Téllez S., López C., Sujka E., Martínez A.

LIPIDOS TOLEDO S.A. (LIPTOSA)

FEED ADDITIVES 2017, Frankfurt 29th September 2017

Antimicrobials are used in livestock production to maintain health and

productivity. These practices contribute to the spread of drug-resistant pathogens

in both livestock and humans, posing a significant public health threat (Van Boeckel

et al., 2015).

GLOBAL AND EMERGING PUBLIC HEALTH CRISIS OF ANTIBIOTIC RESISTANCE NEW

POLICIES

o The practice without using in food animal

production has been implemented in the European Union countries since 2006 and

more countries are expected to follow.

o Increasing regulation of antimicrobial (treatment and

prophylaxis)

Current use of antimicrobial in livestock (THE REVIEW ON ANTIMICROBIAL RESISTANCE, 2015)

Antimicrobials in swine production

Evolution of PCU in EU (EMA, 2016)

New challenges in EU:

o Ban of the use of at medicinal levels in piglet feed.

o Significant reduction of use.

searching for alternatives to antibiotics:

o WITH

→ similar antimicrobial and growth-promoting effects

→ profitable results

o WITHOUT inducing:

→ bacterial resistance and tolerance

→ potential side effects to animals

→ toxicity to humans

→ residues in foods of animal origin

→ environment impact

Organic acids, enzymes, probiotics, prebiotics, antimicrobial peptides and

phytogenic compounds (including essential oils, botanicals and herbal

extracts) have been widely recognized as potential alternatives to antibiotics in

feed (Yang et al., 2015).

Essential oils (EOs) are aromatic oily liquids obtained from plant material (flowers,

buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots) (Burt, 2004; Calo et al.,

2015)

They can be obtained by different methods: cold pressing, fermentation,

extraction or steam distillation.

EOs are highly complex mixtures of individual aroma compounds: terpene

compounds (mono-, sesqui- and diterpenes), alcohols, acids, esters, epoxides,

aldehydes, ketones, amines and sulfides.

Composition of EOs depend on:

o Species and subspecies

o Part of the plant

o Harvesting season

o Geographical source

o Extraction method

~ 3000 EOs are known, of which about

300 are commercially important.

The greatest use of EOs in the EU is in

food/feed (as flavourings), perfumes

and pharmaceuticals.

GRAS (Generally Recognized As Safe)

ANTIMICROBIAL PROPERTIES:

, antiviral,

antimycotic, antitoxigenic,

antiparasitic and insecticidal

o Phenolic components

o Minor components

Major components of some EOs that exhibit antibacterial

properties (Burt, 2004)

MODE OF ANTIBACTERIAL ACTION;

o Still not completely understood

o Hydrophobicity

o Main mechanisms of action:

(Faleiro, 2011)

→ Cell wall and membrane disturbance

→ Leakage of cell contents

→ Alterations on intracellular and

external ATP balance

→ Synthesis of heat shock proteins (HSPs)

→ pH disturbance

→ Intracytoplasmic changes (coagulation)

→ Damage to DNA

→ Activity anti-Quorum sensing

Mechanisms of action of essential oils and their components in a bacterial cell (adapted from Burt 2004).

EOs are more active against Gram-positive than Gram-negative bacteria (Burt, 2004)

Determination of the antimicrobial activity

o Bacteriostatic/ Bactericide

o Difficult (volatile properties, insolubility in water)

o Need for development and implementation of standardized tests in vitro

o Comparison of published data is complicated

In vitro to in vivo extrapolation of results

Protection of EOs for enhancing their antimicrobial effect

SYNERGISM BETWEEN:

o Different components of EOs whole EOs have a greater antibacterial activity

than the sum of the individual effects of the different components

o EOs and antibiotics

o EOs and

To evaluate the efficacy of a complementary feed based on a combination of essential

oils and organic acids against several respiratory, digestive and skin swine bacterial

pathogens.

Combination initially designed against S. suis. Include 6 EOs (including Origanum

vulgare and Syzygium aromaticum oils), organic acids and their salts.

VISAVET Health Surveillance Centre (Complutense University, Madrid)

MIC determination by Broth Micro-Dilution Method (CLSI, 2013)

31 clinical strains (porcine origin):

BACTERIAL PATHOGEN STRAINS ORIGIN DISEASE/LESIONS

Streptococcus suis

Articulation (n=2)

Brain (n=7)

Heart (n=2)

MENINGITIS, ARTHRITIS, PNEUMONIA,

ENDOCARDITIS

Staphylococcus aureus Articulation (n=4) EPIDERMITIS, ARTHRITIS

Staphylococcus hyicus Articulation (n=2)

Brain (n=2) EXUDATIVE EPIDERMITIS

Haemophilus parasuis

Heart (n=1)

Lung (n=1)

Brain (n=1)

GLÄSSER DISEASE

Pasteurella multocida Lung (n=3) PNEUMONIA, PERICARDITIS, PLEURITIS

Escherichia coli Liver (n=2)

Brain (n=2) SEPTICEMIA, COLIBACILOSIS, EDEMA

DISEASE

Actinobacillus pleuropneumoniae Heart (n=1)

Lung (n=1) PNEUMONIA AND PLEURITIS

BACTERIAL PATHOGENS No.

