Eugenol stimulates lactate accumulation yet inhibits volatile fatty acid production and eliminates coliform bacteria in cattle and swine waste

AIM: To determine how eugenol affects fermentation parameters and faecal coliforms in cattle and swine waste slurries stored anaerobically. METHODS AND RESULTS: Waste slurries (faeces:urine:water, 50:35:15) were blended with and without additives and aliquoted to triplicate 1-l flasks. Faecal coliforms were eliminated in cattle and swine waste slurries within 1 or 2 days with additions of eugenol at 10.05 mm (0.15%) and 16.75 mm (0.25%). At these concentrations volatile fatty acids (VFA) were reduced ca 70 and 50% in cattle and swine waste, respectively, over 6-8 weeks. Additionally, in cattle waste, eugenol stimulated the accumulation of lactate (>180 mm) when compared with thymol treatment (20 mm lactate). In swine waste, lactate accumulation did not occur without additives; eugenol and thymol stimulated lactate accumulation to concentrations of 22 and 32 mm, respectively. CONCLUSIONS: Eugenol added to cattle waste may be more beneficial than thymol because not only does it effectively control faecal coliforms and odour (VFA production), it also stimulates lactate accumulation. This in turn, causes the pH to drop more rapidly, further inhibiting microbial activity and nutrient emissions. SIGNIFICANCE AND IMPACT OF THE STUDY: Plant essential oils have the potential to solve some of the environmental problems associated with consolidated animal feeding operations. Thymol and eugenol reduce fermentative activity, thus, have the potential to reduce emissions of greenhouse gases and odour, and curtail transmission of pathogens in cattle and swine wastes.     

Activity and mechanisms of action of selected biocidal agents on Gram-positive and -negative bacteria

AIMS: This study investigates the antimicrobial activity and mode of action of two natural products, eugenol and thymol, a commonly utilized biostatic agent, triclocarban (TCC), and two surfactants, didecyldimethylammonium chloride (DDDMAC) and C10-C16 alkyldimethyl amine N-oxides (ADMAO). METHODS AND RESULTS: Methods used included: determination of minimum inhibitory concentrations (MICs), lethal effect studies with suspension tests and the investigation of sub-MIC concentrations on growth of E. coli, Staph. aureus and Ps. aeruginosa using a Bioscreen microbiological analyser. Leakage of intracellular constituents and the effects of potentiating agents were also investigated. Only DDDMAC was bactericidal against all of the organisms tested. Eugenol, thymol and ADMAO showed bacteriostatic and bactericidal activity, but not against Ps. aeruginosa. TCC was only bacteristatic against Staph. aureus, but like the other agents, it did affect the growth of the other organisms in the Bioscreen experiments. All of the antimicrobial agents tested were potentiated by the permeabilizers to some extent and leakage of potassium was seen with all of the agents except TCC. CONCLUSIONS: DDDMAC was bactericidal against all organisms tested and all compounds had some bacteriostatic action. Low level static effects on bacterial growth were seen with sub-MIC concentrations. Membrane damage may account for at least part of the mode of action of thymol, eugenol, DDDMAC and ADMAO. SIGNIFICANCE AND IMPACT OF THE STUDY: The ingredients evaluated demonstrated a range of bactericidal and bacteriostatic properties against the Gram-negative and -positive organisms evaluated and the membrane (leakage of intracellular components) was implicated in the mode of action for most (except TCC). Sub-MIC levels of all ingredients did induce subtle effects on the organisms which impacted bacterial growth, even for those which had no true inhibitory effects.     

