Effects of adaptation to carvacrol on Staphylococcus aureus in the planktonic and biofilm phases.

The effect of exposure to sub-minimum inhibitory concentrations of carvacrol, for either 3-10 days, on direct (carvacrol) or cross-protection (cinnamaldehyde, eugenol, antibiotics) and the influence on planktonic and biofilm growth of four Staphylococcus aureus strains were reported. The sequential exposure to carvacrol resulted in a direct protection that was more evident in two of the four strains after 10 days. No significant cross-protection against cinnamaldehyde, eugenol and antibiotics was detected. An adaptive response was associated with a prolonged lag phase, a lower yield of bacteria, a colony phenotype likely to be associated to small colony variants and an increase in biofilm production. Generally, the biofilm of the adapted strains was less susceptible to subMICs of carvacrol compared to the biofilms of non-adapted strains. In contrast, it was demonstrated that in the case of mature biofilms the susceptibility was similar. The exposure of S. aureus to carvacrol at concentrations above the MIC resulted in a very low mutation frequency.

Control of biofilm formation by poly-ethylene-co-vinyl acetate films incorporating nisin.

The aim of this study was to evaluate the effect of poly-ethylene-co-vinyl acetate (EVA) films incorporating different concentrations (0.1%, 0.5% and 1%) of nisin on the biofilm-forming ability of Listeria monocytogenes ATCC 7644, Staphylococcus aureus 815 and Staphylococcus epidermidis ATCC 35984. Nisin was incorporated into two grades of EVA (EVA14 and EVA28) in the melt during a common film-blowing operation. The efficacy of EVA/nisin films was evaluated by biofilm biomass measurements and Live/Dead staining in combination with fluorescence microscopy. In order to evaluate whether the nisin incorporation could modify the film surface properties, contact angle measurements and scanning electron microscopy were performed. The results revealed the efficacy of EVA14/nisin films in reducing biofilm formation on their surfaces with more evident effect for S. epidermidis than L. monocytogenes and S. aureus strains. In contrast, EVA28/nisin films showed unsatisfactory activity. Fluorescence microscopy confirmed poor biofilm formation on EVA14/nisin films, also characterised by the presence of dead cells. The data presented in this study offer new potential applications for developing strategies aimed to improve the effect of antimicrobial agents.

Characterization of bacterial lipid profiles by using rapid sample preparation and fast comprehensive two-dimensional gas chromatography in combination with mass spectrometry.

The bacteria fatty acid profile has been extensively studied for taxonomic classification purposes, since bacteria, in general, contain particular and rare fatty acids, compared with animal and plant tissues. As for any real-world sample type, the development of rapid and reliable methods for (i) sample identification (in this case, bacterium type), and (ii) constituent identification (in this instance, the fatty acid profile) is desirable. In this research, a half-an-hour procedure, to analyze bacteria, was developed: a 2-min one-step sample preparation step was followed by a relatively fast comprehensive 2D GC-MS separation (25 min). Furthermore, dedicated MS libraries were constructed for the identification of bacteria and fatty acids. Finally, data processing, only qualitative at this stage, was carried out with the support of a novel comprehensive 2D GC software.

In vitro activity of carvacrol against staphylococcal preformed biofilm by liquid and vapour contact.

Carvacrol is an important component of essential oils and recently has attracted much attention as a result of its biological properties, such as a wide spectrum of antimicrobial activity. The aim of this study was to evaluate the effect of carvacrol in liquid and vapour phase on preformed biofilms of Staphylococcus aureus and Staphylococcus epidermidis by determining biofilm biomass and cultivable cell numbers, and by using epifluorescence and scanning electron microscopy. Carvacrol was able to reduce biofilm biomass and cell viability more effectively when used with liquid contact rather than with vapour phase. The efficacy of treatment with carvacrol vapour was found to be dependent on exposure time. The predominance of red fluorescence using a LIVE/DEAD BacLight Viability kit (Molecular Probes) and the partially destroyed biofilm architecture as determined by microscopy in treated samples provided evidence for the efficacy of carvacrol. The findings of this investigation suggest a potential application for carvacrol in the inactivation of staphylococcal biofilms.

