Sara A. Burt

Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7

Aims: To quantify the antibacterial properties of five essential oils (EO) on a non‐toxigenic strain of Escherichia coli O157:H7 in the presence and absence of a stabilizer and an emulsifier and at three different temperatures.

Carvacrol Induces Heat Shock Protein 60 and Inhibits Synthesis of Flagellin in Escherichia coli O157:H7

ABSTRACT The essential oils of oregano and thyme are active against a number of food-borne pathogens, such as Escherichia coli O157:H7. Carvacrol is one of the major antibacterial components of these oils, and p-cymene is thought to be its precursor in the plant. The effects of carvacrol and p-cymene on protein synthesis in E. coli O157:H7 ATCC 43895 cells were investigated. Bacteria were grown overnight in Mueller-Hinton broth with a sublethal concentration of carvacrol or p-cymene, and their protein compositions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by Western blotting. The presence of 1 mM carvacrol during overnight incubation caused E. coli O157:H7 to produce significant amounts of heat shock protein 60 (HSP60) (GroEL) (P < 0.05) and inhibited the synthesis of flagellin highly significantly (P < 0.001), causing cells to be aflagellate and therefore nonmotile. The amounts of HSP70 (DnaK) were not significantly affected. p-Cymene at 1 mM or 10 mM did not induce HSP60 or HSP70 in significant amounts and did not have a significant effect on flagellar synthesis. Neither carvacrol (0.3, 0.5, 0.8, or 1 mM) nor p-cymene (0.3, 0.5, or 0.8 mM) treatment of cells in the mid-exponential growth phase induced significant amounts of HSP60 or HSP70 within 3 h, although numerical increases of HSP60 were observed. Motility decreased with increasing concentrations of both compounds, but existing flagella were not shed. This study is the first to demonstrate that essential oil components induce HSP60 in bacteria and that overnight incubation with carvacrol prevents the development of flagella in E. coli O157:H7.

Increase in Activity of Essential Oil Components Carvacrol and Thymol against Escherichia coli O157:H7 by Addition of Food Stabilizers

The major components of oregano and thyme essential oils that had previously been shown to inhibit Escherichia coli O157:H7 were determined by high-performance liquid chromatography with UV detection and liquid chromatographic tandem mass spectrometry. The MICs and MBCs of carvacrol, thymol, p-cymene, and gamma-terpinene against a strain of E. coli O157: H7 phage type 34 isolated from bovine feces were determined by microdilution assay. The constituents were then tested in checkerboard assays to detect possible interactions. Carvacrol and thymol displayed bacteriostatic and bactericidal properties with MICs of 1.2 mmol/liter and were additive in combination. p-Cymene and gamma-terpinene displayed no measurable antibacterial activity up to 50 mmol/liter, and neither influenced the activity of carvacrol or thymol. Growth curves in the presence of nonlethal concentrations of carvacrol with the addition of agar (0.05%, wt/vol) or carrageenan (0.125%, wt/vol) as stabilizer were produced by optical density measurement. The stabilizers agar and carrageenan both significantly improved the effectiveness of carvacrol in broth, possibly because of a delay in the separation of the hydrophobic substrate from the aqueous phase of the medium. When carvacrol was dissolved in ethanol before addition to broth, stabilizers were not needed. Carvacrol and thymol, particularly when used in combination with a stabilizer or in an ethanol solution, may be effective in reducing the number or preventing growth of E. coli O157:H7 in liquid foods.

Synergy between essential oil components and antibiotics: a review.

Abstract With the increase in antibiotic-resistant bacteria and the lack of new antibiotics being brought onto the market, alternative strategies need to be found to cope with infections resulting from drug-resistant bacteria. A possible solution may be to combine existing antibiotics with phytochemicals to enhance the efficacy of antibiotics. A group of phytochemicals that is said to have such effects, according to in vitro studies, is essential oils (EOs) and their components. Amongst others, EOs containing carvacrol, cinnamaldehyde, cinnamic acid, eugenol and thymol can have a synergistic effect in combination with antibiotics. Several modes of action have been put forward by which antibiotics and the essential oil components may act synergistically, such as by affecting multiple targets; by physicochemical interactions and inhibiting antibacterial-resistance mechanisms. Many reported assays show additivity or moderate synergism, indicating that EOs may offer possibilities for reducing antibiotic use.