Susceptibility of food-borne bacteria to binary combinations of antimicrobials at selected a(w) and pH

AIM: To evaluate the antibacterial susceptibilities of food-borne bacteria to individual and binary mixtures of a synthetic antimicrobial agent with a natural phenolic compound. METHODS AND RESULTS: Antibacterial susceptibilities of Escherichia coli, Listeria innocua, Salmonella Typhimurium and Staphylococcus aureus to individual and binary mixtures of potassium sorbate with a phenolic compound (thymol, carvacrol, or eugenol) were evaluated, at selected water activity (a(w); 0.99 or 0.97) and pH (5.5 or 4.5). The bacteria studied were susceptible to the action of the antimicrobials individually with minimal inhibitory concentrations that varied from 800-ppm potassium sorbate for Staph. aureus at a(w) 0.99, and pH 5.5 to 100-ppm thymol or carvacrol for the four studied bacteria at a(w) 0.97 and pH 4.5. Several binary mixtures of potassium sorbate with thymol, carvacrol or eugenol inhibited bacterial growth. Antimicrobial agent inhibitory concentrations in the mixture varied among bacteria, additionally depending on the a(w) and the pH tested. CONCLUSIONS: Synergistic binary mixtures with fractional inhibitory concentration index <0.6 include 100- or 200-ppm potassium sorbate with 50- or 100-ppm thymol, carvacrol or eugenol. SIGNIFICANCE AND IMPACT OF THE STUDY: The synergistic combinations could be useful in reducing the amounts of antimicrobials needed to inhibit growth, thus diminishing consumer concerns regarding chemical preservatives.     

Genetic diversity of Ocimum gratissimum L. based on volatile oil constituents, flavonoids and RAPD markers

Morphological, chemical and genetic differences of 12 tree basil (Ocimum gratissimum L.) accessions were studied to determine whether volatile oils and flavonoids can be used as taxonomical markers and to examine the relationship between RAPDs to these chemical markers. Eugenol, thymol, and geraniol were the major volatile oil constituents found in Ocimum gratissimum. Xantomicrol and cirsimaritin were the major external flavones. The accessions morphologically described as O. gratissimum var. gratissimum contained eugenol as the major volatile oil constituent, and cirsimaritin as the major flavone. Ocimum gratissimum var. macrophyllum accessions contained thymol as the major volatile oil constituent, and xantomicrol as the major flavone. A distinct essential oil and flavone chemotype (producing geraniol and a mixture of the flavones cirsimaritin, isothymusin, xanthomicrol, and luteolin) was found in an accession genetically more distant from the other two groups when analyzed by molecular markers. The accessions could be divided based on volatile oil constituents into six groups: (1) thymol: alpha-copaene (ot24, ot25, ot26, and ot28); (2) eugenol:spathulenol (ot17, ot63, and ot52); (3) thymol:p-cymene (ot65); (4) eugenol:gamma-muurolene (ot27 and ot29); (5) eugenol:thymol: spathulenol (ot85); and (6) geraniol (ot84). Cluster analysis of RAPD markers showed that there are three groups that are distinct genetically and highly correlated (r=0.814) to volatile oil constituents.     

Physiochemical and molecular properties of antimicrobial-exposed Staphylococcus aureus during the planktonic-to-biofilm transition

This study was designed to characterize the physicochemical and molecular properties of Staphylococcus aureus cells treated with nisin, allyl isothiocyanate (AITC), thymol, eugenol, and polyphenol during the transition from planktonic to biofilm growth as measured by hydrophobicity, auto-aggregation, and differential gene expression. Thymol exhibited the highest antimicrobial activity against planktonic, biofilm-forming, biofilm, and dispersed cells, showing 0.21, 0.22, 0.46, and 0.26 mg/ml of MIC values, respectively. The lowest hydrophobicity was observed in planktonic cells treated with polyphenol (16 %), followed by thymol (29 %). The auto-aggregation abilities were more than 85 % for nisin, AITC, eugenol, polyphenol, and the control. The cell-to-surface interaction was related positively to biofilm formation by S. aureus. The adhesion-related gene (clfA), virulence-related genes (spa and hla), and efflux-related gene (mdeA) were down-regulated in both planktonic and biofilm cells treated with AITC, thymol, and eugenol. The results suggest that the antimicrobial tolerance and virulence potential were varied in the cell states during the planktonic-to-biofilm transition. This study provides useful information for understanding the cellular and molecular responses of planktonic and biofilm cells to antimicrobial-induced stress.     

