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Year : 2018  |  Volume : 2  |  Issue : 1  |  Page : 9-15

Exploring potential of phage therapy for tuberculosis using model organism

Department of Microbiology, Basic Medical Sciences, Panjab University, Chandigarh, India

Date of Web Publication5-Mar-2018

Correspondence Address:
Prof. Sanjay Chhibber
Department of Microbiology, Basic Medical Sciences, Panjab University, Chandigarh - 160 025
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_93_17

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Antibiotics, one of the miracle discoveries of the 20th century after world war revolutionized the treatment and prophylaxis of infectious diseases. Antibiotics led to an increase in the quality of health-care system but with the emergence of antibiotic-resistant bacterial strains compromised their very potential. The WHO calls bacterial resistance as one of the major threats to global health, food security, and development today. Antibiotic resistance poses a need of alternative therapy to conventional antibiotics. As proved in preantibiotic era phage therapy is effective against a number of successful pathogens and can be used as an alternative strategy to restrain stern infections such as antibiotic resistance tuberculosis (TB). In the present paper, effectiveness of phage therapy and use of model organisms for developing treatment strategy for antibiotic-resistant TB is discussed so as to explore new possibilities in battle against antibiotic resistance.

Keywords: Antibiotic resistance, biofilm, endolysins, phage-therapy, tuberculosis

How to cite this article:
Gondil VS, Chhibber S. Exploring potential of phage therapy for tuberculosis using model organism. Biomed Biotechnol Res J 2018;2:9-15

How to cite this URL:
Gondil VS, Chhibber S. Exploring potential of phage therapy for tuberculosis using model organism. Biomed Biotechnol Res J [serial online] 2018 [cited 2018 Mar 21];2:9-15. Available from: http://www.bmbtrj.org/text.asp?2018/2/1/9/226583

  Introduction Top

Mycobacterial infections are a cause of a variety of health problems such as tuberculosis (TB), leprosy, Searls ulcer, and fish tank granuloma. Although a vast range of mycobacterial infections occurs in humans and other animals, TB is one of the most serious diseases that results in chronic infections and death. If not treated properly, Mycobacterium tuberculosis (Mtb) is considered as one of the most successful pathogens causing latent infection without any clinical manifestation in more than one-third population across the globe. Annually, it is estimated that there are 2 million deaths due to TB and about 8–10 million cases reported annually. According to the WHO TB report 2016, in 2015 the incidence of new cases was 10.6 million worldwide including 1.2 million people HIV-TB positive. Mtb infection led to 1.4 million deaths in 2015 in addition to 0.4 million death following HIV-TB coinfection.[1] TB was declared an international emergency in 1993 by WHO, but unfortunately, 22 years after this declaration, the disease still remains a serious risk to global health. TB is considered among top three fatal diseases along with HIV and malaria, its incidence being higher in immune suppression cases such as HIV coinfection. Due to this, TB is found to be a major cause of mortality in AIDS.[2] Mtb was a primary killer of HIV-positive patients in 2015, 1 out of 3 HIV deaths was due to TB.[1] In addition to HIV coinfection, multidrug-resistant (MDR) TB has been reported, which is resistant to primarily two antibiotics named isoniazid (INH) and rifampicin (RMP). Increasing incidence of infection, coinfection, and antibiotic resistance poses a challenge to scientists to focus on novel antimicrobial agents such as bacteriophages,[3],[4] therapeutic enzymes,[5] phytochemicals,[6] pigments,[7] and silver nanoparticles.[8] The use of bacteriophages as therapeutic agent to treat bacterial diseases is not a new concept for nonmycobacterial bacteria, but limited research and knowledge is available on mycobacterial infections.[9] Initial attempts to treat mycobacterial infections were not successful, but later a decrease in bacterial load in liver, spleen, and lungs of guinea pigs was observed on treatment with phages.[10] Prolonged treatment with mycobacteriophages also showed decreased granuloma formation and decreased pathology in guinea pigs.[11] In earlier days, intracellular delivery of phage to macrophage, which is the site of infection, was postulated to be very difficult, However, in 2002, Broxmeyer showed intracellular delivery of bacteriophages by nonvirulent Mycobacterium smegmatis and this opened up new possibilities for further research.

