Phage Therapy - A Novel Alternative to Antibiotics to Combat Bovine Mastitis.pptx
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Mastitis is the persistent, inflammatory reaction of the udder tissue which is associated with physical changes of udder and chemical or bacteriological changes in the milk of farm/lactating animals. Also known as Garget, Mammitis, mammite, dagadi and agalactiosis. One of the important infectious disease of mammary gland of all mammals that has serious impact on livestock production. Phages are the most abundant biological entities on the planet, around 1031 (Batinovic 2019) Method to combat antibiotic resistance bacteria Alteration of gut microbiota Phage Therapy Antimicrobial peptides (AMP) Importance of phage therapy Phage therapy very important role in treating infections that don't respond to antibiotics Phage therapy is nature’s “antibiotics” and may be a good alternative treatment In developed countries, livestock farming accounts for about 50–80% of total antibiotic
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Phage Therapy - A Novel Alternative to Antibiotics to Combat Bovine Mastitis.pptx
- 2. Speaker: Anil Kumar Puniya Phage Therapy - A Novel Alternative to Antibiotics to Combat Bovine Mastitis
- 3. • Mastitis is the persistent, inflammatory reaction of the udder tissue which is associated with physical changes of udder and chemical or bacteriological changes in the milk of farm/lactating animals. • Also known as Garget, Mammitis, mammite, dagadi and agalactiosis. • One of the important infectious disease of mammary gland of all mammals that has serious impact on livestock production. 10/1/2023 Mammae = mammary gland -itis = Latin suffix for inflammation Swelling pain warm redness www.healthscience.com
- 4. Mastitis is one of the most important diseases of dairy animals which directly or indirectly affect the economy of the farmers and ultimately affect the economy of the country. Mastitis is a global problem and every country including developed ones suffer huge financial losses. It adversely affects: • Animal health • Quality of milk • Quantity of milk • Economics of milk production
- 5. Streptococus spp. E. Coli Staphylococus aureus Bacillus spp. Pasteurella multocida Klebsiella pneumoniae Spherophorus necrophorus Pseudomonas aeruginosa Alcaligenes faecalis Leptospirae Aerobacter aerogenes Brucellae Nocardia asteroides Mycoplasma spp. Actinomyces bovis Campylobacter spp. Mycobacterium tuberculosis Corneybacterium pyogenes Clostridium perfringens Coxiella burneti 10/1/2023 Common causative agent Staphylococcus aureus Streptococcus agalactiae Corynebacteriu m pyogens Pseudomonas aeruginosa Mycobacterium tuberculosis Brucella abortus Escherichia coli
- 6. Bovine Mastitis Bovine mastitis is the most widespread and economically important disease worldwide The prevalence of bovine mastitis resulted in extensive use of antibiotics It was revealed that 90% of antibiotic residues in milk are a consequence of mastitis therapy Cause antibiotic resistance Most common pathogen is staphylococcus aureus, E.coli Sometime streptococcus
- 7. What is an antibiotic resistant bacteria? • Non-resistant bacteria • Multiplication Few bacteria get mutate • Mutation In the presence of drug only drug resistance bacteria can survive • Drug resistance bacteria multiply and thrive
- 8. Global threat (AMR) Centers for Disease Control (CDC) and WHO have declared antibiotic resistance a threat to global health CDC estimates antibiotic-resistant infections result in 2 million illnesses and at least 23000 deaths a year United Kingdom government’s - estimated 700000 people die each year globally from resistant infections Bacterial infections may result in the deaths of over 10 million people by the year 2050 (Ghosh et al., 2018)
- 9. Impact of antibiotics resistance on human life Resistant organisms lead to treatment failure Increased mortality Resistant bacteria may spread in Community Low level resistance can go undetected Added burden on healthcare costs
- 10. Antibiotic Resistance Bacteria Selective Pressure Mutation Gene Transfer Inappropriate Use Inadequate Diagnostics Agricultural Use Cause of antibiotic resistance bacteria