Synergism of thymol,carvacrol and eugenol in larvae of the cattle tick,Rhipicephalus microplus,and brown dog tick,Rhipicephalus sanguineus

The effects of combinations of the monoterpenes thymol and carvacrol and the phenylpropanoid eugenol in larvae of Rhipicephalus microplus (Canestrini, 1888) (Acari: Ixodidae) and Rhipicephalus sanguineus sensu lato (s.l.) (Acari: Ixodidae) were assessed by the larval packet test. The CompuSyn program was used to make qualitative assessments of the effects (synergistic, additive and antagonistic) of the associations. The effects of all combinations tested against R. microplus larvae were synergistic, with combination indices (CIs) <0.70. When tested against R. sanguineus, eight of the mixtures showed a synergistic effect (CI < 0.70); only the carvacrol + thymol mixture at LC₅₀ presented a moderate synergistic effect, with CIs between 0.70–0.90. This study is the first to determine the effects of the interactions of these substances in the control of these two tick species. The combinations of carvacrol + thymol, carvacrol + eugenol and thymol + eugenol have synergistic effects in R. microplus and R. sanguineus s.l. larvae.     

A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol

AIMS: The minimum inhibitory concentration (MIC) of oregano essential oil (OEO) and two of its principle components, i.e. thymol and carvacrol, against Pseudomonas aeruginosa and Staphylococcus aureus was assessed by using an innovative technique. The mechanism of action of the above substances was also investigated. METHODS AND RESULTS: The applied technique uses 100-well microtitre plate and collects turbidimetric growth data. To produce the inhibition profiles, a wide range of concentrations were tested for each of the three compounds, as well as for carvacrol-thymol mixtures. Following a specific mathematical analysis of the observed inhibition profiles from all compounds, it was suggested that mixtures of carvacrol and thymol gave an additive effect and that the overall inhibition by OEO can be attributed mainly to the additive antimicrobial action of these two compounds. Addition of low amounts of each additive: (a) increased permeability of cells to the nuclear stain EB, (b) dissipated pH gradients as indicated by the CFDA-SE fluorescent probe irrespective of glucose availability and (c) caused leakage of inorganic ions. CONCLUSION: Mixing carvacrol and thymol at proper amounts may exert the total inhibition that is evident by oregano essential oil. Such inhibition is due to damage in membrane integrity, which further affects pH homeostasis and equilibrium of inorganic ions. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of extent and mode of inhibition of specific compounds, which are present in plant extracts, may contribute to the successful application of such natural preservatives in foods, since certain combinations of carvacrol-thymol provide as high inhibition as oregano essential oil with a smaller flavour impact.     

Insecticidal activity of selected monoterpenoids and rosemary oil to Agriotes obscurus (Coleoptera: Elateridae)

Acute toxicities of three naturally occurring monoterpenoid essential oil constituents and the essential oil of rosemary were tested against late instars of Agriotes obscurus (L.) (Coleoptera: Elateridae). Both contact and volatile toxicities of thymol, citronellal, eugenol, and rosemary oil were determined. Also, phytotoxicity of these compounds was evaluated on corn germination and seedling development. Thymol had the greatest contact toxicity (LD50 = 196.0 microg/larva), whereas citronellal and eugenol were less toxic (LD50 = 404.9 and 516.5 microg/larva, respectively). Rosemary oil did not show any significant contact toxicity, even at 1,600 microg/larva. In terms of volatile toxicity, citronellal was the most toxic to wireworm larvae (LC50 = 6.3 microg/cm3) followed by rosemary oil (LC50 = 15.9 microg/cm3), thymol (LC50 = 17.1 microg/cm3), and eugenol (LC50 = 20.9 microg/cm3). Thymol, eugenol, and citronellal significantly inhibited corn seed germination and development, whereas rosemary oil had only minimal phytotoxic effects.     

