Bacteriophages of Mycobacterium tuberculosis, their diversity, and potential therapeutic uses: a review

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tuberculosis) is a highly infectious disease and worldwide health problem, with a high mortality rate and nearly ~ 1.6 million recognized deaths in 2021. It has harmed humankind for approximately 9000 years, with the first report dating back more than 3000 years ago in India and China [1, 2]. TB had a notable effect on social health owing to decreased influence and a more negligible therapeutic effect with mycobacterial therapy. The quick prevalence of disease and the warning development of drug resistance, particularly the appearance of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains, have called the alarm to gain novel effective drugs; thus, finding a substitute line for the controlling and management of TB has become essential. One of the main features of Mycobacterium is that it produces highly resistant mutants under selective pressure conditions caused by antibiotics. The following evolutionary achievement of resistant mutants depends mostly on the mutant’s resistance rate and ability and selective elimination owing to antibiotic therapy. Among the various developing antibacterial approaches, phage therapy is thought to be a precise hopeful resolution. Bacteriophages (phages) are a type of viruses that infect bacteria and are very widespread in the environment. Bacteriophages can be used clinically to handle bacterial disease as natural antibacterial agents [3‐6]. In this review, we will introduce mycobacteriophages, their use as a therapeutic approach and diagnosis tools, and their superiority and some challenges and limitations as therapeutic applications.

The review included full-text articles published in different countries over the last 22 years were explained the therapeutic uses of mycobacteriophages in Drug-resistant TB. The review similarly excluded articles that had not been considered by academic counterparts and articles published past the chosen period.

It is evaluated that drug-resistant strains of Mycobacterium will kill more than 75 million people in the next 35 years. According to the World Health Organization Global TB report, Tuberculosis mortality has increased since the COVID-19 pandemic. The Covid-19 pandemic may increase the number of new cases of tuberculosis due to resource constraints and other constraints in TB native areas [7]. Over the last two decades, multi-drug strains (MDR), extensively-drug (XDR), extremely-drug (XXDR), and total-drug resistant (TDR) strains of M. tuberculosis have emerged as a worldwide challenge. One of the main reasons for the prosperity of M. tuberculosis in causing infection and escaping the host immune response is its specific cell envelope, which is mainly composed of lipids and carbohydrates. The presence of these compounds enables the bacterium to adapt to different environmental conditions and protect the bacterium in the presence of drugs [8, 9].

For this reason, in the treatment of tuberculosis, monotherapy is not recommended, and several antibiotics use simultaneously. Standard treatment for susceptible strains of M. tuberculosis includes treatment with four first-line drugs (treatment with isoniazid, rifampicin, ethambutol, and pyrazinamide for 2 months, followed by treatment with isoniazid and rifampicin for 4 months) [10]. However, various factors have led to the spread of drug-resistant strains of M. tuberculosis. These factors include incorrect prescription of drugs, insufficient access to drugs, and poor commitment to treatment [11]. MDR-TB strains are resistant to at least two first-line drugs and should treat for 9–20 months. However, the treatment success rate of these strains is 56% compared to sensitive strains. In addition to being resistant to isoniazid and rifampicin, XDR-TB strains are resistant to fluoroquinolone and one of three second-line injectable drugs (amikacin, capreomycin, or kanamycin). Also, the success rate of treating these strains is 39% [10]. XDR-TB strains are resistant to all first- and second-line antibiotics. In recent years, new antibiotics including bedaquiline have been added to the tuberculosis treatment program. The use of bedaquiline in patients with MDR/XDR TB was able to cure 82% of patients. The recovery rate of patients with MDR TB was 89.9%, but the recovery rate of patients with XDR TB was 71.9% [12]. However, in recent years, in countries such as Iran, Italy, and India, strains resistant to all antibiotics (even resistant to antibiotics under discovery and development) have been reported [13‐15], which the WHO called Drug-resistant TB (TDR-TB). Therefore, due to the emergence of these resistant and incurable strains with available antibiotics, researchers are looking to discover new drugs and even newer methods for treating tuberculosis [16].