  Drug Resistant Mycobacterium Tuberculosis Top

Mtb is one of the top ten causes of death globally as per WHO data.[12] According to the WHO 2016 annual report, about 50% of these cases take place in Brazil, Russia, India, China, and South Africa only (“BRICS” countries). The WHO policy aims to finish the global TB epidemic, with targets to cut TB deaths by 95% and to cut new cases by 90% between 2015 and 2035. Mycobacterial infections are ubiquitous in entire animal kingdom including the veterinary world. Very small inocula (only 10 bacilli) can initiate the disease after inhalation and phagocytosis by alveolar macrophages which provide them protection. Mtb is an intracellular pathogen having specific mechanisms by which it evades host defense system.[13] Usually, in humans, Mtb can persist longer in the body without causing any significant symptoms, known as latent stage of infection. It may also cause active infection in immunocompromised and aged persons. Immunosuppression from other infections such as HIV can escort the Mtb infection from latent to active disease with an increase of 20 fold.[2] According to WHO 2016 report 1.8 million (1.4 million HIV-negative and 0.4 million HIV-positive) people were killed by TB in 2015 Cell-mediated immunity impairment leads to high risk of activation and progression of disease in most cases. The ability of Mtb to grow slowly in latent stage and fast in active stage makes treatment multifaceted and tricky. In early days, TB was easily curable with a combination of antibiotics in the majority of patients, streptomycin and para-aminosalicylic acid were successful against Mtb.[14] However, with time, bacilli have developed resistance to these drugs.[15] After 1960s, there was the introduction of first-line drugs such as isoniazid (IZN), pyrazinamide (PZA), rifampicin (RMP), and ethambutol (EMB), approved by the US food and drug administration and CDC for treatment of mycobacterial infection. These first-line drugs were used for many years, and isoniazid and RMP are still most effective against drug-sensitive strains.[15] The drugs such as IZN, PZA, RMP, and EMB are very effective and act by various mechanisms such as inhibition of cell wall components, inhibiting and depleting the membrane energy and inhibiting the nucleic acid synthesis.[16],[17] In early days, EMB treatment was very lengthy, but the introduction of RMP revolutionized the treatment by decreasing course of treatment to 2–6 months from 18 months.[18] Although DOTS (Directly Observed Treatment Short course) therapy is used worldwide and is successful since last decade emergence of MDR-TB is a serious concern As per the WHO guidelines MDR-TB is caused by organisms that are resistant to most efficient and globally used anti-TB drugs including isoniazid and RMP, known as first-line anti-TB drugs. MDR-TB results from either infection with organisms which are previously drug-resistant or may develop resistance due to patient's ill management, especially due to poor treatment process. There are several reasons that lead to failure of first-line drugs. These include requirement long treatment of 2–6 months with combination of drugs such as PZA, ETH, RMP, and IZN for first 2 months and later INH and RMP for subsequent 4 months. This long regime leads to incompliance of treatment and appearance of MDR-TB. Second, the drug-drug interactions, neurotoxicity, poor water solubility and hepatotoxicity of first-line drugs also accompany treatment failures. Although modified RMP nanoparticles showed improved solubility with slow release rate which may overcome treatment hurdles but these studies are limited toin vitro system only.[19] Third, problems of poor management of TB such as poor quality of drugs, poor medical prescription, and inconsistent supply of drugs in developing countries may also lead to resistance in Mtb. Finally, the presence of MDR-TB and extensively drug resistance-TB (XDR-TB) worsens the situation as they make treatment longer, toxic, and more expensive, 50–200 times costlier than first-line drugs.[20] There were about 480000 new cases of MDR-TB in the year 2015, and 45% of the total incidents were only from three countries, i.e., India, China, and Russia Federation.[1] Another term popularly known as XDR-TB is a type of TB caused by organisms that are resistant to isoniazid and RMP, i.e. MDR-TB in addition to any fluoroquinolone and other second-line anti-TB injectable drugs such as gatifloxacin, ciprofloxacin, moxifloxacin, ofloxacin, amikacin, kanamycin, or capreomycin [Table 1]. The mentioned drugs are used as alternative to first-line anti-TB drugs in MDR TB when generally EMB, RMP, and IZN are incompetent.[21] XDR-TB can reduce the treatment success rate to half.[22] The MDR-TB and XDR-TB do not respond to conventional standard 2–6-month treatment with first-line drugs. It may take years to cure the disease with drugs which are rather more toxic and too expensive to be afforded by people, making it one of the deadly diseases in the third world countries. Conventional laboratory diagnostic procedures are also a hurdle in staring treatment of TB immediately as these techniques do not provide rapid results to clinicians,[23] Hence, there is a pressing need for development of new diagnostic tools which may be helpful in rapid diagnosis of TB infection.[24]
Table 1: The difference between multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis resistance

