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Finding A Novel Cure: Developing Phage Therapy For Urinary Tract Infections
Date : 16/05/2021
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Uploaded by : Harroop
Uploaded on : 16/05/2021
Subject : Medicine
AbstractBacteriophages (phages) are viruses which have developed mechanisms to parasitically infect bacteria using bacterial organelles to internally reproduce, and resultantly lysing[1] the host cell. Therapeutically, it is this biological property that has expressed medical potential as a viable alternative therapy against antibiotic-resistant bacteria, most particularly, Escherichia coli (E. coli). Escherichia coli is responsible for a plethora of diseases, predominantly intestinal[2] and extraintestinal (Urinary Tract Infections) consequently, it is the growing antibiotic-resistance in addition to the prevalence of E. coli-related diseases which makes it an ideal target for phage therapy [6]. Urinary tract infections are ubiquitous in healthcare-associated environments, contributing to over 100,000 annual hospitalizations in the US [4]. Conventionally, phage therapy had been popularised by the Soviet Union, adopting clinical applications throughout Russia and Georgia. Contentiously, phage therapy was not widely principal in the treatment of diseases within the West although because of the burgeoning concern of antibiotic-resistance, research into the therapeutic applications of phage therapy has grown. Currently, there have been reported successes of therapeutic phage in in vitro, and in vivo applications, although no phage products have been approved by the governing bodies of the European un ion or the United States. Therefore, additional research into in vivo models is mandatory to ensure the success of medicinal phage therapy. To achieve this, a repeatable efficacious phage cocktail needs to be developed during in vitro studies of E. coli. This report thus discusses the experimental protocols, primarily the use of Killing Assays in aims to identify a suitable phage cocktail (sourced from Uganda at 109 PFU/ml[3]) which can sufficiently suppress the growth of naturally antibiotic-resistant E. coli PA5. Conclusively, the growth of PA5 was noticeably suppressed, until developing phage-resistance. IntroductionEscherichia coli is a gram-negative, rod-like bacteria (Bacilli), existing as seven distinct types enteropathogenic, enteroinvasive, enteroaggregative, enterotoxigenic, diffusely adherent, adherent invasive, and enterohaemorrhagic[1]. The latter being formidably associated with E. coli 0157:H7, a strain of bacteria notoriously responsible for diarrhoea/haemorrhagic colitis, and a contributor towards haemolytic uremic syndrome (HUS) HUS is accentuated by the consumption of antibiotics [10]. Consequently, adding emphasis towards the cardinal importance of phage therapy as a feasible alternative to antibiotics. However, the effect of phage therapy will be investigated on E. coli PA5, a strain of E. coli which expresses similar effects. PA5 can cause urinary-tract infections (UTIs), UTIs usually occur as a result of the bacteria travelling along an inserted catheter and causing a bladder/kidney infection. A UTI is an infection present in any part of the lower urinary-system, including the kidneys, ureters, bladder and urethra. Perhaps, the seriousness of a UTI is demonstrated as over 50% of women will experience at least 1 UTI during their life time [13], consequently it is pertinent to find an antibiotic alternative to reduce the number or suppress the effects. [4] [6]An important characteristic of E. coli bacteria is the ability to adhere to mucous membranes in the urinary tract. Mucous membranes located in the lower urinary-tract contain a variety of molecules, most predominantly, mannose, a sugar. E. coli adhere to mannose molecules using their fimbriae, this binding mechanism consequently prevents the bacteria from being cleared from the urinary tract by the flow of urine. Once the bacterial cells are bond to the mucous membranes, they can thus infect. Natural antibiotic resistanceAntibiotic resistance is the ability of a microorganic to tolerantly be unaffected to antibiotics. The development of antibiotic resistance occurs as a result of an evolutionary mechanism, through the process of natural selection. Variation within species of E. coli is expressed due to arbitrary mutations that occur during DNA replication, mutations can therefore introduce characteristics that pose certain antibiotic resistance. Consequently, post-antibiotic exposure, non-resistant