The discovery of antibiotics has been a major achievement in combatting bacterial diseases. Unfortunately, their widespread use led to the development and dissemination of a multitude of bacterial strains with resistances to clinically important antibiotics. Especially multi-resistant strains pose a threat to public health due to increasingly limited options in their treatment. To curb this development and guarantee the efficiency of life-saving antibiotics in the future, strategies to minimize the spread of antibiotic resistances need to be employed.
One important aspect in this context is the use of antibiotics in livestock production. In addition to their pharmaceutical application to both prevent and treat bacterial infections, antibiotics have long been used as antimicrobial growth promoters (AGP) in animal nutrition. However, to minimize the risk of antibiotic resistance development within the livestock industry, antibiotics are increasingly banned as AGP. Sweden was the first country to take this step back in 1986 (Casewell et al., 2003), with other countries following, including the member states of the EU in 2006 (1831/2003/EC).
Consequently, alternatives to AGP are increasingly being sought to prevent loss of productivity or an increase of intestinal and systematic bacterial infections in animal husbandry. One group of potential candidates are plant-derived bio-active compounds. This group of substances has no clear definition in the literature and several different terms such as phytogenics, phytochemicals, or botanicals are used for their description. In this study, they are referred to as phytogenics. Many phytogenics have been shown to exhibit antimicrobial properties (Burt, 2004; Reichling et al., 2009), which may be beneficial for animal health by preventing the growth of bacterial pathogens or disturbing the expression of their virulence factors. However, the high dosages of most phytogenics required for these effects are problematic in animal nutrition since many of these compounds are very odor- and taste-intensive, which can lead to feed refusal in animals, especially in pigs or ruminants. In addition, most phytogenics are rather expensive, which leads to uneconomical products when phytogenic feed additives are formulated with bactericidal concentrations. However, phytogenics exhibit a range of effects on bacteria at sub-MIC levels, which offer new strategies and solutions to support public health and animal welfare. In particular, the inhibitory effects of phytogenics on biofilm formation are of great significance as biofilm-associated microorganisms exhibit decreased susceptibility to antimicrobial agents (Donlan, 2001). There are still major gaps in the understanding of the modes of action phytogenics exert on microbial communities and their host, especially since most studies are conducted with type strains of relevant pathogens, which may differ in their response to phytogenics in comparison to strains that were isolated from the natural environment.
The present study aims to determine the effect of different phytogenics on selected
The phytogenics garlic oil (
Major chemical constituents of the studied phytogenics
Tabelle 1. Chemische Hauptbestandteile der untersuchten phytogenen Substanzen
Garlic oil | Diallyl disulfid (59.1%), diallyl trisulfid (11.7%), and methyl propyl trisulfid (1.1%) |
Thymol | Thymol (100%) |
Carvacrol | Carvacrol (100%) |
Thyme oil | |
Cinnamaldehyde |
Based on data from gas chromatography-mass spectrometry analysis provided by Delacon Biotechnik GmbH
Susceptibility testing of all
The test organisms were incubated in 10 ml Mueller-Hinton broth for 16–20 h at 37°C. The cultures were then diluted with fresh Mueller-Hinton broth (approx. 1:500 dilutions) to achieve a final bacterial inoculum of 5 × 105 to 2 × 106 CFU ml−1. 50 μl of the inoculum were added to each well to obtain a total test volume of 100 μl and final substance concentrations of 10000 ppm to 5 ppm. In addition, each microtiter plate contained four replicates with Mueller-Hinton broth alone as negative growth control (NC) and four replicates with bacterial inoculum without test substance as positive growth control (PC). The starting bacterial concentration was verified with colony count method. The microtiter plates as well as the colony count plates were incubated for 16–20 h at 37°C. The MIC was defined as the lowest concentration of the test substance that completely inhibits visible growth (no obvious cell pellet or turbidity).
The biofilm biomass was assessed with the microtiter plate assay by measuring all the attached biomass via crystal violet staining (defined as “CV-biofilm”). Within this definition, the field isolates
All the statistical analyses were performed with SAS 9.4 (SAS Institute, Inc., Cary, NC, USA). Parameters were analyzed for outliers and normal distribution with the UNIVARIATE procedure. To compare the differences among concentrations within different substances and strains, data were subjected to an analysis of variance (ANOVA) using PROC GLIMMIX according to the model:
The MIC of each test substance was defined as the lowest concentrations that completely inhibits visible growth of all the
The MIC values for
Figure 1
CV biofilm and viable counts (CFU ml−1) of
Figure 1. CV-Biofilm und Zellzahlen (KBE ml−1) von

No interaction of the applied concentration and
MIC values determined for
The count of viable cells was determined at the first sub-MIC and at 5 ppm substance concentration as well as for the PC. Generally, cell growth of both
Rapeseed oil, which was used as lipophilic control substance, had no significant effect on CV-biofilm of
Regarding the use of phytogenics as potential AGP alternatives in livestock production especially two issues need to be highlighted: Most studies investigated bacterial strains that are relevant for human medicine but not necessarily for livestock species. And secondly, most MIC values found in literature are values for individual strains.
Therefore, in the present study, garlic oil, cinnamaldehyde, carvacrol, thymol, and thyme oil were tested for their antimicrobial effects on both reference strains and field isolates of
The observed reduction of
In conclusion, the results of the present study show the strong antibacterial activity of cinnamaldehyde, carvacrol, thymol, and thyme oil on bacterial field isolates. The similar response of field isolates and type strains as well as the good comparability to literature values suggests a general effect on the studied bacterial species. The influence of cinnamaldehyde, carvacrol, thymol, and thyme oil on CV biofilm formation at sub-MIC level demonstrate their applicability at sub-lethal and therefore more economical concentrations. In addition, phytogenics may exhibit their effects on bacterial pathogens at sub-MIC level without lethal selection pressure, which probably reduces the chance of resistance formation. These findings may contribute to the development of potential alternatives to AGP in animal feed in order to increase productivity and animal welfare in modern livestock production.
Figure 1

Major chemical constituents of the studied phytogenicsTabelle 1. Chemische Hauptbestandteile der untersuchten phytogenen Substanzen
Garlic oil | Diallyl disulfid (59.1%), diallyl trisulfid (11.7%), and methyl propyl trisulfid (1.1%) |
Thymol | Thymol (100%) |
Carvacrol | Carvacrol (100%) |
Thyme oil | |
Cinnamaldehyde |