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  Phage Therapy Top

Bacteriophages, as name implies are eaters of bacteria, they are parasites on bacteria. Phages are natural antibacterial agents which are ubiquitous, obligate parasites which are very specific to their host. The discovery of phages is still controversial but Felix D' herelle discovered phages officially in 1917 and named them due to their ability of parasitizing bacteria.[25] Over the years, the properties of phages have been explored and based on these observations; a generalized opinion is that phages are harmless to humans unlike their bacterial host. D'herelle was again first to use shiga phage to treat dysentery on 12-year-old boy and after that phages were used to treat staphylococcal skin infections.[26] The time period between 1920s and 1950s is considered as golden time of phage therapy, but with the introduction of antibiotics in 1940s, phage therapy was absolutely replaced by antibiotics. Bacteriophages are very diverse in their origin, genome organization, evolution, and abundance as there are more than 1031 phages present on the earth.[27],[28] Viral ecologists estimated that globally there are about 1023 phage infections per se cond which shows that phage population is extremely large and vibrant.[29] Phages classification is based on their structures, tail type and nucleic acid types according to the International Committee on Taxonomy of Viruses (ICTV). At present, ICTV has characterized over 5000 bacteriophages, attributed to 13 families and 39 genera. Phages can also be classified on the basis of phage genome type which is similar to classification proposed by ICTV [30] and further subclassified by means of genome structure, size and their host range.[31] European Bioinformatics Institute (EMBL-EBI) European nucleotide archive contain sequence, length and known proteins of about 2010 phages in their database as updated on May 5, 2015. Phages show great genetic variability in terms of their host range as well as size and have varying genome organization, for example, Leuconostoc phage L5 has 2435 b in its genome whereas Pseudomonas phage 201phi2-1 contains 316,674 b in its genome. As GenBank repository more than 330 phages have been sequenced for Mycobacterium spp., 43 for Streptococcus spp., 100 for Staphylococcus spp., 61 for  Salmonella More Details spp., 15 for Klebsiella spp., 35 for Vibrio spp., and some of these phages have also been studied for their protein sequence. Phage are being sequenced to characterize and explore their potential application in various fields, as “The Actinobacteriophage database” contains sequenced genome data from 10487 phages, that includes 1410 Mycobacterium phages [Table 2], 219 Gordonia phages, 151 Arthrobacter phages full genome sequence (http://phagesdb.org/). Besides their similar host range, these phages can show a great variability in their genome organization as Salmonella phage SPN3US contains 240,413b in its genome whereas Salmonella phage FSL SP-004 contains 29,742b which is one-tenth of its comparative phage genome. In broader classification, bacteriophages are of two types: lysogenic (temperate) in which bacteriophage integrates their genome into host DNA and lytic (virulent) in which bacteriophage replicates rapidly into cell and subsequently burst the host cell to continue the infection to bacterial cells. Lytic bacteriophages reproduce in a logarithmic manner in host bacterial cell, and released by lysis of the infected bacterium [Figure 1], which includes the holin-endolysin release system.[32],[33] Holins generate an abrasion in the bacterial membrane through which endolysins finds the way to peptidoglycan layer.[33] Endolysins are cell wall hydrolases that degrade the bacterial peptidoglycan, lead to cell lysis and discharge of progeny phages.[34] It was considered that phage lambda lysis is only holin-endolysin-dependent process but another parallel pathway regulated by spanins and Ms6 LysB, an accessory lytic proteins were introduced later on.[35],[36] Phage therapy is still found to be successful against a number of pathogens such as Pseudomonas, Staphylococcus, Klebsiella, and  Escherichia More Details coli, and staphylococcal lung infection.[37],[38],[39],[40] In recent years, phage therapy has shown a significant promise in the treatment of infections caused by pathogens which are resistant to multiple antibiotics. Chhibber [41] showed that phages can be used for treating Klebsiella pneumoniae respiratory tract infection, and a single i.p. dose was enough to rescue all of the i.n. challenged animals. Similarly, phage treatment is also reported to be effective in cerebrospinal meningitis in infants.[42] Phage therapy has shown promise against many infections caused by E. coli including skin infections,[43] recurrent subphrenic and subhepatic abscesses,[44] cystic fibrosis by Pseudomonas aeruginosa,[45] staphylococcal eye infections,[46] Gram-negative mediated neonatal sepsis,[47] inflammatory urinary tract infections [48] and Buruli Ulcer caused by Mycobacterium ulcerans.[49] It was also shown that phage treatment during lethal infection leads to increase in phage titer with time whereas in case of antibiotics the concentration declines.[50],[51] In earlier days, it was assumed that phages can act only against extracellularly multiplying bacterium but a recent study showed that phages are competent of intracellular killing of engulfed methicillin-resistant Staphylococcus aureus. This was demonstrated using host bacteria as a vehicle to deliver phages inside phagocytic cells.[52],[53] A mathematical modeling in population dynamics technique showed that a single dose of phage was more effective rather than multiple doses of antibiotics.[54]
Table 2: Available data of mycobacteriophages on the actinobacteriophage database (http://phagesdb.org/)

