CSI: Investigating and solving disease challenges without antibiotics

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CSI Investigating and solving disease
challenges without antibiotics

• Dan Rosener, DVM
• Agtech Products, Inc.

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Company Specific Inoculant (CSI)

• Tailor-made Direct-fed Microbials (DFMs) designed
  to control a wide assortment of pathogenic
  organisms and enhance productivity using a
  variety of molecular biotechnology tools, in
  vitro inhibition assays and Bio-Numerics
  software

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DFM Mode of Action - Overview

• Competitive exclusion
• Competition for attachment sites /or nutrients
• Prevention of colonization of pathogens by
  production of
• Organic acids
• Hydrogen peroxide
• Antimicrobial peptides (bacteriocins)
• Production of enzymes, vitamins or other growth
  factors
• Metabolism detoxification of undesirable
  compounds
• Immunomodulatory effects

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Bacteriocin Background

• First discovered in 1925
• Protein antibiotics of the colicin type, i.e.
  molecules characterized by lethal biosynthesis,
  predominantly intraspecies killing activity, and
  adsorption to specific receptors on the surface
  of bacteriocin-sensitive cells
• Anti-microbial peptides produced by bacteria
  which kill or inhibit the growth of other
  bacteria
• Bio-preservative in food industry

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Bacteriocin Modes of Action

• Cationic, surface-active agents that disrupt the
  structure of cell membrane phospholipids and
  increase cell permeability by a detergent-like
  action
• Inhibit bacterial protein synthesis by
  interfering with peptidyl transferase activity in
  the ribosome

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Bacillus-based bacteriocins

• Polymyxin (B. polymyxa)
• Bacitracin (B. subtilis)
• Subtilin (B. subtilis)
• Subtilosin A (B. subtilis)
• Bacillocin (B. licheniformis)
• Thuricin (B. thuringiensis)
• Megacin (B. megaterium)

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CSI Investigating and solving disease
challenges without (conventional) antibiotics

• Dan Rosener, DVM
• Agtech Products, Inc.

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CSI Development Process
Isolation and Characterization of target
organisms from Company Farrowing Units
Plate for target organisms
Swabs
Pick 3-5 colonies of each/plate
Isolate DNA
Identify Clusters of Target Organisms
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RAPD PCR
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CSI Development Process
Isolation and Characterization of target
organisms from Company Farrowing Units
Plate for target organisms
Swabs
Pick 3-5 colonies of each/plate
Isolate DNA
Identify Clusters of Target Organisms
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Sample C. difficile Dendrogram
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Sample C. difficile Dendrogram
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The C.S.I. Process
Identify Bacillus Strains to Inhibit Specific
Pathogenic Bacteria
Incubate tubes () control tube clostridia
only Treated tubes clostridia strains
Bacillus supernatant
(-) control tube only broth
Tubes Seeded with Company Specific
Pathogens
Bacillus supernatant A
Control
Treated
Treated
Control
Bacillus supernatant B
Clostridia strain
Bacillus B
Control
Bacillus A
- Control
Bacteriocins
-

Measure optical density of control and treated
wells
Isolates
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An evaluation of various B. subtilis genotypes
against various C. difficile

• Introduction
• Not all C. difficile isolates are alike
• Have varying sensitivity patterns against
  commercial antibiotics and bacteriocins
• Not all B. subtilis isolates are alike
• Different isolates produce different bacteriocins
  with varying spectrums of activity

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Objective

• The objective of this study was to evaluate the
  antagonistic activity of six different
• B. subtilis isolates and their bacteriocins
  against six different C. difficile isolates.

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Methods

• C. difficile strains were isolated from piglets,
  tested using Multiplex PCR to verify the presence
  of toxin genes, and then analyzed using RAPD PCR
  genotyping.
• Random Amplified Polymorphic DNA (RAPD) PCR
  typing yields unique banding patterns which are
  used to DNA fingerprint the various genotypes.
• RAPD sub-typing facilitates the study of
  diversity changes over time and also serves as a
  basis for sub-sampling within large populations.
  
• Bio-Numerics (a software program that uses
  algorithms to study the banding patterns of the
  different DNA fingerprints) was utilized to form
  a dendrogram.

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Methods (cont.)

• Six C.difficile test strains were chosen from the
  six largest clusters in a comprehensive
  dendrogram representing over 1000 isolates.
• Bacteriocins were collected from the supernatant
  of six different B. subtilis fermentations.
• Assays were set up in duplicate 48 well plates
  with positive and negative controls. Plates were
  incubated anaerobically for 24 hours and
  inhibition of clostridial growth was determined
  by light absorbance measured using a microplate
  reader.

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Percent inhibition of C. difficile
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Summary

• When present, most farms have multiple sub-types
  of C. difficile.
• There are numerous different genotypic strains of
  Bacillus subtilis producing a wide variety of
  bacteriocins.
• Bacteriocins produced by B. subtilis are capable
  of inhibiting the growth of C. difficile, but not
  all bacteriocins are equally effective.

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Features

• Heat stable
• Premix stable
• Easy to use
• All natural
• Safe
• Research Proven
• Specific Strains

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• Thank you!