophage therapy as a novel approach is gaining major interest, as antimicrobial resistant pathogens have emerged. They are successfully used in agriculture worldwide. Food and Drug Administration (FDA) has approved the use of certain phages on crops in order to reduce crop diseases.

Co-evolutionary forces have endowed phages with a vast array of counter host defenses such as amended receptor proteins, hydrolyzing enzymes etc. [31]. Phage cocktails could also be used to ensure the adequate coverage of common strains and to reduce the probability of phage resistant bacterial mutants [14]. To circumvent immune response issues, one should carefully study each case and choose the appropriate administration route, phage exposure time, dose and buffers. Development of sequence technology and molecular biology enhanced our basic knowledge of bacteria and bacteriophage interactions and enabled novel possibilities of phage utilization including genetically engineered phage, dual therapy and enzymes derived from phages [32]. Indeed, bacteriophages are the potential agent to be utilized in diverse biotechnological applications, varying from food – animal agriculture, human and veterinary medicine to environmental science (Fig. 2).

Amid the looming crisis of antimicrobial resistant pathogens, dual therapy is considered as a newer strategy to combat global threat. The consolidate result of two therapeutic agents is much greater than sum of their individual effects, a fact that brings significantly higher rates of therapy success achieved by synergistic effects. Less frequent resistance evolves with dual therapy because a strain non-sensitive to any one agent could be destroyed by the second [29]. Several studies have been done to investigate the role of phages in combination with antibacterial drugs to restore antibacterial activities through synergy. P. aeruginosa in vitro growth inhibition through the synergy of bacteriophage and antibiotic (streptomycin) has been demonstrated [30].

. Towards the end of lytic cycle, phage utilizes different set of enzymes to enable host cell lysis and release of progeny virions. Tailed phage system depends on the coordinated action of two types of proteins, namely holins and endolysins for the degradation of bacterial cell wall peptidoglycan [26]. Like entire phage, endolysins are effective and specific bacteriolytic agents at low dosage and are classified into five different groups. Numerous in vitro studies have shown different Staphylococcal endolysins, like MV-L and chimeric lysin – ClyS to be active against S. aureus infections such as MRSA, methicillin-sensitive S. aureus (MSSA), vancomycin-resistant S. aureus (VRSA) and vancomycin-intermediate S. aureus (VISA) [27,28].

Peptidoglycan Hydrolases (VAPGH) and Polysaccharide depolymerases are the two groups of phage proteins, allowing phage to adsorb and eject its genome into the host cell.

Phage encoded enzymes serve as an alternative to the whole phage application. Phage genome encodes numeral enzymes and proteins required to rupture bacterial cells during infection.

Multiple studies have been carried out for successful modification of phage in order to overcome certain obstacles and optimize their advantages as bactericidal agent.

The major goal of genetic engineering is to generate bacteriophage with broader host range, lack of toxin genes and other traits to perform desired task.

Thus, phage therapy demonstrates propitious evidence and safety profile.

The traditional concept of phage therapy is the direct application of naturally isolated virulent phage to the patient with an aim of lysing pathogenic bacteria responsible for causing infectious diseases. Here phages are used as a sole therapeutic agent, this form of phage application is referred to as ‘Conventional phage therapy’ [22]

A spectrum of strategies have been described here ranging across natural and engineered phages, enzymes derived from phages and combination of phages with antimicrobial substances

Bacteriophage therapy effectively reduced bacterial load and enhanced wound healing which was infected with P. aeruginosa and S. aureus, in a study demonstrated in both swine and rodent models by Mendes et al. [21].

Polymicrobial biofilms are recognized as a prime factor in the failure of chronic wound healing in timely fashion.

Wound infections following trauma are grouped as early infections caused by the exposure of wound to bacteria in the battlefield and late nosocomial infections that are acquired during medical care. Implementation of phage therapy as bacteriophage impregnated dressings in the battlefield and appropriate phage usage against nosocomial infections potentially can reduce mortality and morbidity rate from battlefield trauma.