The main risk factors include contact with people who are infected by tuberculosis, living with people infected by human immunodeficiency virus (HIV), HIV co-infection, being a former prisoner, being a smoker, alcoholism, being an immigrant, being male, being middle-aged, health care staff and those with chronic obstructive pulmonary disease (COPD). Hospital-acquired tuberculosis infection is more commonly recognized in High-frequency TB and low and medium-income countries which report 87% annually. The number of TB cases per 100,000 Healthcare workers in some down and average-income countries is further than twofold the frequency level among the general population, and healthcare services are a significant origin of TB transfer in these countries. Another factor involved in the development of TB is urbanization, mostly among high-capacity regions. For many reasons, such as overpopulation, quick improvement, and other environmental features, many cases happened. Close contact and inhalation in the same nearby environs among patients with TB and susceptible persons cause the extent of TB. Numerous studies have revealed that TB patients need reception to the ICU conveys a high mortality level of 25–63% [17, 18]. Delays in treatment or diagnosis could lead to acute disease and higher mortality rates. Several studies have assessed risk factors for death in the treatment of TB. For example, age, sex, bacteriological case, immune and dietary condition of the host, and drug abuse, have been recognized. Effective treatment of TB is essential to treating the patient and decreasing the spread of M. tuberculosis in public places. But an important subject is the widespread occurrence of drug-resistant TB (DR-TB, worries around DR-TB are growing in current years. At least 5% of whole universal cases of TB have several types of drug resistance, that is, resistance to, as a minimum, one first-line anti-TB drug [19‐22].

Bacteriophages, the most plenty organisms on the earth, are the shady subject of the biological world, making an enormous, extremely old, dynamic, and genetically various population. About 10³¹ tailed phage elements join in about 10²³ infections per second worldwide, with the total population changing in a short time. Shortly after Felix d'Herelle discovered bacteriophage, the idea of using phage to treat the infectious disease was introduced [23‐25]. In the 1940s, the use of phage therapy was restrained by the preface of penicillin and other antibiotics. While the concept of phage therapy has been about for approximately a century, it is yet well-thought-out empiric therapy in Western countries and has not been permitted for human practice so far. Although, the emergence of drug-resistant bacteria as MDR-TB and XDR-TB phage therapy is well thought-out to be a significant candidate for substitute therapeutic agents [26, 27]. According to their survival life strategies, phages display three diverse life cycles: lytic, lysogenic, and pseudo-lysogenic when infecting a bacterial host. The phage must first attach to the host cell and then inject its genetic material into the cell (Fig. 1A). A significant dissimilarity among viruses that infect bacteria and eukaryotic cells is the early procedures related to infection. Usually, bacteriophages are challenged by attaching and penetrating a bacterial cell wall. Then, expel their genomic materials the inside of the cell. The intrusion into the bacterial wall is usually related to a cell wall digesting enzyme(s), regularly mentioned as a peptidoglycan hydrolase or endolysin, often found in a tail construction of tailed phages. The phage multiplies during the lytic phase, and progeny phages explode the cell and exit. The phage does not reproduce in the lysogenic cycle, but its genome goes into a quiet state and is generally integrated into the host genome (Fig. 1A). In the pseudo lysogenic step, the phage does not experience lysogeny, nor does it display a lytic cycle, but it stays in a non-active condition. Phages that reproduce through the lytic cycle are called virulent phages, whereas those that replicate via both lytic and lysogenic cycles are identified as temperate phages. The lysogenic stage may be constant for numerous generations, and the bacteriophage could modify the phenotype of the bacterium by gene expression that is not light in the normal period of infection in a procedure identified as lysogenic conversion (Fig. 1A). Phages may have a pseudolysogeny stage in their life cycle. It refers to a condition that a phage has joined a bacterial cell and does not unify in a constant style, then will remain in this manner while situations fall out which trigger them to go into the lytic or lysogenic life cycle. The carrier state defines combinations of bacteria and of bacteriophages that are invariable and stable. A section of bacteria is persistent, but some sensitive alternates’ attendance seems to endure the phage population so that both progress [28‐32].

