Phage Therapy PDF

Phage therapy Nefise Büşra Karaca Özde Deniz Özkan Outline 01 Introduction A. Definition of phage therapy B. Historical background 02 The Science Behind Phage Therapy A. What are bacteriophages? B. How do bacteriophages infect bacteria? C. Strategies for phage therap...

Phage therapy Nefise Büşra Karaca Özde Deniz Özkan Outline 01 Introduction A. Definition of phage therapy B. Historical background 02 The Science Behind Phage Therapy A. What are bacteriophages? B. How do bacteriophages infect bacteria? C. Strategies for phage therapy 03 Advantages of Phage Therapy A. Specificity towards target bacteria B. Ability to evolve alongside bacterial resistance C. Minimal impact on the body's natural microbiota D. Potential for personalized medicine Outline 04 Applications of Phage Therapy A. Treatment of bacterial infections B. Use in food safety C. Environmental applications 05 Challenges and Limitations A. Bacterial resistance to phages B. The risk of contribution to antibiotic-resistant C. Decrease in activity due to immune response 06 Conclusion A. Recap of key points B. Importance of phage therapy in the context of antimicrobial resistance C. Future prospects and potential impact Definition of Phage Therapy Phage therapy is a form of treatment that utilizes bacteriophages, which are viruses that specifically infect and kill bacteria, to combat bacterial infections in humans, animals, or even plants. In phage therapy, these viruses are applied either externally (e.g., topically for skin infections) or internally (e.g., orally or intravenously for systemic infections) to target and eliminate the pathogenic bacteria causing the infection. The goal of phage therapy is to harness the natural ability of bacteriophages to infect and replicate within bacterial cells, ultimately leading to the destruction of the infecting bacteria and resolution of the infection (Kaitlyn et al., 2019). Introduction - Historical Background 01 Discovery of Bacteriophages 01 02 First discovered independently by Frederick Twort in 1915 and Félix d'Hérelle in 1917. D'Hérelle coined the term "bacteriophage," meaning "bacteria eater" Early Experiments 02 03 In the 1920s and 1930s, d'Hérelle conducted experiments demonstrating the ability of bacteriophages to kill bacteria Particularly in the context of dysentery and cholera infections. Initial Clinical Applications In Eastern Europe, particularly in the Soviet Union 03 04 Extensively researched and used to treat various bacterial infections, including dysentery and staphylococci. Eliava Institute of Bacteriophages, Microbiology, and Virology in Tbilisi, Georgia Decline in the West 04 Discovery and widespread use of antibiotics in the mid-20th century. 05 Antibiotics - more convenient to produce, standardize, and administer compared to phages Revival of Interest 05 Due to the rise of antibiotic-resistant bacteria and the limitations of conventional antibiotic treatments  What are Bacteriophages, often referred to simply as phages, are viruses that bacteriophages? specifically infect and replicate within bacteria. They are the most abundant and diverse biological entities on Earth, estimated to outnumber bacteria by at least tenfold. Bacteriophages have a unique structure and life cycle, allowing them to infect bacterial cells and use the bacterial machinery to replicate and produce progeny phages (Kasman & Porter, 2022) Structure A protein coat, or capsid - encapsulation of the genetic material (DNA or RNA) Tail-like structure - surrounds capsid and provides attachment to the bacterial host Host Specificity A specific host range - only infect certain bacterial species or strains Interactions between surface molecules on the bacteriophage and receptors on the bacterial cell surface  What are bacteriophages? Lifecycle 2 primary lifecycle pathways: 1. Lytic Cycle: The phage injects its genetic material into the bacterial cell, hijacks the bacterial machinery to replicate its own DNA and proteins, and eventually lyses (bursts open) the bacterial cell, releasing progeny phages to infect other bacteria. 2. Lysogenic Cycle: The phage integrates its genetic material into the bacterial chromosome, becoming a prophage, and replicates along with the host cell. Under certain conditions, such as stress, the prophage may revert to the lytic cycle and initiate cell lysis.  How do bacteriophages infect bacteria? 3. Replication and 1. Attachment 2. Penetration Transcription Bacteriophages have protein Once attached to the bacterial cell Once inside the bacterial cell, structures on their surface surface, the bacteriophage injects the phage genetic material called tail fibers or tail spikes its genetic material (either DNA or takes over the bacterial that recognize and bind to RNA) into the interior of the machinery. In the case of DNA specific receptors on the bacterial cell. This injection phages, the phage DNA is surface of the bacterial cell. process is facilitated by the replicated by the bacterial These receptors are often contraction of the tail sheath, enzymes, and viral genes are proteins or carbohydrate which acts like a syringe to deliver transcribed into messenger structures located on the the phage DNA into the bacterial RNA (mRNA). bacterial cell wall. cytoplasm.  How do bacteriophages infect bacteria? 