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

Alternatives to Antibiotics:Recent scientific development

Cyril G. Gay, DVM, Ph.DNational Program Leader

Animal Production and ProtectionAgricultural Research Service

United S Department of [email protected]

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• Increased regulation – ban of some

antibiotics

• Examination of alternatives to conventional antibiotics is warranted

www.ars.usda.gov/alternativestoantibiotics

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.

Symposium Objectives

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


What are alternatives to antibiotics?

Drugs, biologics, biotherapeutics, feed additives

1. Alternatives: Lessons from nature2. Immune modulation approaches 3. The gut microbiome – microbial ecology4. Alternatives for growth promotion5. Regulatory pathways to enable licensing

of novel alternatives to antibiotics

Symposium OrganizationFive Major Topics

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.

Alternatives to Antibiotics: Lessons from Nature

SESSION 1


Plant viruse.g., Tobacco mosaic virus

Bacteriophagee.g., T1, T2, T4,

Animal viruse.g., Influenza, HIV

Human

Poultry Swine

BacteriophageBacteriophage

Bacteria Specific Bacteria Specific

BacteriophageBacteriophage

• 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

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 FDA approved E. coli phage for using on food

• Possible therapy for multi-drug-resistant strains of bacteria

Bacteriophages: the alternatives to antibiotics for animal feedJae-Won Kim, CJ Research Institute of Biotechnology, CJ Cheiljedang, Seoul, Republicof Korea

BacteriophageChallenges

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 MechanismsLabrie et al. Nat Rev Micro 8(5):317, 2010

Bacterial cell wall and murein hydrolases from Hermoso et al., Curr Opin Microbiol 10:461, 2007

Amidases

Muramidase (Lysozyme)

Glucosaminidases

Endopeptidases

Holins form lesions inbacterial membranes are involved with release of phage

Autolysins and prophage enzymes present in genomesof bacteria

Bacteriophage Gene Products

Gel Electrophoresis and Spot Assay with Clostridium perfringes Bacteriophage Lysin

Lysin

Lysostaphin Lysozyme

Control

Lane 1 – molecular weight markersLane 2 – induced E. coli lysateLane 3 – nickel purified phage lysin

Spot assays are conducted with E. colilysate or purified recombinant lysin ascompared to lysostaphin and lysozymeNOTE clear zone of complete lysis

20kD

25kD

C. perfringensStrain 39 host

Seal et al. Arch Virol 156:25, 2011

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 cost-effective expression system

Seal et al. Arch Virol156:25, 2011

Schmelcher et al. Appl

Environ Microbiol.

78(7):2297-305. 2012

Animal-derived antimicrobial peptides (APM)

“Innate host defense”

• Present in all organisms

• Ancient Snake: cathelicidin Frog: plastrin Insect: cecropin Bacteria: bacteriocins

• Limited repertoire

• Rapid acting

• Broad specificity

• Constitutive and Stimulated

Cellular defenses

Effector molecules

NeutrophilsMacrophagesNK-cells

EnzymesHost Defense PeptidesCollectins

“The first line of defense against infection”

Chicken Cathelicidin 2 (Cath-2)

• Cationic

• 26 amino acids

• Amphipathic

C-RFGRFLRKIRRFRPKVTITIQGSARF-NH2

Hinge region

N

CVan Dijk et al., 2005Xiao et al., 2006

Henk P. Haagsman Department of Infectious Diseases and Immunology, Utrecht University, The Netherlands

Heterophils

Mononuclear Cells

Giemsa Anti-CATH-2Van Dijk et al., 2009

Chicken Cathelicidin 2 (Cath-2)

Chicken Cathelicidin 2 ConclusionsConclusions

• 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 with CATH-2 significantly reduced Salmonella survival in the crop

Development of Cathelicidin 2 Challengeshallenges

• Improve specific activities of compounds

• Improve stability of lead structures

• Development carrier/delivery systems

• Pharmacokinetics

• Cost-effective large scale production

• 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

• More attention needs to be paid to how to enhance the

expression of endogenous AMPs through nutritional

regulation

Animal-derived antimicrobial peptides Animal-derived antimicrobial peptides (AMPs)(AMPs)

