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Phage therapy for Clostridium difficile infection: An alternative to antibiotics?
Author's Accepted Manuscript
Phage therapy for C. difficile infection: An alternative to antibiotics? William Sangster MD, John P. Hegarty PhD, David B. Stewart MD
www.elsevier.com/locate/yscrs
PII: DOI: Reference:
S1043-1489(14)00037-2 http://dx.doi.org/10.1053/j.scrs.2014.05.014 YSCRS472
To appear in:
Seminars in Colon and Rectal Surgery
Cite this article as: William Sangster MD, John P. Hegarty PhD, David B. Stewart MD, Phage therapy for C. difficile infection: An alternative to antibiotics?, Seminars in Colon and Rectal Surgery, http://dx.doi.org/10.1053/j. scrs.2014.05.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Phage therapy for C. difficile infection: an alternative to antibiotics? William Sangster MD1, John P Hegarty PhD1, David B Stewart MD1
1
Division of Colorectal Surgery, Penn State Milton S. Hershey Medical Center, Hershey, PA
Address Correspondence: David B. Stewart MD Penn State Milton S. Hershey Medical Center 500 University Drive P.O. Box 850, MC 137 Hershey, PA 17033 PH: (717) 531-5164 FX: (717) 531-0646 [email protected] Abstract As a consequence of their widespread use, a critical limitation in current antibiotic therapy is bacterial resistance. In the case of Clostridium difficile infection (CDI), current antibiotic regimens may not necessarily suffer due to drug resistance, though they have become increasingly ineffective due to the dysbiosis they induce, resulting in notoriously high recurrence rates. As a result, interest in alternative treatment modalities has recently surfaced. Amongst these emerging treatments, newer investigations are being invested in the older concept of bacteriophage therapy. This approach, first identified in the early 19th
century, offers a more microbe-specific treatment option that can, theoretically, pointedly target Clostridium difficile while sparing the other bacterial organisms of the human gut. The aim of this article is to explain the intellection behind bacteriophage therapy for the treatment of bacterial infections in humans, to review the historical research on bacteriophage therapy, and to introduce the reader to recent investigations into bacteriophage therapy for the treatment of CDI.
Keywords: Clostridium difficile, Bacteriophage, Phage therapy History A bacteriophage, or phage for short, is a virus that infects and replicates within bacteria. Historically, the existence of bacteriophages was first documented in 1915 by Frederick Twort. [1] In his work with Staphylococcus, Twort noted areas of “glassy transformation” within bacterial lawns, denoting regions where bacteria were not proliferating. As he continued to observe this process, he found that not only did these interruptions in the bacterial lawn represent areas of bacterial destruction, but Twort also discovered that he could transpose these areas on other Staphylococcus colonies and observe a similar phenomenon. In 1917, a microbiologist by the name of Félix d’Herelle noted zones of lysis within bacterial lawns that he referred to as “plaques”. [2] He theorized that within these plaques was an invisible agent that was causing bacteriolysis. He would go on to label these invisible agents “bacteriophages”. d’Herelle’s continued work throughout the early 20th century established much in the way of bedrock information for later
bacteriophage research and provided a conceptual framework for future investigations on the use of bacteriophages for therapeutic purposes. After their discovery, bacteriophages were the subject of multiple studies aimed at treating common bacterial infections. In fact, much of d’Herelle’s early work was related to the use of bacteriophages to treat Shigella dysentery in rabbits and humans. [2] During the 1920s and 1930s, bacteriophage therapy began to become more widespread, and US pharmaceutical companies such as Eli Lilly began producing bacteriophage preparations for the treatment of Staphylococcal infections. [3] For reasons that will be discussed infra, these early commercial products were not effective, and with the eventual discovery of antibiotics such as penicillin, interest in bacteriophage therapy within the Western world rapidly waned. With advances in gene sequencing and with the development of electron microscopy during the 20th century, bacteriophages that were once thought to be invisible have been both photographed and classified. In 1971, the International Committee on Taxonomy of Viruses classified bacteriophages into orders and families according to morphology and nucleic acid composition. [4] As of 2012, 7 orders and 96 families of bacteriophages were recognized. [5] Caudovirales is the order of bacteriophages commonly found within Clostridial species. [6] This order is defined by bacteriophages that have a head containing double stranded DNA and a tail that the bacteriophage uses to adsorb (attach) to bacteria. Within Clostridium difficile, two families of the Caudovirales order that have been frequently isolated are Myoviridae and Siphoviridae. [7,8] Studies analyzing the utility of these bacteriophages as treatment options for CDI are ongoing and will be further discussed in this article.