Phages can be isolated from different sources including soil, water, and sewage [3]. The Mycobacteriophage was primarily isolated in 1954. Through the 1960s and 1970s, phages were used for typing M. tuberculosis clinical isolate in epidemiological research [33]. Most of the lytic phages of M. tuberculosis belong to Order—Caudovirales; the Family—Siphoviridae, and cluster K [34, 35], which is divided into seven sub-clusters (Table 1). Due to the massive genetic multiplicity between bacteriophages, they are classified into clusters and subclusters. Cluster K is one of the known clusters in which all members can lyse M. tuberculosis, and Mycobacteriophage DS6A infects the M. tuberculosis complex. Siphoviridae has a high flexible tail structure that makes it difficult to identify. The pointed capsid layer is occupied with dsDNA. The genomes of Cluster K phage (average genome length, 60 kbp) have some uncommon structures containing start-associated sequences (SAS) and extended start-associated sequences (ESAS). It is believed that the discovery of new mycobacteriophages would help grow the present database and can be conducted to recognition of un-explored infectious phages as a source of hydrolytic enzymes, for example, Endolysins, EPS depolymerase, and Phospholipases/Esterases [33, 36, 37].

In the last phase of the lytic cycle, newly assembled phage particles must be released from the infected host. Therefore, the phage must destroy peptidoglycan, mycolic acid, and cell membrane structures. Phage produces two proteins to release from the cytoplasm of host bacteria: endolysin and holin. Endolysins cut covalent links in the peptidoglycan (PG) and disrupt cell wall integrity that supports the discharge of phage particles from the bacterial host [1‐3].

Holins are a large group of small hydrophobic membrane proteins holding a transmembrane area congested in the inner membrane and cause cell membrane permeability by making perforations that collapse the proton motive force (PMF) of the cell membrane and leading to cell death (Fig. 1B). Holins are thought-out to be the simplest biological device as they control the secreted and availability of endolysins to the cell wall. Holins do a different well-known function, which contains release of gene transferal mediators, having a function in biofilm development, simplifying several procedures essential for differentiation, such as spore germination, [4] aid in diverse responses to stressful situations [5] and release toxins and associated proteins [38‐42].

Mycobacterial envelop contains a cytoplasmic membrane, a peptidoglycan layer covalently connected to the arabinogalactan-peptidoglycan complex, and mycolic acids (Fig. 1B). Mycobacteriophages are the phages that infect mycobacterial species. Mycobacteriophages produce two endolysins, LysinA and LysinB, for overcoming these complex layers. LysinA and LysinB affect peptidoglycan and mycolic acid arabinogalactan separately. LysB is a mycolylarabinogalactan esterase that cuts the ester link among arabinogalactan and mycolic acid (Fig. 1B). Thus, cell lysis occurs following the loss of communication between the Mycobacterium cell wall and the outer membrane. Although a great number of mycobacteriophages have been identified, few studies have been performed on mycobacteriophage endolysins. Most studies have been reported about endolysin of D29 and Ms6 mycobacteriophages. Fraga et al. showed for the first time that recombinant LysB exhibits lytic activity on M. ulcerans isolates [43]. Also, they showed that using LysB for the management of mouse models of M. ulcerans footpad infection inhibits cell proliferation. Pohane et al. carried out a study on the structure and function of the Lysin A of Mycobacteriophage D29. By making several structures, they studied the details of LysinA and obtained the shortest protein sequence with a catalytic domain [44]. Mycobacteriophage lysines are considered a potential alternative treatment for mycobacterial infections caused by MDR and XDR strains [5, 45, 46].

The host range of a bacteriophage is specified as the extent of hosts that it can contaminate. This span is related to host features (e.g., protection and restriction-modification systems and the existence of phage receptors), environmental elements (e.g., temperature and pH), and structures determined by the phage. Specific bacteriophages frequently exhibit a narrow host range and contaminate a narrow spectrum of bacterial strains of similar species. In comparison, common bacteriophages intrinsically exhibit a wide host range. Phage–host interaction is unique, and phages are very precise to their bacterial hosts, and they replicate using the facilities of the host cell. The first stage in the bacteriophage life cycle is its binding to the bacterial cell surface by a receptor on the phage tail or capsid. The capability of a phage to recognize and bind to receptors is one of the factors that influence its host range. Various mycobacteriophages belonging to cluster K (Table 1) can infect a different range of hosts, including slow-growing mycobacteria (e.g., M. tuberculosis) and fast-growing (e.g., M. smegmatis), however comprehensive visions into specific host ranges stay mostly missing because of the aspect that the most common identified mycobacteriophages were isolated via M. smegmatis mc2155. It is commonly well-thought-out, in the situation of their therapeutic usage, that lytic phages by a wide host range (e.g., at genus or species level) are more helpful in fighting bacterial infection than those with a narrow host range (e.g., at strain level) [25, 47, 48].A phylogenic tree of All 159 mycobacteriophage according to Whole-genome sequencing available in Genebank database were analysed using neighbor-joining method. The figure shows that two large clades, which upper one contains a small number of sequence. the lower large clade is divided into two cluster (Fig. 2).

