CHAPTER ONE
1. INTRODUCTION
Although the advent of “Phage therapy” in the early 20th century by the French-Canadian Microbiologist, Felix d’ Herelle, it was met with great enthusiasm. However, it was soon forgotten with the introduction of antibiotics (Altamirano & Barr, 2019) and the little amount of information available on phages at that time. The continuous use and often misuse of antibiotics, has resulted in the formation of antibiotic-resistant bacteria carrying resistance genes (Lin, Koskella, & Lin, 2017) which encode proteins that annul the effect of these antibacterial agents. The alarming increase in antibiotic resistance has led to a renewed interest in phage therapy; especially the therapeutic use of phages to treat bacterial infections. This report, therefore, aims at discussing the uses and benefits of bacteriophages as an alternative to antibiotics, antibiotic resistance and strategies to combat it, the combination of phages and antibiotics, advantages of phages over antibiotics and the challenges and opportunities of phage therapy.
1.1 DISCOVERY OF PHAGES
Phages were discovered independently by Fredrick Twort in 1917 and Felix d’Herelle in 1919. The term “BACTERIOPHAGE” which literally means “BACTERIUM-EATER” was coined by Felix after he successfully treated patients infected with Shigella dysenteriae using what he called “ an invincible, antagonistic microbe of the dysentery bacillus” (Dublanchet & Bourne, 2007, pp. 15-18).
1.2 BIOLOGY OF PHAGES
Bacteriophages are viruses that infect and replicate only inside bacteria. They are composed of a genetic material which is either DNA or RNA and an outer protein layer called CAPSID. Most, if not all, phages are infectious only to bacteria that have complementary receptors to theirs (Lin et al., 2017). The phage binds irreversibly to these receptors using specialized structures such as tail fibres and base pins (found in tailed phages, for example, T4 phages that infect Escherichia coli) or other structures. Thereafter, the genetic material is injected into the target pathogen through an “Injection mechanism”.
Based on the life cycle of phages, the genetic material can replicate using the host cellular machinery to produce multiple progenies which lyse the cell, using phage-derived lytic proteins, and are released to repeat the cycle (lytic/virulent phages).
Alternatively, the genome can be integrated with the host chromosome forming a “prophage” that enters a latent phase within the host. Consequently, the prophage replicates together with the host’s chromosome thereby leading to the vertical transfer of the phage (genome), that is, from mother to daughter cell (lysogenic /temperate phages) (Lin et al., 2017), some of which excise from the bacterial chromosome due to environmental or physiological stressors and later undergo the lytic cycle (Kortright, Chan, Koff, & Turner, 2019). The most common lytic phages associated with human pathogens and gut microflora are in the orders Caudovirales, “tailed phages” which contain double-stranded DNA genomes, and Microvirales, which are tailless, single-stranded DNA viruses (Lin et al., 2017).
Bacteria have evolved anti-phage systems to defend themselves against viral penetration. As listed by Dy, Richter, Salmond, and Fineran (2014), the systems include adsorption, injection blocking, inhibition, toxin-antitoxin and the Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR Associated System (CRISPR/Cas) (Dy et al., 2014; Lin et al., 2017).
However, Safari et al. (2020) reported that phages have also co-evolved to resist these systems, by assessing anti-CRISPR proteins and evading Restriction modification (R/E) systems.
