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].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 receptor-binding domain [42], [43], [44], [45] or modifying phages to express bacterial biofilm-degrading enzymes [46].The evolution of phage resistance in bacteria is inevitable. Bacteria can develop resistance through mutations and other mechanisms. However, acquisition of resistance against phages may be accompanied by a decrease in virulence or a reduction in bacterial fitness, based on the ‘trade-offs’ evolutionary rationale [4], [47]. Bacteria can block phage DNA injection, degrade exogenous DNA from phage vectors using Cas nucleases or enzymes, and interfere with phage DNA replication and phage assembly to achieve phage resistance [48]. The phage cocktail, a combination of different phages, provides a means of decreasing phage resistance.Regarding safety issues, during CRISPR-Cas system delivery, phages may also deliver host mobile genetic elements via hijacked generalised transduction and result in the subsequent spread of virulence genes [49]. Park et al. removed virulence factor genes from their S. aureus host strain to prevent contamination in phage lysates, since staphylococcal virulence factors are commonly located in mobile genetic elements [39]. Safety issues of phage-delivered CRISPR-Cas still require further investigation.With regard to pharmacokinetic issues, similar to traditional phage therapy, the distribution of phages into target tissues or organs is crucial. The use of phage-delivered CRISPR-Cas as an antimicrobial agent against a target bacterium also requires sufficient titre and time. Encapsulation is a method that improves phage adsorption and distribution, increasing the circulation time by evasion of the immune response [50], stomach acidity [51], [52] and enzyme-rich tissue fluids or free radicals [53].6. ConclusionOwing 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 should be conducted in the future.

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].

Phage therapy through intravesical administration has been used to treat urinary tract infections in patients scheduled to undergo transurethral resection of the prostate

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 et al. used a topical phage cocktail on a P. aeruginosa-infected burn wound. The phage cocktail decreased the bacterial load in the wound to a greater extent than that in the control group, which used 1% sulfadiazine silver emulsion cream as standard care [11].

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.

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].

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].

Phage therapy exhibit certain pros and cons like any other curative methods.1)Phage infection resistance strategy: Over millions of years bacteria have coevolved with viruses (Bacteriophages), ergo they have adapted numerous resistance mechanisms.a)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].b)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].c)Bacteria can inhibit phage genome injection by the super injection exclusion (Sie) system (protein-mediated event) encoded by prophage which render bacteria resistant to further phage infections [66].d)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.e)Replication of phage genome can be occluded either by bacteriophage exclusion (BREX) system and abortive system, anti-phage defense system [67].2)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].3)Some phages are antigenic and known to elicit anti-phage antibodies seem to be a major constraint in phage therapy [32].4)Template bacteriophages potentially transfer harmful genes (antimicrobial resistant genes) to their host bacteria, a serious constraint in phage therapy [29].5)Narrow host range of bacteriophages could constitute a crucial impediment to presumptive phage therapy [69].6)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].7)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].

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].Mahichi et al. [62] were able to generate broader host range recombinant E. coli phages that possess strong lytic activity. The long tail fiber genes of T2 phage were replaced with those of phage IP008 through homologous recombination. The resultant chimeric phage acquired the broader host range of IP008 phage while maintaining the bacteriolytic activity of T2 phage. Lu and Collin [63] engineered bacteriophage to express bacterial biofilm degrading enzyme upon infection, simultaneously attacking bacterial host cells within and along the biofilm matrix composed of Extracellular Polymeric Substances (EPS). They modified E. coli T7 phage to express dispersin B (Dsp B), an enzyme capable of degrading bacterial EPS components. The engineered phage potentially reduced bacterial biofilm cell count by > 100 fold compared to that of wild type phage.Bioengineered phage has also found its application in the field of vaccinology. The measures that aid to drastically decrease the burden of infectious diseases are antibiotics, hygiene and vaccination. Despite antimicrobials being the sole lifesaving tool in defending against microbial infections, pathogens quickly acquire phenotype resistance within few years of their introduction. 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].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.

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. Patients with ulcerative colitis (UC) infection caused by C. difficile has been treated for 4.5 years with fecal microbiota transplantation (FMT) – associated with the transfer of intestinal microbiome to restore the homeostasis [51]. Diarrhea due to E. coli in childrens is the most common infection in developing countries which has become a major issue of concern due to the antibiotic resistant strains. Bourdin et al.

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.

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].

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.

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].

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].

Gastrointestinal 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.

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].

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

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.