strains of E. coli will resultantly be terminated by a specific antibiotic, whereas resistant strains withstand the environmental stresses being exerted. The combination of reduced intra-specific competition therefore leads to the exponential growth of resistant strains of E. coli, such as PA5. Furthermore, antibiotics can express adverse effects on the microbiota, disrupting health human bacterial growth and opening an opportunity to E. coli developing. [7]Alternatively, bacteria do not rely on internal mechanisms or mutation rate to acquire antibiotic resistance. Genes encoding antibiotic resistance can be transferred between bacterium. Through horizontal transmission [2], a resistant bacterium can transfer the genes to a susceptible one. Inter-specific horizontal gene transmission can result in multiple resistant genes being transferred to a bacterium although these genes only possess an evolutionary advantage if a selection pressure is applied. Antibiotic exposure will ensure that susceptible bacteria are destroyed, contrastingly bacteria with the acquired resistant will be able to survive and multiple to become prevalent in the environment. The absence of antibiotic exposure can contribute to bacteria being at an evolutionary disadvantage, harbouring additional DNA requires a larger energy commitment to maintain the genetic information encoding for an antibiotic resistance however, this is only beneficial is that genetic material is essential for survival. [7] [12]Plasmids are small, circular and occasionally linear pieces of DNA, containing a variety of genes that encode for a plethora of different proteins and enzymes responsible for giving the bacterial cells different properties. Plasmids can therefore also have genes that express antibiotic resistance, in addition to conferring resistance to toxic heavy metals, including Mercury and silver. Playing an important part in the development of antibiotic resistance, plasmids can be copied, shared and swapped among bacteria of the same or different genera, through a process known as conjugation. Bacteria conjugation is a non-contact process, two bacterial cells are connected by a hollow tubular structure known as a pilus that draws the bacteria closer and allowing a pore to form in the envelops of the cells. The formation of pores enables the plasmid to be passed from a donor to the recipient. [2][7] E. coli studies by Stanley Cohen in 1972 [11] had resulted into alternative method of acquiring antibiotic resistance, bacteria transformation. Bacteria transformation involves bacterial cells taking up DNA fragments within the surrounding environment. The acquisition of DNA fragments can potentially contain genes that encode for proteins and enzymes responsible for antibiotic resistance. DNA fragments are suspended in the environment as a consequence of bacterial death, the cellular structure decomposes, and cellular components are deposited. The stability of DNA ensures that the molecule can comfortably remain in the environment without significant decay, awaiting to be taken up by a recipient bacterium. Multiple plasmids can be accumulating inside a bacterium, thereby leading to a larger variety of antibiotic resistance genes, in addition to amassing virulence genes. However, work by Hedges and Jacob s had discovered the concept of bacterial transposons, pieces of DNA with the ability to integrate and exchange between bacterial genomes independently from conjugation and transformation. [7]
A third mechanism for the exchange of genetic material including genes for antibiotic resistance is known as transduction [7]. Natural antibiotic resistance can be accentuated by bacteriophage. Lysogenic phage infects bacteria, inserting their genome into the host bacterial cell the phage replicates correspondingly to the replication of the bacterium. During an induction event which threatens the viability of the phage, the phage enters the lytic cycle attempting to use the internal bacterial organelles such as the 70s ribosomes to sequence and translate viral proteins (including capsomeres) and RNA/DNA which are then assembled into phage. However, occasionally during the induction event, phages can remove bacterial DNA located within a proximity to the phage DNA attachment site. Bacterial DNA encoding for antibiotic resistance can therefore be extracted into the phage DNA, henceforth during further infection cycles the phage would insert the DNA into the new hosts genome transferring antibiotic resistance. [14]. Fig.1: Diagrammatic illustration of bacterial gene transfer.
