Influence of general stress-response alternative sigma factors σ(S) (RpoS) and σ(B) (SigB) on bacterial tolerance to the essential oils from Origanum vulgare L. and Rosmarinus officinalis L. and pulsed electric fields.

This study assessed the influence of general stress-response alternative sigma factors RpoS (σ(S)) and SigB (σ(B)) on tolerance of Escherichia coli (E. coli MG1655 and its isogenic mutant E. coli MG1655 ΔrpoS) and Listeria monocytogenes (L. monocytogenes EGD-e and its isogenic mutant L. monocytogenes EGD-e ΔsigB) to the essential oils (EOs) from Origanum vulgare L.-oregano (OVEO) and Rosmarinus officinalis L.-rosemary (ROEO), as well as the changes in tolerance of parental and ΔrpoS and ΔsigB mutant strains to OVEO, ROEO and pulsed electric fields (PEF) following overnight exposure to subinhibitory concentrations (1/2×minimum inhibitory concentration-MIC) of each tested EO. MIC values of OVEO and ROEO against the mutant cells were usually lower than those found against the parental cells. Survivor curves showed that mutant cells were more sensitive to these EOs than parental cells. The recovery of survivors in selective media showed a greater proportion of cells sublethally injured at their cell envelopes in the mutant strains compared with the parental strains. Induction of increased direct-tolerance to OVEO and ROEO or cross-tolerance to PEF was not observed after pre-exposure of parental and mutant cells to EOs. Otherwise, parental and mutant cells of E. coli and L. monocytogenes pre-exposed to OVEO or ROEO showed decreased tolerance when further treated with the homologous stressing agent at 2×MIC. Still, mutant cells pre-exposed to OVEO or ROEO showed lower tolerance to PEF than parental strains. These results showed the influence of σ(S) and σ(B) in tolerance of single strains of E. coli and L. monocytogenes, respectively, to OVEO and ROEO. Moreover, the deletion of σ(S) and σ(B) resulted in decreased tolerance to OVEO, ROEO or PEF in tested strains following exposure to OVEO or ROEO at a subinhibitory concentration.

Assessment of tolerance induction by Origanum vulgare L. essential oil or carvacrol in Pseudomonas aeruginosa cultivated in a meat-based broth and in a meat model.

This study assessed the efficacy of Origanum vulgare L. essential oil (OVEO) and carvacrol in inhibiting the growth of Pseudomonas aeruginosa ATCC 9027, as well as the development of direct tolerance and cross-tolerance when this bacterium was challenged with sublethal amounts of these substances in a meat-based broth and in a meat model. OVEO and carvacrol at their minimum inhibitory concentrations (MICs), 1/2 MIC and 1/4 MIC decreased the viable cell counts of P. aeruginosa in meat-based broth. Direct tolerance or cross-tolerance was not induced after exposure of the assayed bacterial strain to sublethal amounts of OVEO or carvacrol in meat-based broth and in an artificially contaminated ground beef. Bacterial cells progressively subcultured in meat-based broth with increasing amounts of the tested substances survived up to the MIC of OVEO and to 1/2 MIC of carvacrol. The results reveal a lack of induction of tolerance in P. aeruginosa by exposure to OVEO or carvacrol in meat-based broth and in a meat model.

Lack of induction of direct protection or cross-protection in Staphylococcus aureus by sublethal concentrations of Origanum vulgare L. essential oil and carvacrol in a meat-based medium.

The capacity of Origanum vulgare L. essential oil (OVEO) and its majority compound, carvacrol (CAR), to induce direct tolerance and cross-tolerance in Staphylococcus aureus against high temperature (45 °C), lactic acid (pH 5.2) and NaCl (10 g/100 mL) was assessed. Overnight exposure of S. aureus to sublethal concentrations (1/2 MIC, 1/4 MIC) of either OVEO or CAR in meat broth revealed no induction of direct protection. S. aureus cells pre-adapted to OVEO or CAR showed no induction of cross-protection to high temperature, lactic acid or NaCl. Cells subjected to 24 h cycles of adaptation in increasing amounts (1/2 MIC to 2 × MIC) of OVEO or CAR showed no increase in direct tolerance. These results revealed a lack of induction of direct protection or cross-protection in S. aureus exposed to sublethal amounts of OVEO or CAR in meat-based broth, as determined by monitoring cell survival and growth behavior.