- 11. (Saha & Mukherjee, 2019) Mechanism of antibiotic resistance
- 12. Bacterial cell wall hydrolases (BCWH) 1 Antimicrobial peptides (AMP) 2 Nano-material 3 Bacteriocins 4 Alteration of gut microbiota 5 Phage Therapy 6 (Sarkar et al., 2018) Method to combat antibiotic resistance bacteria
- 13. Advantages (carrili et al., 2011) Bactericidal agent Auto Dosing Low inherent toxicity Minimal disruption of normal flora Rapid discovery Biofilm Clearance Relatively Low cost
- 14. Phages – Discovery and Mechanism of Action Félix d’Hérelle in his Phages are the most abundant biological entities on the planet, around 1031 (Batinovic 2019). Phage head contains about 20 faces and 30 edges. Follows lytic and lysogenic cycles 14 Frederick William Twort
- 15. Early Phage Therapy Twort - Effect of transparent material that inhibited bacterial growth 1917 - d’Herelle - isolated anti-shigella microbe that the idea of an obligate parasite bacteria was termed bacteriophage or “bacteria-eater” 1919 - d’Herelle - used phage to treat chickens infected with Salmonella gallinarum 1927 - Clinical trials treating cholera in India showed that mortality decreased from 62.8% in control groups to 8.1% in phage-treated group (Kortright et al., 2019)
- 16. Selecting against efflux pump positive MDR pathogens Expunging biofilms with phages Phage Antibiotic Synergy (PAS) Tackling AMR with Phages
- 17. Importance of phage therapy Phage therapy very important role in treating infections that don't respond to antibiotics Phage therapy is nature’s “antibiotics” and may be a good alternative treatment In developed countries, livestock farming accounts for about 50–80% of total antibiotic Gonzalez and Calap, 2021 Phage Therapy Animal Heath and phage
- 18. A B C Drug sensitive bacteria Phages work against both treatable and antibiotic- resistant bacteria. They may be used alone or with antibiotics and other drugs. Phages multiply and increase in number by themselves during treatment They only slightly disturb normal “good” bacteria in the body. Phages are natural and easy to find.
- 19. Phages work against both treatable and antibiotic- resistant bacteria. They may be used alone or with antibiotics and other drugs. Phages multiply and increase in number by themselves during treatment They only slightly disturb normal “good” bacteria in the body. Phages are natural and easy to find. Advantages of Phage Therapy over the antibiotics
- 20. Animals Infection Pathogen Phages Reference Bovines Mastitis S. aureus ISP phage Figueiredo et al., 2017 Bigot et al., 2011; Guenther et al., 2011; Modi et al., 2001; Leverentz et al., 2003; Whichard et al., 2003; Guenther et al., 2012; Bovines Mastitis S. aureus Phage cocktail Metritis E. coli Phage cocktail Haemorragic septicemia P. mutocida PMP-GAD-IND phage Swine Diarrhea Salmonella sp. Phage cocktail Chronic wounds Multibacterial biofilms Phage cocktail Rabbits Cholera-like diarrhoea V. cholerae Phage cocktail Dogs Otitis externa P. aeruginosa Phage cocktail Urinary tract infections P. aeruginosa Pbunavirus PB1-like phages cocktail Urinary tract infections E. coli 5 promising single phages Phages used as remedy to improved the animal health
- 21. PAS effect of phage ØMFP on E-coli MFP on Luria Bertani agar plates Plaque sizes of phage ØMFP on E-coli MFP • Disc containing ß-lactam group of antibiotics aztreonam and cefixime (indicated by ‘‘+’’ symbols) produced large phage plaque • Host strain is resistant to many types of penicillins (amoxicillin, trimethoprim) Phage made quite small plaque with no cefotaxime and remarkably other large one with opt. conc. 50 ng/mL (Comeau et al., 2007)
- 22. In well A, the adherence of EPEC to the surface of HEp-2 cells was thoroughly evident but in well B, there was no sign of adhesion to the cells, indicating the efficacy of the phage in eliminating bacteria Addition of the phage to EPEC-infected HEp-2 cells (106 pfu/ml), the bacteria were completely demolished (Vahedi et al., 2018)