Detailed Overview: Mechanisms of Phage Therapy
Bacteriophages consists of an icosahedral head comprising of capsomere viral proteins, the head encapsulates the DNA- approximately 150 genes in addition to a tail section involving a helical sheath and tail fibres (Fig.2). Infection begins with the phage particle recognising the host bacterium s receptor sites, adhering to the surface by its tail. Consequently, the DNA is injected into the bacterium where it proceeds to prolifically replicate, entering the lytic cycle. Under the influence of viral DNA, new viral tails and heads are synthesised and assembled within the bacterial cell, parasitically utilising the cells organelles and energy resources. With the manufacturing of new bacteriophage, the bacterium undergoes cell lysising- bacteriophages burst through the cell membrane and protoplasm into the environment as virions effectively destroying the bacteria (Fig.3). From previous analysis, the isolated PA5 phage used in this study undergoes the lytic cycle during infection. [1] [6] [7] [16]
Fig.2: Diagrammatic illustration of bacteriophage. However, there is an alternative viral route, the lysogenic pathway. Integrating into the hosts genome, the bacteriophage (prophage) enters in a quiescent state for an indefinite period. Instead of influencing the hosts protein synthesis and replication, the prophage exerts no control. Regarding the prophage latency there can be considered two distinct categorisations, episomal and pro-viral. Episomal latency incorporates inactive viral nucleic acids, to which are freely suspended in the cytoplasm. The latter categorises the integrating property of phage DNA into the bacterium genome, as previously mentioned [8]. Induction events affecting the viability of the prophage can stimulate reactivation and synthesis of viral proteins, the prophage enters the lytic cycle. Therapeutically, lysogenic phages present a medical disadvantage, and under international regulations cannot be used. Despite this, E. coli PA5 can be infected by lytic phages. [7] [15] [16]Fig.3: Illustration of lytic and lysogenic cycles.
Bacterial presence usually corresponds to the habitation of phage, sewage therefore ideally provides a plethora of different phage for experimental use the phages involved in this study were sourced from Ugandan sewage. Phages were made clonal through subjection to 5 rounds of purification, using plaque assays. Identified below are 9 phages with capability to infect E. coli PA5 (Fig.4).Fig.4: Transmission electron microscope images of phages examined in this study
Conclusion: Analysis
Conclusively, the concocted phage cocktails therapeutically expressed medical potential as a viable alternative to antibiotics. Treating E. coli with phage evidently reduced the bacterial population for 4 hours. The optical density (OD600) of the solutions were measured at 0 CFU/mL during this period, therefore, phage therapy can effectively suppress PA5 growth. Though, this is therapeutically not acceptable, since a regular dose must be administered, 4 hours is insufficient time. Treating E. coli with the 4-phage cocktail, 10B, was relatively more effective at suppressing bacterial growth in addition to measuring the lowest final OD600 0.294. However, fundamentally being the most important conclusion, E. coli exposed to phage had developed mechanisms of resistance. Since recovered strains were unaffected by wildtype and supernatant phage, for a viable novel cure, it is pertinent to address this issue.
Evaluation:
With respect to phage-resistance, there are 3 alternative approaches which can further progress the research, as well as circumventing this issue. Firstly, introducing a secondary dose of phage, before E. coli PA5 develops resistance can postpone the exponential growth enabling suppression to occur for a longer period, to allow the human immune system to progressively resolve the infection itself. Secondly, isolating new phages may prove effective, as formulating newer cocktails is the only method of overcoming the evolved PA5. In combination with the first recommendation, the introduction of a subordinate dose of novel phage could completely thaw resistance, since previous opportunistic exposure is non-existing. Thirdly, a popular endorsement, is exploring combined phage-antibiotic therapy. Despite attempts to evade the issue of antibiotic-resistance, incorporating phages to initially suppress PA5, proceeding to use antibiotics to completely disrupt further growth could effectively treat urinary-tract infections. This alternative approach is a method of reducing antibiotic usage, by substituting phages, in addition to there being less potential for PA5 to develop mechanisms of antibiotic-resistance, by already being affected by phage. These are all approaches which can progressively advance this research. In addition to this, repeats need to be conducted to firm the effectiveness of each phage, by narrowing the effect of chance and anomalies.
