Secondary Plant Compounds (SPC’s) to reduce the use of antibiotics?

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Initial in vitro trials give reason for hope

Antibiotic Resistance

Some bacteria, due to mutations, are less sensitive to certain antibiotics than others. This means that if certain antibiotics are used, the insensitive ones survive. Because their competitors have been eliminated, they are able to reproduce better. This resistance can be transferred to daughter cells by means of „resistance genes“. Other possibilities are the intake of free DNA and therefore these resistance genes from dead bacteria 1, through a transfer of these resistance genes by viruses 2 or from other bacteria by means of horizontal gene transfer 3 (see figure 1). Every application of antibiotics causes a selection of resistant bacteria.  A short-term use or an application at a low dosage will give the bacteria a better chance to adapt, promoting the generation of resistance (Levy, 1998).

Antibiotics are promoting the development of resistance:

  • Pathogenic bacteria possessing resistance genes are conserved and competitors that do not possess these genes are killed
  • Useful bacteria possessing the resistance genes are conserved and serve as a gene pool of antibiotic resistance for others
  • Useful bacteria without resistance, which probably could keep the pathogens under control, are killed

Reducing the use of antibiotics
Ingredients from herbs and spices have been used for centuries in human medicine and are now also used in modern animal husbandry. Many SPC’s have antimicrobial characteristics, e.g. Carvacrol and Cinnamon aldehyde. They effectively act against Salmonella, E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Entero and Staphylococcus, and Candida albicans. Some compounds influence digestion, others act as antioxidants. Comprehensive knowledge about the single ingredients, their possible negative but also positive interaction (synergies) is essential for developing solutions. Granulated or microencapsulated products are suitable for addition to feed, liquid products would be more appropriate for an immediate application in the waterline in acute situations.

SPC’s (Activo® Liquid) against livestock pathogens in vitro
In “agar diffusion tests”, the sensitivity of different strains of farm-specific pathogens was evaluated with different concentrations of Activo® Liquid. The effectiveness was determined by the extent to which they prevented the development of bacterial overgrowth. The larger the bacteria-free zone, the higher the antimicrobial effect.

In this trial, Activo® Liquid showed an antimicrobial effect on all bacteria tested. The degree of growth inhibition positively correlated with its concentration.

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

 

Activo® Liquid against antibiotic resistant field pathogens in vitro
It cannot be excluded that resistant pathogens not only acquired effective weapons to render antibiotics harmless to them but also developed general mechanisms to rid themselves of otherwise harmful substances. In a follow-up laboratory trial, we evaluated whether the Activo® Liquid composition is as effective against ESBL producing E. coli and Methicillin resistant S. aureus (MRSA) as to non-resistant members of the same species.

Trial Design: Farm isolates of four ESBL producing E. coli and two MRSA 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.

The efficacy of SPC’s (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)(table 2).

Conclusion:
To contain the emergence and spread of newly formed resistance mechanisms it is of vital importance to reduce the use of antibiotics. SPC’s are a possibility to decrease antibiotic use especially in pro- and metaphylaxis, as they show good efficacy against the common pathogens found in poultry, even against resistant ones.

 

I. Heinzl 




Necrotic enteritis in poultry

chicks waterline

Enteric diseases cause significant economic losses due to decreased weight gain, higher mortality, higher feed conversion, higher veterinary costs and medicine and a higher risk of contamination by poultry products in food production. The losses due to necrotic enteritis mainly occurring in broilers and fattening turkeys in intensive floor or free-range management are put at 2 billion US$ per year.

After the ban of antibiotic growth promoters, the relevance of this formerly well controllable disease reappeared and increased.

Necrotic enteritis is a disease of the gut
It is caused by specific gram-positive, anaerobic bacteria – Clostridium perfringens, mostly Type A. Clostridia are found in litter, faeces, soil, dust and in healthy animals’ guts. These spore forming bacteria are extremely resistant against environmental influences and can survive in soil, feed, and litter for several years and even reproduce.
Clostridium perfringens is a component of the normal gut flora. It occurs in a mixture of diverse strains in a concentration of up to 105 CFU / g intestinal content. In animals suffering from necrotic enteritis particularly one strain of Clostridium perfringens is found in a much more higher concentration of 106-108 CFU / g.
Necrotic enteritis affects chickens and turkeys at the age of 2-16 weeks, proliferating at the age of 3-6 weeks. There is an acute clinical, and a subclinical form.
Birds suffering from the clinical form clearly show symptoms like a poor general state of health and diarrhoea. Mortality rates up to 50 % can occur. Subclinical necrotic enteritis cannot be diagnosed easily, as there are no clear symptoms. This form, however, stays within the flock and causes losses due to decreased growth.