Selection Bacteriophages are widely distributed in areas populated by bacterial hosts and are believed to outnumber bacteria by approximately ten-fold. [9] Though outnumbered, bacteria have continued to thrive through the evolution of bacteriophage defense mechanisms that include inhibiting bacteriophage attachment to cell surface receptors as well as through the induction of cell suicide to abort bacteriophage reproduction. [10,11] However, a large proportion of bacteria still succumb to bacteriophage infection, as bacteriophages have also adapted equally effective strategies to insert their genome into bacterial cells and to reproduce rapidly. [11] This dynamic interaction between bacteriophages and bacteria represents a co-evolutionary relationship that has garnered much interest in an effort to limit the development of bacteriophage-resistant bacteria. Following adsorption on bacterial surface receptors, bacteriophages will insert their genetic material into the bacterial cytoplasm. [12] Depending on the bacteriophage, as well as environmental factors, the genetic material can follow one of two reproductive pathways. One pathway involves the viral genome replicating rapidly within the bacterial cytoplasm, assembling progeny virions, and then exiting the host bacteria, usually with resultant bacteriolysis. [13] This pathway is known as the lytic cycle and has been observed in bacteriophages that infect Escherichia coli, Salmonella, and Bacillus. [14-16] While some bacteriophages are solely or preferentially lytic, others are termed temperate bacteriophages since, depending on ambient conditions, they may favor a different pathway known as lysogeny. In this scenario, once the bacteriophage’s genetic material
enters the bacteria, it is usually able to incorporate itself directly within the bacterial genome and to enter a state of apparent quiescence, at which point the virus is referred to as a prophage. The prophage genetic material is then replicated concomitantly along with the bacterial genome in a direct, linear and symbiotic fashion that can last indefinitely. If during this time the bacterial cell is exposed to any sort of physiologic stress, such as UV radiation or antibiotics, a recombinant event known as excision may occur whereby the prophage will remove itself from the bacterial genome. It is at this point that the prophage enters the lytic reproductive pathway, commandeering the bacteria’s cellular machinery to replicate progeny virions and to exit the bacterial host as previously described. [13] The environmental and ambient factors that influence a temperate bacteriophage to follow a lytic or lysogenic pathway are complex and can affect the utility of using temperate bacteriophages to treat bacterial infections. While infected with a temperate bacteriophage, bacteria are placed under the additional metabolic burden of replicating both their genome as well as that of the prophage. [17] Despite a potential decrease in fitness of the lysogenous bacterial cell, co-existence with temperate bacteriophages can provide a selective advantage to both the bacterium and bacteriophage. For example, certain strains of E. Coli undergoing lysogenic conversion can result in prophage encoded genes being introduced into the bacterial genome that convert a non-virulent strain into a virulent one which now has a selective advantage over non-lysogenic bacteria of the same genus and species. [18] Another classic example of lysogeny providing a selective advantage to the bacterial host is observed in the production of highly virulent toxins by Shigella and Staphylococcal bacteria after introduction of bacteriophage genetic elements. [19, 20] An additional advantage
provided to the bacterial host is that prophage genes protect the bacterial cell from infection by other bacteriophages of the same species, since there is a prophage already occupying the insertion site of a future viral genome of the same type. [17] These increases in fitness are frequently short-lived from a bacterial standpoint, though, as ultimately the bacteriophage will destroy its host; however, the fact that bacteriophages can serve as gene transfer agents has potential implications both in terms of bacteriophage-directed therapies, as well as in terms of safety concerns surrounding fecal microbiota transplant (FMT), since both introduce bacteriophages into the recipient hindgut.
Clinical experiences With the widespread use of antibiotics, drug resistance is not just a theoretical concern or an occasional phenomenon, but is rather an emerging dilemma that can result in the loss of life due to bacteria that have no known treatment. [21-23] Beyond the phenomenon of drug resistance; however, is the fact that antibiotics are not specific to one type of bacteria, and their use results in a dysbiosis within the gut that has a causal role in relapsing and recurrent infections. [24] Antibiotics, therefore, collapse under their own weight, producing unintended disturbances to the bacterial component of the microbiome of the human gut, resulting in a lesser likelihood of eradicating infections such as CDI. As a result, interest in alternative therapies has grown and recent research in the area of bacteriophage therapy has been revitalized, since it offers the prospect of a microbiome sparing, species-specific treatment for diseases such as CDI.