1.3. DESCRIPTION OF PHAGE THERAPY
The extensive work of Felix on phages as antibacterial agents led to a phenomenon called “phage therapy”. This can be defined as the use of specific viruses to treat bacterial infections with the aim of lysing the bacterial cell. Lytic phages are widely used against the lysogenic phages and this is attributed to the ability of these phages to directly kill the bacteria. A disadvantage to the use of lysogenic phages is their inherent ability to mediate gene transfer between bacteria by specialized transduction (Monteiro, Pires, Costa, & Azeredo, 2019). The hindrances faced by many scientists during the earliest trials of phage therapy such as the limited stability of the viruses in solution, a significant drop in phage titre during storage and processing (Malik et al., 2017), contamination from bacterial antigen and delivery of phages to the site of infection (Lin et al., 2017), led to the wide dismissal of this therapeutic method. Moreover, a better understanding of the mechanism of action of antibiotics in the 1940s made antibiotic therapy gain more attention and led to a significant decrease in the interest of phages by most western countries. Exceptions to these include countries in Europe, e.g. Georgia, Poland etc. where phage therapy has been continually practised for decades and used to treat infections caused by most clinical antibiotic-resistant pathogens (Abedon, Garcia, Mullany, & Aminov, 2017; Lin et al., 2017).
CHAPTER TWO
2. ANTIBIOTICS AND ANTIBIOTIC RESISTANCE
Antibiotics are a type of antimicrobial agent. They are produced by microorganisms to inhibit the growth and replication of other microbes and are most effective against bacterial infections. They are recognized as one of the most successful forms of chemotherapy developed in the 20th century (Banin, Hughes, & Kuipers, 2017) and the entire history of Medicine at large. Since the introduction of antibiotics over 70 years ago, they have been used extensively in all fields of medicine and allowed great advancements in Medicine, for example, the use of antibiotics in organ transplantation, Chemotherapy etc. (Altamirano & Barr, 2019).
2.1. DISCOVERY OF ANTIBIOTICS
The discovery of antibiotics is credited to two major scientists which are Alexander Fleming, a physician cum Microbiologist and Paul Ehrlich, a German Scientist. Ehrlich envisioned a “magic bullet” which would selectively destroy a disease-causing microbe without harming the host. In 1909, he came about a compound called ARSPHENAMINE (an organoarsenic derivative) that cured syphilis infected rabbits (Aminov, 2010). The compound marketed as “Salvarsan”, exhibited the potential of curing Syphilis infected patients.
However due to its severe side-effects, from rashes to liver damage, an alternative compound “Neosalvarsan” was developed and preferably used as it was less toxic and more soluble (Kon & Rai, 2016). These two compounds became very successful and were frequently recommended until the introduction of Penicillin in 1940. Penicillin known as the first natural antibiotic produced (Mater methods, 2018) was discovered accidentally by Alexander Fleming in 1928. During one of his studies, he noticed a zone of inhibition of growth on agar plates which he initially thought to be contaminated with the Penicillium mould (Kon & Rai, 2016). He later identified the substance like Penicillin. The isolation of penicillin from the surface of agar plates made it easier, more reliable and required fewer resources as compared to the animal model testing methods such as those used by Ehrlich (Aminov, 2010). As a result, this method became widely used to produce penicillin in large quantities and was made available for use on humans in 1940. Nevertheless, more efficient technologies have been used and are still in use to develop drugs effective against microorganisms. Following the evolutionary trend, microbes, especially bacteria, have evolved to resist the effects of these antimicrobial agents; this would become known as “antibiotic/antimicrobial resistance”.
2.2. ANTIBIOTIC RESISTANCE
Antibiotic resistance can be simply defined, as the ability of bacteria to evade antibiotics designed to kill them. Also known as antimicrobial resistance (AMR), it is “the resistance of microorganisms to antimicrobial agents which they were first sensitive to” (Kon & Rai, 2016). Generally, these bacteria have evolved to produce resistance genes as well as novel mechanisms which govern a higher Minimum Inhibitory Concentration (MIC) than the original wild strain of the organism (Acar & Rostol, 2001).
