Action of carvacrol in Salmonella Typhimurium biofilm: A proteomic study.

Carvacrol presents action in Salmonella Typhimurium biofilms, however the antibiofilm mechanism of this compound has not been fully established yet. In the present study, the aim was to evaluate protein profile changes in S. Typhimurium biofilm treated with carvacrol. Proteomic analysis of treated versus untreated biofilm showed several changes in proteins involved with S. Typhimurium biofilm and antioxidant activity. The proteins DsbA (thiol: disulfide interchange protein DsbA), LuxS (S-ribosylhomocysteine lyase), DksA (RNA polymerase binding transcription factor DksA), and SODs (superoxide dismutases) A, B and C had their synthesis decreased after treatment with carvacrol. These proteins play a key role in S. Typhimurium biofilm formation, demonstrating the dynamic antibiofilm action of carvacrol. The differentially expressed proteins identified provide possible action targets for future studies in order to gain more insight into the mechanism of action of carvacrol on S. Typhimurium biofilm.

Photodynamic inactivation as an emergent strategy against foodborne pathogenic bacteria in planktonic and sessile states.

Foodborne microbial diseases are still considered a growing public health problem worldwide despite the global continuous efforts to ensure food safety. The traditional chemical and thermal-based procedures applied for microbial growth control in the food industry can change the food matrix and lead to antimicrobial resistance. Moreover, currently applied disinfectants have limited efficiency against biofilms. Therefore, antimicrobial photodynamic therapy (aPDT) has become a novel alternative for controlling foodborne pathogenic bacteria in both planktonic and sessile states. The use of aPDT in the food sector is attractive as it is less likely to cause antimicrobial resistance and it does not promote undesirable nutritional and sensory changes in the food matrix. In this review, aspects on the antimicrobial photodynamic technology applied against foodborne pathogenic bacteria and studied in recent years are presented. The application of photodynamic inactivation as an antibiofilm strategy is also reviewed.

Photodynamic Inactivation Mediated by Erythrosine and its Derivatives on Foodborne Pathogens and Spoilage Bacteria.

The purpose of the present study was to evaluate the efficacy of photodynamic inactivation (PDI) mediated by erythrosine (ERY) and its ester derivatives erythrosine methyl ester (ERYMET) and erythrosine butyl ester (ERYBUT) on foodborne pathogens and spoilage bacteria. We evaluated Staphylococcus aureus ATCC 25923, Aeromonas hydrophila ATCC 7966, Salmonella enterica serotype Typhimurium ATCC 14028, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853. The toxicity of all of the compounds was assessed in VERO cells. PDI mediated by ERY and its derivatives combined with a light-emitting diode was performed at different concentrations and exposure times. S. aureus was more photosensitive than Gram-negative bacteria to ERY, ERYMET, and ERYBUT. The ERY-mediated PDI of S. aureus induced a significant reduction of 4.0 log CFU/ml at a light dose of 40 J/cm(2). ERYMET and ERYBUT at lower light doses than ERY completely eradicated S. aureus. When photoirradiated with ERY at light doses of 156 and 234 J/cm(2), A. hydrophila was completely eradicated. ERYBUT was more efficient in the PDI of A. hydrophila than ERYMET, even at 1 x 10(-5) M and lower light doses. Salmonella Typhimurium, E. coli, and P. aeruginosa required higher concentrations of photosensitizers to reduce cell survival. ERYBUT and ERY may be promising photosensitizing agents against A. hydrophila and S. aureus. They were effective at reducing bacterial counts at nontoxic concentrations. The photoinactivation rate of the evaluated bacteria decreased in the following order: S. aureus > A. hydrophila > E. coli > S. Typhimurium > P. aeruginosa.

Multi-targets of antimicrobial photodynamic therapy mediated by erythrosine against Staphylococcus aureus identified by proteomic approach.

