Bacteriophage therapy

“STRATEGY TO SLOW AND REVERSE ANTIBIOTIC RESISTANCE” : BACTERIOPHAGE THERAPY

1. PRESENTED BY- RAJNANDINI SINGHA PHARM D 1 “STRATEGY TO SLOW AND REVERSE ANTIBIOTIC RESISTANCE” PRESENTED BY- RAJNANDINI SINGHA PHARM D

2. OBJECTIVES What is antimicrobial resistance? Why antibacterial resistance is a concern? Factors of AMR Mechanism of antibiotic resistance Alternative approach to antibiotic resistance – “BACTERIOPHAGE THERAPY” Bacteriophage classification and source. Lytic and lysogenic cycle. Preparation and Methods. 2

3. Bacteriophage receptors Mechanism of bacteriophage Clinical studies Complications Bacteriophage therapy over antibiotics Applications PHAGOBURN Conclusion 3

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5. INTRODUCTION Antimicrobial drug resistance is the ability to resist the effects of the medication which was previously used to treat them. This is responsible for millions of death world wide and is consider as a major health care concern nowadays. Antimicrobial resistance (AMR) threatens the effective prevention and treatment of ever increasing range of infections caused by the microbes. The cost of health care for patient with resistance infection is higher. 5

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7. ANTIMICROBIAL RESISTANCE Antimicrobial resistance is the ability of a microorganism (like bacteria, viruses and some parasites) to stop an antimicrobial such as antibiotics, antiviral and antifungals) from working against it. ANTIBIOTIC RESISTANCE= MIC/MCC > TOXIC PLASMA CONCENTRATION 7

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9. WHY RESISTANCE IS A CONCERN ? 9

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15. MECHANISM OF ANTIBIOTIC RESISTANCE INTRINSIC RESISTANCE 1.LACK TARGET 2. EFFLUX PUMPS INHIBITOR DRUG INACTIVATION ACQUIRED RESISTANCE MUTATION PLASMIDS 15

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19. MECHANISM OF RESISTANCE GENE TRANSFER Transfer of r-gene from one bacterium to another Conjugation Transduction Transformation Transfer of r-genes between plasmids within the bacterium By transposons By integrons 19

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23. AN ALTERNATIVE APPROACH TO ANTIBIOTIC RESISTANCE- “BACTERIOPHAGE THERAPY” A ONCE AND FUTURE SOLUTION

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25. BACTERIOPHAGE Bacteriophages are viruses capable of infecting and killing bacteria. They have been referred to as bacterial parasites ,with each phage type depending on a single strain of bacteria act as host. Bacteriophage carry only the genetic information needed for their protein coats. They require precursor, energy generation and require ribosome supplied by their bacterial host cell. 25

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27. BACTERIOPHAGE CLASSSIFICATION At present ,over 5000 bacteriophage have been studied by electron microscopy and have been divided into 13 virus family 27

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29. SOURCES OF BACTERIOPHAGE In human and animal intestine. In running water. In the soil. sewages 29

30. Administration Phages can usually be freeze-dried and turned into pills without materially reducing efficiency. Temperature stability up to 55 °C and shelf lives of 14 months have been shown for some types of phages in pill form. Application in liquid form is possible, stored preferably in refrigerated vials. Oral administration works better when an  antacid  is included, as this increases the number of phages surviving passage through the stomach. Topical administration often involves application to gauzes that are laid on the area to be treated. IV phage drip therapy was successfully used to treat a patient with  MDR   Acinetobacter baumannii  in  Thornton Hospital  at UC San Diego 30

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32. LYTIC AND LYSOGENIC CYCLE 32

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35. EFFLUX PUMPS INHIBITOR Cytoplasmic membrane transport proteins. Major mechanism for resistance in antibiotics like tetracycline. The system pump solutes out of the cell. Efflux pumps allows the microorganism to regulate their internal environment by removing toxic substance , including antimicrobial agents . 35

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40. PREPARATION 40

41. METHODS 1. Phage were mixed with bacterial cells for 10 mins ( long enough for adsorption but too short for further infection). 2. The mixture is diluted by 10,000.( only those cells thst are bound phage in the initial incubation will contribute to the infected population ; progeny phage produced from those infections will not find host cells to infect). 3. Incubate the dilution. At intervals, a sample can be remoced from the mixture and the number of free phage counted using a plaque assay. 41

42. PHAGES CROSSES THE COMPLEMENTATION TEST MIXED INFECTION: A single cell is infected with two phage particles at once . MIXED INFECTION (CO-INFECTION) 1. It allows one to perform phage crosses. If two different mutants of the same phage co- infect a cell, recombination can occur between the genomes. The frequency of the genetic exchange can be used to order genes on the genome. 2.It allows one to assign mutation to complementation group. If two different mutant phage co- infect the same cell as a result each provides the function that the other was lacking, the two mutants must be in different genes(complementation group). If not, the two mutation are likely in the same gene. 42

43. TRANSDUCTION AND RECOMBINANT DNA During infection, a phage might pick up a piece of bacterial DNA ( Mostly happens when a prophage excises from the bacterial chromosome ). The resulting recombinant phage can transfer the bacterial DNA from one host to another, known as specialized transduction. 43