2.3. RESISTANCE: NATURAL OR MAN-MADE?
Bacterial resistance to antibiotics can either be a natural (innate) attribute or man-made (acquired) mechanism (Acar & Rostol, 2001). The latter includes anthropogenic activities which are majorly the clinical and industrial use of antibiotics (Altamirano & Barr, 2019). From the use of antibiotics in agriculture to enhance the growth of livestock, treating animals, or to increase the nutrients in the soil, to prolonged use for treating infectious diseases of humans (Ventola, 2015). In contrast, natural resistance arises due to horizontal transfer of genes; the transfer or movement of genes through the 3 stages “transformation, transduction and conduction” (Altamirano & Barr, 2019). In addition, Transposons that transmit mobile integrons allow pathogens to share resistance mechanisms. This attribute is very beneficial to organisms that are capable of resisting only one type of antibiotic. With this, they can acquire numerous antibiotics from transposons that transfer different antibiotic resistance genes (Kon & Rai, 2016). Furthermore, innate resistance is also a result of the spontaneous mutations that occur in chromosomal genes as an answer to selection pressure exerted by antibiotics, synthetics, antiseptics and other drugs (Acar & Rostol, 2001; Altamirano & Barr, 2019). According to Acar and Rostol (2001), no strict separation exists between chromosomal and transferable resistance. These attributes are used solely as an epidemiological tool.
The different events that occur in a bacterial life leading to resistance are summarized in the table below:
TABLE : EVENTS LEADING TO RESISTANCE IN BACTERIA
SOURCE: Acar, J. and Röstel, B. (2001). Why and How Resistance Develops. Antimicrobial Resistance: An Overview, 20(3), 801.
Microorganisms utilize numerous resistance mechanisms. As listed by Kon and Rai (2016), they are;
1) Porin modification.
2) Efflux pumps.
3) PBP modification.
4) Enzymatic inhibition.
2.4. EXTENT AND CONSEQUENCES
The excessive use of antibiotics has resulted in the emergence of Multidrug-resistant organisms (MDR) also known as “superbugs”. Other examples of resistant pathogens are Extensively drug-resistant (XDR) and Pan drug-resistant (PDR) pathogens (Altamirano & Barr, 2019). Many bacteria have been classified as presenting serious economic, societal and health threats by the Centre of Disease Control (CDC) (Ventola, 2015) particularly the ESKAPE pathogens; Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumonia, Actinobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp which cause serious nosocomial infections (Lin et al., 2017) and with the characteristic ability to evade the biocidal activity of antibiotics using highly developed resistance mechanisms (Altamirano & Barr, 2019).
The burden of antimicrobial resistance has led to an increased raging to return to the “pre-antibiotic era” (Lin et al., 2017) that is, the treatment methods used before the introduction of antibiotics. Public health organizations such as CDC and WHO has estimated that a total of 2 million illnesses are caused by MDR pathogens with at least 23,000 deaths per year (Lin et al., 2017). Among the deadliest MDRs is the Methicillin-Resistant Staphylococcus aureus (MRSA) which alone accounts for more deaths yearly than HIV/AIDS, Parkinson’s disease and homicide combined (Ventola, 2015; Lin et al., 2017). According to Altamirano and Barr (2019), a review carried out on AMR predicts that by 2050 an estimated cost of 10 trillion USD would have been spent on treatment with over 10 million deaths occurring. However, these statistics do not fully encompass the extent of AMR as the economic burden is hard to quantify. In addition, several consequences of AMR must be taken into account such as reduced possibilities of treating infections due to the increasing amounts of less efficacious agents (Friedman, Temkin, & Carmeli, 2015), loss of productivity, lower economic output and increased costs associated with highly expensive antibiotics (Friedman et al., 2015; Prestinaci, Pezzotti, & Pantosti, 2015). So a reversal to older treatment techniques such as disinfection, amputation, isolation etc. (Michael, Dominey-Howes, & Labbate, 2014) which are not only less effective, but also have a longer time of action, may lead to prolonged infections and finally, death.
