A simple and rapid lysis method for preparation of genomic DNA from Gram-negative bacteria

A very efficient, simple and economic lysis method for rapid preparation of genomic DNA from Gramnegative bacteria was developed. This method includes a novel step lysis by treatment with carvacrol followed by simple ethanol precipitation. The procedure was realised without using of detergents or enzymes. Moreover, the resultant genomic DNA was in good quantity and quality and can be used successfully for restriction endonucleases digestion, PCR amplification and others types of molecular biology manipulations.Keywords: Genomic DNA, lysis, carvacrol, Gram-negative bacteria, Escherichia coli, Erwinia chrysanthem

Comparative study of the antifungal activity of some essential oils and their major phenolic components against Aspergillus niger using three different methods

This study aimed to evaluate the antimould activity of oregano, thyme, rosemary and clove essential oils and some of their main constituents: eugenol, carvacrol and thymol against Aspergillus niger. This antifungal activity was assessed using broth dilution, disc diffusion and micro atmosphere methods. In both agar diffusion and micro atmosphere tests, all the investigated agents showed no inhibitory effect on Aspergillus niger growth at concentrations lower than 10% (v/v). However, broth dilution test showed the highest sensitivity. Using this method, the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values of the tested agents were between 0.025 and 1%. The anti-Aspergillus effect of oregano and thyme oils was more potent than that of clove and rosemary oils. Concerning the phenolic compounds, thymol and carvacrol proved to have better anti-Aspergillus effect than eugenol. Accordingly, we can say that the antifungal efficacy of these agents is better appreciated when they are applied directly into liquid medium than when they are applied as volatiles or diffused in solid medium. Therefore, as these agents are active at low concentrations, they could be used in the formulation of natural preparations, and thereby could be proposed in therapeutic or hygienic contexts.Key words: Essential oils, thymol, carvacrol, antifungal activity, Aspergillus niger, micro atmosphere, agar diffusion, broth dilution

Metabolisme des phospholipides dans les plaquettes du rat spontanement hypertendu

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Effet de l’adjonction d’un additif naturel riche en huiles essentielles de thym sur l’émission du méthane entérique et sur les performances de production des bovins

The objective of the present study is to evaluate the effect of the incorporation, in the feed ration, of natural additive rich in phenolic essential oils based on thyme, on the emission of enteric methane in dairy cows. In a second step, the study consists in evaluating the effect of the same additive on the performances of cattle, namely milk production yield in lactating cows and the average daily gain in feedlot cattle. The study measured methane production before and after the addition of the feed additive to the diet of five Holstein cows. After two weeks of adaptation to the diet, methane production was measured by the respiratory mask method. Then, 50 g of the product rich in thyme essential oils was added to the ration, which means 7.15 g/kg dry matter, and a second methane measurement was performed after two weeks of adaptation. The addition of the product to the cows' diet resulted in an average reduction of 21% in the amount of enteric methane emitted. In a second step, the same product was used to test its effect on live weight gain in feedlot bulls. In this respect, ten bulls from a fattening farm were tested. Five received 70 g of the additive in their feed ration and five served as controls. The bulls that received the feed additive showed a higher average daily gain compared to the control group. The results obtained were highly significant (p<0.01), with means of 1.55 ± 0.058 kg for the control group vs 1.88 ± 0.177 kg for the group that received the additive. To test the effect of the same feed additive on milk production, a third trial was conducted. Eight Holstein cows were divided into two homogeneous groups. Four received 100 g of the product rich in essential thyme oil in their feed ration and four served as a control group. An increase in daily milk production was noted in all cows receiving the additive compared to the control cows. The results of the average amounts of milk produced per liter per day were statistically significant (p<0.05), with averages of 15.38±1.32 l/d for the control group and 19.17±1.96 l/d for the group with the additive. Milk fat was also improved in the cows supplemented with the additive, with an average of 3.54±0.26 g/100 g in the control group vs 3.66±0.34 g/100 g of milk in the group with the additive. However, the results obtained for the milk fat showed no statistic significance (p>0.05). The trials undertaken during this study allowed to verify the interest and the relevance of the use of the tested natural feed additive, not only for the decrease of enteric methane emission and the preservation of the environment, but also for its beneficial effects on cattle productions.  Keywords: Cattle, essential oils, thyme, greenhouse effect, methane, performanceL’objectif de la présente étude est d’évaluer l’effet de l’incorporation dans la ration alimentaire d’un additif naturel riche en huiles essentielles phénoliques à base de thym, sur l’émission du méthane entérique chez la vache laitière. L’étude évalue également l’effet du même additif sur les performances des bovins, à savoir la production laitière chez des vaches en lactation et le gain de poids vif chez des taurillons à l’engraissement. L’étude a porté sur la mesure de la production du méthane avec ou sans l’ajout de l’additif alimentaire à la ration de cinq vaches de race Holstein. Après deux semaines d’adaptation au régime alimentaire, la production de méthane a été mesurée par la méthode du masque respiratoire. Ensuite, 50 g du produit riche en huiles essentielles de thym a été ajouté à la ration, soit 7,15 g/kg de matière sèche, et une deuxième mesure de méthane a été effectuée après deux semaines d’adaptation. L’addition du produit à l’alimentation des vaches a été à l’origine d’une réduction moyenne de 21% de la quantité de méthane entérique émise. Dans un second essai, le même produit a été utilisé pour tester son effet sur le gain de poids vif chez des taurillons d’engraissement. A ce propos, dix taurillons d’un atelier d’engraissement ont fait objet de l’expérimentation. Cinq ont reçu 70 g de l’additif dans leur ration alimentaire et cinq ont servi de témoins. Les taurillons qui ont reçu l’additif alimentaire ont montré un gain de poids vif moyen plus important, comparé au lot témoin. Les différences quant aux gains moyens quotidiens (GMQ) sont hautement significatives (p<0,01), avec des moyennes de 1,55 ± 0,058 kg pour le lot témoin et 1,88 ± 0,177 kg pour le lot qui a reçu l’additif. Afin de tester également l’effet du même additif alimentaire sur la production laitière, un troisième essai a été mené. Ainsi, huit vaches de race Holstein ont été scindées en deux groupes homogènes. Quatre ont reçu 100 g du produit riche en huiles essentielles de thym dans leur ration alimentaire et quatre ont servi de lot témoin. Une augmentation de la production laitière journalière a été notée chez toutes les vaches qui ont reçu l’additif par rapport aux vaches témoins. La production laitière des vaches ayant reçu l’additif a été de 19,2 ±1,96 l/j et celle du lot témoin a été de 15,4 ± 1,32 l/j. Cette différence de production laitière entre les deux lots a été statistiquement significative (p<0,05). Le taux butyreux a aussi connu une amélioration significative chez les vaches supplémentées avec l’additif (p>0,05). Ainsi, une moyenne de 3,54 ± 0,26 g/100 g de lait a été noté chez le lot témoin contre 3,66 ± 0,34 g/100 g de lait chez le lot avec additif. Les essais entrepris durant cette étude ont permis de vérifier l’intérêt et la pertinence de l’utilisation de l’additif alimentaire naturel testé, non seulement pour la réduction de l’émission du méthane entérique, et donc de la préservation de l’environnement, mais également pour ses effets bénéfiques sur les productions bovines. Mots clés: Bovins, huiles essentielles, thym, effet de serre, méthane, performance