Much of the ineffectiveness of initial trials of bacteriophage treatments during the mid19th century was due to an incomplete understanding of the nature of viruses in general. For instance, in d’Herelle’s work, he believed that one bacteriophage could be active against a wide number of bacterial genera. [2] It is now appreciated that bacteriophages have a narrower host range than antibiotics and can only infect a specific scope of bacteria, which varies in latitude based on the bacteriophage in question. [25] The specificity of this interaction limits the application of certain bacteriophages in therapy; however, the advantage is that this same characteristic also ensures that the administered bacteriophage will eradicate the targeted bacterial strain while preserving commensal bacterial flora. Another limitation of early bacteriophage preparations was related to what was, in earlier times, a sparse understanding of bacteriophage pharmacokinetics when administered to a eukaryotic subject. It has been subsequently discovered that bacteriophages are recognized by the immune system of eukaryotes as a foreign entity, and that without alterations to their native viral structure, they are usually rapidly eliminated from the systemic circulation by the reticuloendothelial system. [26] In order to address this issue, one study, performed in mice, serially passed E. Coli bacteriophages through the rodent’s circulatory system, demonstrating that variant bacteriophages arose that were capable of evading the reticuloendothelial system while retaining their bacterial infectivity, offering hope that systemic administration of bacteriophages is feasible. [27] The relationship between bacteriophages and the reticuloendothelial system is complex and a better understanding of bacteriophage delivery methods will be important in creating viable phage therapies.
Current research into the use of bacteriophage therapy for the control of CDI is extremely limited. As mentioned earlier in the article, a number of bacteriophages that infect Clostridium difficile have been identified, and in a few cases, the genomic clusters which constitute the prophage form of the virus have been partially sequenced. [28] These bacteriophages have been found to represent either the Myoviridae or Siphoviridae families. Thus far, a principle limitation in their in vivo testing has been that these bacteriophages are temperate in nature; no purely lytic bacteriophage specific for Clostridium difficile has been isolated to date. It has been suggested that this phenomenon may be related to frequently harbored prophage genes within Clostridium difficile that impart resistance to infections from lytic bacteriophages, or to the fact that Clostridium difficile frequently exists as spores in the environment which would favor bacteriophages that integrate into the bacterial genome. [29] Even though temperate in nature, studies using bacteriophages to treat CDI have shown promising, albeit seminal, proof-of-principle as an alternative treatment modality and give plausibility to bacteriophage therapy for CDI. Recently, in vitro studies using Myovirus resulted in a significant reduction of vegetative Clostridium difficile colonies with no detrimental impact on other non-pathogenic bacteria. [29, 30] Also noted in this study [30], was a significant reduction of Clostridium difficile toxin production after exposure to bacteriophages, suggesting that bacteriophage therapy could be beneficial either by eradicating toxigenic CDI, or by at least mitigating the production of toxins which instigate this form of infectious colitis. Due to the limitations in using temperate bacteriophages as a therapeutic option for CDI, researchers have continued to identify and isolate the major components that enable a
bacteriophage to lyse bacteria. For instance, to degrade the peptidoglycan bacterial cell wall, bacteriophages utilize a peptidoglycan hydrolase commonly referred to as an endolysin. [31] One group of researchers [32] have been able to utilize a bacteriophage endolysin to specifically inhibit and even lyse strains of Clostridium difficile, offering a novel approach to the treatment of CDI that is phage- related.
Phage tail- like particles An interesting permutation on the subject of CDI phage therapy is the use of phage taillike particles (PTLP’s). It has been described in multiple research laboratories, including those of the authors of this article, that upon induction of bacteriophages from C. difficile, a viral-like structure is sometimes produced which has a contractile tail, a base plate, and tail fibers, similar to a phage but without a capsid, and thus without viral genetic material. Therefore, the origin of these particles is one that likely became divergent from bacteriophages in the past, with a distinct phylogenetic lineage to that of viruses. The production of such particles has been well studied in Pseudomonas aeruginosa where they are referred to as pyocins and are produced as a means of killing neighboring bacteria, thus providing a selective advantage. [33] The lack of genetic material prevents these particles from inadvertently serving as gene transfer agents, which if their refinement were to demonstrate effective killing of C. difficile, would provide a safety advantage compared to either traditional bacteriophage therapy or FMT. More recently, a phage tail-like particle was identified within Clostridium difficile, its genomic cluster was partially sequenced, and in vitro studies demonstrated bactericidal
activity against certain strains of C. difficile, including ribotype 027. [34] The identified PTLP’s within Clostridium difficile are considered to be R-type, since they very closely resemble Myoviridae in that they have a contractile body and non-flexible tail fibers. This is in contrast to a F-type PTLP that can be found in Pseudomonas which morphologically resemble Siphoviridae bacteriophages as they have a non-contractile body and flexible tail fibers [35]; F-type PTLP’s have not been identified in C. difficile to date. Similar to the referenced study above, our group has also induced and exposed R-type PTLP’s to lawns of Clostridium difficile and we have observed that PLTP’s will result in bacteriolysis within the bacterial lawn. [unpublished data] Within our PTLP genomic cluster sequencing data, we have also identified a cell wall hydrolase gene, translocase genes, and permease genes that may encode functions within PTLP’s that enable them to be released from bacteria. [unpublished data] By utilizing site-directed mutagenesis, the mechanism that each of these features provides to lysing C. difficile can be further assessed. With further development, we anticipate PTLP’s will represent another alternative therapy for CDI.
