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
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
www.ars.usda.gov/alternativestoantibiotics
What are alternatives to antibiotics?
Drugs, biologics, biotherapeutics, feed additives
1. Alternatives: Lessons from nature
2. Immune modulation approaches
3. The gut microbiome – microbial ecology
4. Alternatives for growth promotion
5. Regulatory pathways to enable licensing
of novel alternatives to antibiotics
Symposium Organization
Five 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
www.ars.usda.gov/alternativestoantibiotics
Plant virus e.g., Tobacco mosaic virus
Bacteriophage e.g., T1, T2, T4,
Animal virus e.g., Influenza, HIV
Human
Poultry Swine
Bacteriophage
Bacteria Specific
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
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 feed
Jae-Won Kim, CJ Research Institute of Biotechnology, CJ Cheiljedang, Seoul, Republic
of Korea
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
Bacterial cell wall and murein hydrolases from Hermoso et al., Curr Opin Microbiol 10:461, 2007
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
Bacteriophage Gene Products
Gel Electrophoresis and Spot Assay with
Clostridium perfringes Bacteriophage Lysin
Lysin
Lysostaphin Lysozyme
Control
Lane 1 – molecular weight markers
Lane 2 – induced E. coli lysate
Lane 3 – nickel purified phage lysin
Spot assays are conducted with E. coli
lysate or purified recombinant lysin as
compared to lysostaphin and lysozyme
NOTE clear zone of complete lysis
20kD
25kD
C. perfringens
Strain 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 Virol
156:25, 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
Neutrophils
Macrophages
NK-cells
Enzymes
Host Defense Peptides
Collectins
“The first line of defense against infection”
Chicken Cathelicidin 2 (Cath-2)
• Cationic
• 26 amino acids
• Amphipathic
C-RFGRFLRKIRRFRPKVTITIQGSARF-NH2
Hinge region
N
C Van Dijk et al., 2005
Xiao et al., 2006
Henk P. Haagsman
Department of Infectious Diseases and Immunology, Utrecht University, The Netherlands
Heterophils
Mononuclear
Cells
Giemsa Anti-CATH-2 Van Dijk et al., 2009
Chicken Cathelicidin 2 (Cath-2)
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 with
CATH-2 significantly reduced Salmonella survival in the crop
Development of Cathelicidin 2
Challenges
• 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 (AMPs)
Conclusions
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 88:562-566 Lee, 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 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.
www.ars.usda.gov/alternativestoantibiotics
SESSION 2
Immune Modulation Approaches to Enhance
Disease 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 receptors PRRs TLRs NLRs RIG-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 resistance Inflammation
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 ppm carvacrol
3 ppm 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
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
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
www.ars.usda.gov/alternativestoantibiotics
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.
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
Metabolomics
DNA Microbiome or
Virome
RNA Metagenomics
Metatranscriptomics
16s Survey
Microbiome and Metagenomic Analysis
The ruminal microbiome and animal health
John Wallace, Rowett Institute of Nutrition and Health, UK
Microbial 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.
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.
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
www.ars.usda.gov/alternativestoantibiotics
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-microbial Anti-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)
How do they work?
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
www.ars.usda.gov/alternativestoantibiotics
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:
Antibiotics and their alternatives
Allen HK, et al.Trends in Microbiology March 2013, Vol. 21, No. 3
Future 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 used to develop
strategies to enhance the health, productivity and welfare
of animals
FUTURE DIRECTIONS
Future Direction
• 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
Future Direction
• 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!
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