TB control is confined by present detection methods. Clinicians use X-rays, microscopy, and cultures as widespread implements to identify TB. Using molecular methods such as the GeneXpert system, TB is detected in a short time and with high sensitivity, but so far, this device has not been widely used. Culturing of Mycobacterium is known as the gold standard diagnostic, but many mycobacterial species are slow-growing, such as M. tuberculosis and M. bovis. Phage-based diagnosis generally comprised of two overall extents: phage amplified biologically (PhAB) assay and phage reporter assays (PRAs). The PhAB uses a definite characteristic of the phage’s natural capacity to infect, strengthen, and disrupt the cells to identify the mycobacteria. PRAs usually encompass genetically altered bacteriophages or their hosts with the aim of a fluorescent, luminescent or different signal can be identified. Previously, phage-based kits existed and were mainly considered for M. tuberculosis recognition in human sputum samples. Nowadays, it is probable to use an in-house alternative test, which founds a laboratory-established phage amplification assay (PA) not expressively diverse from the commercial one. This might characterize an appropriate substitute for PCR tests, particularly in low-income countries, because it depends on only simple microbiological methods. The defect of PA can be an ineffective infection in a significant number of bacteria in the specimen, which can limit from half to four-fifths of the measured CFU, and could be triggered by some reasons; e.g., phage replication does not happen in dormant bacteria [49, 50].

After in vitro examinations, each new treatment candidate must be evaluated for efficacy and safety in an animal model and then performed in human experiments. Each TB therapy choice will requirement to overwhelming defies the infection plans (tissue/granuloma diffusion, penetration to host cell, drug interface with HIV treatment). Moreover, they must be rare in toxicity and confrontational properties on microflora, short in time, and will have to be made accessible in the countryside and poor regions [51]. The key benefits of phage therapy are low charge of manufacturing, no side effects on microflora, and auto-adjustment of phage levels in the patient. The negative impacts of phage and chemical medicines have not been recognized. The phages could not entirely remove a bacterial pathogen alone because they would lose the bacterial host devices. However, effective phage management could expressively decrease the number of targeted bacteria. Finally, the mammalian immune system entirely removes pathogen remains from the tissue. In this procedure called “Immunophage Synergy”, the act of the immune system is required and counterparts the phage antimicrobial activity, seen in neutrophil-phage collaboration [52]. Moreover, phages have the potential to stimulate anti-inflammatory cytokines over their contact with host immune cells, helping to decrease inflammation and tissue injury. For example, bacteria were used as a carrier to transport lytic phages into the macrophages of the mouse to destroy methicillin-resistant S. aureus inward of the cells. About M. tuberculosis, Mycobacteriophage sending into macrophages has been reached using M. smegmatis or liposomes. As well as being fast-growing and non-virulent, M. smegmatis can similarly render as host bacterial storage for Mycobacteriophage multiplying phage titers before attainment of the targeted M. tuberculosis. However, M. smegmatis intervened in mycobacteriophage transfer has been confirmed in vitro. Owing to its pathogenicity in mice models, it could not be a proper strain to achieve phage transport training in vivo. The high specificity of DS-6A creates it a noteworthy candidate for TB therapy. Sula et al. gained incentive results causing treatment by DS-6A and a decrease in lesions in the spleen, lungs, and livers of guinea pigs [53]. In a study by Nieth et al., a non-bacterial vector was used to send bacteriophages into infected cells. They tried to encapsulate bacteriophages into liposomes. Additionally, they showed that liposome-associated bacteriophages are driven up into eukaryotic cells more capably than free bacteriophages (Fig. 3) [54]. These are important indications in the progress of an intracellular bacteriophage therapy that may be beneficial in combat contrary to multi-drug-resistant intracellular pathogens such as M. tuberculosis [25, 51].