Future Even though bacteriophage therapy has been studied since the early 19th century, its use in the treatment of CDI is still in its infancy. While numerous studies continue to identify and sequence bacteriophages within Clostridium difficile, studies focused on the use of bacteriophages as a treatment for CDI are limited. The temperate nature of Clostridium difficile bacteriophages poses a potential challenge for their use as therapy for CDI, and
further work is needed before bacteriophages become a viable option to treat CDI. Phagerelated therapies, such as endolysins and PTLP’s, offer interesting alternatives but also require further development, existing in only a nascent form at present. Our group, and several others, plan on continuing to work with PTLP’s to better define their structure and functions using cyro-electron microscopy and eventual in vivo testing.
References 1. Twort FW: An investigation on the nature of the ultra-microscopic viruses. Lancet II:1241-1243, 1915 2. d’Herelle F: Sur un microbe invisible antagoniste des bacilles dysenteriques. C.R. Acad. Sci 165:373-375, 1917 3. Summers WC: Phage and the early development of molecular biology, in Abedon ST, Calendar RL (eds): The Bacteriophages. Oxford University Press, 2005, pp 37 4. Ackermann HW: Classification of bacteriophages, in Abedon ST, Calendar RL (eds): The Bacteriophages. Oxford University Press, 2005, pp 8-16 5. Leuven: Virus Taxonomy: 2012 Release. International Committee on Taxonomy of Viruses, July 2012. Web Nov 2013 6. Ackermann HW: Tailed bacteriophages the order caudovirales. Adv in Virus Res 51:135-201, 1998 7. Fortier LC, Moineau S: Morphological and genetic diversity of temperate phages in Clostridium difficile. Appl Environ Microbiol 73:7358-7366, 2007
8. Nale JY, Shan J, Hickenbotham PT, et al: Diverse temperate bacteriophage carriage in Clostridium difficile 027 strains. PLoS One 7:e37263, 2012 9. Brüssow H, Hendrix RW: Phage genomics: small is beautiful. Cell 108:13–16, 2002 10. Fortier LC, Sekulovic O: Importance of prophages to evolution and virulence of bacterial pathogens. Virulence 4(5): 354-365, 2013 11. Samson JE, Magadán AH, Sabri M, et al: Revenge of the phages: defeating bacterial defences. Nat Rev Microbiol 11(10):675-87, 2013 12. Yacoby I, Shamis M, Bar H, et al: Targeting antibacterial agents by using drugcarrying filamentous bacteriophages. Antimicrob Agents Chemother 50:20872097, 2006 13. Echols, H: Developmental pathways for the temperate phage: lysis vs lysogeny. Annual Review of Genetics 6:157-190, 1972 14. Ferguson S, Roberts C, Handy E, et al: Lytic bacteriophages reduce Escherichia coli O157: H7 on fresh cut lettuce introduced through cross-contamination. Bacteriophage 3(1):e24323, 2013 15. Woolston J, Parks AR, Abuladze T, et al: Bacteriophages lytic for Salmonella rapidly reduce Salmonella contamination on glass and stainless steel surfaces. Bacteriophage 3(3):e25697, 2013 16. El-Arabi TF, Griffiths MW, She YM, et al: Genome sequence and analysis of a broad-host range lytic bacteriophage that infects the Bacillus cereus group. Virol J 10:10-48, 2013
17. Brüssow H: Prophage genomics, in Abedon ST, Calendar RL (eds): The Bacteriophages. Oxford University Press, 2005, pp 17-23 18. Ohnishi M, Kurokawa K, Hayashi T: Diversification of Escherichia coli genomes: are bacteriophages the major contributors? Trends Microbiol 9:481-5, 2001 19. Herold S, Karch H, Schmidt H: Shiga toxin-encoding bacteriophages: Genomes in motion. Int J Med Microbiol 294:115-121, 2004 20. Yoshizawa Y, Sakurada J, Sakurai S, et al: An exfoliative toxin A-converting phage isolated from Staphylococcus aureus strain ZM. Microbiol Immunol 44(3):189-91, 2000 21. Spellberg B, Powers JH, Brass EP, et al: Trends in antimicrobial drug development: Implications for the future. Clin Infect Dis 38:1279-1286, 2004 22. Canton R, Morosini MI: Emergence and spread of antibiotic resistance following exposure to antibiotics. FEMS Microbiol 35:977-991, 2011 23. Round JL, Mazmanian SK: The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol 9:313-323, 2009 24. Skraban J, Dzeroski S, Zenko B, et al: Gut microbiota patterns associated with colonization of different Clostridium difficile ribotypes. PLoS One 8(2):e58005, 2013 25. Merril CR, Scholl D, Adhya, S: Phage therapy, in Abedon ST, Calendar RL (eds): The Bacteriophages. Oxford University Press, 2005, pp 725-741 26. Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska B, et al: Learning from bacteriophages - advantages and limitations of phage and phage-encoded protein applications. Curr Protein Pept Sci 13(8):699-722, 2012