Ultimately, in vivo studies (emulating biological environments), need to be considered before the application of medicinal phage [9]. In vitro environments operate under sterile conditions, however human environments have a plethora of factors which impact the efficacy of phage, caused either by anatomical or physiological interactions. The research conducted during this experiment is the first step of a larger multi-process investigation.
Bibliography: (References)
1: Abedon, T.S., et al. (2011). Phage Treatment for Human Infections. Bacteriophage, Vol.1, pp.66-85. Available [Online]: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278644/pdf/bact0102_0066.pdf [Accessed 10 Jul. 2018]
2: Arber, W. (2014). Horizontal Gene Transfer among Bacteria and Its Role in Biological Evolution. Life (Basel), Vol. 4, pp.217-224. Available [Online]: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187160/ [Accessed 18th Jul. 2018]
3: Baer, A., Kehn-Hall, K. (2014). Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems. Journal of Visualized Experiments. Available [Online]: http://doi.org/10.3791/52065 [Accessed 24 Jul 2018]
4: Barber, A.E., et al. (2013). Urinary Tract Infections: Current Emerging Management Strategies. Clinical Infectious diseases, Vol. 57, pp.719-724. Available [Online]: https://academic.oup.com/cid/article/57/5/719/310850 [Accessed 17th Jul. 2018]
5: Bdbiosciences.com. (2018). Plaque Assay. Available [Online]: http://www.bdbiosciences.com/sg/resources/baculovirus/protocols/plaque_assay.jsp [Accessed 24 Jul. 2018].
6: Bolocan, A.S., et al. (2016). Phage therapy targeting Escherichia coli- a story with no end? Microbiology letters, Vol. 363, Issue. 22. Available [Online]: https://doi.org/10.1093/femsle/fnw256 [Accessed 16th Jul. 2018]
7: Bowater, L., (2017). The Microbes Fight Back: Antibiotic Resistance. Royal Society of Chemistry.
8: Brussow, H., Canchaya, C. and Hardt, W. (2004). Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion. Microbiology and Molecular Biology Reviews, Vol.68, pp.560-602. Available [Online]: https://mmbr.asm.org/content/mmbr/68/3/560.full.pdf. [Accessed 16th Jul. 2018].
9: Bruttin, A. and Brussow, H. (2003). Human Volunteers Receiving Escherichia coli Phage T4 Orally: A Safety Test of Phage Therapy. American Society of Microbiology, Antimicrobial Agents and Chemotherapy, Vol. 49, pp. 2874-2878. Available [Online]: https://aac.asm.org/content/aac/49/7/2874.full.pdf [Accessed 17th Jul. 2018].
10: Capparelli, R., et al. (2006). Selection of an Escherichia coli O157:H7 bacteriophage for persistence in the circulatory system of mice infected experimentally. Clinical Microbiology and infection, Vol. 12, pp.248-253. Available [Online]: https://ac.els-cdn.com/S1198743X14639185/1-s2.0-S1198743X14639185-main.pdf?_tid=2a1cd82c-3d80-4c2b-9b43- ceababc0fce9acdnat=1534088573_348ff8ebcdbdcef4f7729c851fdab044 [Accessed 17th Jul. 2018].
11: Cohen, S.N., et al. (1973). Construction of Biologically Functional Bacterial Plasmids In Vitro. Proceedings of National Academy of Sciences of the United States of America, Vol. 70, pp. 3240-3244. Available [Online]: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC427208/pdf/pnas00138-0206.pdf [Accessed 18th Jul. 2018]
12: Davies, J., and Davies, D. (2010). Origins and Evolution of Antibiotic Resistance. Microbiology and Molecular Biology Reviews, Vol. 74, pp 417-433. Available [Online]: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937522/pdf/0016-10.pdf [Accessed 16th Jul. 2018]
13: Dielubanza E.J., and, Schaeffer A.J. (2011). Urinary Tract Infections in Women. Medical Clinics of North America, Vol. 95, pp.27-41. Available [Online]: https://www.scholars.northwestern.edu/en/publications/urinary-tract-infections-in-women-4 [Accessed 17th Jul. 2018]
14: Lederberg, J. (1956). Genetic Transduction. American Scientist, Vol. 44, pp.264-279. Available [Online]: https://profiles.nlm.nih.gov/ps/access/BBABGL.pdf [Accessed 18th Jul. 2018]
15: Lees, E., (2015). Edexcel A Level Biology Student book 1. Hodder Education: London, pp-89-90.