Release of protein, lipid, and vitamin E from almond seeds during digestion.

The evaluation of the bioaccessibility of almond nutrients is incomplete. However, it may have implications for the prevention and management of obesity and cardiovascular disease. This study quantified the release of lipid, protein, and vitamin E from almonds during digestion and determined the role played by cell walls in the bioaccessibility of intracellular nutrients. Natural almonds (NA), blanched almonds (BA), finely ground almonds (FG), and defatted finely ground almonds (DG) were digested in vitro under simulated gastric and gastric followed by duodenal conditions. FG were the most digestible with 39, 45, and 44% of lipid, vitamin E, and protein released after duodenal digestion, respectively. Consistent with longer residence time in the gut, preliminary in vivo studies showed higher percentages of nutrient release, and microscopic examination of digested almond tissue demonstrated cell wall swelling. Bioaccessibility is improved by increased residence time in the gut and is regulated by almond cell walls.

Effects of oregano, carvacrol and thymol on Staphylococcus aureus and Staphylococcus epidermidis biofilms.

The aim of this study was to evaluate the effect of oregano essential oil, carvacrol and thymol on biofilm-grown Staphylococcus aureus and Staphylococcus epidermidis strains, as well as the effects of the oils on biofilm formation. For most of the S. aureus (n=6) and S. epidermidis (n=6) strains tested, the biofilm inhibitory concentration (0.125-0.500 %, v/v, for oregano, and 0.031-0.125 %, v/v, for carvacrol and thymol) and biofilm eradication concentration (0.25-1.0 %, v/v, for oregano and 0.125-0.500 %, v/v, for carvacrol and thymol) values were twofold or fourfold greater than the concentration required to inhibit planktonic growth. Subinhibitory concentrations of the oils attenuated biofilm formation of S. aureus and S. epidermidis strains on polystyrene microtitre plates.

Interaction of four monoterpenes contained in essential oils with model membranes: implications for their antibacterial activity.

The present article reports the antimicrobial efficacy of four monoterpenes (thymol, carvacrol, p-cymene, and gamma-terpinene) against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli. For a better understanding of their mechanism of action, the damage caused by these four monoterpenes on biomembranes was evaluated by monitoring the release, following exposure to the compounds under study, of the water-soluble fluorescent marker carboxyfluorescein (CF) from large unilamellar vesicles (LUVs) with different lipidic composition (phosphatidylcholine, PC, phosphatidylcholine/phosphatidylserine, PC/PS, 9:1; phosphatidylcholine/stearylamine, PC/SA, 9:1). Furthermore, the interaction of these terpenes with dimyristoylphosphatidylcholine multilamellar vesicles as model membranes was monitored by means of differential scanning calorimetry (DSC) technique. Finally, the results were related also with the relative lipophilicity and water solubility of the compounds examined. We observed that thymol is considerably more toxic against S. aureus than the other three terpenes, while carvacrol and p-cymene are the most inhibitory against E. coli. Thymol and carvacrol, but not gamma-terpinene and p-cymene, caused a concentration-dependent CF leakage from all kinds of LUVs employed; in particular, thymol was more effective on PC and PC/SA LUVS than on PC/PS vesicles, while carvacrol challenge evoked a CF leakage from PC/PS LUVs similar to that induced from PC/SA LUVs, and lower than that measured with PC vesicles. Concerning DSC experiments, these four terpenes caused a decrease in Tm and (especially carvacrol and p-cymene) DeltaH values, very likely acting as substitutional impurities. Taken together, our findings lead us to speculate that the antimicrobial effect of thymol, carvacrol, p-cymene, and gamma-terpinene may result, partially at least, from a gross perturbation of the lipidic fraction of the plasmic membrane of the microorganism. In addition to being related to the physicochemical characteristics of the compounds (such as lipophilicity and water solubility), this effect seems to be dependent on the lipidic composition and net surface charge of the microbic membranes. Furthermore, the compounds might cross the cell membranes, thus penetrating into the interior of the cell and interacting with intracellular sites critical for antibacterial activity.