Rosmarinus officinalis L. essential oil and its majority compound 1,8-cineole at sublethal amounts induce no direct and cross protection in Staphylococcus aureus ATCC 6538.

In this study, the inhibitory efficacy of Rosmarinus officinalis essential L. (ROEO) and 1,8-cineole (CIN) in inhibiting the growth and survival of Staphylococcus aureus ATCC 6538 and the induction of direct and bacterial cross protection (lactic acid pH 5.2; NaCl 100 g/L; high temperature 45°C) were evaluated following exposure to sublethal and increasing amounts of these treatments in meat broth. All of the concentrations of the ROEO and CIN examined in this study (minimum inhibitory concentration [MIC], 1/2 MIC, and 1/4 MIC) inhibited the viability of S. aureus throughout the 120 min of exposure. The overnight exposure of S. aureus to sublethal amounts of both ROEO or CIN in meat broth did not result in direct or cross protection. Cells progressively subcultured (24-h cycles) in meat broth with increasing amounts of ROEO or CIN showed no increased direct tolerance. These results reveal the antimicrobial efficacy of ROEO and CIN for use in food conservation systems as anti-S. aureus compounds given their efficacy at inhibiting bacterial growth, in addition to their lack of induction for the development of homologous and heterologous resistance.

Combination of Origanum vulgare L. essential oil and lactic acid to inhibit Staphylococcus aureus in meat broth and meat model

This study assessed the occurrence of an enhancing inhibitory effect of the combined application of Origanum vulgare L. essential oil and lactic acid against Staphylococcus aureus by the determination of Fractional Inhibitory Concentration (FIC) index and cell viability in meat broth and meat model. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the oil was 0.6 and 1.25 µL.mL-1, respectively. Lactic acid showed MIC and MBC of 2.5 and 5µL.mL-1, respectively. FIC indices of the combined application of the oil and lactic acid were 0.5 showing a synergic interaction. The essential oil and lactic acid showed similar (p>0.05) anti-S. aureus effect in meat broth over 96 h of exposure. Treatment with essential oil or lactic acid presented a smaller anti-staphylococcal effect in meat in comparison to meat broth. No significant difference (p>0.05) was found for the microbial counts in meat treated with each antimicrobial alone or in mixture. These results could arise as an interesting approach for the improvement of food preservation using more natural procedures, considering the current demand of consumer and sensory quality of foods.

Rosmarinus officinalis L. essential oil and the related compound 1,8-cineole do not induce direct or cross-protection in Listeria monocytogenes ATCC 7644 cultivated in meat broth.

Listeria monocytogenes has the capability of adapting to 1 or more antimicrobial compounds or procedures applied by the food industry to control the growth and survival of microorganisms in foods. In this study, the effects of Rosmarinus officinalis essential oil (EO) and the related compound 1,8-cineole on the inhibition of the growth and survival of L. monocytogenes ATCC 7644 were determined. The ability of the R. officinalis EO and 1,8-cineole to induce direct and cross-protection of bacteria against various stresses (lactic acid, pH 5.2; NaCl, 3 g/100 mL; high temperature, 45 °C) was also determined. At all concentrations tested (minimum inhibitory concentration (MIC), ½ MIC, and » MIC), both compounds inhibited the cell viability of L. monocytogenes over 120 min of exposure. Overnight exposure of L. monocytogenes to sublethal amounts of either the R. officinalis EO or 1,8-cineole in meat broth revealed no induction of direct or cross-protection against lactic acid, NaCl, or high temperature. Similarly, cells subjected to 24 h cycles of adaptation with increasing amounts (½ MIC to 2× MIC) of the EO and 1,8-cineole showed no increase in direct tolerance, as they were able to survive in growth medium containing up to ½ MIC of either substance. These results show the antimicrobial efficacy of R. officinalis EO and 1,8-cineole for use in systems, particularly as anti-L. monocytogenes compounds.

Evidence for lack of acquisition of tolerance in Salmonella enterica serovar Typhimurium ATCC 14028 after exposure to subinhibitory amounts of Origanum vulgare L. essential oil and carvacrol.