Staphylococcus aureus is a global challenge to the clinical field and food industry. Therefore, the development of antimicrobial photodynamic therapy (aPDT) has become one of the valuable methods to control this pathogen. The antibacterial activity of photoinactivation by erythrosine (Ery) against S. aureus has been reported, but its modes of action are unclear. This study aimed to employ a proteomic approach to analyze modes of action of Ery-aPDT against S. aureus. We determined the antibacterial effect by Ery-aPDT assays, quantified reactive oxygen species (ROS) and injury to the cell membrane, and determined protein expression using a proteomic approach combined with bioinformatic tools. Ery-aPDT was effective in reducing S. aureus to undetectable levels. In addition, the increment of ROS accompanied the increase in the reduction of cell viability, and damage to cellular membranes was shown by sublethal injury. In proteomic analysis, we found 17 differentially expressed proteins. These proteins revealed changes mainly associated with defense to oxidative stress, energy metabolism, translation, and protein biosynthesis. Thus, these results suggest that the effectiveness of Ery-aPDT is due to multi-targets in the bacterial cell that cause the death of S. aureus.

Synergistic inhibition of Salmonella Typhimurium and Staphylococcus aureus in apple jam by cinnamaldehyde and potassium sorbate.

The objective of this study was to evaluate the antimicrobial effectiveness of cinnamaldehyde (CIN) and potassium sorbate (P.S.), alone and in combination, against Salmonella Typhimurium and Staphylococcus aureus in vitro and in apple jam. Antimicrobial activity in vitro was investigated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), time-kill assay and determination of fractional inhibitory concentration index. CIN MIC and MBC was 312 µg/mL. P.S. MIC and MBC were 2500 and 5000 µg/mL, respectively, against S. Typhimurium; and 10,000 and 20,000 µg/mL, respectively, against S. aureus. The compounds combined exhibited a synergistic effect (FIC < 0.5), inhibiting S. Typhimurium growth after 12 h and S. aureus after 24 h. The effect of CIN and P.S., at sub-inhibitory concentrations, against bacterial strains in apple jam was evaluated during storage. Physicochemical and sensory analyses were also performed. No cultivable S. Typhimurium or S. aureus cells were recovered in apple jam supplemented with CIN + P.S. on the third day of storage. The addition of CIN and P.S. did not affect the physicochemical properties and sensory evaluation showed a score above 7.0. CIN and P.S. association at sub-inhibitory concentrations was effective in controlling foodborne pathogens and improved the shelf life of apple jam.

Proteomic Investigation over the Antimicrobial Photodynamic Therapy Mediated by Rose Bengal Against Staphylococcus aureus.

In order, understanding the antimicrobial action of photodynamic therapy and how this technique can contribute to its application in the control of pathogens. The objective of the study was to employ a proteomic approach to investigate the protein profile of Staphylococcus aureus after antimicrobial photodynamic therapy mediated by rose bengal (RB-aPDT). S. aureus was treated with RB (10 nmoL L-1 ) and illuminated with green LED (0.17 J cm-2 ) for cell viability evaluation. Afterward, proteomic analysis was employed for protein identification and bioinformatic tools to classify the differentially expressed proteins. The reduction in S. aureus after photoinactivation was ~2.5 log CFU mL-1 . A total of 12 proteins (four up-regulated and eight down-regulated) correspond exclusively to alteration by RB-aPDT. Functionally, these proteins are distributed in protein binding, structural constituent of ribosome, proton transmembrane transporter activity and ATPase activity. The effects of photodamage include alterations of levels of several proteins resulting in an activated stress response, altered membrane potential and effects on energy metabolism. These 12 proteins required the presence of both light and RB suggesting a unique response to photodynamic effects. The information about this technique contributes valuable insights into bacterial mechanisms and the mode of action of photodynamic therapy.

Anti-mycotoxigenic and antifungal activity of ginger, turmeric, thyme and rosemary essential oils in deoxynivalenol (DON) and zearalenone (ZEA) producing Fusarium graminearum.

This study aimed to evaluate the antimycotoxigenic effect of essential oils (EOs) obtained from four different aromatic plants on the production of deoxynivalenol (DON) and zearalenone (ZEA) by Fusarium graminearum. The EOs from ginger (GEO), turmeric (TEO), thyme (ThEO) and rosemary (REO) were obtained by hydrodistillation and identified by gas chromatography/mass spectrometry (GC/MS). The major compounds found were mostly monoterpenes and sesquiterpenes. The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were 11.25, 364, 366 and 11,580 µg mL-1 for ThEO, GEO, REO and TEO, respectively. The results evidenced that the assessed EOs inhibited DON and partially ZEA production by F. graminearum. ThEO and GEO were the EOs with most potent antimycotoxigenic action for DON and ZEA, respectively. These EOs have shown promising results in vitro regarding inhibition of mycotoxin production and might be used in the future as substitutes for synthetic fungicides.

