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Introduction
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 Escherichia coli field isolates from diseased farmed animals and Salmonella enterica subsp. enterica serotypes associated with either feed materials, infected laying hen flocks and food or isolated from diseased humans. Garlic oil, cinnamaldehyde, carvacrol, thymol and thyme oil were chosen due to their practical relevance as phytogenic feed additives and for comparability to existing literature data. Studying the effect of these phytogenics on biofilm formation of field isolates at sub-MIC levels may contribute to the development of alternatives to traditional AGPs in animal feed in order to support productivity and subsequently public health.
Materials and Methods
Phytogenic substance
The phytogenics garlic oil (Allium sativum), cinnamaldehyde, carvacrol, thymol, and thyme oil (Thymus vulgaris) were provided by Delacon Biotechnik GmbH, Engerwitzdorf, Austria. The major metabolites were identified and quantified by gas chromatography-mass spectrometry (GC-MS) analysis (Table 1). Rapeseed oil was used as lipophilic control substance for CV-biofilm assay (VFI GmbH, Wels, Austria).
Major chemical constituents of the studied phytogenics
Tabelle 1. Chemische Hauptbestandteile der untersuchten phytogenen Substanzen
Based on data from gas chromatography-mass spectrometry analysis provided by Delacon Biotechnik GmbH
Test organisms
E. coli field isolates were provided by the veterinarian pathology department of AGES GmbH Linz, Austria, and serotyped by the national reference laboratory for Escherichia coli of AGES GmbH Graz, Austria. Due to their high-level growth in pure culture in various organs and their mucoid or hemolytic properties, the field isolates O54:H21, O88:H8, O149:H10 (all three isolated from pig), and O8:H2 (isolated from cattle) were classified as pathogenic. E. coli O174:H2 was isolated from cattle and carries verotoxin genes (vtx1 and vtx2) as well as the enterohemolysin gene (E-hly). Orough:Hrough was isolated from piglet and is positive for verotoxin gene 2 (vtx2). E. coli ONT:H10 is a field isolate from small intestine of calf and possesses the heat-stable enterotoxin A gene (estA). E. coli O157:H7 was provided by the national reference laboratory for Escherichia coli of AGES GmbH Graz and was tested positive for both verotoxin genes (vtx1 and vtx2), the enterohemolysin gene (E-hly), and the intimin gene (eae). E. coli reference strain ATCC 8739 was purchased from Microbiologics Inc., Minnesota, USA (WDCM number 00012).
Salmonella enterica subsp. enterica field isolates were sero-typed by the national reference laboratory for Salmonella of AGES GmbH Graz. Salmonella Mbandaka was isolated from a process control sample of an Austrian feed manufacturer. This serotype was also detected in laying hen feed, flocks, and eggs as well as human fecal samples and traced back to an oil mill in Italy (personal communication). Salmonella Senftenberg was isolated from a soybean meal sample imported from Italy (RASFF notification No. 2013.1738). Salmonella Typhimurium (multi-resistant field isolate from human stool sample), Salmonella Enteritidis (field isolate from human stool samples), and Salmonella Infantis (multi-resistant field isolate from mature poultry) were provided by the national reference laboratory for Salmonella of AGES GmbH Graz. Salmonella Typhimurium reference strain ATCC 14028 was purchased from Microbiologics Inc. (WDCM number 00031).
AST (Antimicrobial susceptibility testing)
Susceptibility testing of all E. coli and Salmonella serotypes was performed with broth micro-dilution method according to DIN EN ISO 20776-1:2019 (ISO, 2019). Stock solutions (200000 ppm) of garlic oil, cinnamaldehyde, carvacrol, thymol, and thyme oil were prepared in absolute ethanol (VWR International GmbH, Vienna, Austria) and serially diluted (1:1 dilutions) in Mueller-Hinton broth (Oxoid GmbH, Wesel, Germany) to obtain concentrations of 20000 ppm to 10 ppm. Each substance concentration was pipetted horizontally into Nunc® polystyrene 96-well microtiter plates (Thermo Scientific, Waltham, Massachusetts, USA) in four technical replicates (50 μl per well).