2.5. STRATEGIES TO COMBAT ANTIBIOTIC RESISTANCE
Antibiotic resistance is caused mainly by the excessive use and/or misuse of antibiotics, in treating viral and fungal infections. If any strategy is to be utilized, “it has to be multidimensional, multidisciplinary and global” (Altamirano & Barr, 2019, p. 5). Some of them are policies promulgated by the CDC, for example, prohibiting the unnecessary use of antibiotics on agriculture and livestock (Singhai, 2016) and creating adequate awareness to the general public on the health detriments of self-medication and improper dosing. In addition, stewardship programs have been provided to educate clinicians on following evidence based guidelines in prescribing antibiotics for the right infections after proper diagnosis (Manning, Pfeiffer, & Larson, 2016). Also, antibiotic combination therapy is an emerging alternative which offers great effects on pathogens as opposed to the individual effect of each antibiotic (Maheshwari, 2004). Although these strategies do not completely eradicate the rise of antibiotic resistant pathogens, they slow it down. It is of great importance to note that old therapeutic methods, new methods, and the neglected ones such as phage therapy are also encouraged.
CHAPTER THREE
3. BACTERIOPHAGES AS THERAPEUTIC AGENTS
Phage therapy is an age long phenomenon, although introduced over a century ago, it was abandoned after the emergence of antibiotic therapy which became more successful. However, the rise in antibiotic resistance has led to a renewed interest in phages as a promising therapeutic agent. This interest is attributed to a better understanding of the mechanism of action of phages, their biology and genetics (Altamirano & Barr, 2019). An ideal anti-bacterial drug should exhibit a wide range of activity, lack a specific target that would limit its application i.e. it should be multidimensional, remain viable when administered orally, maintain activity in biofilms, accumulate in macro-forms, penetrate through bacteria cell walls and be usable on sensitive patients (Guo et al., 2020). Phages, however, are natural anti-bacterial agents. Due to their specificity, they possess a narrow range of bactericidal activity. Thus far, no adverse effects have been recorded on the oral administration of phages (Guo et al., 2020). Phages also lack the ability to accumulate in macro-forms, however, preparations of phages called “phage cocktails” are used to target multiple receptors on the host cell surface thereby expanding the bactericidal spectrum. Notwithstanding, more information about the host receptors for each phages should be established as well as the use of combination therapy to limit the advent of phage resistant hosts (Altamirano and Barr, 2019).
Lytic phages are strictly used for phage therapy. Temperate phages are not advised due to the high risk of lysogenic conversion which is one of the ways which bacteria acquire resistant traits and genes that increases their virulence (Altamirano & Barr, 2019). These phages can be administered through intra-peritoneal, intra-venous, oral routes and even sprayed on the site of infection (Sagor et al., 2005). Additionally, lytic phages have been shown to be good bio-control agents against food borne pathogens and have several advantages over sanitizers, detergents etc. (Sagor et al., 2005). They have been used successfully for both therapeutic and prophylactic purposes in surgery and treating gastro-enteric infections especially those caused by Escherichia coli, Salmonella typhi, and Pseudomonas aeruginosa (Lin et al., 2017). Also, “BIOPHAGE-PA; a preparation of phages that target multidrug resistant Pseudomonas aeruginosa has been used to treat patients with chronic otitis” (Guo et al., 2020).
Research has been, and still is, carried out to determine the effectiveness of phages many of which were done in countries that embraced phage therapy before it was dismissed. Moreover, the first test to determine the safety of administering phages was carried out in Switzerland in 2005 (Altamirano & Barr, 2019; Guo et al., 2020). This trial was to determine the effect of a T4 phage targeted against Escherichia coli for the treatment of diarrhoea. No adverse effect was recorded neither was T4 specific antibodies found in the serum of the volunteers (Guo et al., 2020), phages did not multiply in the gut of the individuals and only a small fraction of phages was recovered from the faeces of the individuals (Altamirano and Barr, 2019). With the innovations on technology, the genetics of phages and how to genetically engineer them to deliver CRISPR/Cas programmed to destroy antibiotic resistant genes and plasmids in bacteria (Yosef, Manor, Kiro, & Qimron, 2015; Lin et al., 2017) are in study. Research is being conductedon the efficacy, safety, route of administration, side effects etc. of phage therapy. There are also arising complications which limits the scope of phage therapy such as liberation of endotoxins due to the breakdown of bacterial cell wall, phage resistance, limited host range (Guo et al., 2020), susceptibility to host restriction enzymes etc. (Sagor et al., 2005).