Mycobacteriophages mainly have a limited host spectrum (narrow) which can be solved by having a rich phage database and also by using bioengineering methods. Due to the intracellular nature of M. tuberculosis, phage access to the bacterium is difficult. However, using carriers such as M. smegmatis and phage encapsulation [55‐58], the phage can be transported and reach the bacteria. Another challenge with mycobacteriophage therapy is phage resistance. Due to the widespread use of phages as therapeutic and ecological bio-control agents, selective pressure could lead to the expansion of resistant bacteria. Long interaction between phages and bacteria has caused bacteria to develop a variety of mechanisms to escape from phages, and in phages emerging certain approaches to escape the antiviral systems [59]. Although the appearance of resistant bacteria, phages will find an approach to confirm their dispersion. Based on data from various studies, phage resistance may be due to damage or altering their external receptors over mutation of genes accountable for the production of these receptors, so inhibiting phage incorporation, prevention of phage DNA diffusion, hindering of the receptor(s) inhibition of intracellular phage association and hydrolyses phage genome by the production of restriction endonuclease enzymes. Phage-derived enzymes can destroy cell surface receptors. So far, it was believed phage therapy, like antibiotics, just decreases the number of bacteria. But treatment breaks happen when bacteria are provided to improve phage resistance through phage management. Therefore, some strategies have been suggested to inhibit phage resistance in bacteria, including using phage cocktails instead of monotherapy and phage engineering that goes beyond simple phage monotherapy to preclude resistance, such as multi-phage cocktails, phage engineering, and combining phages with antibiotics [60, 61]. Some studies have warned of the possibility of phage toxicity to humans. But the genes with the potential for toxicity can be eliminated using genetic bioengineering techniques. It is desirable to identify all genes and protein functions before using phages in clinical trials to prevent such complications [62, 63].

The emergence of drug-resistant bacterial pathogens such as MDR-TB and XDR-TB has become an intense challenge for scientists and the health of the community. The absence of efficient therapeutic procedures for MDR-TB and XDR-TB isolates needs alternative and innovative ways. The long treatment period, side effects, and high cost in unindustrialized countries have caused unfortunate agreement regarding using treatment procedures, additional operations the occurrence of drug-resistant strains. Novel antimicrobial agents, including bedaquiline, have been progressive; however, the necessity for novel therapeutic plans is inevitable [64]. AK15, a small mycobacteriophage-derived peptide, and its isomer AK15-6 exhibit effective anti-M. tuberculosis activity. Both AK15 and AK15-6 directly prevented M. tuberculosis by membrane interruption. Also, they displayed cell selectivity and synergistic properties with rifampicin. They proficiently decreased the mycobacterial load in the lungs of mice infected by M. tuberculosis [65]. Carlos et al. prepared a cocktail of five phages that reduces the occurrence of phage resistance and cross-resistance and powerfully destroys the M. tuberculosis strains [2].

Additionally, these phages act without antagonistic effect on antibiotics and infect equally isoniazid-resistant and -sensitive strains [66]. Yeswanth et al. evaluated the effect of phage cocktails on mycobacterium growth. In their 5-phage cocktail, two of them (D29 and TM4) were identified to infect M. tuberculosis isolates. These two phages and DS6A were grown via M. tuberculosis (H37Ra) as a host. Mycobacteriophages displayed synergy with antimicrobial agents, for instance, rifampicin and isoniazid. Finally, it was determined that mycobacteriophages are effective in inhibiting M. tuberculosis equally in the lag and log phase for some weeks. These results have significant effects on developed phage therapy for Mycobacterium [67].