The prime route of bacterial infection in underprivileged countries is from contaminated food and water through fecal-oral route [10]. Infections are usually treated with antibiotics but due to the emergence of resistant strains alternative therapy options are required. Bacteriophages play vital roles in shaping the composition and diversity of the bacterial communities. Myoviridae, Siphoviridae and Podoviridae are the three abundant virus families in the human gut [16]. Several studies have evaluated phage therapy potentials for the treatment of gastrointestinal infections caused by V. cholera, C. difficile, E. coli and S. enterica. Nale et al. [17] demonstrated that phage therapy significantly reduced C. difficile biofilm and prevented colonization in a G. mellonella model when used solely or in combination with antibiotics (vancomycin). Prophylactic phage treatment 2 h prior to bacterial challenge showed 100% survival rate while simultaneous application of phage & bacteria showed 72% survival rate and phage application 2 h post bacterial challenge showed 30% survival rate.

2.2.2. Gastrointestinal infectionsGastrointestinal tract (GIT) is a complex environment containing billions of microorganisms. Of these microbes, bacteria form the vast majority of biomass. Enteric pathogens form principal part of the medical burden around the globe. The most common intestinal infections causing bacteria are E. coli causing gastroenteritis, Helicobacter pylori causing chronic gastritis, Salmonella enterica causing salmonellosis and Clostridium difficile causing diarrhea.

Cocktail of phages are developed against different species of bacteria present within the biofilms and predominant in purulent infections. P. aeruginosa lung infection in mouse and sepsis in Galleria mellonella (wax moth) model was successfully treated with cocktail of six phages [14]. Bactericidal activity of spray-dried phage formulations were assessed against MDR P. aeruginosa in a lung infected mouse model [15].

Many pulmonary pathogens are able to form biofilm in which bacteria are encased in extracellular matrix and metabolically less active [12]. Mycobacterium tuberculosis (M. TB) is an agent of tuberculosis (TB) which is the global health problem. Antimicrobial resistant M. tuberculosis strains are non-sensitive to two of the frontline drugs (rifampicin and isoniazid) used in the treatment of TB. Therefore, there is an urgent need of novel therapies for TB [13]. Many in vivo phage therapy studies have been undertaken against P. aeruginosa, S. aureus, H. influenzae, B. cepacia and TB infection in animal models [10,12,13].

These bacteria infect the patients with weakened immune system [10]. P. aeruginosa acts as an opportunistic pathogen in cystic fibrosis (CF) patients. Lung CF is an autosomal recessive genetic disease which results in reduced hydration and secretion of highly viscous mucus that covers the respiratory epithelium causing chronic inflammation and bacterial infection [11]. S. aureus and H. influenzae are the two common strains isolated from sputum of infected lungs. Burkholderia cepacia is another opportunistic bacterial pathogen in the lung CF causing acute pulmonary diseases and sepsis or chronic infection.

Patients in intensive care unit (ICU) settings often suffer from ventilator associated pneumonia (VAP) infections caused by MDR Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Acinetobacter baumanni and Haemophilus influenzae.

Many in vitro and in vivo studies have demonstrated that phage therapy has been used for the successful treatment of antibiotic resistant pulmonary infections (Pneumonia, and cystic fibrosis), gastrointestinal infections, and topical and wound infections.

Based on the biological cycles, phages can be divided into two types, namely lytic (virulent) phages and lysogenic (temperate) phages. Lytic phages multiply within the bacterial host cell and assembled progeny are released by the lysis of host cell. Lysogenic phages are able to integrate their genetic material into the bacterial host genome and remain dormant. They replicate together with the bacterial genome and are passed to the successive bacterial generations. In later stages due to the external stimuli lysogenic cycle is switched to active lytic cycle. A pseudolysogenic cycle has been reported, an alternate lysogenic cycle in which the phage genome remains inactive within the bacterial host [4].

The classifications are based upon the evaluation of diverse phage properties such as genome composition, morphology, host range, sequence similarity and pathogenicity (Table 1). Due to lack of universal genomic marker and high diverse structure, creation of viral phylogenetic tree experienced limited success [7]. Development of techniques and availability of phage genomic sequence data gave rise to a variety of grouping schemes including phage proteomic tree, Kmer based grouping, phage network clusters and many more [8]. Currently, ICTV has described 19 phage families within the order Caudivirales among which Myoviridae, Podoviridae, Siphoviridae, Microviridae, Inoviridae are the most-well characterized ones, Herelleviridae and Ackermannviridae are the recently traced families [9].