Phages are abundant in nature and are natural bacteria pathogens., making it an effective alternative to antibiotic therapy. Moreover, the low rate of antibiotic recovery called the “dry pipeline” aggravates the problem of antibiotic resistance (Altamirano & Barr, 2019), phage therapy has proven to be the best alternative yet.
3.1. COMPARING ANTIBIOTICS TO PHAGE THERAPY
Phages and antibiotics are regarded as bactericidal agents, the former being a natural killer of bacteria and the latter synthetic or semi-synthetic compounds, however, involving lysis and inhibition respectively (Lin et al., 2017). The following points elaborate on some of the differences between both antibacterial agents.
3.1.1. SPECIFICITY:
The major difference between phages and antibiotics is the specificity of phages to the host which is due to the specific receptors found on their envelopes or capsids. Unlike antibiotics with a much wider spectrum, they are not known to cause side effects and or secondary infections (Romero-calle, Benevides, Goes-Neto, & Billington, 2019) such as mucosa candidiasis, antibiotic-associated diarrhoea, pseudomembranous colitis caused by Clostridium difficile (Altamirano & Barr, 2019; Lin et al., 2017). Although the specificity to strain and species is an added advantage, it comes with its drawbacks. For instance, the fact that phage therapy aims directly at a particular pathogen limits its effect on infections (Altamirano & Barr, 2019) caused by more than one pathogen (Lin et al., 2017). Notwithstanding, this can be sorted using phage cocktails and accurate diagnosis of the causative agent. Latz et al. (2016) reported that phages that target antibiotic-resistant bacteria are more likely to be found in areas with high numbers of individuals infected with resistant bacteria. This provides an easy way to access phages infective to a particular resistant pathogen (Lin et al., 2017).
3.1.2. SAFETY:
Phage therapy has recently garnered attention by western medicine and while much of the information on its safety is new and underdeveloped (Lin et al., 2017), oral administration of phages has been proven to be safe. Antibiotic therapy, on the other hand, is very well documented to cause anaphylaxis and other hypersensitivity reactions, nephro-toxicity, toxic necrolysis and more. especially in immunocompromised patients. This is accounted for by the synthetic nature of antibiotics, particularly the beta lactams which are notorious triggers of anaphylactic reactions (Altamirano & Barr, 2019). While the pros of phage therapy significantly outweigh the cons, there are hypothetical cases whereby the administration of phages worsens the patient’s condition especially if they are immunodeficient (Lin et al., 2017). Scientists still argue the possibility of this as no evidence has been reported to support this claim.
3.1.3. BIOFILM PENETRATION:
Biofilms are made up of a matrix of polysaccharide secreted by bacteria to serve as a barrier against external forces such as resisting the penetration of antibiotics. Normally, large doses of antibiotics are required to penetrate the biofilm. This results in regrowth of the pathogen after antibiotic therapy is stopped and also poses serious health risks (Lin et al., 2017). In contrast, phages are equipped with enzymes on their capsid (Extracellular Polymeric Substance) that degrades extracellular polymers (biofilms) (Lin et al., 2017) thereby granting them the access to the target bacteria. Upon the release of progeny virions, the biofilm is gradually broken down as they infect and lyse each bacteria cell (Kon & Rai, 2016). Parasion, Kwiatek, Gryko, Mizak, and Malm (2013) reported that the in-vitro application of phages on the colonies of Pseudomonas aeruginosa not only degraded existing biofilm but also prevented additional biofilm formation. This suggests that phage therapy can also be used to reduce infections caused by biofilm-coated pathogens on the surface of medical devices (Lin et al., 2017).