A One-Pot Ultrasound-Assisted Almond Skin Separation/Polyphenols Extraction and its Effects on Structure, Polyphenols, Lipids, and Proteins Quality

Almond skin is an important by-product in the almond processing industry, rich in potentially health-promoting phenolic compounds. The objective of this present study is to separate the skin from the almond and extract its polyphenol contents using Ultrasound-Assisted Extraction (UAE) at room temperature. Optimization was performed according to a two-variable central composite design (CCD), and the optimum combination of ultrasonic intensity and extraction temperature was obtained through multi-response optimization: ultrasonic intensity (UI), 9.47 W.cm−2; and temperature, 20 °C for an extraction time of 20 min. Under the above-mentioned conditions, total phenolic content was 258% higher with UAE than silent experiment. Mathematic modelling and microscopic investigations were achieved to enable understanding physical and structural effects of ultrasound on almond skins and comprehension of the mechanism behind the enhancement of mass transfer phenomena. Scanning Electron Microscopy (SEM) showed different acoustic cavitation impacts including fragmentation, sonoporation, and erosion. Extracts were analyzed by ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS), identifying a combination of flavanols, flavanones and non-flavonoids. UAE shows no negative effect on almond proteins and lipids when compared to natural almonds (NS)

In Vitro and In Vivo Comparison of Changes in Antibiotics Susceptibility of E. coli and Chicken’s Intestinal Flora after Exposure to Amoxicillin or Thymol

This study aims at verifying, in vitro, the extent to which the use of amoxicillin or thymol induces the selection of resistant bacteria and at evaluating in vivo their effects on the development of antimicrobial resistance in the intestinal flora of poultry. E. coli strain was subcultured on agar plates containing increasing concentrations of either amoxicillin or thymol. Thereafter, minimal inhibitory concentrations (MICs) of thymol, amoxicillin, and two other antibiotics, tylosin and colistin, were determined using the microdilution method. Groups of chicks were subjected to a 2-week regime of either amoxicillin or thymol added to their drinking water. During the treatment with either thymol or amoxicillin, the total aerobic mesophilic flora (TAMF) was counted on thymol-gradient plates or amoxicillin-gradient plates and the MICs of antibiotics and thymol for E. coli isolates were determined. The in vitro test showed that for E. coli, which had been serially subcultured on increasing concentrations of amoxicillin, a 32-fold increase in MIC values for amoxicillin and a 4-fold increase for colistin and tylosin were noted. However, the MIC of thymol for this strain remained constant. For the E. coli, which had been serially subcultured on increasing concentrations of thymol, no change in the MIC values for antibiotics and thymol was observed. The in vivo test confirmed the in vitro one. It demonstrated that exposure to amoxicillin induced a selection of antimicrobial resistance in TAMF and intestinal E. coli, whereas exposure to thymol did not. The results showed that the group receiving thymol had a lower consumption index compared to the other groups. This study demonstrates the feasibility of this natural product as an alternative solution to the current use of antibiotics in poultry farming