16: Roberts, M.B.V. (1986). Biology, a functional approach. 4th ed. Middlesex: Nelson, pp.499-501.
17: Soothhil, J.S., (1992). Treatment of experimental infections of mice with bacteriophages. Journal of Medical Microbiology, Vol. 37, pp.258-261. Available [Online]: http://www.microbiologyresearch.org/docserver/fulltext/jmm/37/4/medmicro-37-4-258.pdf?expires=1533990124id=idaccname=guestchecksum=1F853051DA0109E199A499EA00BC73E7 [Accessed 16th Jul. 2018]
18: Wong, C.S., et al. (2000). The Risk of Haemolytic-uremic Syndrome after Antiobitic Treatment of Escherichia Coli 0157:H7 Infections. New England Journal of Medicine, Vol. 342, pp. 1930-1936. Available [Online]: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3659814/pdf/nihms460007.pdf [Accessed 17th Jul. 2018]
A third mechanism for the exchange of genetic material including genes for antibiotic resistance is known as transduction [7]. Natural antibiotic resistance can be accentuated by bacteriophage. Lysogenic phage infects bacteria, inserting their genome into the host bacterial cell the phage replicates correspondingly to the replication of the bacterium. During an induction event which threatens the viability of the phage, the phage enters the lytic cycle attempting to use the internal bacterial organelles such as the 70s ribosomes to sequence and translate viral proteins (including capsomeres) and RNA/DNA which are then assembled into phage. However, occasionally during the induction event, phages can remove bacterial DNA located within a proximity to the phage DNA attachment site. Bacterial DNA encoding for antibiotic resistance can therefore be extracted into the phage DNA, henceforth during further infection cycles the phage would insert the DNA into the new hosts genome transferring antibiotic resistance. [14]. Fig.1: Diagrammatic illustration of bacterial gene transfer.
Detailed Overview: Mechanisms of Phage Therapy
Bacteriophages consists of an icosahedral head comprising of capsomere viral proteins, the head encapsulates the DNA- approximately 150 genes in addition to a tail section involving a helical sheath and tail fibres (Fig.2). Infection begins with the phage particle recognising the host bacterium s receptor sites, adhering to the surface by its tail. Consequently, the DNA is injected into the bacterium where it proceeds to prolifically replicate, entering the lytic cycle. Under the influence of viral DNA, new viral tails and heads are synthesised and assembled within the bacterial cell, parasitically utilising the cells organelles and energy resources. With the manufacturing of new bacteriophage, the bacterium undergoes cell lysising- bacteriophages burst through the cell membrane and protoplasm into the environment as virions effectively destroying the bacteria (Fig.3). From previous analysis, the isolated PA5 phage used in this study undergoes the lytic cycle during infection. [1] [6] [7] [16]
Fig.2: Diagrammatic illustration of bacteriophage. However, there is an alternative viral route, the lysogenic pathway. Integrating into the hosts genome, the bacteriophage (prophage) enters in a quiescent state for an indefinite period. Instead of influencing the hosts protein synthesis and replication, the prophage exerts no control. Regarding the prophage latency there can be considered two distinct categorisations, episomal and pro-viral. Episomal latency incorporates inactive viral nucleic acids, to which are freely suspended in the cytoplasm. The latter categorises the integrating property of phage DNA into the bacterium genome, as previously mentioned [8]. Induction events affecting the viability of the prophage can stimulate reactivation and synthesis of viral proteins, the prophage enters the lytic cycle. Therapeutically, lysogenic phages present a medical disadvantage, and under international regulations cannot be used. Despite this, E. coli PA5 can be infected by lytic phages. [7] [15] [16]Fig.3: Illustration of lytic and lysogenic cycles.
