Cyril gay oie international symposium on prudent use of antimicrobials march 2013


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  • These novel biocontrol approaches should employ strategies specifically geared to reduce or eliminate drug resistance development, and include demonstrated synergistic approaches that could both reduce costs and increase efficacy while reducing the risk of drug resistance development.
  • Feeding 5 ppm cavacrol, 3 ppm cinn and 2 capsacin exerted significant gene changes in the gut. 74, 62 and 254 gene changes were observed with feeding cavacrol. Cinn and capsacin respectively with most gene change occurring with capsacin feeding. Functionally, general metabolism , protein especially anf immunity and defense genes were signifcantly altered by feeding these phytochemicals. Most importantly, 7, 6 and 16% genes ourt of 45,000 genes interoggated showed signifcant changes after feeding with cavacrol, cinn and capsacin respectively.
  • Employing a set of meta-omics techniques to gain a global perspective on the in-situ ecology and function of the microbiome and trying to tie the knowledge gained from each of those technologies together to answer fundamental questions on microbiome structure, ecology and evolution and how that all relates to host health and disease
  • Probiotics is initially known as beneficial bacteria which enhance gut immunity. Although in nature reused litter in the poultry farm provides “starter-cultyre”, in production agriculture, commercial probiotics have proven to be quite effective to promote young chicken’s gut health. With increasing understanding that gut homeostasis of commensal bacteria is important in maintaining gut health and animal well-being different types of bacteria including lactobacillus, bifidobacteria and yeast including saccharomyces, and aspergillus have widely used to improve gut health. Probiotics have been regarded as alternative to AGP with their digestion-promoting, anti-microbial activity, and immune enhancing activity. Probiotics in conjunction with other alternative strategies have shown excellent results as AGP alternatives.
  • Cyril gay oie international symposium on prudent use of antimicrobials march 2013

    1. 1. Alternatives to Antibiotics: Recent scientific development Cyril G. Gay, DVM, Ph.D National Program Leader Animal Production and Protection Agricultural Research Service United S Department of Agriculture
    2. 2. Antibiotic Use in Food Animals • Therapeutic - treatment of diseased animal • Prophylactic - disease prevention • Metaphylactic - therapeutic and prophylactic use • Growth promotion – accelerate growth of animals • Regardless of personal opinions: • Increasing concern with antibiotic resistance
    3. 3.
    4. 4. Symposium Objectives 1) highlight promising research results on alternatives to antibiotics, 2) assess challenges associated with their commercialization and use, and 3) provide actionable strategies to enable the development of alternatives.
    5. 5. What are alternatives to antibiotics? Drugs, biologics, biotherapeutics, feed additives Vaccines, probiotics, prebiotics, phytochemicals, essential oils, heavy metals, organic acids, bacteriophage, bacteriophage gene products, host-derived antimicrobials, small interfering RNAs, naturally occuring antibacterial lytic enzymes, recombinant and hyperimmune therapeutic antibodies, immune enhancers
    6. 6. Symposium Organization Five Major Topics 1. 2. 3. 4. 5. Alternatives: Lessons from nature Immune modulation approaches The gut microbiome – microbial ecology Alternatives for growth promotion Regulatory pathways to enable licensing of novel alternatives to antibiotics
    7. 7. SESSION 1 Alternatives to Antibiotics: Lessons from Nature Aim of this session was to review novel alternative approaches for preventing and/or treating bacterial pathogens (and where applicable viral and parasitic pathogens) in food animal production.