Synergistic Action of AMX Associated with 1,8-Cineole and Its Effect on the ESBL Enzymatic Resistance Mechanism

The purpose of the present study is twofold. First, it aims to evaluate the synergistic action of the ß-lactam antibiotic; AMX is associated with 1,8-cineole on six clinical isolates of ESBL-producing Escherichia coli and Klebsiella pneumoniae strains. Second, it aims to determine the effect this association has on the ESBL enzymatic resistance mechanism. The synergistic action of AMX/1,8-cineole was evaluated using partial inhibitory concentrations (PIC), determined by a microplate, a checkerboard and time–kill assays. The effect of AMX/1,8-cineole associations on the ESBL enzymatic resistance mechanism was evaluated using a new optimized enzymatic assay. This assay was based on the determination of the AMX antibacterial activity when combined with 1,8-cineole (at subinhibitory concentrations) in the presence or absence of the ß-lactamase enzyme toward a sensitive E. coli strain. The results of both checkerboard and time–kill assays showed a strong synergistic action between AMX and 1,8-cineole. The results of the enzymatic assay showed that the combination of AMX with 1,8-cineole notably influences the enzymatic resistance of the reaction by decreasing the affinity of the β-lactam antibiotic, AMX, to the β-lactamase enzyme. All obtained results suggested that the AMX/1,8-cineole association could be employed in therapy to overcome bacterial resistance to AMX while reducing the prevalence of resistance

The Combination of Amoxicillin and 1,8-Cineole Improves the Bioavailability and the Therapeutic Effect of Amoxicillin in a Rabbit Model

In this study, the effectiveness of the combination therapy of 1,8-cineole with amoxicillin (AMX) and clavulanic acid (Clav) was investigated. For this, the pharmacokinetic behaviors of AMX in rabbits were studied after a single oral dose. The animals were divided randomly into two groups: the reference group (received AMX/Clav (50/12.5 mg/kg)) and the test group (received AMX/Clav/1,8-cineole (50/12.5/10 mg/kg)). Blood samples were collected prior to administration and after T1h, T2h, T3h, and T6h post-administration. Plasma concentrations of AMX were quantified using a validated HPLC method. The antibacterial activity of plasma and cerebrospinal fluid (CSF) of treated rabbits was tested against Escherichia coli ESBL-producing a strain by microdilution method. The obtained results showed significant differences in pharmacokinetic parameters between the two groups. The resulting AUC0–6h and Cmax mean values of the AMX reference group were 14.74 µg.h/mL and 3.49 µg/mL, respectively. However, those of the AMX test group were 22.30 µg.h/mL and 5.79 µg/mL, respectively. The results showed that the antibacterial activity of the plasma and CSF test group was significantly higher than that of the reference group. The effectiveness of this combination (Olipen: AMX/Clav/1,8-cineole) was demonstrated by increasing the level of the antibiotic and by improving the bioavailability

The Antistaphylococcal Activity of Amoxicillin/Clavulanic Acid, Gentamicin, and 1,8-Cineole Alone or in Combination and Their Efficacy through a Rabbit Model of Methicillin-Resistant Staphylococcus aureus Osteomyelitis

The aim of this research paper is to test the antistaphylococcal effect of 1,8-cineole, amoxicillin/clavulanic acid (AMC), and gentamicin, either separately or in combination against three Staphylococcus aureus strains isolated from patients suffering from osteomyelitis. This activity was tested in vitro by using the microdilution method and the checkerboard assay. The efficacy of these three antibacterial agents was then tested in vivo by using an experimental model of methicillin-resistant S. aureus osteomyelitis in rabbits. This efficacy was assessed after four days of treatment by counting the number of bacteria in the bone marrow. The obtained results in vitro showed that the combination of the AMC with gentamicin did not induce a synergistic effect, whereas the combination of the two antibiotics with 1,8-cineole did. This effect is stronger when AMC is combined with 1,8-cineole as a total synergistic effect was obtained on the three strains used (FIC ≤ 0.5). In vivo, a significant reduction was noted in the number of colonies in the bone marrow when rabbits were treated with AMC associated with either 1,8-cineole or gentamicin compared to rabbits treated with AMC, gentamicin, or 1,8-cineole alone. These results demonstrated that 1,8-cineole showed a synergistic effect in combination with both AMC and gentamicin, which offer possibilities for reducing antibiotic usage. Also, the AMC associated with 1,8-cineole could be used to treat MRSA osteomyelitis