Antifungal activity of essential oils evaluated by two different application techniques against rye bread spoilage fungi

AIMS: To study how antifungal activity of natural essential oils depends on the assay method used. METHODS AND RESULTS: Oils of bay, cinnamon leaf, clove, lemongrass, mustard, orange, sage, thyme and two rosemary oils were tested by two methods: (1) a rye bread-based agar medium was supplemented with 100 and 250 microl l-1 essential oil and (2) real rye bread was exposed to 136 and 272 microl l-1 volatile oil in air. Rye bread spoilage fungi were used for testing. Method 1 proved thyme oil to be the overall best growth inhibitor, followed by clove and cinnamon. On the contrary, orange, sage and rosemary oils had very limited effects. Mustard and lemongrass were the most effective oils by the volatile method, and orange, sage and one rosemary showed some effects. Oil compositions were analysed by gas chromatography-mass spectrography. CONCLUSIONS: Antifungal effects of the essential oils depended on the application method. Larger phenolic compounds such as thymol and eugenol (thyme, cinnamon and clove) had best effect applied directly to medium, whereas smaller compounds such as allyl isothiocyanate and citral (mustard and lemongrass) were most efficient when added as volatiles. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proves that the method used for screening essential oils as potential antimicrobials should correspond with the application sought.     

The antibacterial mechanism of carvacrol and thymol against Escherichia coli

Aims:  To investigate the antibacterial mechanism of carvacrol and thymol against Escherichia coli.
Methods and Results:  The time-kill curve results showed that carvacrol and thymol at 200 mg l⁻¹ could inhibit the growth of E. coli . Flow cytometry and fluorescent dyes were used to explore the effect of two components on membrane permeability and membrane potential. In membrane permeability experiment, the mean fluorescence intensity of cells treated with 200 mg l⁻¹ carvacrol or thymol were lower than nonexposed cells. The ratio of red to green fluorescence intensity of DiOC₂(3) reflected the change of membrane potential. Carvacrol and thymol at 200 mg l⁻¹ caused the ratio of red/green decreasing from 0·42 of control to 0·08 and 0·07, respectively.
Conclusions:  Carvacrol and thymol had desired antimicrobial effect on E. coli . The antibacterial effects were attributed to their ability to permeabilize and depolarize the cytoplasmic membrane.
Significance and Impact of the Study:  This study showed the potential use of flow cytometry as a suitable method to investigate the mode of antibacterial action of essential oil components.     

Bioactivity of selected plant essential oils against listeria monocytogenes

Ninety-three different commercial essential oils were screened for activity against 20 Listeria monocytogenes strains in vitro and the results correlated against the actual chemical composition of each oil. There was a substantial difference in the activity between different essential oils as expected, but there was also a difference in activity between different samples of the same essential oil. Strong anti- Listeria activity was often correlated with essential oils containing a high percentage of monoterpenes, eugenol, cinnamaldehyde, thymol, and sometimes with citronellol, limonene and geraniol. However, as there was often no correlation between the anti- Listeria activity and the main chemical components, it is possible that either there is a more complex relationship with the chemical composition (which includes the minor components) or that substantial adulteration had occurred in some essential oil samples. The possible use of a number of essential oils in a dual role as flavours and antimicrobials is discussed.     