Factors promoting an infection with necrotic enteritis should be avoided!
In general, factors have to be cited that create an intestinal environment favourable for the facultative anaerobic Clostridium perfringens or weaken the immune status of the host:

  1. Feed:
    Here NSP’s have to be mentioned. Undigested NSP’s serve as substrate and some of them cause higher production of mucus also serving as substrate and providing ideal anaerobic conditions. Undigested proteins due to high contents in the diet also serve as substrates. Animal protein and fat are worse than vegetable variants and a homogeneous size of particles in the diet is better than an inhomogeneous mixture.
  2. Stress
    Stresses such as feed change or high stocking density favour NE
  3. Diseases
    Immunosuppressive diseases such as infective chicken anaemia, Gumboro or Marek’s decrease resistance against intestinal infections and facilitate their colonisation. Some pathogens exert pressure on the gut and prepare the way for clostridia. Here Cryptosporidia and salmonella have to be mentioned.

New approaches
Secondary plant compounds show good results against the two microorganisms just mentioned. In a trial conducted with free range broilers in France, a combination of a vaccination against coccidia and a mixture of secondary plant compounds (Activo® liquid) resulted in a reduced occurrence of necrotic enteritis in the trial group compared to the control. Additionally due to an improved feed conversion, the margin per animal in the trial group was 5 Cent higher than in the control (1,44 € vs 1,39 €).
In an in vitro test, Activo® liquid also showed bactericidal efficacy against field isolated Salmonella pulmorum and Salmonella gallinarum at a 2 % concentration.
The trials show that combined with a good feeding and stress management, secondary plant compounds, could be a good tool to eliminate predisposing factors for necrotic enteritis and could therefore help control this economically important disease.




Secondary plant compounds are the new frontier in poultry nutrition

BROILER

Why should you read another story about phytogenics? Or, is it botanicals, spices, herbs, and extracts? No matter what we call them, scientists have named them “secondary plant compounds”, and if we are to follow the American tradition we can call them SPC. Then, here is the first interesting thing we can discuss about this plant-derived class of active compounds. They are “secondary” in nature, but not insignificant. They play no role in normal metabolism, but they help plants (and now animals) survive under adverse conditions. Perhaps, this is why some experts consider them as the next frontier in poultry nutrition. With poultry that are raised in less than ideal conditions, especially when we consider the movement towards antibiotic reduction (for growth promoting reasons, not complete removal of all medicines), we understand that such natural compounds can be of significant help.

As it happens, the majority of poultry specialists in Europe and increasingly in the Americas consider SPC as an almost-essential element in diets for broilers and layers (and turkeys, ducks, and all poultry for that matter) when birds are raised without antibiotics. Some go even further and use them along with antibiotics because, as we all know, antibiotics are never 100% efficient as bacteria sooner or later develop some form of resistance. Such resistance has not yet been observed with SPC. So if one is to use SPC in poultry feds, which ones to buy? A quick glance at the market will reveal more commercial products than can possibly be imagined. Some must be better than the rest, but how can we separate the wheat from the chaff? Price alone is not always a good indicator. A high quality product must be expensive – for there is no such thing as a free lunch – but all expensive products are not always of the highest possible quality!

There are three basic criteria, which we can mention briefly here:

  1. SPC are volatile – at least most of them. As such, unprotected products will soon evaporate if left in the open air as it happens with feed prepared in commercial farms. So, some form of protecting SPC is essential.
  2. SPC are innumerable – so finding the right mix for the job required is important. You cannot get the same results with any kind of mix. So, in designing an SPC mix, the manufacturer must declare and have knowledge of the target to be accomplished.
  3. SPC are powerful – meaning you cannot just keep adding as much as possible. Here finding the exact dosage for the right purpose is a difficult balancing exercise. So, the right mix and the right dosage must be combined, otherwise animals will refuse the feed (worst case scenario) or just fail to benefit from SPC inclusion.

There is so much more to learn about this exciting class of compounds that can replace the growth promoting action of antibiotics that it is worth spending time learning more about them.

 




Secondary plant compounds against antibiotic-resistant E. coli

Poultry drinking line

Due to incorrect therapeutic or preventive use of antibiotics in animal production as well as in human medicine, occurrence of antibiotic resistant pathogens has become a widespread problem. Enterobacteria in particular (e.g. Salmonella, Klebsiella, E. coli) possess a special mechanism of resistance. By producing special enzymes (ß-lactamases), they are able to withstand the attack of so-called ß-lactam antibiotics. The genes for this ability (resistance genes) can also be transferred to other bacteria resulting in a continuously increasing problem. Divers point mutations within the ß-lactamase genes lead to the occurrence of „Extended-Spectrum-Beta-Lactamases“ (ESBL), which are able to hydrolyse most of the ß-Lactam-antibiotics. AmpC Beta-Lactamases (AmpC) are enzymes, which express a resistance against penicillins, cephalosporins of the second and third generation as well as cephamycins.