    8. 8. Bacteriophage Human Animal virus e.g., Influenza, HIV Poultry Plant virus e.g., Tobacco mosaic virus Bacteriophage e.g., T1, T2, T4, Bacteria Specific Swine
    9. 9. Bacteriophage • Discovered by Felix d’Herelle in 1917 • Infect and replicate in bacterial cells • Host specific infections • Must enter and exit bacterial host cell • Lytic cycle and lysogenic cycle • DNA and RNA phages
    10. 10. Bacteriophage as an Intervention • Felix d’Herelle explored using phage solutions to treat dysentery in humans during early 1900’s • Phages supplied to Russian soldiers during World War II • Republic of Georgia using phage therapy since the 1940s • In 2006, US FDA approved Listeria phage solution for using in ready-to-eat meat & poultry • In 2011, US alternatives to antibiotics for animal feed phage for Bacteriophages: the FDA approved E. coli Jae-Won Kim, CJ Research Institute of Biotechnology, CJ Cheiljedang, Seoul, Republic using on food of Korea • Possible therapy for multi-drug-resistant strains
    11. 11. Bacteriophage Challenges Restriction modification – degradation of phage DNA upon infection CRISPR – clustered regularly interspaced short palindromic repeats Immunogenicity – antibodies developed against phage Resistance – mutations in bacterial genes for adsorption and lysogeny Lateral Gene Transfer – virulence factors & antibiotic resistance Bacteriophage Resistance Mechanisms Labrie et al. Nat Rev Micro 8(5):317, 2010
    12. 12. Bacteriophage Gene Products Amidases Muramidase (Lysozyme) Glucosaminidases Endopeptidases Holins form lesions in bacterial membranes are involved with release of phage Autolysins and prophage enzymes present in genomes of bacteria Bacterial cell wall and murein hydrolases from Hermoso et al., Curr Opin Microbiol 10:461, 2007
    13. 13. Gel Electrophoresis and Spot Assay with Clostridium perfringes Bacteriophage Lysin C. perfringens Strain 39 host Lysin Control Lysostaphin Lysozyme 25kD 20kD Lane 1 – molecular weight markers Lane 2 – induced E. coli lysate Lane 3 – nickel purified phage lysin Seal et al. Arch Virol 156:25, 2011 Spot assays are conducted with E. coli lysate or purified recombinant lysin as compared to lysostaphin and lysozyme NOTE clear zone of complete lysis
    14. 14. Bacteriophage Gene Products • Phage genomics-proteomics to search for potential novel antimicrobials • Bacteriophage lytic enzymes • Amidase • Muramidase • Endopeptidase • Recombinant phage enzymes lytic against Clostridium perfringens • Chimeric phage lysins act synergistically against Staph. Aureus • Challenges - Need efficient and costeffective expression system Seal et al. Arch Virol 156:25, 2011 Schmelcher et al. Appl Environ Microbiol. 78(7):2297-305. 2012
    15. 15. Animal-derived antimicrobial peptides (APM) “Innate host defense” • • Present in all organisms Cellular defenses Neutrophils Macrophages NK-cells Ancient     Snake: cathelicidin Frog: plastrin Insect: cecropin Bacteria: bacteriocins • Limited repertoire • Rapid acting • Broad specificity • Constitutive and “The Stimulated first Effector molecules Enzymes Host Defense Peptides Collectins line of defense against infection”
    16. 16. Chicken Cathelicidin 2 (Cath2) N • Cationic • 26 amino acids • Amphipathic C-RFGRFLRKIRRFRPKVTITIQGSARF-NH2 α Hinge region α C Van Dijk et al., 2005 Xiao et al., 2006 Henk P. Haagsman Department of Infectious Diseases and Immunology, Utrecht University, The Netherlands
    17. 17. Chicken Cathelicidin 2 (Cath2) Heterophils Mononuclear Cells Van Dijk et al., 2009 Giemsa Anti-CATH-2
    18. 18. Chicken Cathelicidin 2 Conclusions • Salmonella enteritidis infections of chickens accumulation of CATH-2 heterophils at site of infection • CATH-2 is microbiocidal against Gram (-), Gram (+) bacteria, yeasts and fungi • Truncated CATH-2 analogs are also antimicrobial • CATH-2 and derived peptides induce cytokine production in a chicken macrophage cell line • CATH-2 reduces the LPS-induced inflammatory response • Prophylactic or therapeutic treatment of chickens
    19. 19. Development of Cathelicidin 2 C hallenges • Improve specific activities of compounds • Improve stability of lead structures • Development carrier/delivery systems • Pharmacokinetics • Cost-effective large scale production
    20. 20. Animal-derived antimicrobial peptides (AMPs) Conclusions • Most tested AMPs kill bacteria by membrane disruption. Some AMPs may have intracellular targets • Some AMPs show strong activity with high cytotoxicity, while some AMPs show low cytotoxicity with weak activity • AMPs with more potent activity and lower cytotoxicity will be obtained through screening and molecular design • The expression level of recombinant peptides needs to be improved dramatically in order to apply AMPs into animal production
    21. 21. Passive immunity using hyperimmune IgY Treatment strategy that will allow the maintenance of high-titer antibodies against parasites in the gut showed protective effects 420 415 410 body 405 weight (gm) 400 395 390 385 Control Eimeria IgY Low IgY High Lee, SH., Lillehoj, HS. et al., 2009 Poultry Science 88:562-566 Lee, SH., Lillehoj, HS. et al., 2009 Veterinary Parasitology 163-123-126
    22. 22. SESSION 2 Immune Modulation Approaches to Enhance Disease Resistance and Treat Animal Infections Aim of this session was to address novel drug-free strategies to enhance innate defense mechanisms and/or adaptive immune responses by modulation of immune pathways through immune sensing molecules.