Contribution of the Glucosinolate Fraction to the Overall Antioxidant Potential, Cytoprotection against Oxidative Insult and Antimicrobial Activity of Eruca sativa Mill. Leaves Extract.

BACKGROUND: Eruca sativa Mill. (Brassicaceae) is commonly utilized as an ingredient in salads and also as a folk remedy to treat various diseases. OBJECTIVE: The objective of this study was to establish the contribution of the glucosinolate (GLS) fraction to the overall antioxidant, cytoprotection against oxidative insult and antimicrobial properties of the hydro-alcoholic extract of E. sativa leaves from Sicily (Italy), characterized phytochemically. MATERIALS AND METHODS: The antioxidant activity was evaluated by different in vitro systems. The cytoprotective effect against hydrogen peroxide (H2O2)-induced oxidative stress was tested in human peripheral blood mononuclear cells (PBMCs). The antimicrobial potential against bacteria and fungi was assayed by standard methods. RESULTS: E. sativa extract exhibited both radical scavenging (50% inhibitory concentration [IC50] 1.04 ± 0.04 mg/mL) and ferrous ions-chelating activity (IC50 0.327 ± 0.0032 mg/mL) and mild reducing power; the GLS fraction showed chelating ability only (IC50 0.225 ± 0.009 mg/mL). In the experimental model of H2O2-induced oxidative stress in human PBMCs, a significant cytoprotective effect and a suppression of reactive oxygen species production by both extract and GLS fraction were observed (P < 0.001). E. sativa extract displayed moderate antimicrobial activity against Gram-positive bacteria, and Staphylococcus aureus was the most sensitive strain (minimum inhibitory concentration 0.125 mg/mL), whereas the GLS fraction was not active. CONCLUSION: GLSs are not involved in the primary antioxidant activity of E. sativa leaf extract but they are, almost in part, responsible for its ferrous ion-chelating properties. Iron-chelating compounds in E. sativa extract may protect cells under conditions of oxidative stress, and GLSs might play a chief role in this effect. SUMMARY: Eruca sativa Mill. leaf extract exhibited antioxidant activity in different in vitro systems, whereas the glucosinolate (GLS) fraction showed Fe2+-chelating ability onlyA significant cytoprotective effect and a suppression of intracellular reactive oxygen species production by both extract and GLS fraction were observed in human peripheral blood mononuclear cellsE. sativa extract displayed moderate antimicrobial activity against Gram-positive bacteria, whereas the GLS fraction was not active. Abbreviations used: GLS: Glucosinolate; H2O2: Hydrogen peroxide; PBMCs: Peripheral blood mononuclear cells; IC50: 50% inhibitory concentration; MIC: Minimum inhibitory concentration.

Triterpenoid saponins from Pteleopsis suberosa stem bark.

Thirteen oleanane saponins (1-13), four of which were new compounds (1-4), were isolated from Pteleopsis suberosa Engl. et Diels stem bark (Combretaceae). Their structures were determined by 1D and 2D NMR spectroscopy and ESI-MS spectrometry. The compounds were identified as 2alpha,3beta,19alpha,23,24-pentahydroxy-11-oxo-olean-12-en-28-oic acid 28-O-beta-D-glucopyranosyl ester (1), 2alpha,3beta,19beta,23,24-pentahydroxy-11-oxo-olean-12-en-28-oic acid 28-O-beta-D-glucopyranosyl ester (2), 2alpha,3beta,19alpha,23-tetrahydroxy-11-oxo-olean-12-en-28-oic acid 28-O-beta-D-glucopyranosyl ester (3), and 2alpha,3beta,6beta,19alpha,24-pentahydroxy-11-oxo-olean-12- en-28-oic acid 28-O-beta-D-glucopyranosyl ester (4). The presence of alpha,beta-unsaturated carbonyl function was not common in the oleanane class and the aglycons of these compounds were not found previously in the literature. Moreover, the isolated compounds were tested against Helicobacter pylori standard and vacA, and cagA clinical virulence genotypes. Results showed that compound 6 has an anti-H. pylori activity against three metronidazole-resistant strains (Ci 1 cagA, Ci 2 vacA, and Ci 3).

