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Animal Health
Thursday, February 23, 2017 6:59:51 PM
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Secondary plant compounds and their antimicrobial properties to inhibit the growth of antibiotic resistant bacteria

 

T. Rothstein and I. Heinzl, EW Nutrition GmbH

 

 

Abstract


After the European ban of antibiotics as growth promoters in animal production, there is higher interest in naturally derived and generally recognised as safe substances as an appropriate replacement. The emergence of antibiotic resistant pathogens makes the demand for alternatives even more urgent.


In contrast to plant extracts, that can vary considerably in the content of bioactive compounds and thus also in their efficacy, purified secondary plant compounds allow the production of a standardised blend and thus a reproducible mode of action. Appropriate mixtures should be composed with a broad efficacy against major gram negative and positive pathogens.


In an attempt to determine the antimicrobial efficacy of Activo® Liquid, a water soluble mixture of standardized secondary plant compounds, we conducted in vitro experiments on major poultry pathogens by Agar Diffusion Tests. Here, the blend of secondary plant compounds showed an antimicrobial effect on all tested bacteria occurring in farm animals. The degree of growth inhibition positively correlated with the concentration of Activo® Liquid.


To further determine the antimicrobial efficacy in liquid medium, we tested Activo® Liquid against ESBL producing E.coli and Methicillin resistant S. aureus field isolates compared to their corresponding reference strains in a MIC assay.


The efficacy of Activo® Liquid against the tested strains could be demonstrated in a concentration dependent manner with antimicrobial impact at higher concentrations and bacteriostatic effects in concentrations up to 0,1% (ESBL) and 0,2% (MRSA).


These encouraging results give reason for further investigations with Activo® Liquid in animal production.

 

Introduction


Antibiotics were and still remain the method of choice against bacterial diseases. Due to their additional positive effect on growth performance they increasingly have been used in animal husbandry. But every use of antibiotics is a selection of resistant bacteria and especially this prophylactic use at low dosage gives bacteria a better chance to adapt. For this very prophylactic and metaphylactic use secondary plant compounds could be an alternative: they are generally recognized as safe and a lot of them show antimicrobial characteristics, e.g. Carvacrol and Cinnamon aldehyde. They effectively act against Salmonella, E. coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Entero- and Staphylococcus and Candida albicans (Baiju and Modak, 2007; Khan et a., 2009; Yossa et al., 2011) but also against multi drug resistant strains of Escherichia coli, Klebsiella pneumoniae and Candida albicans (Khan et al., 2009). Some compounds influence digestion and others act as antioxidants. An optimal combination has both - positive influence on health and performance. As secondary plant compounds act via several modes, the question was if they can also have an effect against antibiotic resistant bacteria like ESBL producing E. coli or MRSA.


Material and Methods


Trial I: in so called "agar diffusion tests" (method available on request), the sensitivities of reference strains –representative for the different species of pathogens - were evaluated with different concentrations of a specific blend of secondary plant compounds. The effectiveness of the active substances was determined by the extent to which they prevent the development of bacterial overgrowth. The diameter of the bacteria free zone around an applied component can be translated into the antimicrobial efficacy against a densely grown bacterial population on a petri dish - the bigger the bacteria free zone, the higher the antimicrobial effect.

 

Trial II: Farm isolates of four ESBL producing E. coli and two Methicillin resistant Staphylococcus aureus strains were compared to nonresistant reference strains of the same species with respect to their sensitivity against Activo® Liquid. In a Minimal Inhibitory Concentration Assay (MIC) under approved experimental conditions (Vaxxinova Diagnostic, Muenster, Germany) the antimicrobial efficacy of Activo® Liquid in different concentrations was evaluated.

 

Results


Trial I: In this trial, the blend of secondary plant compounds and organic acids (Activo® Liquid) showed an antimicrobial effect on all tested bacteria occurring in farm animals. The degree of growth inhibition positively correlated with the concentration of Activo® Liquid (see table 1).

 

Table 1:  Inhibition of field isolated standard pathogens by different concentrations of Activo® Liquid

 
   
Secondary Plant Compounds
(Activo® Liquid)
  Central Poultry Diagnostic Laboratory, Kondapur, Hyderabad (India)

10%

2%

1%

 

E. coli(reference strains)

++

+

+

 

Proteus vulgaris(reference strains)

+

+

+

 

Pseudomonas fluorescens

++

+

-

 

Salmonella pulmorum

++

++

+

 

Salmonella gallinarum

++

++

+

 

Staphylococcus aureus(reference strains)

+++

++

++

  +++ 22 – 29 mm ZOI (zone of inhibition)
   ++ 15 – 21 mm ZOI
    + 10 – 14 mm ZOI
-     < 10 mm ZOI.

 

Trial II: The efficacy of Activo® Liquid against the tested strains could be demonstrated in a concentration dependent manner with antimicrobial impact at higher concentrations and bacteriostatic efficacy in dilutions up to 0,1% (ESBL) and 0,2% (MRSA)(see table 2).

 

Table 2:  Efficacy of secondary plant compounds (Activo® Liquid) against ESBL producing E. coli and MRSA

 

   

Secondary Plant Compounds
(Activo® Liquid)

 

Laboratory: Vaxxinova, Münster, Germany

0.10%

0.20%

0.40%

1%

 

E.coliReference ATCC25922

+

++

++

++

 

ESBL 1 (Pig)

-

++

++

++

 

ESBL 2 (Pig)

+

++

++

++

 

ESBL 3 (Poultry)

+

++

++

++

 

ESBL 4 (Poultry)

-

++

++

++

 

S. aureusReference ATCC29213

-

+

++

++

 

MRSA 1 (Pig)

-

+

++

++

 

MRSA 2 (Pig)

-

+

+

++

    -   no effect 
  +  growth inhibiting
 ++  bactericide  

 

Conclusions


In order to contain the emergence and spread of newly formed resistance mechanisms it is of vital importance to reduce the use of antibiotics. A general rethinking is necessary to rise to the challenge and give new approaches a chance. These approaches however, will only be successful in combination with good management practices. Antibiotics must not be used for growth promotion or metaphylactic treatment, but only as a pure curative instrument. In In vitro-trials the liquid blend of secondary plant compounds and organic acids (Activo® Liquid) showed very antimicrobial effects against prevalent livestock pathogens. This gives reason for further investigations with secondary plant compounds in animal production in order to use them for pro- and  metaphylaxis and to reduce the use of antibiotics with their help in the future. The positive influence on performance parameters, as shown in many other trials, is an additional incentive for farmers to use these flavoring substances in the feed.


The high efficacy of secondary plant compounds against ESBL Producing Escherichia coli and Methicillin resistant Staphylococcus aureus (MRSA) could be a further step towards the reduction of antibiotic use. Due to the specific mode of action of antibiotics, resistance mechanisms are more likely to emerge. To support antibiotic treatment, Activo® Liquid,  a blend of natural compounds with broad spectrum efficacy probably can be a safe supplement for the control of pathogenic organisms.

 

 

For more of the article, please click here.

 

Article made possible through the contribution of T. Rothstein, I. Heinzl and EW Nutrition GmbH

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