What are ß-lactam antibiotics?
The group of ß-lactam antibiotics consists of penicillins, cephalosporins, monobactams and carbapenems. A characteristic of these antibiotics is the lactam ring (marked in orange):

Mode of action of ß-lactam antibiotic
If a bacterial cell is growing, the cell wall also has to grow. For this purpose, existing conjunctions are cracked and new components are inserted. ß-lactam-antibiotics disturb the process of cell wall construction by blocking an enzyme needed, the transpeptidase. If crosslinks necessary for the stability of the cell wall cannot be created, the bacteria cannot survive. Resistant bacteria, which are able to produce ß-lactamases, destroy the ß-lactam antibiotics and prevent their own destruction.

Secondary plant compounds
Secondary plant compounds and their components are able to prevent or slow down the growth of moulds, yeasts, viruses and bacteria. They attack at various sites, particularly the membrane and the cytoplasm. Sometimes they change the whole morphology of the cell. In the case of gram-negative bacteria, secondary plant compounds (hydrophobic) have to be mixed with an emulsifier so that they can pass the cell wall which is open only for small hydrophilic solutes. The modes of action of secondary plant compounds depend on their chemical composition. It also depends on whether single substances or blends (with possible positive or negative synergies) are used. It has been observed that extracts of spices have a lower antimicrobial efficacy than the entire spice.

The best explained mode of action is the one of thymol and carvacrol, the major components of the oils of thyme and oregano. They are able to incorporate into the bacterial membrane and to disrupt its integrity. This increases the permeability of the cell membrane for ions and other small molecules such as ATP leading to the decrease of the electrochemical gradient above the cell membrane and to the loss of energy equivalents of the cell.

Trial (Scotland)

Design
Two strains of ESBL-producing and AmpC respectively, isolated from the field, a non-resistant strain of E. coli as control. Suspensions of the strains with 1×104 KBE/ml were incubated for 6-7 h at 37°C together with different concentrations of Activo® Liquid or with cefotaxime, a cephalosporin. The suspensions were then put on LB-Agar plates and bacteria colonies were counted after a further 18-22h incubation at 37°C. Evaluation of the effects of Activo® Liquid on ESBL-producing as well as on E. coli resistant for aminopenicillin and cephalosporin (AmpC)

Results
The antimicrobial efficacy of the blend of secondary plant compounds depended on concentration with bactericidal effect at higher concentrations and bacteriostatic at dilutions up to 0,1%. It is also possible that bacteria could develop a resistance to secondary plant compounds; the probability is however relatively low, due to the fact that essential oils contain hundreds of chemical components (more than antibiotics) making it difficult for bacteria to adapt.

 

 

 




Phytogenics can positively influence the efficacy of antibiotics

phytgenics spice powder

Many veterinary antibiotics are applied via the waterline, where they are dosed in combination with other feed additives. Amongst those are mixtures of secondary plant compounds with a proven antimicrobial efficacy against veterinary pathogenic bacteria. However, little research has been done to evaluate any effect that antibiotics and phytogenics may have on each other. A possible influence of phytogenics on the efficacy of antibiotics through the combined administration would require a change in application recommendations of antibiotics and phytogenic feed additives. In the case of no interaction, no changes would be necessary. If they were to interact in a positive way, the dosages could be lowered and if they interact in a negative way, a combined application would be avoided.

Antibiotics and SPC’s in co-incubation
There are different groups of antibiotics depending on the chemical structure and on the pathogen they target. Some impair the cell wall or the cytoplasmic membrane (polymyxins, ß-lactam antibiotics) and some affect protein synthesis (macrolides, Chloramphenicol, Lincospectin, tetracyclines, aminoglycosides). Others compromise DNA and RNA synthesis (fluorchinolones, ansamycines) and some disturb the metabolism of e.g. folic acid (Trimethoprim).
The intention of a trial with these different groups of antibiotics was to evaluate possible interactions they may have with a combination of secondary plant compounds. Four ESBL producing E. coli field isolates from poultry flocks were experimentally assessed  as well as a ß-lactamase positive and a ß-lactamase negative reference strain as quality control strains for antimicrobial susceptibility testing.
Two-fold serial dilutions of antibiotics and the liquid product based on secondary plant compounds were co-incubated in a checkerboard assay. The highest concentration of the antibiotic was chosen according to CLSI standard recommendations. The control of the serial dilution of SPC’s was made without antibiotics and vice versa.