Antiviral and immunoregulatory effect of a novel exopolysaccharide from a marine thermotolerant Bacillus licheniformis.

EPS-1 is a novel extracellular polysaccharide produced by a strain of thermotolerant Bacillus licheniformis, isolated from a shallow marine hot spring of Vulcano Island (Italy). In this paper, antiviral and immunomodulatory effects of EPS-1 were evaluated. It was found that EPS-1 treatment impaired HSV-2 replication in human peripheral blood mononuclear cells (PBMC) but not in WISH cells. Since several cytokines modulate the immune response to viruses, Th1- and Th2-type cytokines were assayed in supernatants of PBMC in different experimental conditions. EPS-1 induced IL-12, IFN-gamma, IFN-alpha, TNF-alpha and IL-18, but not IL-4. Thus, the antiviral effect of EPS-1 on PBMC seems to be related to the pattern of cytokines induced.

Characterization of flavonoids and pectins from bergamot (Citrus bergamia Risso) peel, a major byproduct of essential oil extraction.

Bergamot peel is an underutilized byproduct of the essential oil and juice-processing industry. As with other Citrus peels, it still contains exploitable components, such as pectins and flavonoids. Commercial glycoside hydrolases, specifically a combination of pectolytic and cellulolytic enzymes, solubilized a high percentage of the material (81.94%). The flavonoid profile of the peel consisted of characteristic Citrus species flavanone rutinosides and neohesperosides derived from naringenin, eriodictyol, and hesperetin. In addition, a number of minor flavanone and flavone glycosides, not found in orange and lemon peels, were identified. The majority of flavonoids were extracted in the two 70% v/v EtOH extractions. Processing this material clearly has economic potential leading to low environmental impact.

Enzymatic hydrolysis of flavonoids and pectic oligosaccharides from bergamot (Citrus bergamia Risso) peel.

Pectinolytic and cellulolytic enzymes (Pectinase 62L, Pectinase 690L, and Cellulase CO13P) were used to evaluate the solubilization of carbohydrates and low molecular weight flavonoids from bergamot peel, a major byproduct of the essential oil industry. The enzymes were characterized for main-chain and side-chain polysaccharide hydrolyzing activities and also against pure samples of various flavonoids previously identified in bergamot peel to determine various glycosidase activities. The addition of Pectinase 62L or 690L alone, or the combination of Pectinase 62L and Cellulase CO13P, was capable of solubilizing between 70 and 80% of the bergamot peel, and up to 90% of the flavonoid glycosides present were cleaved to their aglycones. Cellulase CO13P alone solubilized 62% of the peel but had no deglycosylating effect on the flavonoid glycosides. Over a 24-h time course, a rapid release of cell wall carbohydrates was observed after treatment with Pectinase 62L, with a concurrent gradual hydrolysis of the flavonoid glycosides. Size-exclusion chromatography of the solubilized extract showed that after 24-h incubation, the majority of the solubilized carbohydrates were present as monosaccharides with a smaller proportion of oligosaccharides.

Ex vivo efficacy of gemifloxacin in experimental keratitis induced by methicillin-resistant Staphylococcus aureus.