Antifungal activity of thyme (Thymus vulgaris L.) essential oil and thymol against moulds from damp dwellings

AIMS: To characterize antifungal activities of essential oil of thyme (Thymus vulgaris L.) and pure thymol, as comparative substance, on different mould species isolated from damp dwellings. METHODS AND RESULTS: Fifty samples of wall scrapes were collected from damp dwellings in Zagreb, the capital of Croatia. The members of the following mould genera were recovered from the samples: Aspergillus (44%), Penicillium (18%) Alternaria, Ulocladium, Absidia and Mucor (8%) Cladosporium, Trichoderma and Rhizopus (6%), and Chaetomium (2%). Two strains of Stachybotrys chartarum were isolated from damp dwellings in Slovakia. Antifungal activities of the thyme essential oil, which contains p-cymene (36.5%), thymol (33.0%) and 1,8-cineole (11.3%) as main components, and pure thymol were determined by the dilution method and exposure to vaporous phase of the oil. Minimum inhibitory concentrations (MIC) of both thymol and essential oil were bellow 20 microg ml(-1), except for Mucor spp. (50.20 microg ml(-1)). Thymol exhibited approximately three-times stronger inhibition than essential oil of thyme. The vaporous phase of the thyme essential oil (82 microg l(-1)) in glass chambers strongly suppressed the sporulation of moulds during 60 days of exposure. CONCLUSION: The thyme essential oil possesses a wide range spectrum of fungicidal activity. The vaporous phase of the oil exhibited long-lasting suppressive activity on moulds from damp dwellings. SIGNIFICANCE AND IMPACT OF THE STUDY: Essential oil of thyme and thymol could be used for disinfection of mouldy walls in the dwellings in low concentration.     

Cch1p Mediates Ca2+ Influx to Protect Saccharomyces cerevisiae against Eugenol Toxicity

Eugenol has antifungal activity and is recognised as having therapeutic potential. However, little is known of the cellular basis of its antifungal activity and a better understanding of eugenol tolerance should lead to better exploitation of eugenol in antifungal therapies. The model yeast, Saccharomyces cerevisiae, expressing apoaequorin was used to show that eugenol induces cytosolic Ca²⁺ elevations. We investigated the eugenol Ca²⁺ signature in further detail and show that exponentially growing cells exhibit Ca²⁺ elevation resulting exclusively from the influx of Ca²⁺ across the plasma membrane whereas in stationary growth phase cells Ca²⁺ influx from intracellular and extracellular sources contribute to the eugenol-induced Ca²⁺ elevation. Ca²⁺ channel deletion yeast mutants were used to identify the pathways mediating Ca²⁺ influx; intracellular Ca²⁺ release was mediated by the vacuolar Ca²⁺ channel, Yvc1p, whereas the Ca²⁺ influx across the plasma membrane could be resolved into Cch1p-dependent and Cch1p-independent pathways. We show that the growth of yeast devoid the plasma membrane Ca²⁺ channel, Cch1p, was hypersensitive to eugenol and that this correlated with reduced Ca²⁺ elevations. Taken together, these results indicate that a cch1p-mediated Ca²⁺ influx is part of an intracellular signal which protects against eugenol toxicity. This study provides fresh insight into the mechanisms employed by fungi to tolerate eugenol toxicity which should lead to better exploitation of eugenol in antifungal therapies.     

Assessing essential oil components as plant-based preservatives against fungi that deteriorate herbal raw materials

This study assesses the antifungal efficacy of 14 essential oil (EO) components and some of their combinations as inhibitory to the growth of the aflatoxigenic fungus Aspergillus flavus LHPA₉ isolated from biodeteriorating Asparagus racemosus herbal raw materials. The aim was to determine whether they could be recommended as plant-based preservatives for enhancement of the shelf life of herbal raw materials. Thymol, eugenol, menthol, and their combinations were highly efficacious as their minimum inhibitory concentration (MIC) for inhibition of fungal growth as well as aflatoxin B₁ secretion was less than 1.0 μl ml⁻¹. Geranyl acetate, linalool, β-asarone, 1, 8-cineol, and E-citral were moderately antifungal as their MIC ranged between 1.0 and 5.0 μl ml⁻¹. During antioxidant activity 2, 2-diphenyl-1-picrylhydrazyl assay, thymol, eugenol, and β-caryophyllene showed strong radical scavenging activity, whereas β-asarone and p-cymene showed moderate activity. Some combinations of EO components showed synergism while others exhibited an additive or antagonism effect in their activity. The findings point to a recommendation that EO components are good alternatives to synthetic preservatives to prevent deterioration of stored herbal raw materials by fungal and aflatoxin contamination and free-radical oxidation.     