    23. 23. Diversity of stimuli can bind host conserved recognition receptors (PRRs= Pattern Recognition Receptors) and stimulate immune response Innate sensing receptors PRRs TLRs NLRs RIG-1 Bali Pulendran and David Artis. 2012, New Paradigms in Type 2 Immunity. Science 337, 431.
    24. 24. Some phytochemicals (essential oils) interact with host PRRs and initiate inflammatory immune response Bioactive phytochemicals PAMPs (Pathogens, diets) DAMPs PRRs and downstream signaling components are molecular targets for dietary intervention strategies using phytochemicals to reduce PRR-mediated inflammation Innate immune response -Host resistance PRRs: -Wound healing TLRs, NODs (Necrotic cell products) Level of PRR activation Inflammation Dysregulated immune response -Cancer -Atherosclerosis -Insulin resistance
    25. 25. Phytochemicals – Plant Extracts Dietary supplementation with phytonutrients such as safflower leaf, plum, anethol, mushroom, capsaicin, cinnamaldehyde, tumeric , garlic, etc. Enhance innate immunity Lee, S.H. et al., 2008, 2009, 2010, 2011,2012
    26. 26. Functional Genomics Gene Ontology categories 5 ppm 3 ppm carvacrol cinnamaldehyde 2 ppm capsicum N of genes which expression… - is altered 74 62 254 - is up regulated 26 31 98 - is down regulated 48 31 156 - General metabolism 13 12 51 - protein metabolism, 8 16 26 - Signal transduction 7 10 31 - Nucleic acid metabolism 5 14 25 - Immunity and defense 7 6 16 Examples of GO Categories for: KIM, DK, Lillehoj, HS, et al., 2010. Poul Sci 89:68-81
    27. 27. Phytochemicals Challenges • Conducting a “general” evaluation of the effect of phytonutrients on production performance is difficult • Few published studies are available; often papers provide minimal description of additive composition or active ingredients • Phytochemicals are very distinct molecules, with different effects, doses and mechanisms of action, and need to be developed accordingly
    28. 28. SESSION 3 The Gut Microbiome and Immune Development, Health and Disease Recent advances are demonstrating that the gut microbiota plays a key role in health and disease. Aim of this session was to capture state-of-the-art results in farm animals and humans to assess how the gut microbiome analysis is helping to solve disease problems.
    29. 29. Bacteriotherapy • Pathological imbalances within the intestinal microbiota, termed dysbiosis, are often associated with chronic Clostridium difficile infections in humans • Fecal transplantation, the administration of homogenized feces from a healthy donor, is a promising alternative therapy • Bacteriotherapy was shown to be a viable therapeutic target to treat chronic C. difficile infections and potentially other forms of persistent dysbiosis Lawley T.D et al. PLoS Pathog. 2012 October; 8(10): e1002995
    30. 30. Microbiome and Metagenomic Analysis Metatranscriptomics Metagenomics RNA Microbiome or Virome 16s Survey DNA The ruminal microbiome and animal health John Wallace, Rowett Institute of Nutrition and Health, UK Metabolomics
    31. 31. Microbial genomics • Genomics and metagenomics approaches to understand hostenvironment-microbe interactions • Evaluation of host response patterns to a single pathogen versus commensal flora • Assess the role of the commensal flora as a protective barrier against invading pathogens • Elucidate how commensal organisms or probiotics can be used to prevent or treat infections Demuth A et al., Infect Genet Evol. 2008 May;8(3):386-93. Epub 2008 Jan 18. Review.