Bacterial presence usually corresponds to the habitation of phage, sewage therefore ideally provides a plethora of different phage for experimental use the phages involved in this study were sourced from Ugandan sewage. Phages were made clonal through subjection to 5 rounds of purification, using plaque assays. Identified below are 9 phages with capability to infect E. coli PA5 (Fig.4).Fig.4: Transmission electron microscope images of phages examined in this study
Conclusion: Analysis
Conclusively, the concocted phage cocktails therapeutically expressed medical potential as a viable alternative to antibiotics. Treating E. coli with phage evidently reduced the bacterial population for 4 hours. The optical density (OD600) of the solutions were measured at 0 CFU/mL during this period, therefore, phage therapy can effectively suppress PA5 growth. Though, this is therapeutically not acceptable, since a regular dose must be administered, 4 hours is insufficient time. Treating E. coli with the 4-phage cocktail, 10B, was relatively more effective at suppressing bacterial growth in addition to measuring the lowest final OD600 0.294. However, fundamentally being the most important conclusion, E. coli exposed to phage had developed mechanisms of resistance. Since recovered strains were unaffected by wildtype and supernatant phage, for a viable novel cure, it is pertinent to address this issue.
Evaluation:
With respect to phage-resistance, there are 3 alternative approaches which can further progress the research, as well as circumventing this issue. Firstly, introducing a secondary dose of phage, before E. coli PA5 develops resistance can postpone the exponential growth enabling suppression to occur for a longer period, to allow the human immune system to progressively resolve the infection itself. Secondly, isolating new phages may prove effective, as formulating newer cocktails is the only method of overcoming the evolved PA5. In combination with the first recommendation, the introduction of a subordinate dose of novel phage could completely thaw resistance, since previous opportunistic exposure is non-existing. Thirdly, a popular endorsement, is exploring combined phage-antibiotic therapy. Despite attempts to evade the issue of antibiotic-resistance, incorporating phages to initially suppress PA5, proceeding to use antibiotics to completely disrupt further growth could effectively treat urinary-tract infections. This alternative approach is a method of reducing antibiotic usage, by substituting phages, in addition to there being less potential for PA5 to develop mechanisms of antibiotic-resistance, by already being affected by phage. These are all approaches which can progressively advance this research. In addition to this, repeats need to be conducted to firm the effectiveness of each phage, by narrowing the effect of chance and anomalies.
Ultimately, in vivo studies (emulating biological environments), need to be considered before the application of medicinal phage [9]. In vitro environments operate under sterile conditions, however human environments have a plethora of factors which impact the efficacy of phage, caused either by anatomical or physiological interactions. The research conducted during this experiment is the first step of a larger multi-process investigation.
Bibliography: (References)
1: Abedon, T.S., et al. (2011). Phage Treatment for Human Infections. Bacteriophage, Vol.1, pp.66-85. Available [Online]: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278644/pdf/bact0102_0066.pdf [Accessed 10 Jul. 2018]
2: Arber, W. (2014). Horizontal Gene Transfer among Bacteria and Its Role in Biological Evolution. Life (Basel), Vol. 4, pp.217-224. Available [Online]: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187160/ [Accessed 18th Jul. 2018]
3: Baer, A., Kehn-Hall, K. (2014). Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems. Journal of Visualized Experiments. Available [Online]: http://doi.org/10.3791/52065 [Accessed 24 Jul 2018]
4: Barber, A.E., et al. (2013). Urinary Tract Infections: Current Emerging Management Strategies. Clinical Infectious diseases, Vol. 57, pp.719-724. Available [Online]: https://academic.oup.com/cid/article/57/5/719/310850 [Accessed 17th Jul. 2018]
5: Bdbiosciences.com. (2018). Plaque Assay. Available [Online]: http://www.bdbiosciences.com/sg/resources/baculovirus/protocols/plaque_assay.jsp [Accessed 24 Jul. 2018].