3.1.4. EFFECT OF BACTERIAL RESISTANCE:
It is widely acknowledged that pathogens are evolving to resist most of the antibiotics available. Although phages are not an exception to this, they are not as worrisome as with antibiotics (Kon & Rai, 2016). The best- known anti-phage mechanisms include but is not limited to prevention of viral DNA entry, eluding viral receptors, CRISPR/Cas systems that recognize and degrade previously encountered DNA (Altamirano & Barr, 2019). Besides, most of the bacteria resistant mechanisms can be avoided using phage cocktails or a combination of phage and antibiotic therapy called “combination therapy” (Golkar, 2014).
3.1.5. RELATIONSHIP WITH THE IMMUNE SYSTEM
Phages have the distinct ability to spread systematically and self-replicate at the site of infection, however, they do not completely eradicate the infection. This is because the complete removal of the pathogens results in the termination of the viral replicative cycle (Altamirano & Barr, 2019). This means that the immune system is needed to remove lingering bacterial populations in order for phage therapy to be effective—a collaboration termed “Immuno-phage synergy” (Altamirano & Barr, 2019). This is a major disadvantage of phage therapy as it presents serious health threats to immunocompromised patients. In contrast, antibiotics work independent of the patient’s immune system and are therefore preferred in some cases. Further research on the immune-phage phenomenon is still required especially with clinical trials as regards human health, as much of the current research is limited to animal models.
CHAPTER FOUR
4. APPROACHES TO PHAGE THERAPY DELIVERY
Generally, there are two known types of phage therapy based on the spectrum of activity and host range the phages possess, they are mono-phage and poly-phage therapy. The former involves the use of phages with very narrow host range, that is, specific to single or a few bacteria strain(s) within a species (Chan & Abedon, 2012). It, however, has its own limitations; the evolutionary changes that occur between phages, although an advantage over antibiotics, does not decrease the possibilities of bacteria resisting phage attacks (Altamirano & Barr, 2019).
Moreover, due to the narrow spectrum, there is a constant need to match the phages with the pathogens to determine their suitability for clinical trials (Altamirano & Barr, 2019; Chan & Abedon, 2012). To address these limitations, poly-phage therapy such as “phage cocktails” are used—this involves the combination of different phage types, usually mono-phages, to improve the phage spectrum of activity.
4.1. PHAGE COCKTAILS
Phage cocktails are preparation of phages directed towards different strains of bacteria and ultimately different species. Some drawbacks of it are the potential of the mixed phages to co-infect a bacterium (Chan & Abedon, 2012) and a longer purification process (Altamirano & Barr, 2019). However, phage cocktails have been used to successfully treat diarrhoeic and pyogenic infections (Sarker et al., 2012). Phage therapy can be delivered through different routes, the common ones being oral and parental routes. Others are topical, inhalation and otic routes.
4.2. ORAL PHAGE DELIVERY
This is the most studied route of phage therapy delivery, proven to be safe and successfully used to treat gastrointestinal tract infections (Ryan, Gorman, Donnelly, & Gilmore, 2011) mainly caused by MDR pathogens (Zelasko, Gorski, & Dabrowska, 2017). The acidity of the stomach greatly limit phages by deactivating them, however, it can be prevented by encapsulating them in a liposome or mannitol-alginate macrospheres to enhance entry and shield them from neutralizing antibodies (Singla, Harjai, Katare, & Chhibber, 2016).
4.3. TOPICAL PHAGE DELIVERY
In recent years, attention has been directed towards the use of phages in wound infections particularly caused by MDR bacteria (Chang, Morales, Okamoto, & Chan, 2020). In a study conducted by Ryan et al. (2011) to test the efficacy of phage therapy on wound infected by Salmonella spp and Campylobacter jejuni, phage cocktails were cultured, Salmonella enterica serovar enteritis phage type 4 strain P125589 was inoculated on the wound and spread around the wound surface using a glass spreader. The results showed that the efficacy of the therapy depended on the MOI ratio of phage particles to bacteria cells, the higher the number of phage particles the lower the bacteria concentration (Ryan et al., 2011). Furthermore, the phages can be administered in: liquid forms such as phosphate-buffered solutions (PBS) (dripped or sprayed on the infection site) and semisolid forms such as hydrogels (agarose), organogels, creams etc. (Chang et al., 2020) which are more easily applied and never runoff.