Thymol and eugenol derivatives as potential antileishmanial agents

In Northeastern Brazil visceral leishmaniasis is endemic with lethal cases among humans and dogs. Treatment is toxic and 5–10% of humans die despite treatment. The aim of this work was to survey natural active compounds to find new molecules with high activity and low toxicity against Leishmania infantum chagasi. The compounds thymol and eugenol were chosen to be starting compounds to synthesize acetyl and benzoyl derivatives and to test their antileishmanial activity in vitro and in vivo against L. i. chagasi. A screening assay using luciferase-expressing promastigotes was used to measure the growth inhibition of promastigotes, and an ELISA in situ was performed to evaluate the growth inhibition of amastigote. For the in vivo assay, thymol and eugenol derivatives were given IP to BALB/c mice at 100 mg/kg/day for 30 days. The thymol derivatives demonstrated the greater activity than the eugenol derivatives, and benzoyl-thymol was the best inhibitor (8.67 ± 0.28 μg/mL). All compounds demonstrated similar activity against amastigotes, and acetyl-thymol was more active than thymol and the positive control drug amphotericin B. Immunohistochemistry demonstrated the presence of Leishmania amastigote only in the spleen but not the liver of mice treated with acetyl-thymol. Thus, these synthesized derivatives demonstrated anti-leishmanial activity both in vitro and in vivo. These may constitute useful compounds to generate new agents for treatment of leishmaniasis.     

Larvicidal activity of essential oils extracted from commonly used herbs in Lebanon against the seaside mosquito, Ochlerotatus caspius

This study investigates the potential of essential oils from commonly used medical and culinary herbs in Lebanon as an environmentally safe measure to control the seaside mosquito, Ochlerotatus caspius. The composition of essential oils extracted from parsley seeds and leaves, alpine thyme inflorescences, anis seeds, and coriander fruits were analyzed by GC-MS, and the major components of these oils were found to be thymol, sabinene, carvacrol, anethole, and linalool, respectively. Mosquito larvicidal assays were conducted to evaluate the LC(50) and LC(90) after 24 and 48h of the essential oils and their major constituents. All of the tested oils proved to have strong larvicidal activity (LC(50): 15-156ppm) against Oc. caspius fourth instars, with the most potent oil being thyme inflorescence extract, followed by parsley seed oil, aniseed oil, and then coriander fruit oil. Toxicity of each oil major constituent was also estimated and compared to a reported larvicidal compound, eugenol.     

Mechanisms underlying the toxicity of lactone aroma compounds towards the producing yeast cells

AIMS: To study the fundamental mechanisms of toxicity of the fruity aroma compound gamma-decalactone, that lead to alterations in cell viability during its biotechnological production by yeast cells; Yarrowia lipolytica that is able to produce high amounts of this metabolite was used here as a model. METHODS AND RESULTS: Lactone concentrations above 150 mg l-1 inhibited cell growth, depolarized the living cells and increased membrane fluidity. Infrared spectroscopic measurements revealed that the introduction of the lactone into model phospholipid bilayers, decreased the phase transition temperature. Moreover, the H+-ATPase activity in membrane preparations was strongly affected by the presence of the lactone. On the other hand, only a slight decrease in the intracellular pH occurred. CONCLUSIONS: We propose that the toxic effects of gamma-decalactone on yeast may be initially linked to a strong interaction of the compound with cell membrane lipids and components. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings may enable the elaboration of strategies to improve yeast cell viability during the process of lactones bioproduction.     

