Myrtus communis leaf compounds as novel inhibitors of quorum sensing-regulated virulence factors and biofilm formation: In vitro and in silico investigations.

Antibiotic resistance of the Gram-negative bacterium Pseudomonas aeruginosa and its ability to form biofilm through the Quorum Sensing (QS) mechanism are important challenges in the control of infections caused by this pathogen. The extract of Myrtus communis (myrtle) showed strong anti-QS effect on C hromobacterium . violaceum 6267 by inhibiting 80 % of the production of violacein pigment at a sub-MIC concentration of 1/8 (31.25 µg/mL). In addition, the extract exhibited an inhibitory effect on virulence factors of P. aeruginosa PAO1 at half MIC (125 µg/mL), significantly reducing the formation of biofilms (72.02 %), the swarming activity (75 %), and the production of protease (61.83 %) and pyocyanin (97 %). The active fraction also downregulated the expression of selected regulatory genes involved in the biofilm formation and QS in the P. aeruginosa PAO1 strain. These genes included the autoinducer synthase genes (lasI and rhlI), the genes involved in the expression of their corresponding receptors (lasR and rhlR), and the pqsA genes. The analysis of the active fraction by HPLC/UV/MS and NMR allowed the identification of three phenolic compounds, 3,5-di-O-galloylquinic acid, myricetin 3-O-α-l-rhamnopyranoside (myricitrin), and myricetin 3-O-(2″-O-galloyl)-ß-d-galactopyranoside. In silico studies showed that 3,5-di-O-galloylquinic acid, with an affinity score of -9.20 kcal/mol, had the highest affinity to the active site of the CviR protein (3QP8), a QS receptor from C. violaceum. Additionally, myricetin 3-O-α-l-rhamnopyranoside (myricitrin) and myricetin 3-O-(2″-O-galloyl)-ß-d-galactopyranoside interact to a lesser extent with 3QP8. In conclusion, this study contributed significantly to the discovery of new QS inhibitors from M. communis leaves against resistant Gram-negative pathogens.

Chestnut Honey Is Effective against Mixed Biofilms at Different Stages of Maturity.

The irresponsible overuse of antibiotics has increased the occurrence of resistant bacterial strains, which represents one of the biggest patient safety risks today. Due to antibiotic resistance and biofilm formation in bacteria, it is becoming increasingly difficult to suppress the bacterial strains responsible for various chronic infections. Honey was proven to inhibit bacterial growth and biofilm development, offering an alternative solution in the treatment of resistant infections and chronic wounds. Our studies included chestnut honey, valued for its high antibacterial activity, and the bacteria Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and S. epidermidis, known to form multi-species biofilm communities. Minimum inhibitory concentrations (MIC) of chestnut honey were determined for each bacterial strain. Afterwards, the mixed bacterial biofilms were treated with chestnut honey at different stages of maturity (incubation times: 2, 4, 6, 12, 24 h). The extent of biofilm inhibition was measured with a crystal violet assay and demonstrated by scanning electron microscopy (SEM). As the incubation time increased and the biofilm became more mature, inhibition rates decreased gradually. The most sensitive biofilm was the combination MRSA-S. epidermidis, with a 93.5% inhibition rate after 2 h of incubation. Our results revealed that chestnut honey is suitable for suppressing the initial and moderately mature stages of mixed biofilms.

Activity of Binary Combinations of Natural Phenolics and Synthetic Food Preservatives against Food Spoilage Yeasts.

Natural compounds are a suitable alternative to synthetic food preservatives due to their natural origin and health-promoting properties. In the current study, phenolic-phenolic and phenolic-synthetic combinations were tested for their antibiofilm formation, anti-planktonic growth, and anti-adhesion properties against Debaryomyces hansenii, Wickerhamomyces anomalus (formerly Pichia anomala), Schizosaccharomyces pombe, and Saccharomyces cerevisiae. The phenolics were vanillin and cinnamic acid, while the synthetic preservatives were sodium benzoate, potassium sorbate, and sodium diacetate. The vanillin-cinnamic acid combination had synergistic effect in all the tested yeasts for the biofilm inhibition with a fractional inhibitory concentration index (FICI) of ≤0.19 for W. anomalus, 0.25 for S. pombe, 0.31 for S. cerevisiae, and 0.5 for D. hansenii. Most of the phenolic-synthetic combinations had indifferent interaction regarding biofilm formation. The vanillin-cinnamic acid combination also had higher activity against spoilage yeasts adhesion on the abiotic surface and planktonic growth compared to the phenolic-synthetic combinations. For the phenolic-synthetic anti-planktonic activity, synergistic interaction was present in all the vanillin-synthetic combinations in S. pombe, vanillin-sodium benzoate and vanillin-potassium sorbate in S. cerevisiae, vanillin-sodium benzoate in W. anomalus, and cinnamic acid-sodium diacetate in S. pombe. These results suggest a novel antimicrobial strategy that may broaden the antimicrobial spectrum and reduce compound toxicity against food spoilage yeasts.