In recent years, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) strains has been observed in ocular infections. Resistance of MRSA to second- and third-generation fluoroquinolones has increased interest in the fourth-generation fluoroquinolones. In this study, the antibacterial activity of gemifloxacin against MRSA ocular isolates in vitro and in a modified ex vivo rabbit keratitis model was investigated. In vitro susceptibility test results indicated that the minimum inhibitory concentrations (MICs) of gemifloxacin were lower than the MICs of other fluoroquinolones, including moxifloxacin (MIC50 range, 0.016-0.032 µg/mL; MIC90 range, 0.047-0.094 µg/mL). Results from the ex vivo keratitis model showed a statistically significant decrease in MRSA counts (0.5-2 log10 CFU/g; P <0.05) in corneas treated with 0.3% gemifloxacin every 30 min for 7 h. Moreover, the dose-response effect of different concentrations of gemifloxacin (3-3000 µg/mL) demonstrated that a dose of 30 µg/mL had the same efficacy as the highest dose of 3000 µg/mL against all S. aureus strains. Possibly, gemifloxacin reached a steady-state level in the cornea, as the fourth-generation fluoroquinolones have better anterior chamber penetration. This study demonstrated that 0.3% gemifloxacin ophthalmic solution may be an effective topical therapy for the treatment of MRSA keratitis. In addition, this reproducible, ethical and economic ex vivo infection model can be used as a mechanistically-based alternative to in vivo animal testing, bridging the gap between in vitro and in vivo results.

Antimicrobial activities, toxicity and phenolic composition of Asphodeline anatolica E. Tuzlaci leaf extracts from Turkey.

The antimicrobial activity of acetone, methanol and aqueous extracts of Asphodeline anatolica E. Tuzlaci leaves was evaluated against American type culture collection, food and clinical isolates (Listeria monocytogenes and Staphylococcus aureus including methicillin-resistant strains-MRSA). Biofilm formation, toxicity and characterisation of the polyphenolic content were analysed. The acetone extract demonstrated a higher antibacterial activity against S. aureus including MRSA strains, L. monocytogenes and Pseudomonas aeruginosa than against other extracts. No effect was observed in biofilm formation. The extracts resulted non-toxic against Artemia salina Leach. The phytochemical screening of extracts indicated that they mainly contained six polyphenols identified as catechin 3-O-gallate, protocatechuic acid, diosmin, rutin, cirsimaritin and kaempferol glucoside. This study is the first report on antimicrobial activity and phenolic content of A. anatolica and contributes to enrich the literature data on the biological properties of this plant. A. anatolica leaves have a potential as source of natural antimicrobial compounds.

In vitro activity of plant extracts against biofilm-producing food-related bacteria.

The identification of effective antimicrobial agents also active on biofilms is a topic of crucial importance in food and industrial environment. For that purpose methanol extracts of Turkish plants, Ficus carica L., Juglans regia L., Olea europaea L., Punica granatum L. and Rhus coriaria L., were investigated. Among the extracts, P. granatum L. and R. coriaria L. showed the best antibacterial activity with minimum inhibitory concentrations (MIC) of 78-625µg/ml for Listeria monocytogenes and Staphylococcus aureus and 312-1250µg/ml for Escherichia coli and Pseudomonas aeruginosa. SubMICs produced a significant biofilm inhibition equal to 80-60% for L. monocytogenes and 90-80% for S. aureus. The extracts showed also the highest polyphenol content and the strongest antioxidant activity. Bioassay-guided and HPLC procedures demonstrated the presence of apigenin 4'-O-ß-glucoside in P. granatum L. and myricetrin and quercitrin in R. coriaria L. Antigenotoxicity of plant extracts was also observed The present findings promote the value-adding of P. granatum L. and R. coriaria L. leaves as natural antimicrobial/antioxidant agents for control of food-related bacterial biofilms.

TLR2 activation in corneal stromal cells by Staphylococcus aureus-induced keratitis.