4.4. INHALATION OF BACTERIOPHAGES
Respiratory tract infections e.g. tuberculosis and chronic pneumonia are among the leading causes of death and public health decline (Bodier-Montagutelli et al., 2017). The recent advances in technology provides an opportunity for further research into the use of phage inhalation in treating lung infections. One method is aerosolisation using the nebulizer (Malik et al., 2017) whereby the liquid cocktails are inhaled directly into the lungs; this provides a fast action rate and maintains the phage titre (Bodier-Montagutelli et al., 2017). In a study to determine the efficacy of phage therapy administered by inhalation targeting Pseudomonas aeruginosa, ten weeks old mice infected with bioluminescent P. aeroginosa suspended in 50ml PBS were used (Ryan et al., 2011). This was followed by the phage administration, using a nebulizer, after two hours. Results reported showed that 90% of the mice survived with a very remarkable drop in bacteria count (Ryan et al., 2011). However, these studies are not sufficient to correctly dictate the efficiency of phage therapy. Further studies are required in both infected and uninfected models to aid clinical trials in humans (Bodier-Montagutelli et al., 2017) and to determine the best route and regimen for phage therapy.
CHAPTER FIVE
5. PHAGE AND ANTIBIOTICS COMBINED
With an increase in antimicrobial resistance and MDR bacteria, different alternatives are sought-after to fight against these pathogens, one of which is phage therapy. The efficacy of phage therapy is still questioned by scientists as bacteriophages are also prone to resistance by bacteria. However, to avoid replacing antibiotics, phages and antibiotics can be combined to provide an effect greater than the sum of their independent effects (Tagliaferri, Jansen, & Horz, 2019). Furthermore, combining different therapeutic methods has proven to be a great strategy against infections caused by MDR and opportunistic pathogens (Altamirano & Barr, 2019). Multidrug (combinational) therapy have been successfully used in treating HIV (Highly Active Anti-Retroviral Therapy), malaria and tuberculosis treatments (Altamirano & Barr, 2019). In light of this, Comeau, Tétart, Trojet, Prère, and Krisch (2007) reported that in the presence of sub-lethal concentrations of antibiotics phage production is increased leading to a remarkable drop in bacterial cells; a phenomenon called “Phage Antibiotic Synergy”.
5.1. PHAGE ANTIBIOTIC SYNERGY
Phage antibiotic synergy (PAS) is a phenomenon whereby “sub-lethal doses of certain antibiotics substantially stimulate the host cell’s production of lytic phages” (Altamirano & Barr, 2019; Kim et al., 2018). The PAS is one of the mechanisms which enables phages to adapt to and benefit from their environment: the presence of small concentrations of antibiotics in the environment poses a threat to bacterial cells, subsequently, phages take advantage of this “stressed state” of the cells to propagate efficiently (Comeau et al., 2007). This process would help to reduce antibiotic use and resistance levels. To show PAS, Oechslin et al. (2016) combined 10^8 PFU/mL of phage cocktail with 2.5 MIC of 0.9μg/mL of Ciproflaxin. The antibiotic was highly synergistic with the phages as indicated by ≥ log 3 CFU/ml decrease in bacteria count (Pseudomonas aeruginosa) after 24 hours. The synergistic activity is however dependent on the antibiotic used (Segall, Roach, & Strathdee, 2019). The PAS effects, higher burst size and plaque assays, observed when a combination of siphovirus and ciproflaxin was used against Escherichia coli isolated from urine samples, were not detected with other antibiotics such as tetracyclin (Tagliaferri et al., 2019). Furthermore, Ryan, Alkawareek, Donnelly, and Gilmore (2012) reported complementary results using phage T4 and a low concentration of cefotaxime against Escherichia coli strain 11303. Besides the expected results, the bacteria biofilms were significantly reduced compared to using either treatments (Tagliaferri et al., 2019; Ryan et al., 2012).