27. Merril CR, Biswas B, Carlton Ret al: Long-circulating bacteriophage as antibacterial agents. Proc Natl Acad Sci USA 93:3188-3192, 1996 28. Shan J, Patel KV, Hickenbotham PT, et al: Prophage carriage and diversity within clinically relevant strains of Clostridium difficile. Appl Environ Microbiol 78(17):6027-34, 2012 29. Meader E, Mayer MJ, Gasson MJ, et al: Bacteriophage treatment significantly reduces viable Clostridium difficile and prevents toxin production in an in vitro model system. Anaerobe 16:549-554, 2010 30. Meader E, Mayer MJ, Steverding D, et al: Evaluation of bacteriophage therapy to control Clostridium difficile and toxin production in an in vitro human colon model system. Anaerobe 22:25-30, 2013 31. Schmelcher M, Donovan DM, Loessner MJ: Bacteriophage endolysins as novel antimicrobials. Future Microbiol 7(10): 1-25, 2012 32. Mayer MJ, Narbad A, Gasson MJ: Molecular characterization of a Clostridium difficile bacteriophage and its cloned biologically active endolysin. J Bacteriol 190(2):6734-6740, 2008 33. Michel-Briand Y, Baysse C: The pyocins of Pseudomonas aeruginosa. Biochimie 84:499-510, 2002 34. Gebhart D, Williams SR, Bishop-Lilly KA, et al: Novel high-molecular-weight, R-type bacteriocins of Clostridium difficile. J Bacteriol 194(22):6240-7, 2012 35. Nakayama K, Takashima K, Ishihara H, et al: The R-type pyocin of Pseudomonas
aeruginosa is related to P2 phage, and the F-type is related to lambda phage. Mol Microbiol 38(2):213-31, 2000
Phage therapy for C. difficile infection: An alternative to antibiotics? William Sangster MD, John P. Hegarty PhD, David B. Stewart MD
www.elsevier.com/locate/yscrs
PII: DOI: Reference:
S1043-1489(14)00037-2 http://dx.doi.org/10.1053/j.scrs.2014.05.014 YSCRS472
To appear in:
Seminars in Colon and Rectal Surgery
Cite this article as: William Sangster MD, John P. Hegarty PhD, David B. Stewart MD, Phage therapy for C. difficile infection: An alternative to antibiotics?, Seminars in Colon and Rectal Surgery, http://dx.doi.org/10.1053/j. scrs.2014.05.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Phage therapy for C. difficile infection: an alternative to antibiotics? William Sangster MD1, John P Hegarty PhD1, David B Stewart MD1
1
Division of Colorectal Surgery, Penn State Milton S. Hershey Medical Center, Hershey, PA
Address Correspondence: David B. Stewart MD Penn State Milton S. Hershey Medical Center 500 University Drive P.O. Box 850, MC 137 Hershey, PA 17033 PH: (717) 531-5164 FX: (717) 531-0646 [email protected] Abstract As a consequence of their widespread use, a critical limitation in current antibiotic therapy is bacterial resistance. In the case of Clostridium difficile infection (CDI), current antibiotic regimens may not necessarily suffer due to drug resistance, though they have become increasingly ineffective due to the dysbiosis they induce, resulting in notoriously high recurrence rates. As a result, interest in alternative treatment modalities has recently surfaced. Amongst these emerging treatments, newer investigations are being invested in the older concept of bacteriophage therapy. This approach, first identified in the early 19th
century, offers a more microbe-specific treatment option that can, theoretically, pointedly target Clostridium difficile while sparing the other bacterial organisms of the human gut. The aim of this article is to explain the intellection behind bacteriophage therapy for the treatment of bacterial infections in humans, to review the historical research on bacteriophage therapy, and to introduce the reader to recent investigations into bacteriophage therapy for the treatment of CDI.
