Synergy of antibiotics and phytogenics

Many veterinary antibiotics are applied via the waterline, where they are dosed in combination with other feed additives. Amongst those there are mixtures of secondary plant compounds with a proven antimicrobial efficacy against veterinary pathogenic bacteria. However, there is only sparse literature available if and to which extent antibiotics and phytogenics influence each other. There are several scenarios possible:

• No interaction: if the bacterial strain is sensitive to both, antibiotics and phytogenics, and they do not influence each other in their antimicrobial efficacy, then a combined application with their recommended dosages is possible without restrictions and limitations.
• Negative interaction: if the combined use of antibiotics with phytogenic feed additives attenuates the efficacy of antibiotics, a combined application would not be recommended.
• Positive interaction: if phytogenics increase the effectiveness of antibiotics, a combined application of both might generate a synergistic antimicrobial effect on certain veterinary pathogenic bacteria.

The objective of this study was to investigate whether and how a defined composition of secondary plant compounds influences the efficacy of diverse classes of antibiotics against E. coli reference laboratory strains and respective field isolates. Veterinary antibiotics were selected based on the official data of the German Federal Office of Consumer Protection and Food Safety (BVL) about amounts of applied veterinary antibiotics from 2011 to 2014.

Inhibitors of cell wall synthesis (penicillins and cephalosporins), polypeptide antibiotics interfering with bacterial cell membrane integrity (polymyxins), inhibitors of protein synthesis (tetracyclines, aminoglycosides, phenicols, macrolides and lincosamides), gyrase inhibitors (enrofloxacin) and inhibitors of RNA synthesis (ansamycins), were included in the analysis (Figure 1).

Figure 1 – Points of action for different classes of antibiotics.

Influence of secondary plant compounds

Four E. coli field isolates from poultry flocks were selected to determine their antibiotic resistance profile and their susceptibility towards a defined mixture of secondary plant compounds (SPC Mix). As quality control strains for antimicrobial susceptibility testing, the beta-lactamase negative E. coli strain ATCC 25922 (DSM1103) and the beta-lactamase positive E. coli strain DSM 22664 were used.

To redetermine antimicrobial susceptibility of the selected E. coli strains, microdilution procedures according to Clinical and Laboratory Standards Institute, document M07-A09 (ISO 20776-1) were performed with the following modifications: in addition to serial two-fold dilutions of respective antibiotics, a coincubation with 2-fold serial dilutions of a defined composition of secondary plant compounds (Activo Liquid) was also applied to determine the influence of these mixtures on the effective antibiotic dose. The quality control ranges for the antibiotics mentioned were chosen according to Clinical and Laboratory Standards Institute, document M100-S24 (Performance Standards for Antimicrobial Susceptibility Testing), or determined individually for respective E. coli strains.

Efficacy when used in combination

As shown in Figure 2, minimum bacterial concentration (MBC) determination for individual antibiotics confirmed the sensitivity of E. coli ATCC 25922 and the resistance of E. coli DSM 22664 against penicillins and cephalosporin. All four E. coli field isolates were shown to be resistant against the mentioned beta-lactam antibiotics. Strain DSM 22664 and field isolates 2 and 4 were also determined resistant against tetracyclines and the macrolide antibiotic tylosin. Aminoglycoside resistance was also observed for DSM 22664 and field isolate 1.

In all cases, where resistance against a certain antibiotic was determined, the SPC Mix was not able to recover sensitivity in the respective strain. However, the SPC Mix itself always maintained the same in vitro efficacy, irrespective of the concentration of the antibiotic against which a selected strain was resistant. Thus, no negative influence of the antibiotics on the efficacy of the SPC’s was observed even with high concentrations of beta-lactams, tetracylines, aminoglycosides and tylosin. No influence on the effective antimicrobial dose was observed when beta-lactam antibiotics or gentamicin were combined with the SPC Mix, even if the selected strain was sensitive against above mentioned antibiotics. In all other cases of antibiotic sensitivity, a positive effect with the SPC Mix could be quantified. For most antibiotic/SPC combinations the effective dosage of the respective antibiotic could be reduced by 2-fold, when the concentration of the SPC Mix was also reduced to half the amount, especially for tetracylines, tobramycin, chloramphenicol, tylosin and rifampicin.

Figure 2 – Influence of an SPC mix on the efficacy of selected veterinary antibiotics.

Grey boxes indicate no change in sensitivity, when antibiotics and SPC mix are applied in combination. Resistance towards a given antibiotic is indicated with R in red colour. Green boxes indicate an increase in sensitivity towards an antibiotic, when combined with the SPC mix. Grey to green boxes indicate, that not for all antibiotics of a given class, the sensitivity could be increased.

The most prominent beneficial effect was observed, when polypeptide antibiotics were combined with the SPC Mix. For the reference strains E. coli ATCC 25922, DSM 22664 and E. coli field isolate 1 an at least four-fold reduction of the effective antibiotic dose could be achieved with half or less of the effective dose of the SPC Mix. For the field isolates 3 and 4 the antibiotic concentration could be reduced by half the normal effective dose, even, when the concentration of the SPCs was reduced by 4-fold.

Combining makes sense

The in vitro results demonstrate, that for all tested antibiotics that represent a substantial share in veterinary use, no negative influence of both compound classes on each other was observed. Also 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 additional benefits to the already known improvements in animal performance and health, for which a number of phytogenic feed additives have already proven effective.

Author: Thilo Borchardt, Nadine Scholz-Neumann And Inge Heinzl, Ew Nutrition, Visbek, Germany