STRAINS

Number of strains with MIC (mg/ml) of MIC

(average)

(mg/ml) 0.5 1 1.5 1.75 2 2.5 3 > 3

Staphylococcus aureus 4 4 3

Staphylococcus hyicus 4 4 3

Streptococcus suis 11 5 1 3 1 1 1,88

Haemophilus parasuis 3 1 1 1 1,16

Pasteurella multocida 3 3 1

Escherichia coli 4 4 > 3

A. pleuropneumoniae 2 2 > 3

MIC ≤ 2 mg/ml MIC = 3 mg/ml MIC > 3 mg/ml

Pasteurella multocida Haemophilus parasuis Streptococcus suis **

Staphylococcus hyicus Staphylococcus aureus

Escherichia coli Actinobacillus pleuropneumoniae

Note: Gram +; Gram -

Diversity of published data: (i) evaluation method, (ii) bacteria spp and isolates (clinical or

reference) and (iii) EO/EO component.

PLANT OF ORIGIN MICRO-ORGANISM MIC VALUE (mg/ml) REFERENCE

Origanum vulgare (Oregano)

E. coli S. aureus

1.6-1.8 0.8-0.9

Chouhan et al., 2017

S. aureus 1.25 De Souza et al., 2009 (from Yang et al., 2015)

S. aureus E. coli

5 5

Da Silviera et al, 2011 (from Yang et al., 2015)

E. coli S. aureus

0.5-1.2 0.5-1.2

Burt, 2004

Syzygium aromaticum (Clove)

E. coli S. aureus

1 1

Jagadeesh Babu et al., 2011

E. coli 0.062 Seongwei et al., 2009 (from Yang et al., 2015)

E. coli S. aureus

0.4-2.5 0.4-2.5

Burt, 2004

No data found about specific swine pathogens

The combination is effective against specific species of Gram + and - bacteria

Considerations to take into account:

→ In vitro to in vivo extrapolation of MIC values

→ Protection of EOs and organic acids

→ Pharmacokinetics and pharmacodynamics (oral route)

→ Bacterial pathogenesis

→ Selection of in vivo parameters to determine efficacy:

• Prevalence of disease/lesions/symptoms

• Production indices

• Others

Good results in vivo (data not shown)

→ Reducing incidence of S. suis and H. parasuis in farms

→ Improving production parameters

→ Trials currently running

LIVER

GALL BLADDER

ESOPHAGUS

STOMACH pH 1.4-2.1

LARGE INTESTINE pH 6.5-7.4

SMALL INTESTINE pH 4.4-6.6

RECTUM

APPENDIX

PANCREAS

To evaluate the efficacy of a complementary feed based on a combination of essential

oils and organic acids against Brachyspira hyodysenteriae.

Include 5 EOs (including Origanum vulgare and Cinnamomum zeylanicum oils),

organic acids and their salts.

Animal Disease Diagnostics Center, College of Veterinary Medicine, NCYU (Taiwan)

MIC determination by Agar Dilution Method (CLSI, 2016)

Strains: 56 clinical isolates of Brachyspira hyodysenteriae + B. hyodysenteriae B-78

(ATCC 27164)

BACTERIAL PATHOGEN No.

STRAINS

Number of isolates with MIC (μg/ml) of

62.5 125 250 500 1000 2000 4000 8000

Brachyspira hyodysenteriae 57 0 13 21 18 5

0

10

20

30

40

50

60

70

80

90

100

% iso

late

s

8000 4000 2000 1000 500 250 125 62.5

MIC50 = 250 μg/ml

MIC90 = 500 μg/ml

Few published data to compare (due to its fastidious growth characteristics)

o Vande Maele et al., 2015

o MIC data expressed in mM.

o Best results: MCFA (capric and lauric acid) and essential oil components (thymol,

carvacrol and cinnamaldehyde).

o Verlinden et al., 2013 (Brachyspira intermedia)

o Best results: Cinnamaldehyde (MIC= 80 μg/ml), carvacrol and thymol (MIC=160-320 μg/ml)

Good results in vivo (data not shown)

→ Reducing incidence of disease in farms

→ Improving production parameters

→ Trials currently running

Eubiotics and phytogenic compounds have a good potential as an alternative

to antibiotics in feed for food animal production (Yang et al., 2015).

→ The present study confirms the efficacy of a combination of essential oils

and organic acids against swine pathogens.

→ Further studies are crucial in order to determine the most efficient

combination of protected essential oils and organic acids, according to

bacteria species and their pathogenesis.

It is difficult to conduct systematic and comprehensive evaluations toward the

efficacy and safety of phytogenic compounds due to their complex

composition (Yang et al., 2015).

→ Better understanding of their mechanisms of action and interaction with gut

microbiota, gut physiology and microbiology.

→ Standardized efficacy methods in vitro, in vivo

→ GRAS evaluation

Regulation EC 1831/2003

o Flavourings (2b):

o natural or corresponding synthetic chemically define

flavourings (f.e.: carvacrol)

o natural products – botanically defined (f.e: Oregano oil)

o Other zootechnical additives (4d)

RE-AUTHORIZATION maximum and recommended

levels, incompatibilities, contraindications, target species, etc.

HOLDER SPECIFIC AUTHORISATION

References available upon request