resistance?
Bacteriophages are obligate intracellular parasites which infect and replicate inside bacteria. They are an abundant part of our ecosystem, as there are 1031 on the planet comprising of 13 families and 34 genera. Phages are involved in the transduction of bacteria up to “20 billion times per second”. Phages have always had the potential to fight bacterial infections. However, due to a rise in popularity of antibiotics, particularly penicillin in the 1940’s, their ability to fight bacterial infection has not been fully investigated until now, in the face of increasing antimicrobial resistance. Antimicrobials kill microorganisms or stop their growth, however due to the increasing overuse of antibiotics, the development of multi-resistant bacterial chains known as ‘superbugs’ pose a huge risk to our society. Three ways in which bacteria evade the effects of antimicrobials are by using enzymes to cause the breakdown of antimicrobials, changing the permeability of antimicrobials and changing proteins that the antimicrobial wants to target. Bacteria can produce B-lactamases, that can modify the pathway for enzymes, making it possible for them to resist or evade the effects of the antimicrobials.
Following the discovery of bacteriophages by Felix d’ Herelle in 1917, he used them to treat a 12 year old boy who was suffering from dysentery in 1919. Following just one dosage, the patient’s symptoms cleared up. In 1921, Bruynoghe and Maisin used phage therapy to treat staphylococcus furuncles and carbuncles and there was clear evidence to prove the success of the bacteriophage in reducing symptoms. Phages are already a part of the innate immune system as they compete on mucosal surfaces in animals against bacteria, reducing the ability of bacteria to multiply. Is this only the beginning of their potential in fighting antimicrobial resistance? Due to an increase in disease and health care acquired infections the number of antibiotics we use has steadily risen following WWII. Moreover, across the world more people than ever before have access to healthcare and thanks to advances in medicine we are living for longer. A rise in antimicrobial resistance is a result of the prescription of unnecessary medication, patients not finishing the prescribed course, poor personal hygiene and poor hygiene practices in hospitals along with the use of antibiotics in farming. Indeed, it is thought the use of antibiotics in farming has been a key contributor in aiding antimicrobial resistance. As humans, we eat the meat of cows and chickens who have been fed these antibiotics. These antibiotics do not kill all microorganisms and lead to the emergence of resistant pathogens, that are now inside the human following the digestion of meat. These microorganisms therefore expose the human to resistant strains which is how antimicrobial resistance has spread.
What are bacteriophages and how do they work?
Bacteriophages are bacterial viruses that cause the death of bacterium. They can be classified by their genetic material, either ssDNA, dsDNA, dsRNA or ssRNA. There are known to be six morphological groups of phages,
1) Phages with a contractile sheath and long tail
2) Phages with a rigid sheath and long tail
3) Phages with a capsid and short tail
4) Phages with fibrous structures that have a grand-sized nucleocapsid
5) Phages with a singular nucleocapsid
6) Filamentous phages
The Caudovirales order consisting of Myoviridae, Siphoviridae and Podoviridae families of tailed phages make up 96% of all studied phages.
Phages attach to bacteria that are expressing a particular receptor, they are specific to a certain pathogen. Phages form an irreversible bond with the bacterial cell receptor. Attachment of the phage onto the bacterium can occur via LPS, flagella or the bacteriophages can adhere to bacteria using enzymes, which break down substances in order to reach the bacterial cell wall. In both the lysogenic and lytic pathway viral DNA is replicated. The production of virions occurs, following the packaging of phage particles which aids the destruction of the bacterial cell and the spread of virions around the body.
Lytic Pathway
Lytic bacteriophages have the potential to fight antimicrobial resistance. The lytic pathway leads to the development of multiple virus particles and the lysis of the host cell. Firstly, the bacteria needs to attach to cell surface receptors on the outside of the bacterial cell wall. This occurs as there are complementary receptors on the surface of the host cell. Some bacteriophages have enzymes that can degrade the exopolysaccharide capsules, before they bind to their receptor. Phages insert their DNA into the host, following the degradation of peptidoglycan and creation of pores in the bacterial cell which leads to an expression of genes. These genes cause the production of viral nucleic acids and proteins. Phage particles are then packed together and host cell lysis then occurs. RNA polymerase attaches to a promoter sequence, and transcription processes create viral mRNA which produces viral proteins. This process allows genetic material to replicate and inhibits transcription in the bacterium, allowing the genetic material from the phage to take over as well as causing lysis of the cell wall.
Lysogenic Pathway
Viral DNA integrates into host DNA which replicates inside the host. Lysis of the host cell does not occur when the phage replicates. A prophage exists in the bacterium that lies dormant until ‘stress’ occurs to the host which causes the lytic pathway.