The effects of PAS has further been studied using different combinations of antibiotics and phages to fight against highly resistant, notably the ESKAPE, pathogens. More studies are still required as this is a relatively new phenomenon first discovered in 2007 and a majority of the findings are speculative. Nevertheless, the encouraging results obtained so far proves that Phage/antibiotic combination would likely provide the remedy for infections caused by MDR pathogens.
CHAPTER SIX
6.OPPORTUNITIES AND CHALLENGES OF PHAGE THERAPY
Phage therapy has a wide range of advantages sometimes regarded as phage properties. However, it has minor disadvantages that can readily be resolved. Although few, the cons should not be overlooked as phages are susceptible to being resisted by bacteria. Some of the opportunities for phage therapy includes:
- Phage therapy can potentially limit the dependence on antibiotics which would reduce resistance,
- Only a single dose of phage therapy may be required to produce an effect as they are live agents and replicate within the bacterial cell, achieving “active therapy” and auto-dosing (Loc-Carrillo & Abedon, 2011),
- The low cost of isolation; phages are abundant in nature and easily isolated (Loc-Carrillo & Abedon, 2011),
- Phages are versatile; they can be applied in liquid forms, creams gels, etc. and also combined with antibiotics (Chang et al., 2020).
According to Oliveira, Sillankorva, Merabishvili, Kluskens, and Azeredo (2015), the potential challenges of phage therapy and its development are divided into two factors namely
A) Intrinsic factors include:
- Specificity/narrow host range of the phages (Loc-Carrillo & Abedon, 2011; Wu et al., 2013).
- Phage type: the phages used are “obligately lytic” because temperate phages can convert phage-sensitive bacteria into insensitive ones thereby conferring superinfection immunity on the bacteria (Loc-Carrillo & Abedon, 2011; Wu et al., 2013) and also possibly transfer toxin-encoding genes among bacteria (transduction) (Sulakvelidze, 2011).
- The inherent ability to induce resistance.
B) Extrinsic factors include:
- Incomplete removal of contaminations due to insufficient purification methods of phage preparations (Pires, Costa, Pinto, Meneses, & Azeredo, 2020; Sulakvelidze, 2011),
- Development of phage neutralizing antibodies, although the kinetics of phages are faster than the production of antibodies (Oliveira et al., 2015),
- Treatment of intracellular pathogens; these pathogens multiply within cells and are shielded from external forces including phages (Oliveira et al., 2015).
Other major limitations include: The accessibility of phages (Altamirano & Barr, 2019), Lack of awareness, and regulatory constrains: although phage therapy is accepted as an alternative treatment, it is not approved for use in Europe and USA mainly because of the uncertainty surrounding its production and use (HealthLine, 2019).
Figure 3: Factors that limit phage therapy.
SOURCE: Oliveira, H., Sillankorva S., Merabishvili, M., Kluskens,L.D. and Azeredo J. (2015).
Unexploited Opportunities for Phage Therapy. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561355/
CHAPTER SEVEN
7. CONCLUSION
In an era of antibiotic resistance, phage therapy has proven to be an efficient and effective alternative to antibiotics. Bacteriophages are natural predators of bacteria and therefore possess properties better suited as an antimicrobial agent compared to the chemical counterpart. Phage therapy can be administered to the site of infection either directly (locally) or through the bloodstream. Furthermore, phages can be combined with antibiotics to produce a better therapeutic effect than when used independently. Although the transfer of phage therapy from the laboratory to the bedside may seem challenging due to limited experiments and studies, if explored effectively it would, no doubt, put an end to the dependence on antibiotics as well as cure infections caused by MDR pathogens.
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