The Toll-Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus-induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air-interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub-groups exposed to UV-killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL-8 mRNA expressions were analyzed by quantitative real-time RT-PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL-8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial-corneal tissue interactions and their inflammatory consequences.

In vitro antimycoplasmal activity of oleuropein.

The activity of oleuropein, a phenolic glycoside contained in olive oil, was investigated in vitro against Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma pneumoniae and Mycoplasma pirum. Oleuropein inhibited mycoplasmas at concentrations from 20 to 320 mg/l. The MICs of oleuropein to M. pneumoniae, M. pirum, M. hominis and M. fermentans were 160, 320, 20 and 20 mg/l, respectively.

Staphylococcal biofilm formation as affected by type acidulant.

Staphylococcal growth and biofilm formation in culture medium where pH was lowered with weak organic (acetic and lactic) or strong inorganic (hydrochloric) acids were studied. The effects were evaluated by biomass measurements, cell-surface hydrophobicity, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). The results demonstrated that the inhibition was related to type of acidulant and pH value. At pH 5.0, the antibacterial effect was more pronounced in the presence of acetic acid (58-60% growth reduction) compared with that in the presence of lactic (7-16% growth reduction) and hydrochloric acids (23-24% reduction). The biofilm biomass of Staphylococcus aureus and Staphylococcus epidermidis was reduced by 92, 85, 63, and 93, 87, 81% after exposition to acetic, lactic, and hydrochloric acids, respectively. Increasing the pH from 5.0 to 6.0 resulted in a noticeable reduction in the effectiveness of acids. A minor cells hydrophobic character was also documented. The SEM and CLSM revealed a poorly structured and thinner biofilm compared with the dense and multilayered control. Acidic environment could have important implications for food-processing system to prevent bacterial colonization and control biofilm formation. The findings of this study lead to consider the rational use of the type of acid to achieve acidic environments.

Polymethoxylated, C- and O-glycosyl flavonoids in tangelo (Citrus reticulata×Citrus paradisi) juice and their influence on antioxidant properties.

A separation/identification protocol based on RP-LC-DAD-ESI-MS-MS has been employed for the characterisation of the flavonoid fraction of the juice from tangelos (Citrus reticulata×Citrus paradisi) grown in Southern Italy. Eleven compounds were identified in a single chromatographic course. Of these, two C-glycosyl flavones (lucenin-2 and vicenin-2) and an O-triglycosyl flavanone (narirutin 4'-O-glucoside) were identified for the first time. Fruit juice antioxidant activity was evaluated on the basis of its ability to scavenge DPPH, O2(-), OH and ABTS(+) radicals, and to reduce iron (FRAP). Moreover, the influence of the identified polymethoxylated, C- and O-glycosyl flavonoids on the total antioxidant activity has been elucidated. We also checked the antimicrobial activity of a broad fraction, containing all the detected flavonoids obtained by preparative HPLC, in terms of MICs for Staphylococcus aureus.

Resveratrol role in Staphylococcus aureus-induced corneal inflammation.

The aim of this study was to evaluate the role of trans-resveratrol on Staphylococcus aureus-induced keratitis. Rabbit corneas (intact corneas, abraded corneas and abraded corneas exposed to inactivated S. aureus strains) were placed in an ex vivo culture model. The abraded corneas exposed to S. aureus were divided into two 1-h-treatment sub-groups: corneas treated with trans-resveratrol and corneas treated with vehicle. The tissues were examined by immunohistochemical analyses and quantitative real-time RT-PCR to determine whether resveratrol could reduce TLR2-mediated recognition of S. aureus on epithelial cells and, if so, whether this reduction repressed the expression of inflammatory cytokines. The results demonstrated that resveratrol treatment effectively downregulated cell surface TLR2 on cells stimulated by S. aureus and reduced the expression of interleukin-8 gene. In addition, the corneal culture model tested, which is simple and reproducible, could be an alternative to in vivo animal testing for the development of novel specific therapies.