Lowering the antibiotic dosage by the use of SPC’s
In the experiment all field isolates proved resistant against the ß-lactam antibiotics, two field isolates and one reference strain were resistant against tetracyclines and macrolides and one field isolate and one reference strain against aminoglycocides.
The results showed that there was no negative influence of the antibiotics on the SPC’s and vice versa. Moreover, for several classes of antibiotics an additive to synergistic effect was observed to such an extent that an antibiotic effect could be achieved with half or even one quarter of the former effective dosage. The dosage of the SPC-mixture could also be reduced. Based on the results of this in vitro experiment it can be stated that in the case of antibiotic resistance, the option exists to apply a phytogenic product with broad antimicrobial efficacy. Even more, for most combinations between antibiotics and Activo® Liquid, a defined mixture of secondary plant compounds, their combined use potentiates the individual efficacy of either compound class against E.coli strains in vitro. This adds further benefits to the improvements in animal performance and health, for which a number of phytogenic feed additives have already proven effective.




A powerful alternative to antibiotics for Aquaculture

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Global aquaculture has grown dramatically and shrimp cultivation areas, in particular, have expanded. Unfortunately, the shrimp industry, in particular, faces major problems with bacterial diseases. One of the most important diseases in shrimp is vibriosis. Mortality rates of up to 100 % are possible, so economic losses can be devastating.

Characteristics of vibriosis
Vibriosis generally occurs in all life stages, but mainly in hatcheries. Vibrios are found as normal flora in the hepatopancreas of the healthy crustacean. They can turn from tolerated to pathogenic, if environmental conditions are compromised: e.g. over / underfeeding, overcrowding or decreased levels of oxygen.
The animals can be infected orally, through wounds in the exoskeleton or pores, the gills or the midgut. There are different expressions of the disease depending on which parts of the animal are affected (e.g. appendage and cuticular vibriosis).

Negative effects on the ecology
As antibiotics for shrimps are applied orally together with the feed, not all of them reach their target. An estimated 15-40 % are not ingested due to feeding falling to the bottom. A fraction of the ingested antibiotics is also not absorbed in the body and is excreted. All of these antibiotics stay in the water or sink to the bottom. The number of antibiotics that remain in the water or sediment varies from 1 % (chloramphenicol) up to 90 % (Oxytetracycline).
It is estimated that 70-90 % of antibiotics used in the therapy of farmed organisms end up in the environment and sediment and lead to the development of antibiotic resistance.

Secondary Plant Compounds (SPCs) – a good tool to reduce the use of antibiotics?
SPCs and their components are able to slow down or prevent the growth of molds, viruses, and bacteria. They impair them by acting at different parts/mechanisms of the cells (e.g. cell membrane, transport systems, cell contents, flagella development, quorum sensing…). The best-explained mode of action is one of thymol and carvacrol extracted from thyme and oregano. These substances are able to penetrate the bacterial membrane and disrupt its integrity causing loss of ions or energy equivalents.

Several trials conducted show the high efficacy of secondary plant compounds in aquaculture.

1. Scientific Trial (Kasetsart University, Thailand)

Design
a) 4 groups (6 replicates each) of White Leg Shrimp (L. vannamei) were housed in 100 L aquaria with 10 animals each.

Control: Standard feed, no additive
AB-Group: Standard feed + 10 ppm Enrofloxacin
Activo® Group 1: Standard feed + 100 g Activo®/t of feed
Activo® Group 2: Standard feed + 200 g Activo®/t of feed

Evaluation of mortality and specific growth

a) End of the feeding trial: stressing of the shrimp (high water temperature, 33°C for 1 hour), then challenge with Vibrio parahaemolyticus (7,6 x 106 cfu/ml) by subcutaneous injection.

Evaluation of mortality

a) Survival rates in the AB-Group (93,3 %) and in the Activo® Group 2 (90,0 %) were similar. The specific growth rate of the AB-Group (2,32 %/day) and the Activo® Group 2 (2,22 %/day) were higher than the control (1,94 %/day). The Activo® Group 1 (2,18 %/day) ranged performance-wise between the control and AB-Group.

b) After the challenge, mortality in the control group (43,3%) was approximately twice as high as in the AB-Group (20 %) and in the two Activo® groups (both 23,3%).

2. Field Trial (Shrimp farm Ecuador)

Design
Two ponds with 80.000 shrimps/ha
Control (3 ha): standard feed
Activo® Aqua Group (5 ha): standard feed + 2 kg Activo® Aqua (Activo® upgraded by immune system stimulating- mannan-oligosaccharides) /t of feed on top

Evaluation of average shrimp weight at regular intervals

Results
Activo® Aqua Group showed a consistently better development of body weight compared to the control, resulting in a shorter cultivation period until harvesting (112 compared to 123) and therefore a higher turnover of animals. Feed conversion in the Activo® Aqua group was better in every growth stage.


Both trials present secondary plant compounds as a good alternative to antibiotic growth promoters. In the case of disease, they decrease mortality. Under standard conditions, the improved development shortens time to harvest and increases the turnover. The improved feed conversion lowers feeding costs.