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 E. coli O88:H8, O54:H21, and O174:H2 as well as Salmonella Mbandaka, Senftenberg, Infantis, and Typhimurium showed CV-biofilm formation and were exposed to garlic oil, cinnamaldehyde, carvacrol, thymol, thyme oil, and rapeseed oil as described in section “AST (Antimicrobial susceptibility testing)”. However, nutrient broth instead of Mueller-Hinton broth was used for serial dilutions and to adjust the bacterial inoculum to 2 x 106 to 5 x 106 CFU ml−1, which was verified with colony count method. After incubation for 16–20 h at 37°C, the amount of bacterial growth of the PC, at the fist sub-MIC, and at 5 ppm of each test substance was determined by OD600 measurement with the NanoDrop One photometer instrument (NanoDrop Micro-UV/Vis-Spectral photometer, Thermo Scientific, Waltham, Massachusetts, USA). The microtiter plates were then rinsed two times with water to remove the unattached cells and media components and dried for about 30 min at room temperature. For crystal violet staining, 125 μl of 0.1% (w/v) crystal violet solution (Alfa Aesar by Thermo Fisher GmbH, Kandel, Germany) in water was added to each well and incubated at room temperature for 10–15 min. Subsequently, the plates were rinsed 3–4 times with water and completely dried at room temperature. To quantify the adherent bio-mass, 125 μl 30% acetic acid solution (Merck KGaA, Darmstadt, Germany) in water (v/v) were added to the wells to solubilize the adherent crystal violet. The resulting solution was transferred to a new microtiter plate. Absorbance was measured at 550 nm with the PHOmo micro-plate reader (Anthos Mikrosysteme GmbH, Friesoythe, Germany). The assay was carried out on three different days with four technical replicates per day for each combination of test strain and substance concentration.
Statistical analysis
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:
\matrix{{{Y_{ijk}} = \mu + {\alpha _i} + {\beta _j} + \alpha {\beta _{ij}} + {day}_k + {\varepsilon _{ijk}}} \hfill \cr {i = 1, \ldots,a;j = 1, \ldots,b;k = 1 \ldots,n} \hfill}
where yijk is the observation in concentration i and strain j on day k; μ the overall mean; αi the fixed effect of concentration i; βj the fixed effect of strain j; αβij the interaction effect of concentration i and strain j; day k the random effect of day k, and εijk the random error with mean 0 and variance σ2. In addition, a is the number of concentrations; b the number of strains; and n the number of days.
Results
AST: Determination of MIC
The MIC of each test substance was defined as the lowest concentrations that completely inhibits visible growth of all the E. coli or Salmonella serotypes. The highest potential for antimicrobial effects in E. coli field isolates and reference strain was observed with thymol at a value of 150 ppm, followed by carvacrol and cinnamaldehyde at 300 ppm and thyme oil at 600 ppm. MIC values of Salmonella field isolates and reference strain exposed to the tested phytogenics were comparable to those of E. coli, except for the MIC of carvacrol, which was in line with the value of thymol in E. coli. Garlic oil showed no inhibition of bacterial growth within the concentration range tested (up to 10000 ppm).
Biofilm formation under static conditions
The MIC values for E. coli and Salmonella field isolates examined in the CV-assay were mostly in line with the MIC values from AST, with cinnamaldehyde, thymol, and carvacrol inhibiting growth at 300 ppm, thyme oil at 600 ppm and garlic oil showing no inhibition within the concentration range tested. The CV-biofilms for E. coli and Salmonella were calculated separately and are illustrated in Figure 1.
Figure 1
CV biofilm and viable counts (CFU ml−1) of E. coli and Salmonella exposed to the phytogenics garlic oil, cinnamaldehyde, carvacrol, thymol, and thyme oil. The different letters above the bars (CV absorption) indicate significant differences (P ≤ 0.05) within a chart. The viable counts of the PCs as well as those at the first sub-MIC and at 5 ppm were quantified and plotted as dots.
Figure 1. CV-Biofilm und Zellzahlen (KBE ml−1) von E. coli und Salmonella behandelt mit den phytogenen Substanzen Knoblauchöl, Zimtaldehyd, Carvacrol, Thymol und Thymianöl. Die unterschiedlichen Buchstaben über den Balken (CV-Absorption) zeigen signifikante Unterschiede (P ≤ 0,05) innerhalb eines Diagramms. Die Zellzahlen der Wachstumskontrollen (PC), der ersten sub-MICs und jene bei 5 ppm wurden quantifiziert und als Punkte dargestellt.
No interaction of the applied concentration and E. coli strains has been observed for any of the five phytogenics (P > 0.050). Cinnamaldehyde proved to be the most effective substance against E. coli CV-biofilm formation and showed a reduction of CV absorbance compared to the PC control even at a concentration as low as 5 ppm (−25.5%, P = 0.012). Carvacrol, thymol and thyme oil reduced E. coli CV-biofilm starting at a sub-MIC of 40 ppm (−47.8%, P = 0.001; −33.1%, P = 0.025; and −32.4%, P = 0.002, respectively). Garlic oil reduced E. coli CV-biofilm at a concentration of 10000 ppm by −80.9% (P < 0.001) compared to the PC.