4. Translation 5. Assembly of 6. Cell Lysis and and Assembly New Viral Particles Release The viral mRNA directs the The newly synthesized viral Once assembly is complete, the synthesis of viral proteins components are assembled into bacteriophage triggers the lysis using the bacterial ribosomes. complete virions (fully formed (rupture) of the bacterial cell. These proteins include phage particles) within the This is typically achieved by the structural components of the bacterial cell. This process often expression of lytic proteins that phage, such as capsid proteins occurs in a coordinated manner, degrade the bacterial cell wall, and tail proteins. Additionally, with viral proteins and DNA causing the cell to burst open enzymes required for phage coming together to form mature and release the newly formed replication and maturation are phage particles. phage particles into the produced. surrounding environment. Strategies for Phage Therapy 1. Phage Cocktails Rather than relying on a single phage strain, a mixture of different bacteriophages (phages), each targeting a specific strain or species of bacteria is used (Liu et al., 2020). Selection Criteria: Lytic activity Ability to penetrate biofilms Host range Advantages: Broader spectrum of activity Reduced emergence of phage resistance Synergistic effects, improved treatment outcomes Strategies for Phage Therapy 2. Phage-Antibiotic Synergy The term "phage-antibiotic synergy" (PAS) was initially used in 2007 Certain antibiotics at sub-lethal concentrations can greatly accelerate the propagation of lytic bacteriophages in the host bacteria Accelerated host cell cleavage and rapid diffusion of progeny phages (Comeau et al., 2007) Lytic phage-producing depolymerase + ciprofloxacin to treat the biofilm of Klebsiella pneumonia (Verma et al., 2010) P. Aeruginosa infection rat model, phage-ciprofloxacin: highly synergistic (Oechslin et al., 2016) Strategies for Phage Therapy 3. Phage-Derived Enzymes Virion-associated peptidoglycan hydrolases (VAPGH) Mostly located on the phage base plate Punch holes through degrading the peptidoglycan of the bacterial cell wall (Rodriguez-Rubio et al., 2013) Endolysins First discovered by Jacob et al. (1958) A class of peptidoglycan hydrolases that can directly destroy the peptidoglycan of a bacterial cell wall (Cahill & Young, 2019) Depolymerases Hydrolysis of polysaccharide compounds of bacteria, such as capsule, lipopolysaccharide (LPS), or extracellular polysaccharides of biofilms (Maciejewska et al., 2018; Latka et al., 2017) Strategies for Phage Therapy 4. Phage Engineering Increased phage therapeutic potential, By the expansion or change of lysis spectrum or/and By the delivery of exogenous genes and proteins. Engineering of genes encoding receptor- binding proteins (RBPs) in the tail fibers or spikes of phages (Dams et al., 2019) Strategies for Phage Therapy 5. Phage Combined with CRISPR-Cas System Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-Cas system in bacteria and archaea provides sequence-based adaptive immunity against mobile genetic elements such as viruses and plasmids ( Barrangou et al., 2007). a. Integration of the short fragments of nucleic acid of viruses or plasmids into the CRISPR array and production of short RNA sequences complementary to them (known as CRISPR RNAs, namely crRNAs), b. Guidance of Cas protein complex by crRNAs to specifically target invading foreign genetic elements for degradation (Koonin et al., 2017). Strategies for Phage Therapy 5. Phage Combined with CRISPR-Cas System Precise site interference of CRISPR-Cas + high infection efficiency of a phage Delivery of the CRISPR-Cas system with phage to target bacterial genomic DNA (or RNA) for the elimination of pathogenic bacteria and prevention of the emergence of bacterial resistance (Greene, 2018) Active endogenous CRISPR-Cas system included in the target bacteria is exploited by the phage genome with a mini-CRISPR array to attack the host cell (Li & Peng, 2019). An additional & exogenous CRISPR nuclease for the pathogens without a CRISPR-Cas system Advantages of Phage Therapy 01 Specificity Highly specific to their target bacterial species or strains Allows for targeted treatment while sparing beneficial bacteria in the microbiota 02 Adaptability Phages can evolve rapidly to overcome bacterial resistance mechanisms Effective against antibiotic-resistant bacteria, including multidrug- resistant strains 03 Diversity Selection of phages with optimal lytic activity, host specificity, and other desirable characteristics for therapeutic applications. 04 Low toxicity They specifically target bacterial cells and do not infect or harm human cells, minimizing the risk of adverse effects associated with treatment. 05 Environmental Friendliness A potentially eco-friendly alternative to conventional antibiotic therapy. 06 Personalized Medicine & Treatment Based on the type of bacterial infection, antibiotic susceptibility profiles, and patient-specific considerations. Treatment in Bacterial Infections Bacteriophages have been utilized to address bacterial infections across diverse bodily locations through different formulations. The majority of research in this area has focused on employing bacteriophages for surface-level treatment of bacterial skin infections. Positive outcomes have been observed following the administration of bacteriophages in managing localized infections such as wounds, burns, and trophic ulcers (Principi et al.,2019) Diabetic foot ulcers infected by S. aureus strains can be treated by S. aureus-specific phage even if some strains of S. aureus are resistant to methicillin (Fish et al., 2016) Pseudomonas aeruginosa is the cause of Cystic Fibrosis (CF), an inherited genetic