Keywords: Clostridium difficile, Bacteriophage, Phage therapy History A bacteriophage, or phage for short, is a virus that infects and replicates within bacteria. Historically, the existence of bacteriophages was first documented in 1915 by Frederick Twort. [1] In his work with Staphylococcus, Twort noted areas of “glassy transformation” within bacterial lawns, denoting regions where bacteria were not proliferating. As he continued to observe this process, he found that not only did these interruptions in the bacterial lawn represent areas of bacterial destruction, but Twort also discovered that he could transpose these areas on other Staphylococcus colonies and observe a similar phenomenon. In 1917, a microbiologist by the name of Félix d’Herelle noted zones of lysis within bacterial lawns that he referred to as “plaques”. [2] He theorized that within these plaques was an invisible agent that was causing bacteriolysis. He would go on to label these invisible agents “bacteriophages”. d’Herelle’s continued work throughout the early 20th century established much in the way of bedrock information for later
bacteriophage research and provided a conceptual framework for future investigations on the use of bacteriophages for therapeutic purposes. After their discovery, bacteriophages were the subject of multiple studies aimed at treating common bacterial infections. In fact, much of d’Herelle’s early work was related to the use of bacteriophages to treat Shigella dysentery in rabbits and humans. [2] During the 1920s and 1930s, bacteriophage therapy began to become more widespread, and US pharmaceutical companies such as Eli Lilly began producing bacteriophage preparations for the treatment of Staphylococcal infections. [3] For reasons that will be discussed infra, these early commercial products were not effective, and with the eventual discovery of antibiotics such as penicillin, interest in bacteriophage therapy within the Western world rapidly waned. With advances in gene sequencing and with the development of electron microscopy during the 20th century, bacteriophages that were once thought to be invisible have been both photographed and classified. In 1971, the International Committee on Taxonomy of Viruses classified bacteriophages into orders and families according to morphology and nucleic acid composition. [4] As of 2012, 7 orders and 96 families of bacteriophages were recognized. [5] Caudovirales is the order of bacteriophages commonly found within Clostridial species. [6] This order is defined by bacteriophages that have a head containing double stranded DNA and a tail that the bacteriophage uses to adsorb (attach) to bacteria. Within Clostridium difficile, two families of the Caudovirales order that have been frequently isolated are Myoviridae and Siphoviridae. [7,8] Studies analyzing the utility of these bacteriophages as treatment options for CDI are ongoing and will be further discussed in this article.
Selection Bacteriophages are widely distributed in areas populated by bacterial hosts and are believed to outnumber bacteria by approximately ten-fold. [9] Though outnumbered, bacteria have continued to thrive through the evolution of bacteriophage defense mechanisms that include inhibiting bacteriophage attachment to cell surface receptors as well as through the induction of cell suicide to abort bacteriophage reproduction. [10,11] However, a large proportion of bacteria still succumb to bacteriophage infection, as bacteriophages have also adapted equally effective strategies to insert their genome into bacterial cells and to reproduce rapidly. [11] This dynamic interaction between bacteriophages and bacteria represents a co-evolutionary relationship that has garnered much interest in an effort to limit the development of bacteriophage-resistant bacteria. Following adsorption on bacterial surface receptors, bacteriophages will insert their genetic material into the bacterial cytoplasm. [12] Depending on the bacteriophage, as well as environmental factors, the genetic material can follow one of two reproductive pathways. One pathway involves the viral genome replicating rapidly within the bacterial cytoplasm, assembling progeny virions, and then exiting the host bacteria, usually with resultant bacteriolysis. [13] This pathway is known as the lytic cycle and has been observed in bacteriophages that infect Escherichia coli, Salmonella, and Bacillus. [14-16] While some bacteriophages are solely or preferentially lytic, others are termed temperate bacteriophages since, depending on ambient conditions, they may favor a different pathway known as lysogeny. In this scenario, once the bacteriophage’s genetic material