The natural antimicrobial carvacrol inhibits quorum sensing in Chromobacterium violaceum and reduces bacterial biofilm formation at sub-lethal concentrations

The formation of biofilm by bacteria confers resistance to biocides and presents problems in medical and veterinary clinical settings. Here we report the effect of carvacrol, one of the major antimicrobial components of oregano oil, on the formation of biofilms and its activity on existing biofilms. Assays were carried out in polystyrene microplates to observe (a) the effect of 0–0.8 mM carvacrol on the formation of biofilms by selected bacterial pathogens over 24 h and (b) the effect of 0–8 mM carvacrol on the stability of pre-formed biofilms. Carvacrol was able to inhibit the formation of biofilms of Chromobacterium violaceum ATCC 12472, Salmonella enterica subsp. Typhimurium DT104, and Staphylococcus aureus 0074, while it showed no effect on formation of Pseudomonas aeruginosa (field isolate) biofilms. This inhibitory effect of carvacrol was observed at sub-lethal concentrations (<0.5 mM) where no effect was seen on total bacterial numbers, indicating that carvacrols bactericidal effect was not causing the observed inhibition of biofilm formation. In contrast, carvacrol had (up to 8 mM) very little or no activity against existing biofilms of the bacteria described, showing that formation of the biofilm also confers protection against this compound. Since quorum sensing is an essential part of biofilm formation, the effect of carvacrol on quorum sensing of C. violaceum was also studied. Sub-MIC concentrations of carvacrol reduced expression of cviI (a gene coding for the N-acyl-L-homoserine lactone synthase), production of violacein (pigmentation) and chitinase activity (both regulated by quorum sensing) at concentrations coinciding with carvacrols inhibiting effect on biofilm formation. These results indicate that carvacrols activity in inhibition of biofilm formation may be related to the disruption of quorum sensing.

Low temperature and binding to food components inhibit the antibacterial activity of carvacrol against Listeria monocytogenes in steak tartare.

Carvacrol is a major component of thyme and oregano essential oils and has potential uses as a food preservative. The effect of carvacrol on the growth of Listeria monocytogenes was investigated in vitro and in steak tartare. Carvacrol had strong antilisterial activity in growth medium (MIC = 1.6 mM), but no effect was observed when carvacrol was tested in steak tartare. There were two reasons for this reduced activity: the antilisterial activity of carvacrol was strongly reduced at lower temperatures (10 versus 30 degrees C), and the presence of food components interfered with the activity of carvacrol. Both bovine serum albumin and egg yolk inhibited carvacrol activity at > 0.2% (wt/vol) in growth medium. For the first time, carvacrol was found to bind to albumin, suggesting that the reduced antilisterial activity of carvacrol in foods such as dairy products and uncooked meats is the result of fewer free unbound carvacrol molecules available to interact with bacteria.

Sub-lethal levels of carvacrol reduce Salmonella Typhimurium motility and invasion of porcine epithelial cells

The European ban on the use of antibiotic growth promotors has increased the search for new alternatives to prevent pig intestinal microbial diseases and to stimulate growth. The addition of essential oils or components thereof, such as carvacrol, to pig feed is a promising alternative. In this report we determined the effect of sub-lethal concentrations of carvacrol on Salmonella Typhimurium. At concentrations where growth of Salmonella was not inhibited, carvacrol completely inhibited motility of the bacterium. This loss of motility was not due to the loss of the flagellum or to ATP shortage upon carvacrol treatment. Adhesion of Salmonella to IPEC-J2, porcine intestinal epithelial cells, was not affected by carvacrol but invasion was significantly reduced. In addition, the epithelial gene expression of porcine β-defensin 2, an innate immune response to Salmonella infection, was reduced when Salmonella was exposed to carvacrol. This indicates that invasion but not adhesion of Salmonella triggers the porcine β-defensin 2 expression of porcine epithelial cells.