ConclusionsConclusions

Treatment strategy that will allow the maintenance of high-titer antibodies against parasites in the gut showed protective effects

Lee, SH., Lillehoj, HS. et al., 2009 Poultry Science 88:562-566Lee, SH., Lillehoj, HS. et al., 2009 Veterinary Parasitology 163-123-126

385

390

395

400

405

410

415

420

body weight (gm)

Control Eimeria IgY Low IgY High

Passive immunity using hyperimmune IgYPassive immunity using hyperimmune IgY

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.


SESSION 2

Immune Modulation Approaches to EnhanceDisease Resistance and Treat Animal Infections

Bali Pulendran and David Artis. 2012, New Paradigms in Type 2 Immunity. Science 337, 431.

Diversity of stimuli can bind host conserved recognition receptors (PRRs= Pattern Recognition Receptors) and stimulate immune response

Innate sensing receptorsPRRs TLRs NLRsRIG-1

Some phytochemicals (essential oils) interact with host PRRs and initiate inflammatory immune response

PRRs:TLRs, NODs

Bioactive phytochemicals

DAMPs(Necrotic cell products)

PAMPs(Pathogens, diets)

Innate immune response

Dysregulated immune response

-Host resistance-Wound healing

-Cancer-Atherosclerosis-Insulin resistanceInflammation

Level of PRR activation

PRRs and downstream signaling components are molecular targets for dietary intervention strategies using phytochemicals to reduce PRR-mediated inflammation

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

Phytochemicals – Plant Extracts

Gene Ontology categories 5 ppmcarvacrol

3 ppmcinnamaldehyde

2 ppmcapsicum

N of genes which expression…

- is altered 74 62 254

- is up regulated 26 31 98

- is down regulated 48 31 156

Examples of GO Categories for:

- 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

KIM, DK, Lillehoj, HS, et al., 2010. Poul Sci 89:68-81

Functional Genomics

PhytochemicalsPhytochemicalsChallengesChallenges

• 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


SESSION 3

The Gut Microbiome andImmune 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.

BacteriotherapyBacteriotherapy

• 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

Metabolomics

DNAMicrobiome or

Virome

RNAMetagenomics

Metatranscriptomics

16s Survey

Microbiome and Metagenomic Analysis

The ruminal microbiome and animal healthJohn Wallace, Rowett Institute of Nutrition and Health, UK

Microbial genomicsMicrobial genomics

• Genomics and metagenomics approaches to understand host-environment-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.

ProbioticsProbiotics

• 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.

Gastrointestinal Microbiome

• Bacteria, fungi, viruses, protozoa, helminths

• Bacteria 1011 cells/gram• Firmicutes• Bacteroidetes• Proteobacteria

• Bacteria primarily associated with mucus and macromolecular food matrix (fiber)

• Composition varies• In different section of

GI• In different animals

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 resistance


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.

Antibiotics growth promoters (AGP): How do they work?

Anti-microbialAnti-inflammatory

Dibner, J.J., & Richards, J.D. 2005. Poul. Sci. 84:634-643.Niewold, T.A. 2007. Poul. Sci. 86:605-609.

Antibiotics growth promoters (AGP) Antibiotics growth promoters (AGP) How do they work? How do they work?

Phytochemicals for growth Phytochemicals for growth promotionpromotion

ConclusionsConclusions• 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


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

• 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

CONCLUSIONS FROM SESSION 5

Conclusion:Conclusion:Antibiotics and their alternativesAntibiotics and their alternatives

Allen HK, et al.Trends in Microbiology March 2013, Vol. 21, No. 3

Future DirectionFuture Direction

• 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

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

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.

FUTURE DIRECTIONS

Thank you!Thank you!

[email protected]@ars.usda.gova.gov

www.usda.ars.gov


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

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