enters the bacteria, it is usually able to incorporate itself directly within the bacterial genome and to enter a state of apparent quiescence, at which point the virus is referred to as a prophage. The prophage genetic material is then replicated concomitantly along with the bacterial genome in a direct, linear and symbiotic fashion that can last indefinitely. If during this time the bacterial cell is exposed to any sort of physiologic stress, such as UV radiation or antibiotics, a recombinant event known as excision may occur whereby the prophage will remove itself from the bacterial genome. It is at this point that the prophage enters the lytic reproductive pathway, commandeering the bacteria’s cellular machinery to replicate progeny virions and to exit the bacterial host as previously described. [13] The environmental and ambient factors that influence a temperate bacteriophage to follow a lytic or lysogenic pathway are complex and can affect the utility of using temperate bacteriophages to treat bacterial infections. While infected with a temperate bacteriophage, bacteria are placed under the additional metabolic burden of replicating both their genome as well as that of the prophage. [17] Despite a potential decrease in fitness of the lysogenous bacterial cell, co-existence with temperate bacteriophages can provide a selective advantage to both the bacterium and bacteriophage. For example, certain strains of E. Coli undergoing lysogenic conversion can result in prophage encoded genes being introduced into the bacterial genome that convert a non-virulent strain into a virulent one which now has a selective advantage over non-lysogenic bacteria of the same genus and species. [18] Another classic example of lysogeny providing a selective advantage to the bacterial host is observed in the production of highly virulent toxins by Shigella and Staphylococcal bacteria after introduction of bacteriophage genetic elements. [19, 20] An additional advantage
provided to the bacterial host is that prophage genes protect the bacterial cell from infection by other bacteriophages of the same species, since there is a prophage already occupying the insertion site of a future viral genome of the same type. [17] These increases in fitness are frequently short-lived from a bacterial standpoint, though, as ultimately the bacteriophage will destroy its host; however, the fact that bacteriophages can serve as gene transfer agents has potential implications both in terms of bacteriophage-directed therapies, as well as in terms of safety concerns surrounding fecal microbiota transplant (FMT), since both introduce bacteriophages into the recipient hindgut.
Clinical experiences With the widespread use of antibiotics, drug resistance is not just a theoretical concern or an occasional phenomenon, but is rather an emerging dilemma that can result in the loss of life due to bacteria that have no known treatment. [21-23] Beyond the phenomenon of drug resistance; however, is the fact that antibiotics are not specific to one type of bacteria, and their use results in a dysbiosis within the gut that has a causal role in relapsing and recurrent infections. [24] Antibiotics, therefore, collapse under their own weight, producing unintended disturbances to the bacterial component of the microbiome of the human gut, resulting in a lesser likelihood of eradicating infections such as CDI. As a result, interest in alternative therapies has grown and recent research in the area of bacteriophage therapy has been revitalized, since it offers the prospect of a microbiome sparing, species-specific treatment for diseases such as CDI.
Much of the ineffectiveness of initial trials of bacteriophage treatments during the mid19th century was due to an incomplete understanding of the nature of viruses in general. For instance, in d’Herelle’s work, he believed that one bacteriophage could be active against a wide number of bacterial genera. [2] It is now appreciated that bacteriophages have a narrower host range than antibiotics and can only infect a specific scope of bacteria, which varies in latitude based on the bacteriophage in question. [25] The specificity of this interaction limits the application of certain bacteriophages in therapy; however, the advantage is that this same characteristic also ensures that the administered bacteriophage will eradicate the targeted bacterial strain while preserving commensal bacterial flora. Another limitation of early bacteriophage preparations was related to what was, in earlier times, a sparse understanding of bacteriophage pharmacokinetics when administered to a eukaryotic subject. It has been subsequently discovered that bacteriophages are recognized by the immune system of eukaryotes as a foreign entity, and that without alterations to their native viral structure, they are usually rapidly eliminated from the systemic circulation by the reticuloendothelial system. [26] In order to address this issue, one study, performed in mice, serially passed E. Coli bacteriophages through the rodent’s circulatory system, demonstrating that variant bacteriophages arose that were capable of evading the reticuloendothelial system while retaining their bacterial infectivity, offering hope that systemic administration of bacteriophages is feasible. [27] The relationship between bacteriophages and the reticuloendothelial system is complex and a better understanding of bacteriophage delivery methods will be important in creating viable phage therapies.