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Figure 1: Steps involved in phage mediated bacterial lysis (a) attachment of phage to host bacterium (b) multiplication of phages in host bacterium (c) lysis of host bacterium (d) image showing clear plaques after phage infection

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  Phage Therapy and Mycobacterial Biofilm Top

Most of the drug-resistant bacterial infections such as K. pneumoniae, S. aureus, P. aeruginosa, E. coli are associated with the formation of three-dimensional well-defined adherent bacterial structure called biofilm which makes the treatment difficult with antibiotics. Biofilm are basically complex adherent microbial communities comprising of exopolysaccharide matrix. Biofilm also possess water channels in glycocalyx enclosed structures showing mushroom like structure.[55] Human pathogens such as P. aeruginosa, S. aureus, Staphylococcus epidermis, Candida albicans, Haemophilus influenza, and E. coli form biofilm on biological materials such as cell, tissue, or on medical devices including catheters in chronic infections. Low penetration of antibiotics in biofilm [56],[57] or production of antibiotic inactivating enzymes such as β-lactamase and their accumulation in matrix [58] and in some organisms presence of multidrug efflux pump, can result in increased development of antibiotic resistance in a biofilm.[59] Biofilm lowers the antibiotic susceptibility of an organism, as 1000-fold higher antibiotic dose is required than that required for planktonic cell.[60] Although in TB, there is no clear evidence of biofilm formation, especially in MDR-TB infection, expression of pilin protein in Mtb which is actively engaged in binding to extracellular matrix has been reported.[61] This gives a clue that pathogen surface might be involved in surface attachment.[62] Besides this M. smegmatis glycopeptidolipids (GPL) have been found to be important molecules that help in binding to polyvinyl chloride to form biofilm, the GPL molecules make the mycobacterial surface hydrophobic which helps in attachment to poly vinyl chloride or sliding mobility on hydrophilic surface.[63] In addition, other non-TB bacteria such as Mycobacterium avium and Mycobacterium ulcerans have also been reported to form intracellular biofilm.[64]In vitro biofilm of Mtb and M. smegmatis show a higher degree of drug resistance than growing bacilli or bacilli in planktonic form.[65],[66] In context to drug resistance, the complex architecture of biofilm needs to be explored especially in its contribution inin vivo system and its importance as a target for clearance of mycobacterial infection.[66] The long-time perseverance of infection and antibiotic resistance in mycobacterial infections shows similarity with biofilm-forming other drug-resistant organisms. Non-TB bacteria isolated from potable water, Mycobacterium abscessus, Mycobacterium chelonae, M. smegmatis, and M. avium also showed biofilm forming ability.[67]M. marinum showed biofilm formation on hydrophobic structures such as silicon and on liquid air interfaces.[68] Subsequent studies have also revealed that Mtb is a biofilm forming organism such as other Mycobacteria. Mtb biofilm showed genetic variation from planktonic cell and exhibited higher degree of drug tolerance.[66] Biofilm forming ability of this organism and increased antibiotic resistance has prompted scientists to look for therapies other than antibiotics. Phage therapy can be considered for this purpose as earlier studies has shown its potential against antibiotic-resistant Gram-negative biofilm-mediated infections in humans and experimental models. Bacteriophages that infect Mycobacteria are generally known as mycobacteriophages.[69] Kiefer and Dahl [70] showed that mycobacteriophages have the ability to disrupt M. smegmatis biofilms alone or in combination with mechanical forces such as water flow and sonication. However, over the years, it has been thought that phages are active against only extracellular bacteria. Mtb is an intracellular pathogen which grows inside macrophages and thus makes phages inaccessible to pathogen. Although intracellular targeting of phage by nonvirulent, M. smegmatis showed a model for intracellular targeting of Mycobacterium by phages [71] yet phage therapy remained almost unexplored in mycobacterial infections due to its long generation time and high risk associated with its handling. Model organisms can therefore, be used to explore new possibilities of phage therapy in treating Mycobacterial infections.