MIC values determined for Salmonella with the CV-assay were identical to those of E. coli. The effects of phytogenics on Salmonella CV-biofilm formation were comparable, although an interaction of applied concentration and strain was found for garlic oil (P = 0.027) and thymol (P = 0.001), but not for cinnamaldehyde, carvacrol, and thyme oil (P > 0.050). Cinnamaldehyde was the most effective substance, showing reductions of Salmonella CV-biofilm at 40 ppm (−23.2%, P = 0.043). Carvacrol, thymol, and thyme oil reduced Salmonella CV-biofilms at 80 ppm by −65.6% (P < 0.001), −56.9% (P < 0.001) and −42.7% (P < 0.001), respectively. Garlic oil did not significantly affect CV-biofilm of Salmonella within the concentration range tested.
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 E. coli and Salmonella is influenced at the fist sub-MIC, with Salmonella being subject to higher fluctuations.
Rapeseed oil, which was used as lipophilic control substance, had no significant effect on CV-biofilm of E. coli and Salmonella.
Discussion
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 E. coli and Salmonella species from feed, humans, pigs, poultry, and cattle. The results suggest that Salmonella and E. coli field isolates as well as type strains are rather insensitive to the garlic oil tested. Similar findings are described in the literature (Ross et al., 2001; Dussault et al., 2014). The main compounds of the garlic oil used in the present study are diallyl disulfide and diallyl trisulfide. El-Azzouny et al. (2018) demonstrated that aqueous garlic extract with its main compound allicin has a high antibacterial effect on Salmonella. Therefore, the differences in chemical composition, which resulted from different extraction procedures, may have high impact on the antibacterial efficacy of garlic extracts. All other phytogenics tested showed strong antibacterial effects, with MIC-values in the range of those found in the literature. For example, Ghosh et al. (2013) reported cinnamaldehyde MIC values of 263 ppm and 131 ppm with E. coli and Salmonella Typhi, respectively, while Pei et al. (2009) found cinnamaldehyde, thymol, and carvacrol MIC values at 400 ppm with E. coli. However, slightly higher or lower MIC values of 1250 ppm for thyme oil (Burt & Reinders, 2003; Dussault et al., 2014) or 80 to 100 ppm for carvacrol (García-Heredia et al., 2016) were also observed with different E. coli strains. Based on the data obtained in this study, it is reasonable to state that field isolates do not show relevant differences in susceptibility to the phytogenics studied in comparison to the type strains most commonly analyzed in literature. The CV-biofilm assay was used to investigate the effects of garlic oil, cinnamaldehyde, carvacrol, thymol, and thyme oil on field isolates at sub-MIC concentrations. Cinnamaldehyde proofed to be the most effective substance in reducing the CV-biofilm, showing an effect on E. coli bio-film formation already at the lowest applied concentration (5 ppm). Similar low inhibitory concentration levels of cinnamaldehyde (10 ppm) were observed with E. coli O157:H7 reference strain in the work of Kim et al. (2015). Yuan and Yuk (2019) also worked with the reference strain E. coli O157:H7 but found biofilm reduction at 160 ppm cinnamaldehyde, carvacrol, and thymol. In the present study, carvacrol shows CV-biofilm reduction at 40 ppm and 80 ppm with E. coli and Salmonella field isolates, respectively. Burt et al. (2014) achieved significant reductions of biofilm formation with S. Typhimurium reference strain at concentrations slightly higher (110 ppm) than those found to disturb CV-biofilms of Salmonella field isolates. García-Heredia et al. (2016) achieved a significant reduction of biofilm formation with an E. coli field isolate at 25 to 75 ppm carvacrol and no reduction with type strains. Caceres et al. (2020) investigated effects of thyme oil with E. coli O157:H7 and O33 and found reductions of biofilms at 375 ppm. Overall, the concentrations of the investigated phytogenics required to reduce CV-biofilm formation in both E. coli and Salmonella field isolates are comparable to those found in the literature.
The observed reduction of E. coli CV-biofilm with garlic oil at the highest applied concentration (10000 ppm) was associated with large variations in growth. This may be a general effect that disturbs cell growth but does not specifically affect biofilm formation. Similarly, cinnamaldehyde, carvacrol, thymol, and thyme oil also caused high variations of cell growth one dilution step below the MIC value. However, the lowest concentrations affecting CV-biofilms have been observed at least two dilution steps below the MIC for cinnamaldehyde, carvacrol, thymol and thyme oil.
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.