Viral taxonomic classifications are the responsibility of International Committee on Taxonomy of Viruses (ICTV) and Bacterial and Archaeal Subcommittee (BAUS) within the ICTV

In 1896 Ernest Hanbury Hankin discovered bacteriophages displaying antibacterial properties against Vibrio cholerae from the water of Indian rivers [6]. Phages were discovered in 1915 and 1917 by Fredrick William Twort and Felix d’Herelle respectively. It was Felix d’Herelle French Canadian microbiologist who coined the name “Bacteriophage” (viruses that kill bacteria) for the first time. After his discovery he suggested that phages could serve as a therapeutic tool. He used phage preparations to successfully treat Shigella dysentery patients in France and Cholera outbreak in India. Since then phage therapy was considered as a significant therapeutic tool and the exclusive treatment for bacterial infections [4].

Bacteriophages are viruses, the most abundant organisms and the natural predators of bacteria. They are self-replicating, obligatory intracellular parasites and inert biochemically in extracellular environment. They control the biosynthetic machinery of bacterial host and behest them to produce different viral proteins. They are considered as particles outside the host cell containing nucleic acid (DNA or RNA) which encode necessary information required for their replication. They are primordial ubiquitous organisms found in diverse environment such as soil, water, feces etc [4,5]. Typically, bacteriophage morphology exhibit well defined three-dimensional structure. The genetic material is enclosed in an icosahedral protein capsid head, a tail (spiral contractile sheath surrounding a core pipe and a baseplate with tail fibers) and surface receptor proteins responsible to recognize specific surface molecules on the host bacterium [5].

Phage therapy is an age old practice of pre-antibiotic era in which bacteriophages are harnessed as bio-agents against bacterial infections. The advent of effective chemical antimicrobials has curtailed phage therapy in most of the countries. However, phage clinical utilization continued in place of antibiotics in Eastern Europe and the former Soviet Union. Currently, the phage therapy is gaining much attention as more and more bacteria become resistant to current antibiotics. Phage therapeuatic efficacy has been demonstrated against a wide range of bacterial pathogens such as Staphylococcus species, Pseudomonas spp., and Escherichia coli [2,3]. Thus, phage therapy applications have noted significant progress in broader clinical practices. In fact, phages can be used for different applications ranging from human antibiotherapy to environment disinfection. This review discusses the therapeutic use of bacteriophages as novel antimicrobial agents to vanquish antimicrobial resistance and several modern approaches to phage therapy, thereby encouraging further research with an ultimate goal to utilize phage as therapeutic agent in clinical practices.

Development of drugs has revolutionized the treatment methods of bacterial infectious diseases that have had killed millions of humans in pre-antibiotic era. Indiscriminate use of antibiotics practiced in clinical setup, industry, animal husbandry and agriculture has resulted in the surge of resistance in microorganisms. This emergence and dissemination of antimicrobial drug resistance among pathogens poses an enormous challenge to the public health professionals globally. Antimicrobial resistance may occur naturally, however the process might be accelerated due to the irrational and overuse of antibiotics leading to the mushrooming of resistant pathogens [1]. Infections caused by resistant bacteria are frequent and many of them are multidrug resistant (MDR) and biofilm forming pathogens which are ineffective to the standard treatments resulting in high mortality and morbidity rate.

Antimicrobial resistance is a global health problem and one of the leading concerns in healthcare sector. Bacteriophages are antibacterial agents ubiquitous in nature. With increase in antibiotic resistance, use of bacteriophages as therapeutics has become resurgent in recent times. This review focuses on the recent developments in phage therapy and its applications with respect to human infections, animal, food and environment. Moreover, use of phage proteins, bioengineered bacteriophages, and phage derived vaccines is also highlighted. Additionally, the limitations and challenges with regard to implementation of phage therapy, host safety and immune responses are also reviewed in this article.