The Natural Antimicrobial Carvacrol Inhibits Campylobacter jejuni Motility and Infection of Epithelial Cells

Background Natural compounds with anti-microbial properties are attractive reagents to reduce the use of conventional antibiotics. Carvacrol, the main constituent of oregano oil, inhibits the growth of a variety of bacterial foodborne pathogens. As concentrations of carvacrol may vary in vivo or when used in animal feed, we here investigated the effect of subinhibitory concentrations of the compound on major virulence traits of the principal bacterial foodborne pathogen Campylobacter jejuni. Methods/Principal Findings Motility assays revealed that subinhibitory concentrations of carvacrol inhibited the motility of C. jejuni without affecting bacterial growth. Immunoblotting and electron microscopy showed that carvacrol-treated C. jejuni still expressed flagella. The loss of motility was not caused by reduced intracellular ATP levels. In vitro infection assays demonstrated that subinhibitory concentrations of carvacrol also abolished C. jejuni invasion of human epithelial cells. Bacterial uptake of invasive Escherichia coli was not blocked by carvacrol. Exposure of C. jejuni to carvacrol prior to infection also inhibited cellular infection, indicating that the inhibition of invasion was likely caused by an effect on the bacteria rather than inhibition of epithelial cell function. Conclusions/Significance Bacterial motility and invasion of eukaryotic cells are considered key steps in C. jejuni infection. Our results indicate that subinhibitory concentrations of carvacrol effectively block these virulence traits by interfering with flagella function without disturbing intracellular ATP levels. These results broaden the spectrum of anti-microbial activity of carvacrol and support the potential of the compound for use in novel infection prevention strategies.

Vermin on pig farms are vectors for Clostridium difficile PCR ribotypes 078 and 045.

Clostridium difficile is a gram positive, spore forming, toxin producing, anaerobic bacteria and an opportunistic pathogen for Man and many animal species, causing diarrhea in young piglets. Piglets probably become colonized from the environment. To investigate the possible spread and transmission of C. difficile by vermin, vermin samples were collected on a pig farm in the Netherlands and investigated for the presence of C. difficile. Samples of house mice (n=53), drain flies (n=39), lesser houseflies (n=95), and yellow mealworms (n=11) were found positive for C. difficile in 66%, 97%, 56% and 100% of cases respectively. C. difficile PCR ribotype 078 was found in all categories of vermin and ribotype 045 was found in two samples from the skeletal muscle of mice. House sparrows found dead on the premises (n=35) and bird droppings (n=26) were also investigated and carried C. difficile in 66% and 4% of cases respectively. PCR ribotype 078 was identified in bird and droppings samples but ribotype 045 was not. We conclude that vermin can play a role in the spread and transmission of C. difficile types 078 and 045 within pig farms and to other locations.

Cinnamaldehyde, Carvacrol and Organic Acids Affect Gene Expression of Selected Oxidative Stress and Inflammation Markers in IPEC-J2 Cells Exposed to Salmonella typhimurium

Essential oils and organic acids are used as feed additives to improve health status and reduce colonization with pathogens. Although bactericidal in vitro, concentrations achieved in the animal gut are probably not lethal to pathogens. The aim of this study was to investigate the effects of cinnamaldehyde, carvacrol and cinnamic, lactic and propionic acids on the ability of Salmonella typhimurium ATCC 14028 (ST) to invade intestinal epithelial cells (IPEC‐J2) and on the expression levels of immune related genes in the cells. The minimum inhibitory concentration (MIC) and non‐inhibitory concentration (NIC) were determined and influence on the invasion capacity of ST was investigated. The structure of fimbriae and flagella was analysed by electron microscopy, and expression levels of HSP70, IkBa, IL‐8 and IL‐10 in the IPEC‐J2 cells were carried out by q‐PCR. Cinnamaldehyde, carvacrol and cinnamic and propionic acids inhibited ST invasion but not cell viability, bacterial viability and motility or the development of flagella. Propionic acid and cinnamaldehyde in combination with cinnamic acid caused structural impairment of fimbriae. Cinnamaldehyde up‐regulated expression of HSP70 irrespective of the presence of organic acids or ST; exposure to carvacrol induced HSP70 only in the presence of propionic acid and ST.