- 23. staphylococcus phage M8 (A) and staphylococcus phage B4 (B) Staphylococcus phage M8 on bacterial growth in milk after 8 h of incubation. B: control (no phage); T: test (bacterial culture + Staphylococcus phage M8
- 24. Mice in the A. baumannii-infected group exhibited higher bacterial load 1.46 × 109 CFU/lung, while the bacterial load in phage- rescue group were decreased to 1.28 × 106 CFU/lung phage SH-Ab15519 administered intranasally can effectively rescued the mice from lethal A. baumannii lung infections without deleterious side effects. (Yunfen et al., 2018)
- 25. Therapeutic efficacy of ΦSA012 in a mouse model of S. aureus mastitis. (a) Photographs of the mouse mastitis model after administration of SA003 (105 CFU, n = 5) and phage treatment (105 PFU (MOI = 1, n = 7) and 107 PFU (MOI = 100, n = 6)). Arrows indicates abscesses. (b) S. aureus CFUs in the mammary glands on day 2 after challenge Iwano et al., 2018
- 26. • A spot test and a subsequent plaque assay were performed to determine the lytic efficacy (host range) of three phages (STA1.ST29, EB1.ST11, and EB1.ST27) against 92 S. aureus isolates from quarter foremilk samples of subclinical and clinical mastitis cases Titze et al., 2020 S. aureus isolates 7142 and 10614, for the control culture without phages (blue: 7142; orange: 10614), the assays with the addition of 0.1 mL phage mixture at a final concentration of 1.2 × 108 pfu/mL (grey: 7142; violet:10614) and 1 mL phage mixture at a final concentration of 1.2 × 109 pfu/mL (yellow: 7142; green: 10614). S. aureus at a concentration of averagely 1.1 × 105 cfu/mL.
- 27. Effectiveness of phages treatments Neutralization Phages may be neutralized by antibodies or other compounds (sulakvelidze et al., 2001) Specificity Phage must be lytic and able to infect target bacteria (Guenther et al., 2009) Resistance to phage Bacteria may become resistance to phage Use a cocktail of phage isolate a new phages (Pirnay et al., 2009) Phages concentration (MOI) It is necessity to validate MOI (multiplicity of infection; the average no. of phage per bacterium) It maximize the efficacy of phage solution Environmental conditions The survival of bacteriophages are affected by physio-chemical factor (pH, aw and temperature) Proliferation of phages is limited when pH 4.5 (Jonczyk et al., 2011) Phages administration Phage treatment by: oral, topical, intravenous and intranasal administration depending on site of infection (chilamban et al., 2004) (Oliveria et al., 2015)
- 28. Bacteriophages - against pathogens Bacteriophage Pathogenic host Clinical symptoms CDHM1-6 Clostridium difficile Bacteremia, UTI, wound infection, and endocarditis Msa Staphylococcus aureus Pneumonia and flesh eating disease NK5 Klebsiella pneumoniae Lung infections and pneumonia M4 Pseudomonas aeruginosa Pneumonia TM4, DS-6A Mycobacterium tuberculosis Tuberculosis EC200pp Escherichia coli Diarrhea, UTI, and meningitis A25 Streptococcus pyogenes Sore throat and skin disorder (Shah et al., 2019)
- 29. Case reports Infection Complicating Conditions Antibiotic Courses Phage Dose (PFU) and Duration Outcome Case 1 (Duplessis et al., 2017) Pseudomonas aeruginosa bacteremia DiGeorge syndrome and congenital heart disease with pacemaker Meropenem Aztreonam Polymyxin-B 3.5×105 36 h(six dose total) After phage treatment positive improvement observed Case 2 (LaVergne et al., 2018) Acinetobacter baumanii surgical site infection Craniectomy colistin, azithromycin and rifampin 8.56×107 8 days Initial improvements observed Case 3 (Schooley et al., 2017) Acinetobacter baumanii infected pseudocyst Necrotizing pancreatitis Azithromycin, colistin, and rifampin 5×109 84 days Clinical improvement and resolution of infection