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49. BACTERIOPHAGE THERAPY OVER ANTIBIOTICS Bacteriophage treatment offers a possible alternative to conventional  antibiotic  treatments for bacterial infection. It is conceivable that, although bacteria can develop resistance to phage, the resistance might be easier to overcome than resistance to antibiotics. Bacteriophages are very specific, targeting only one or a few strains of bacteria.  Traditional antibiotics have more wide-ranging effect, killing both harmful bacteria and useful bacteria such as those  facilitating food digestion . The species and strain specificity of bacteriophages makes it unlikely that harmless or useful bacteria will be killed when fighting an infection. 49

50. 50 A few research groups in the West are engineering a broader spectrum phage, and also a variety of forms of  MRSA  treatments, including impregnated wound dressings, preventative treatment for burn victims, phage-impregnated sutures. Enzybiotics  are a new development at Rockefeller University that create enzymes from phage. Purified recombinant phage enzymes can be used as separate antibacterial agents in their own right. Phage Therapy also has the potential of preventing or treating infectious  diseases  of  corals . This could assist with decline of coral around the world.

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52. APPLICATION 1. IN FOOD INDUSTRY: Bacteriophage bioprocessing is practiced in the factory particularly as means of reducing food bacterial loads. Phage based control of pathogens is a non thermal intervention, and has been demonstrated to control the growth of campylobacter and salmonella on chicken skin. Phage application can be also use to control the presence of biofilm in the food processing environment. 2.IN AGRICULTURE AND FISHERIES- Bacteriophage have been considered to control salmonella infestation of cut fruit. The most successful use of phage therapy , already in practice ,has been in the control of fish pathogens. To avoid contamination of food products with listeria monocytogenes, salmonella on cut fruit and vegetable or the pathogenic E.Coli . 52

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54. EXAMPLES NO:1 PHAGE THERAPY REDUCES CAMPYLOBACTER JEJUNI COLONIZATION IN BROILERS Attempts to prevent campylobacter colonization of chickens by biosecurity measures have proven extremely difficult. Probiotic treatment with lactic acid bacteria and competitive exclusion with beneficent micro flora was only partially effective. A preventive group was infected with C. jejuni at day 4 of a 10 day phage treatment. A therapeutic group was phage treated for 6 days , starting 5 days after C . jejuni Colonization of the broilers had been established. Treatment was monitored by enumerating campylobacter colony forming units (CFU) and phage plaque forming units (PFU) from caecal content. A clear log decline in C . Jejuni counts in both pfeventive and therapeutic group were observed. 54

55. EXAMPLE NO :2 KILLING OF MYCOBACTERIUM AVIUM AND MYCOBACTERIM TUBERCULOSIS BY A MYCOBACTERIOPHAGE DELIVERED BY A NON – VIRILENT MYCOBACTERIUM. Mycobacterium smegmatis, an a virulent mycobacterium , is used to delivered the lytic phage TM4 where both M. avium and M. Tuberculosis reside within macrophages This results showed that treatment of M. avium infected , as well as M. Tuberculosis infected , RAW 264.7 MACROPHAGES, with M. smegmatis transiently infected with TM4, resulted in the number of viable intracellular bacilli. 55

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58. SCIENTIFIC REASON BEHIND THE GANGA WATER PURITY!! 58

59. PROBLEMS PROBLEM NO 1 : Host range Solution: cocktails of bacteriophage PROBLEM NO 2 : Bacterial debris present in phage preparation. Solution : Modern technology allows density centrifugation and other methods of purification. PROBLEM NO 3 : Lysogeny Solution: Use only lytic phage that are strong candidates for clinical trails PROBLEM NO 4 : Anti- phage antibodies. Solution: In treating chronic infection, it may be possible to administer higher dose of phage, to compensate for those that are cleared by interaction with neutralizing antibodies. 59

60. Phagoburn  was a European Research & Development (R&D) project funded by the European Commission under the  7th Framework Programme for Research and Development . The project was launched in June 2013 and ended in February 2017.  Phagoburn aimed at evaluating  phage therapy  for the treatment of burn wounds infected with bacteria  Escherichia coli  and  Pseudomonas aeruginosa . 60

61. CONCLUSION? Multi drug resistant bacteria have opened a second window for phage therapy. Phage therapy can then serve as a stand – alone therapy for infection that are fully resistant. It will also then be able to serve as co-therapeutic agent for infection that are still susceptible to antibiotics, by helping to prevent the emergencies of bacterial mutants against either agent. 61

62. REFERENCES 1.Lima-Mendez, G., Toussaint, A. & Leplae, R. A modular view of the bacteriophage genomic space: identification of host and lifestyle marker modules.  Res. Microbial.   162 , 737–746 (2011). Zinder, N. D. & Lederberg, J. Genetic exchange in  Salmonella .  J. Bacteriol .   64 , 679–699 (1952). Phage therapy: An alternative to antibiotics in the age of multi-drug resistance Derek M Lin ,  Britt Koskella , and  Henry C Lin 62