Antifungal and antiaflatoxigenic activity of rosemary essential oil (Rosmarinus officinalis L.) against Aspergillus flavus.

The increased risk to health by diverse pathologies, such as cancer, liver diseases, and endocrine alterations, caused by chemical residues in food, has led to the search for sustainable agricultural management alternatives, such as the use of essential oils for the development of natural and eco-friendly fungicides. The aim of this study was to evaluate the antifungal and antiaflatoxigenic activity of Rosmarinus officinalis L. essential oil (REO) against Aspergillus flavus Link. REO was obtained by hydrodistillation and its major components were identified as 1,8-cineole (eucalyptol, 52.2%), camphor (15.2%) and α-pinene (12.4%) by GC/MS and NMR. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were both 500 µg/mL. REO reduced the mycelial growth of A. flavus at a concentration of 250 µg/mL (15.3%). The results obtained from scanning electron microscopy (SEM) demonstrated a reduction in the size of conidiophores and in the thickness of hyphae in A. flavus caused by treatment with REO (250 µg/mL). The production of ergosterol and the biomass of mycelium were both reduced as the REO treatment concentration increased. The production of aflatoxins B1 and B2 was inhibited after treatment with 250 µg/mL REO, a concentration below the MIC/MFC, indicating that the antiaflatoxigenic effect of REO is independent of its antifungal effect and is likely due to its direct action upon toxin biosynthesis. The data demonstrated that REO may be used as an alternative to synthetic fungicides.

Xanthene Dyes and Green LED for the Inactivation of Foodborne Pathogens in Planktonic and Biofilm States.

This study evaluated the rose bengal- and erythrosine-mediated photoinactivation against Salmonella Typhimurium and Staphylococcus aureus planktonic and sessile cells using green LED as a light source. The free-living or 2-day-old biofilm cells were treated with different concentrations of the photosensitizing agents and subjected to irradiation. Only 5 min photosensitization with rose bengal at 25 nmol L-1 and 75 µmol L-1 completely eliminated S. aureus and S. Typhimurium planktonic cells, respectively. Erythrosine at 500 nmol L-1 and 5 min of light exposure also reduced S. aureus planktonic cells to undetectable levels. Eradication of S. aureus biofilms was achieved when 500 µmol L-1 of erythrosine or 250 µmol L-1 of rose bengal was combined with 30 min of irradiation. Scanning electron microscopy allowed the observation of morphological changes in planktonic cells and disruption of the biofilm architecture after photodynamic treatment. The overall data demonstrate that rose bengal and erythrosine activated by green LED may be a targeted strategy for controlling foodborne pathogens in both planktonic and sessile states.

Cinnamaldehyde induces changes in the protein profile of Salmonella Typhimurium biofilm.

The effect of cinnamaldehyde against biofilm cells of Salmonella Typhimurium ATCC 14028 was evaluated. We also assessed differential protein patterns that were expressed by biofilms compared with planktonic cells and protein expression by cinnamaldehyde-treated biofilms cells. This compound decreased biofilm biomass and metabolic activity of biofilms at both concentrations tested. Cinnamaldehyde treatment reduced the number of attached cells in polypropylene, reflected by colony count and scanning electron microscopy. The proteomic analysis of biofilms compared with planktonic cells indicated that several proteins were upregulated or downregulated, especially proteins that are involved in energy metabolism. Peroxiredoxin, ATP synthase alpha chain protein, conjugal transfer nickase/helicase TraI and elongation factor G were upregulated in untreated-biofilm cells, and their expression decreased as a function of cinnamaldehyde treatment. Cinnamaldehyde had antibiofilm activity, and several differentially expressed proteins identified provide potential and interesting targets to explore new control strategies for S. Typhimurium biofilms.