The concerns about phage therapy as antibacterial agents mostly contain safety and effectiveness subjects and an increase in a possible immune response to any ordered phage. The collected information shows gaps in our considerate clinical association between the reaction among phages and the immune system. Development optimization and purification plans of phages are additional problems required to discuss. Progresses in molecular biology and biotechnology can resolve the difficulties that humans are facing now [68, 69]. Phages have been revealed to be able to transfer genes encoded antibiotic resistance and toxins into host bacterial cells through transduction procedure. Thus, such hazardous genes should be screened through phage therapy. The main goal of phage therapy is to increase the number of phages in the bacterial hosts, which occurs by using host conveniences, but few studies have been done on the side effects of this occurrence. Moreover, Industrial manufacture is a considerable issue in the therapeutic use of phage-encoded proteins. Safety procedures are the essential worries which must be taken into attention through the production procedure [48, 70].

Bacteriophages lyse the bacterial hosts with complex mechanisms, of which little has been studied so far. Thus, more studies are needed to understand their enzymatic machinery, regulatory methods, and biochemical properties. A typical feature of mycobacteria is their complex cell envelop required for intracellular survival. So, inhibition of its formation can be an effective manner in treating tuberculosis. The lysis enzymes produced by Mycobacteriophage appear to target the main structure of the cell envelope and seem to be hopeful candidates for spoiling mycobacteria [45, 46]. Many recent studies are investigating the potential of phage-derived LysA and LysB to kill Mycobacterium, and it has been found that purified recombinant of two enzymes will be more effective. Although the novelty and tendency to use these proteins as a substitute for antibiotics, additional investigation is still required for their medical practice of them. A substitution might be the application of mycobacteriophages prophylactically instead of therapeutic goals. For instance, family or colleagues of patients newly detected with respiratory tuberculosis can consume aspirated phages to inhibit the spread and acquirement of the illness [44, 71]. Despite the numerous advantages of phage therapy in the treatment of infectious diseases, there are obstacles regarding this treatment method. For example, we can mention the lack of regulation for this method and the lack of sufficient scientific evidence [72]. With the increase of in vitro and in vivo studies, we can learn about various aspects of mycobacteriophage therapy and the interaction between phage and the host body and immune system.

In cases where antibiotics alone cannot eliminate the infection, mycobacteriophages can be used along with antibiotics. By studying the mycobacteriophage structure and its enzymes extensively, mycobacteriophage therapy can be personalized [73], and the combination of antibiotics and personalized phage therapy can be a promising method in the treatment of drug-resistant tuberculosis.

The emergence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) M. tuberculosis strains has become a global concern. Among infectious diseases, tuberculosis has the most mortality rate and is increasing. Mycobacterium genome undergoes mutations that subsequently can avoid the drugs generally used to prevent them. The prevalence of resistant strains through the control procedures and treatment of the disease is more complicated than, mainly, when the patient is co-infected by HIV. So, efforts have begun to use an old method to treat bacterial infection that is phage therapy. Phage and Phage-derived proteins could become novel sources of antimicrobial agents. But phage therapy is relatively in its early stages and is full of complications [5, 26]. All studies linking phage management were led to a target to effective treatment of patients more than to resource indication of phage-mediated therapeutic effectiveness; thus, antimicrobial agents besides the phages are often used in their treatments. On the other hand, it is challenging to agree that phage therapy combine with antibiotics [74]. Phage cocktails can be intended to improve the range of activity extent by little active attention and improve the range of activity depth. With only chemotherapeutic combination therapies, in comparison, the main importance in the treatment particularly of special, recognized pathogens, for instance, M. tuberculosis, as a substitution is commonly on improving spectrum of activity profundity. So, to formulize phage cocktails to also fight the development of resistance, more consideration is necessary [75]. The development of nanomedicine has been considered a biological vehicle to perform new theranostics (therapeutics and diagnostics) programs. In current years, bacteriophage investigation notices this course, which has opened up novel paths in drug and gene transfer investigations. Phage endolysins as a new therapeutic scheme has received noteworthy consideration. So far, various endolysins are described, which display-worthy results in the treatment of antibiotic-resistant bacteria. Yet, endolysin also has some challenges. One limitation of endolysin is its limited in-vivo half-life because of the output of cytokines’ inflammatory reaction and the neutralizing antibodies in contrast to it. Novel approaches are required to improve widespread chimeric lysin, to dominate these immunological reactions against endolysin. Though endolysins are demonstrated to be helpful as new therapeutics, additional investigation is essential to study their construction and engineerability in clinical trials [76, 77].