Overnight exposure of Salmonella enterica serovar Typhimurium to sublethal amounts of Origanum vulgare essential oil (OV) and carvacrol (CAR) did not result in direct and cross-bacterial protection. Cells subcultured with increasing amounts of OV or CAR survived up to the MIC of either compound, revealing few significant changes in bacterial susceptibility.

Inhibition of Staphylococcus aureus in broth and meat broth using synergies of phenolics and organic acids.

This study assessed the occurrence of an enhancing inhibitory effect of the combined application of thymol and carvacrol with lactic and acetic acid against Staphylococcus aureus using the determination of Fractional Inhibitory Concentration (FIC) index and kill-time assay in meat broth and in a food model (meat). Minimum Inhibitory Concentration (MIC) of carvacrol and thymol were 1.25 and 0.6 microL/mL, respectively. FIC indices of the combined application of the phenolic constituents and organic acids were 0.5 suggesting a synergic interaction. No difference (p>0.05) was found among the bacterial counts for the meat broth added of the phenolics alone or in combination with lactic acid at sub-inhibitory concentrations. Contrarily, the bacterial counts found for the meat broth added of the phenolics alone were significantly lower (p<0.05) than the counts obtained for the broth added of the combination of phenolics and acetic acid. No difference (p>0.05) was found among the bacterial counts for the meat model added of phytochemicals alone and in combination with the organic acids. The tested antimicrobials alone or in mixture provided smaller anti-staphylococcal effect in meat broth than in meat model. These results could arise as an interesting approach for the improvement of food preservation using more natural procedures, considering the current demand of consumer and sensory quality of foods.

Combined application of origanum vulgare l. essential oil and acetic acid for controlling the growth of staphylococcus aureus in foods / Aplicação combinada do óleo essencial de Origanum vulgare L. e ácido acético para o controle do crescimento de Staphylococcus aureus em alimentos

This study evaluated the occurrence of an enhancing inhibitory effect of the combined application of Origanum vulgare L. essential oil and acetic acid against Staphylococcus aureus by the determination of Fractional Inhibitory Concentration (FIC) index and kill-time assay in nutrient broth, meat broth and in a food model (meat pieces). Acetic acid showed MIC and MFC of 0.6 and 1.25 µL.mL-1, respectively. For O. vulgare essential oil MIC and MBC were 1.25 and 2.5 µL.mL-1, respectively. FIC indexes of the mixture of essential oil and acetic acid at MIC x ½ were £ 1.0, showing an additive effect. No synergy was found at kill-time study. Anti-staphylococcal effect of the antimicrobials alone or in mixture (MIC x ½) was lower in meat than in nutrient and meat broths. The effective combination of essential oils and organic acids could appear as an attractive alternative for the food industry, as the doses to inhibit the microbial growth in foods can be lowered.

Este estudo avaliou a ocorrência de um efeito inibitório potencializado quando da aplicação combinada do óleo essencial de O. vulgare e ácido acético sobre Staphylococcus aureus através da determinação Concentração Inibitória Fracional (FIC) e de ensaios de tempo de morte em caldo nutriente, caldo base carne e em um modelo alimentar (pedaços de carne). O ácido acético mostrou um valor de CIM e CBM de 0,6 e 1,25 µL.mL-1, respectivamente. Estudos prévios encontraram valores de CIM e CBM para o óleo essencial de O. vulgare sobre as cepas teste de S. aureus de 1,25 e 1,5 µL.mL-1, respectivamente. Valores de índices de CIF da mistura do óleo essencial e ácido acético na concentração de CIM x ½ foram £ 1,0 caracterizando uma interação de adição. Nenhum efeito sinérgico foi encontrado nos ensaios de tempo de morte. O efeito anti-estafilocócico dos antimicrobianos isolados ou em combinação (CIM x ½) foi menor quando aplicado em carne em comparação a sua adição em caldo nutriente e caldo carne. A efetiva combinação de óleos essenciais e outros agentes preservativos pode ser reconhecida como uma alternativa promissora para a indústria de alimentos, podendo viabilizando a diminuição de doses de antimicrobianos aplicadas para inibir o crescimento microbiano em alimentos.