disease that chronically affects the lungs and digestive system of children and adults. Pseudomonas is antibiotic-resistant and forms a biofilm. Therefore, phages are effective in killing this pathogen (Alemayehu et al.,2012) Use in Food Safety Campylobacter Because of the ideal body temperature, poultry has emerged as a natural habitat for Campylobacter species, serving as the primary origin of human infections. In chicken skin, the 2-log decrease is seen in fresh samples In raw and cooked beef, Campylobacter levels have been significantly reduced. (Połaska & Sokołowska, 2019) Use in Food Safety Salmonella Salmonella spp. is the most significant pathogen causing foodborne illnesses worldwide (Duc et al., 2018). According to the CDC (2023), Salmonella leads to about 1.35 million infections, 26,500 hospitalizations, and 420 deaths in the United States every year. Salmonella levels were declined by 1.0–2.0 log in raw and pasteurized cheddar cheese created using milk treated with phage. In turkey meats, 5.0-log reduction at 15 °C and 3.0-log reduction at 8 °C is seen. Remarkable decrease by 3.9 and 2.2 log CFU/g for S. Typhimurium and S. Enteritidis, respectively is seen in packaged lettuce. (Połaska & Sokołowska, 2019) Use in Food Safety Listeria monocytogenes L. monocytogenes poses a significant public health threat as a prominent foodborne pathogen, particularly impacting vulnerable populations such as pregnant women, newborns, those with weakened immune systems, and elderly individuals, leading to high mortality rates. Consequently, safeguarding the food chain's integrity, particularly concerning ready-to-eat (RTE) foods, becomes paramount. Reduction of 2.0–4.6 log in melons and only 0.4 log in apples is ensured in fresh-cut fruits. Reducing Listeria counts below the detection limit is ensured in RTE sliced pork ham. (Połaska & Sokołowska, 2019) Use in Food Safety Escherichia coli O157:H7 E. coli O157:H7, known for producing Shiga toxin, has the capability to infiltrate the human gastrointestinal system, leading to illness characterized by symptoms like abdominal cramps and bloody diarrhea. Recent studies have shown that a specialized preparation of bacteriophages targeting E. coli successfully inhibited the growth of this particular strain. Levels of E. coli declined by >94% and 87% on the surface of beef and lettuce, respectively. 4.5-log reduction of E. coli after 2 hours of phage addition is ensured in spinach. (Połaska & Sokołowska, 2019) Environmental Applications  1. The Risk of Emergence of 2. The Risk of Contribution of 3. Decrease in Activity Due to Bacterial Resistant to Bacteriophages to the Immune Response Bacteriophages Development of Antibiotic Resistance Bacteria have the capability to possess Lysogenic phages integrate their DNA 3. Replication and and their components Bacteriophages or develop various mechanisms aimed into the 2. Penetration bacterial genome, potentially Transcription are foreign substances to the body, at preventing viral infections. These facilitating the horizontal transfer of capable of triggering an immune Once attached to the bacterial cell mechanisms may include evasion genetic material and contributing to the surface, the bacteriophage injects reaction that could potentially diminish tactics such as hiding, altering, or losing spread of antibiotic its genetic resistance material (either DNA or genes. the effectiveness of administering receptors, secreting substances to ThroughRNA)transduction, into the interior ofthisthe process bacteriophages. Immune reactions to deter phage attachment to the bacterial could bacterial cell. This injection theoretically lead to the bacteriophages have been observed in process is facilitated by the pathogen, activating defenses to block emergence of of novel contraction microbes or the tail sheath, both animal studies and human trials. phage DNA injection into the cell, and bacteria whichwith actsheightened like a syringe toresistance. deliver inhibiting phage replication and release. the phage DNA into the bacterial cytoplasm. (Principi et al., 2019)  Conclusion 01 Conclusions Because bacteriophages are relatively harmless for eukaryotic cells and have the ability to precisely lyse bacteria, they present a promising alternative for phage therapy. Antibiotics have been crucial in combating infections and saving lives since their discovery and use; however, the growing challenges in creating novel antibiotics and the emergence of bacterial resistance represent a severe threat to human health. Conclusion 02 Challenges: Identification of phage-resistant bacteria, understanding of phage pharmacokinetics and interactions with human tissues, and optimization of treatment protocols. Conclusion 03 The reduction in the usage of antibiotics and the enhanced control of antibiotic-resistant bacteria can be achieved by recent developments in phage therapy, such as phage engineering and CRISPR-Cas.  Conclusion 04 Conclusions Phage therapy is essential in killing or reducing pathogens that cause bacterial infections, preventing foodborne diseases, and contributing to the environment. Conclusion 05 However, the risk of bacterial resistance to phages, the risk of immune system opposition and the risk of phages contributing to antibiotic resistance are serious problems in developing phage therapy methods. THANK YOU  References Alemayehu, D., Casey, P. G., McAuliffe, O., Guinane, C. M., Martin, J. G., Shanahan, F.,... & Hill, C. (2012). 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