Plant-derived compounds as natural antimicrobials to control paper mill biofilms

Biofilms can cause severe problems in industrial paper mills, particularly of economic and technological types (clogging of filters, sheet breaks or holes in the paper, machine breakdowns, etc.). We present here some promising results on the use of essential oil compounds to control these biofilms. Biofilms were grown on stainless-steel coupons with a microbial white water consortium sampled from an industrial paper mill. Five essential oil compounds were screened initially in the laboratory in terms of their antimicrobial activity against planktonic cells and biofilms. The three most active compounds were selected and then tested in different combinations. The combination finally selected was tested at the pilot scale to confirm its efficiency under realistic conditions. All the compounds tested were as active against biofilms as they were against planktonic cells. The most active compounds were thymol, carvacrol, and eugenol, and the most efficient combination was thymol–carvacrol. At a pilot scale, with six injections a day, 10 mM carvacrol alone prevented biocontamination for at least 10 days, and a 1 mM thymol–carvacrol combination enabled a 67 % reduction in biofilm dry matter after 11 days. The use of green antimicrobials could constitute a very promising alternative or supplement to the treatments currently applied to limit biofilm formation in the environment of paper mill machines.     

Calcium Dependence of Eugenol Tolerance and Toxicity in Saccharomyces cerevisiae

Eugenol is a plant-derived phenolic compound which has recognised therapeutical potential as an antifungal agent. However little is known of either its fungicidal activity or the mechanisms employed by fungi to tolerate eugenol toxicity. A better exploitation of eugenol as a therapeutic agent will therefore depend on addressing this knowledge gap. Eugenol initiates increases in cytosolic Ca²⁺ in Saccharomyces cerevisiae which is partly dependent on the plasma membrane calcium channel, Cch1p. However, it is unclear whether a toxic cytosolic Ca²⁺elevation mediates the fungicidal activity of eugenol. In the present study, no significant difference in yeast survival was observed following transient eugenol treatment in the presence or absence of extracellular Ca²⁺. Furthermore, using yeast expressing apoaequorin to report cytosolic Ca²⁺ and a range of eugenol derivatives, antifungal activity did not appear to be coupled to Ca²⁺ influx or cytosolic Ca²⁺ elevation. Taken together, these results suggest that eugenol toxicity is not dependent on a toxic influx of Ca²⁺. In contrast, careful control of extracellular Ca²⁺ (using EGTA or BAPTA) revealed that tolerance of yeast to eugenol depended on Ca²⁺ influx via Cch1p. These findings expose significant differences between the antifungal activity of eugenol and that of azoles, amiodarone and carvacrol. This study highlights the potential to use eugenol in combination with other antifungal agents that exhibit differing modes of action as antifungal agents to combat drug resistant infections.     

Activity of natural antimicrobial compounds against Escherichia coli and Salmonella enterica serovar Typhimurium

AIMS: The objective of this study was to evaluate the inhibitory activity of several natural organic compounds alone or in combination with nisin against Escherichia coli and Salmonella Typhimurium. METHODS AND RESULTS: The minimum inhibitory concentration (MIC) of five natural organic compounds were determined, and the effect of their combinations with nisin was evaluated by the checkerboard assay using the Bioscreen C. As expected, nisin by itself showed no inhibition against either of the Gram-negative bacteria. Thymol was found to be the most effective with the lowest MIC values of 1.0 and 1.2 mmol 1-1 against Salm. Typhimurium and E. coli, respectively. After thymol, the antimicrobial order of the natural organic compounds was carvacrol > eugenol > cinnamic acid > diacetyl. However, the combination of nisin with the natural organic compounds did not result in the enhancement of their antimicrobial activities. On the contrary, combination of nisin with diacetyl against Salm. Typhimurium resulted in an antagonism of diacetyl activity. CONCLUSIONS: While the individual natural organic compounds showed inhibitory activity against the two Gram-negatives, their combinations with nisin showed no improvement of antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the potential of the natural organic compounds to control E. coli and Salm. Typhimurium.