    32. 32. Probiotics • Work through the competitive exclusion of pathogenic bacteria • Promoting epithelial barrier integrity • Influence the host and its gut microbiota by affecting many aspects of the immune system • Regulation of local mucosal cell-mediated immune responses • Increasing antibody production • Reducing epithelial programmed cell death (apoptosis) • Enhancing dendritic cell-induced T cell responses • Improving T cell homing to mesenteric lymph nodes • Augmenting toll-like receptor signaling Lee et al., 2010. J. Poul. Sci 47:106-114.
    33. 33. Gastrointestinal Microbiome • Bacteria, fungi, viruses, protozoa, helminths • Bacteria 10 11 cells/gram • Firmicutes • Bacteroidetes • Proteobacteria • Bacteria primarily associated with mucus and macromolecular food matrix (fiber) • Composition varies • In different section of GI
    34. 34. Integrating nutrition, health and disease research • Science of nutrition • Improved feed efficiency • Improved growth rates • Assess alternative feeds • Science of disease • Prevent intestinal diseases • Reduce inflammation • Prevent colonization of foodborne pathogens • Reduce shedding of pathogens • Increase disease
    35. 35. SESSION 4 Alternatives to Antibiotics to Promote Growth in Livestock, Poultry, and Aquaculture Production Aim of this session was to explore novel approaches that can be used as alternatives for antimicrobial growth promoters during food-animal production. Another key aim was to improve knowledge on mechanisms of action of the growth-promoting characteristics of the proposed approaches.
    36. 36. Antibiotics growth promoters (AGP): Antibiotics growth promoters (AGP) How do they How do they work? work? Anti-inflammatory Anti-microbial Dibner, J.J., & Richards, J.D. 2005. Poul. Sci. 84:634-643. Niewold, T.A. 2007. Poul. Sci. 86:605-609.
    37. 37. Phytochemicals for growth promotion Conclusions • The use of phytochemicals in animal feeds to enhance growth and feed efficiency is a potential alternatives strategy to antimicrobial growth promoters • Phytochemicals are very diverse molecules and an increasing number of publications are documenting their efficacy • Research to understand their mode of action is paramount to achieve improved product consistency, consumer acceptance, and global use
    38. 38. SESSION 5 Regulatory Pathways to Enable the Licensing of Alternatives to Antibiotics Aim of this session was to review regulatory pathways in different regions of the world to license alternatives to antibiotics. Regulatory challenges that are faced in taking new molecules from discovery to commercial production was reviewed. Session also covered how to seek approval for labeling claims that a product is able to reduce use of antibiotics
    39. 39. CONCLUSIONS FROM SESSION 5 • Alternatives to antibiotics will be regulated as a drugs, biologics, feed additives • Alternatives to antibiotics must be developed according to national and international standards and meet requirements for efficacy, safety, and quality • Regulatory processes are in place to enable and facilitate the licensing of alternatives to antibiotics • Need to engage regulatory agencies early in the process
    40. 40. Conclusion: Antibiotics and their alternatives Allen HK, et al.Trends in Microbiology March 2013, Vol. 21, No. 3
    41. 41. Future Direction FUTURE DIRECTIONS • Integrating nutrition, health, and disease research will be driven by technological advances and the application of the “omic” fields in animal agriculture research • These technological advances will include new research tools and opportunities that afford scientists a hitherto unprecedented ability to discern the mechanisms by which alternatives to antibiotics can be effectively used to treat and prevent animals diseases
    42. 42. Future Direction FUTURE DIRECTIONS • There is a critical need for developing novel antimicrobials and alternative strategies for preventing and treating infectious diseases to safeguard the use of currently available antibiotics but importantly meet the challenges of antimicrobial resistance • We need to establish partnerships between academic and government researchers with the feeds and pharmaceutical industries and their regulators to enable the development of effective and safe alternatives to antibiotics
    43. 43. Future Direction FUTURE DIRECTIONS • Understanding the ecology of antimicrobial resistance will remain a critical endeavor • But we need to garner the support of stakeholders, funders, public and government institutions to pay equal attention to problem-solving, such as developing technologies that are more resillient to antimicrobial resistance and alternative strategies to enhance the health and welfare of animals.
    44. 44. Thank you! gov