  Mycobacterium Marinum as a Model Organism to Work on Tuberculosis Top

Working with Mtb is considered to be quite difficult due to its high virulence, ability to form aerosols and requirement of a bio safety level 3 (BSL −3) laboratory. In addition to these, long generation time (20 h) is another hurdle in its study. The use of model organisms that are closely related to Mtb are supposed to be used to uncover new alternatives For example, Salmonella enterica subsp. Typhimurium is used as a model for studies on Salmonella typhi, due to the ease to work with this organism under laboratory conditions.[72]M. marinum, has been proposed as an alternative organism for Mtb-related studies as it overcomes the high virulence and slow growth of Mtb. It is photochromogenic bacterium that grows relatively faster than Mtb, i.e. its generation time is 4 h which is relatively 5 times lesser than Mtb. Till now, there are no reports that M. marinum forms aerosols unlike Mtb. It has a BSL −2 requirement and hence can be easily manipulated on bench top. Since it has a lower optimum temperature requirement of 30°C–35°C for its growth hence, the chance of infection on cooler extremities is rare, and as a result the infection rarely progresses into deeper body organs. All these observations suggest that it is convenient to work with this organism as a model for Mtb. M. marinum shows granulomaous infection which is very similar to Mtb dermal infection and hence, shows a similar type of pathogenesis.[73] Besides this M. marinum forms caseating or noncaseating granulomas depending on the status of the infected host. M. marinum is shown to exhibit orthologous DOS R regulon-encoded antigens to induce T cell immunity which also provides cross-reactivity to Mtb.[74] ESAT 6, a mycobacterial virulence factor is actively involved in cell to cell spread, cell lysis, and phagosomal escape. The expression of this factor augments the infection process.[75],[76] The late granuloma formation in both Mtb and M. marinum is related with decline in expression and secretion of ESAT-6.[77] DOS R and ESAT 6 show a highly conserved antigenic and virulence resemblance between M. marinum and Mtb. Genome studies also show that integrating vectors that use L5 site for attachment are similar in both the organisms. The plasmid and cosmid shuttle which were developed and used for Mtb were also successful in M. marinum.[78] Singlet oxygen sensitive genes, found to be moderately independent of yellow pigment production are common in both M. marinum and Mtb.[79] Differential tracking data suggests that M. marinum shows pathogenesis by endocytic pathway of macrophages and is capable of persistence and replication with in macrophage cell line in vitro, showing similarity with Mtb infection.[80] The phylogenic tree data based on 16S rRNA partial sequence showed that M. marinum and M. ulcerans have very high homology with Mtb. The other methods based on DNA-DNA hybridization, gas chromatography of fatty acids and alcohols have also supported this observation.[81] Genome comparison of Mtb (4.4 mb), M. avium (4.8 mb), and M. marinum (6.6 mb) showed that difference in genome size may be due to gene rearrangements and gene deletions. The other data from CDS also showed that M. marinum and Mtb are diverged from common ancestor.[82] All these observations suggest that the two are closely related organisms and data obtained with M. marinum can be extrapolated for the understanding of different mechanisms operative in virulent Mtb.

  Summary Top

There is a great need to explore and focus on novel treatments like phage therapy due to the fast advent of MDR TB. Phage therapy can be considered as an adjunct to antibiotic treatment alone or in combination for drug-resistant TB. Phage therapy includes the use of reported or newly isolated mycobacteriophages to treat TB. Intracellular nature of mycobacterial infection complicates the phage delivery which can be conquered either using nonpathogenic mycobacterium infected with mycobacteriophage or employing delivery system such as liposome-mediated phage delivery. Mtb is one of the toughest organisms to handle because of aerosol production, long generation time, and requirement of BSL 3 facilities and this makes the handling of the organism difficult. However, M. marinum can be used as surrogate model due to its high genetic and pathogenic similarity with Mtb. M. marinum shows opportunistic infection in humans with very low prevalence and it is easy to manipulate this organism than Mtb. This organism therefore can be used to reveal new pathways in TB treatment studies.

Financial support and sponsorship

Authors are highly obliged to University Grants Commission, New Delhi, India for providing financial assistance to Mr. Vijay Singh Gondil.

Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2]


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