Current research into the use of bacteriophage therapy for the control of CDI is extremely limited. As mentioned earlier in the article, a number of bacteriophages that infect Clostridium difficile have been identified, and in a few cases, the genomic clusters which constitute the prophage form of the virus have been partially sequenced. [28] These bacteriophages have been found to represent either the Myoviridae or Siphoviridae families. Thus far, a principle limitation in their in vivo testing has been that these bacteriophages are temperate in nature; no purely lytic bacteriophage specific for Clostridium difficile has been isolated to date. It has been suggested that this phenomenon may be related to frequently harbored prophage genes within Clostridium difficile that impart resistance to infections from lytic bacteriophages, or to the fact that Clostridium difficile frequently exists as spores in the environment which would favor bacteriophages that integrate into the bacterial genome. [29] Even though temperate in nature, studies using bacteriophages to treat CDI have shown promising, albeit seminal, proof-of-principle as an alternative treatment modality and give plausibility to bacteriophage therapy for CDI. Recently, in vitro studies using Myovirus resulted in a significant reduction of vegetative Clostridium difficile colonies with no detrimental impact on other non-pathogenic bacteria. [29, 30] Also noted in this study [30], was a significant reduction of Clostridium difficile toxin production after exposure to bacteriophages, suggesting that bacteriophage therapy could be beneficial either by eradicating toxigenic CDI, or by at least mitigating the production of toxins which instigate this form of infectious colitis. Due to the limitations in using temperate bacteriophages as a therapeutic option for CDI, researchers have continued to identify and isolate the major components that enable a
bacteriophage to lyse bacteria. For instance, to degrade the peptidoglycan bacterial cell wall, bacteriophages utilize a peptidoglycan hydrolase commonly referred to as an endolysin. [31] One group of researchers [32] have been able to utilize a bacteriophage endolysin to specifically inhibit and even lyse strains of Clostridium difficile, offering a novel approach to the treatment of CDI that is phage- related.
Phage tail- like particles An interesting permutation on the subject of CDI phage therapy is the use of phage taillike particles (PTLP’s). It has been described in multiple research laboratories, including those of the authors of this article, that upon induction of bacteriophages from C. difficile, a viral-like structure is sometimes produced which has a contractile tail, a base plate, and tail fibers, similar to a phage but without a capsid, and thus without viral genetic material. Therefore, the origin of these particles is one that likely became divergent from bacteriophages in the past, with a distinct phylogenetic lineage to that of viruses. The production of such particles has been well studied in Pseudomonas aeruginosa where they are referred to as pyocins and are produced as a means of killing neighboring bacteria, thus providing a selective advantage. [33] The lack of genetic material prevents these particles from inadvertently serving as gene transfer agents, which if their refinement were to demonstrate effective killing of C. difficile, would provide a safety advantage compared to either traditional bacteriophage therapy or FMT. More recently, a phage tail-like particle was identified within Clostridium difficile, its genomic cluster was partially sequenced, and in vitro studies demonstrated bactericidal
activity against certain strains of C. difficile, including ribotype 027. [34] The identified PTLP’s within Clostridium difficile are considered to be R-type, since they very closely resemble Myoviridae in that they have a contractile body and non-flexible tail fibers. This is in contrast to a F-type PTLP that can be found in Pseudomonas which morphologically resemble Siphoviridae bacteriophages as they have a non-contractile body and flexible tail fibers [35]; F-type PTLP’s have not been identified in C. difficile to date. Similar to the referenced study above, our group has also induced and exposed R-type PTLP’s to lawns of Clostridium difficile and we have observed that PLTP’s will result in bacteriolysis within the bacterial lawn. [unpublished data] Within our PTLP genomic cluster sequencing data, we have also identified a cell wall hydrolase gene, translocase genes, and permease genes that may encode functions within PTLP’s that enable them to be released from bacteria. [unpublished data] By utilizing site-directed mutagenesis, the mechanism that each of these features provides to lysing C. difficile can be further assessed. With further development, we anticipate PTLP’s will represent another alternative therapy for CDI.
Future Even though bacteriophage therapy has been studied since the early 19th century, its use in the treatment of CDI is still in its infancy. While numerous studies continue to identify and sequence bacteriophages within Clostridium difficile, studies focused on the use of bacteriophages as a treatment for CDI are limited. The temperate nature of Clostridium difficile bacteriophages poses a potential challenge for their use as therapy for CDI, and
further work is needed before bacteriophages become a viable option to treat CDI. Phagerelated therapies, such as endolysins and PTLP’s, offer interesting alternatives but also require further development, existing in only a nascent form at present. Our group, and several others, plan on continuing to work with PTLP’s to better define their structure and functions using cyro-electron microscopy and eventual in vivo testing.
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