6: Bolocan, A.S., et al. (2016). Phage therapy targeting Escherichia coli- a story with no end? Microbiology letters, Vol. 363, Issue. 22. Available [Online]: https://doi.org/10.1093/femsle/fnw256 [Accessed 16th Jul. 2018]
7: Bowater, L., (2017). The Microbes Fight Back: Antibiotic Resistance. Royal Society of Chemistry.
8: Brussow, H., Canchaya, C. and Hardt, W. (2004). Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion. Microbiology and Molecular Biology Reviews, Vol.68, pp.560-602. Available [Online]: https://mmbr.asm.org/content/mmbr/68/3/560.full.pdf. [Accessed 16th Jul. 2018].
9: Bruttin, A. and Brussow, H. (2003). Human Volunteers Receiving Escherichia coli Phage T4 Orally: A Safety Test of Phage Therapy. American Society of Microbiology, Antimicrobial Agents and Chemotherapy, Vol. 49, pp. 2874-2878. Available [Online]: https://aac.asm.org/content/aac/49/7/2874.full.pdf [Accessed 17th Jul. 2018].
10: Capparelli, R., et al. (2006). Selection of an Escherichia coli O157:H7 bacteriophage for persistence in the circulatory system of mice infected experimentally. Clinical Microbiology and infection, Vol. 12, pp.248-253. Available [Online]: https://ac.els-cdn.com/S1198743X14639185/1-s2.0-S1198743X14639185-main.pdf?_tid=2a1cd82c-3d80-4c2b-9b43- ceababc0fce9acdnat=1534088573_348ff8ebcdbdcef4f7729c851fdab044 [Accessed 17th Jul. 2018].
11: Cohen, S.N., et al. (1973). Construction of Biologically Functional Bacterial Plasmids In Vitro. Proceedings of National Academy of Sciences of the United States of America, Vol. 70, pp. 3240-3244. Available [Online]: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC427208/pdf/pnas00138-0206.pdf [Accessed 18th Jul. 2018]
12: Davies, J., and Davies, D. (2010). Origins and Evolution of Antibiotic Resistance. Microbiology and Molecular Biology Reviews, Vol. 74, pp 417-433. Available [Online]: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937522/pdf/0016-10.pdf [Accessed 16th Jul. 2018]
13: Dielubanza E.J., and, Schaeffer A.J. (2011). Urinary Tract Infections in Women. Medical Clinics of North America, Vol. 95, pp.27-41. Available [Online]: https://www.scholars.northwestern.edu/en/publications/urinary-tract-infections-in-women-4 [Accessed 17th Jul. 2018]
14: Lederberg, J. (1956). Genetic Transduction. American Scientist, Vol. 44, pp.264-279. Available [Online]: https://profiles.nlm.nih.gov/ps/access/BBABGL.pdf [Accessed 18th Jul. 2018]
15: Lees, E., (2015). Edexcel A Level Biology Student book 1. Hodder Education: London, pp-89-90.
16: Roberts, M.B.V. (1986). Biology, a functional approach. 4th ed. Middlesex: Nelson, pp.499-501.
17: Soothhil, J.S., (1992). Treatment of experimental infections of mice with bacteriophages. Journal of Medical Microbiology, Vol. 37, pp.258-261. Available [Online]: http://www.microbiologyresearch.org/docserver/fulltext/jmm/37/4/medmicro-37-4-258.pdf?expires=1533990124id=idaccname=guestchecksum=1F853051DA0109E199A499EA00BC73E7 [Accessed 16th Jul. 2018]
18: Wong, C.S., et al. (2000). The Risk of Haemolytic-uremic Syndrome after Antiobitic Treatment of Escherichia Coli 0157:H7 Infections. New England Journal of Medicine, Vol. 342, pp. 1930-1936. Available [Online]: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3659814/pdf/nihms460007.pdf [Accessed 17th Jul. 2018]
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