Advantages of Phage Therapy
If bacteriophages became a viable treatment for diseases, it would be much more difficult for bacteria to mutate and overcome a bacteriophage, due to the specificity of phages that target a particular bacterium. This provides such vast opportunity to prepare so many phages against all different kinds of pathogens. Only a small amount of bacteriophage is required to treat an antimicrobial infection. Indeed this small amount of phage alongside antimicrobials may be able to overcome resistance. Phages are also able to penetrate the skin. Moreover, phages act upon the bacterium whereas antimicrobials are not concentrated at the site of infection. Also, phages grow exponentially. Therefore, phages may not need to be delivered as frequently into the body as they will continue to replicate until the bacteria has been lysed. . Selecting new phages would be a simpler process than developing new antimicrobials, as they are easy to isolate and have natural presence on Earth and would continue to evolve through natural selection. Bacteriophage therapy would also solve the issue for those who are allergic to antibiotics and the number of side effects reported following phage therapy is much less than that compared with antimicrobials. Bacteriophages have no effect on eukaryotic cells, making them specific for certain bacteria without affecting microflora. Bacteriophages have the potential to cross the blood-brain barrier and therefore the potential to treat diseases such as Parkinson’s. The possible potential of phages is much greater than that of just fighting antimicrobial resistance, it could also be useful in food safety and agriculture.
Disadvantages of Phage Therapy
However, there is much still unknown about phages and their potential. For example, phage free salmonella is harmless, but bacteriophages can move DNA from one bacterium to another. This can introduce exotoxins or new virulence factors which can cause the bacteriophage to become dangerous. It is this unknown capacity of phages, as well questions as how they would be administered, their safe dosage size and the frequency of treatment which makes them off limits as a replacement for antimicrobials until further research is done. Clinical trailing of phages proves to be difficult, as the bacteria need to be identified before being able to test how successful phages are at killing a bacterium. Moreover, the preparation of phages is time consuming and expensive, as they must be isolated, not contain potentially harmful bacteria and should be stored correctly. Since only lytic phages can be used, the potential number of phages to be used in therapy is dramatically reduced. Bacteriophages must be tested on animal models, as we do not know how they would react in vivo. Furthermore, phage therapy can stimulate the immune response which could limit the effect of the antimicrobial if applied at the same time. Antibiotics are prescribed based upon symptoms the patient is feeling, but phage therapy could not be done in the same way, until there is full understanding of the biology and mechanism of the phage which is causing the actual infection. Furthermore, pharmacokinetics of the phage therapy are poorly understood and differ from that of antimicrobials. The initial dosage, phage absorption rate and timings of dosage would all need to be considered.
Moreover, bacteria can become resistant to bacteriophages, which would make them ineffective in the fight against antimicrobial resistance. Resistance can occur if the receptor on the phage mutates and no longer recognises the receptor on the bacterium. Furthermore, phages can become resistant if mutations occur in genes that are vital for phage replication or if they acquire genes that cipher resistance. However, the threat of phage resistance is not as great a risk as that of antimicrobial resistance, as combining phages and creating a “cocktail” is a solution for this. Moreover, since phages are constantly undergoing natural selection, they can counter act the resistance from any bacterial strains by evolving themselves.
Clinical Trials
Due to a lack of support from pharmaceutical companies and governments in the West, only recently did the first clinical trials take place with phages with remarkable success. William Smith carried out preclinical studies and found that bacteriophage therapy was successful in treating E.coli infections in mice.
However, in Georgia and Poland specific research into the potential of phages has already begun, with the desire to examine bacteriophages as possible treatments for bacterial infections. Indeed, Kochetkova et al. proved that by use of staphylococcus and pseudomonas agents on infected wounds on cancer patients following surgery, bacteriophage treatment was 82% successful whereas the use of antimicrobials was successful in 61% of cases. Moreover, Cislo et al. used agents of pseudomas, staphlylococcus and klebsiella to treat 31 patients with skin infections with a 74% success rate. These are just two examples showing the success of phage therapy in treating bacterial infection.
Lysin Therapy
Lysins are enzymes that cleave peptidoglycan from the gram positive bacterial cell wall, leading to lysis of the cell and the spread of infection. They are produced as one of the final stages in the lytic cycle. They are effective in treating gram positive bacterial infections due to their lack of outer membrane. Recently CF-301 lysin was effective in phase 1 clinical trials against methicillin resistant Staphylococcus aureus. Similarly to bacteriophages, lysins are specific for a particular bacterium, do not affect commensal flora and act at the bacterial site of infection. Lysins are categorized by their catalytic activity. Lysozymes and amidases are most commonly produced by phages. No bacterial resistance has occurred to lysin therapy, making it a possible form of therapy in the fight against antimicrobial resistance.
Ultimately, phages are extremely specific which makes them advantageous in killing a particular pathogen without damaging commensal flora, yet the bacterium must be identified and its mechanisms understood before it can be used in treating a bacterial infection. Moreover, the naturally occurring abundance of bacteriophages means the possibility of pathogens that can be eradicated is huge. On the other hand, the potential of a phage to become virulent following the addition of virulence factors makes them currently unstable to be used instead of antimicrobials. It is possible that bacteriophages combined with antibiotics may be useful in solving the issue of antimicrobial resistance. Further clinical trials are needed, yet it is safe to say the potential of phage therapy is vast and it may help in solving the issue of antimicrobial resistance.
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