- 30. Clinical trial Infection Trial Treatment Group Placebo Group Phage Dose (PFU) Outcome Trial 1 (Wright et al., 2009) Pseudomonas aeruginosa otitis Placebo controlled, double blind for safety and effectiveness 12 individuals received phage cocktail 12 individuals received a single dose of glycerol- PBS buffer 109 Three individuals from each group had undetectable levels of P. aeruginosa at the end of the trial Trial 2 (Sarker et al., 2016 E-coli diarrheal diseases Placebo controlled, double blinded trial 40 individuals received phage cocktail 41 individuals received oral rehydration solution 1.4×109 No significant difference observed between phage treatment and placebo group Trial 3 (Jault et al., 2018) Pseudomonas aeruginosa burn wound infection Placebo controlled, blinded trial 12 individuals received a phage cocktail 13 individuals received 1% sulfadiazin e silver 2×107 (expect- ed) 200- 2000 PFU(act ual) Trial halted due to insufficient efficacy This was likely due to lower applied dose of phage than expected
- 31. Bacteriophage that are approved or are undergoing clinical trials for Human use Product Condition Company ListShield Food Industry(Listreria monocytogenes) Food industry(Escherichia Coli) Intralytix SalmoShield Burn infections(Pseudomonas aeruginosa and E.coli) Intralytix Phagedys Treatment and prophylaxis of dysentry Biochimpharm PhagoBurn E. Coli or P. aeruginosa Multiple centres (Haldar et al., 2018)
- 32. Limitations of phage therapy Limitations Phage Selection Phage host range limitation “Uniqueness” of phages as pharmaceuticals Unfamiliarity with phages (Carrili et al., 2011)
- 33. Future prospects Multidrug-resistant bacteria have opened a second window for phage therapy Mixed therapy of phage and antibiotics could be ideal combination for mastitis treatment
- 34. Bacteriophage is one such alternative which can be developed as a solution for the multidrug resistance crisis They are highly specific, effective, and safe for the treatment and prevention of infectious diseases Phage therapy is an attractive option to prevent and control mastitis Humanity take advantage of the natural predators of bacteria to fight against pathogen and to increase the quality life Conclusions
Editor's Notes
- Bacteria ( ~ 70%) Yeasts and molds ( ~ 2%) Unknown ( ~ 28%) physical trauma weather extremes
- The ability of bacteria or other mo’s to resist the effect of antibiotics to which they were once sensitive In other words the bacteria which are not killed or controlled by antibiotics
- The term bacteriophage was introduced by French-Canadian microbiologist Felix d’Herelle in 1917 upon his isolation of an invisible microbe from the stools of dysentery patients. During the following two decades, several commercially available phage products emerged, although their application for the elimination of infections was often inconsistent owing largely to the lack of phage biology knowledge at the time. The discovery of penicillin in 1928 heralded the beginning of the antibiotic era, and by the 1940s the application of phages as antibacterials had diminished in much of the world (Kuchment 2012). Nevertheless, these applications have continued in regions of the former Soviet Union. Significantly, the emerging multidrug resistance problem in bacterial pathogens has prompted a renewed interest in the exploitation of phages as therapeutic antibacterial agents. Mechanism of action Phages are considered to be the most abundant biological entities on the planet, with estimated particle numbers of more than 1031 (Hendrix 2003). They are usually found where their specific bacterial hosts thrive, given that they are obligate intracellular parasites and unable to propagate without a bacterial cell. They are broadly classified as either virulent or temperate. In the case of virulent phages, following phage attachment and injection of its genome into the host cell, the host DNA replication and protein synthesis machinery is exploited to synthesize new phage particles, which are released from the cell, usually culminating in the death of the bacterium. This is termed the lytic infection cycle. Although temperate phages are capable of replicating in this way, they also have the ability to replicate using the lysogenic cycle in which their DNA can become part of the bacterial genome as a prophage. Induction, or the excision of the prophage from the genome, can occur spontaneously or in response to cellular internal or external triggers (Nanda et al. 2015). This results in lysis and release of progeny phages that may proceed to lyse or lysogenize other susceptible bacteria.