Eradication of multiple-species biofilms from food industrial and domestic surfaces using essential oils.

Microbial biofilm formation represents a serious problem for both food industry and households. Natural biofilms are formed mostly by multiple species, and show resistance against most of the usual sanitizers. In this study, the effects of cinnamon (Cinnamomum zeylanicum), marjoram (Origanum majorana) and thyme (Thymus vulgaris) essential oils (EOs) and their main components (cinnamaldehyde, terpinene-4-ol, and thymol) were investigated on four-species biofilms of Escherichia coli, Listeria monocytogenes, Pseudomonas putida and Staphylococcus aureus. Minimum bactericide concentration (MBC) and killing time were determined by means of the microdilution method. MBC of the investigated EOs and components was between 0.5 mg/mL (cinnamaldehyde) to 25 mg/mL (terpinene-4-ol). Killing times for the four-species suspension were 5 or 10 min, time spans usable in the food industry. For eradication of the mixed-population biofilm from stainless steel (SS), polypropylene (PP), tile and wood surfaces, EO- or EO component-based disinfectant solutions were developed, and their effects were compared to a peracetic acid-based industrial sanitizer (HC-DPE). Total eradication of biofilms (99.9%) was achieved, with solutions containing cinnamon and thyme EO and EO components, from SS and PP, but not from tile or wood surfaces. Apparently, cinnamon EO, terpinene-4-ol and thymol have better disinfectant activity than HC-DPE.

Hydrolysis of Edible Oils by Fungal Lipases: An Effective Tool to Produce Bioactive Extracts with Antioxidant and Antimicrobial Potential.

Hydrolysis of olive, rapeseed, linseed, almond, peanut, grape seed and menhaden oils was performed with commercial lipases of Aspergillus niger, Rhizopus oryzae, Rhizopus niveus, Rhizomucor miehei and Candida rugosa. In chromogenic plate tests, olive, rapeseed, peanut and linseed oils degraded well even after 2 h of incubation, and the R. miehei, A. niger and R. oryzae lipases exhibited the highest overall action against the oils. Gas chromatography analysis of vegetable oils hydrolyzed by R. miehei lipase revealed about 1.1 to 38.4-fold increases in the concentrations of palmitic, stearic, oleic, linoleic and α-linolenic acids after the treatment, depending on the fatty acids and the oil. The major polyunsaturated fatty acids produced by R. miehei lipase treatment from menhaden oil were linoleic, α-linolenic, hexadecanedioic, eicosapentaenoic, docosapentaenoic and docosahexaenoic acids, with yields from 12.02 to 52.85 µg/mL reaction mixture. Folin-Ciocalteu and ferric reducing power assays demonstrated improved antioxidant capacity for most tested oils after the lipase treatment in relation to the concentrations of some fatty acids. Some lipase-treated and untreated samples of oils, at 1.25 mg/mL lipid concentration, inhibited the growth of food-contaminating bacteria. The lipid mixtures obtained can be reliable sources of extractable fatty acids with health benefits.

In Vitro Activity of Selected Phenolic Compounds against Planktonic and Biofilm Cells of Food-Contaminating Yeasts.

Phenolic compounds are natural substances that can be obtained from plants. Many of them are potent growth inhibitors of foodborne pathogenic microorganisms, however, phenolic activities against spoilage yeasts are rarely studied. In this study, planktonic and biofilm growth, and the adhesion capacity of Pichia anomala, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Debaryomyces hansenii spoilage yeasts were investigated in the presence of hydroxybenzoic acid, hydroxycinnamic acid, stilbene, flavonoid and phenolic aldehyde compounds. The results showed significant anti-yeast properties for many phenolics. Among the tested molecules, cinnamic acid and vanillin exhibited the highest antimicrobial activity with minimum inhibitory concentration (MIC) values from 500 µg/mL to 2 mg/mL. Quercetin, (-)-epicatechin, resveratrol, 4-hydroxybenzaldehyde, p-coumaric acid and ferulic acid were also efficient growth inhibitors for certain yeasts with a MIC of 2 mg/mL. The D. hansenii, P. anomala and S. pombe biofilms were the most sensitive to the phenolics, while the S. cerevisiae biofilm was quite resistant against the activity of the compounds. Fluorescence microscopy revealed disrupted biofilm matrix on glass surfaces in the presence of certain phenolics. Highest antiadhesion activity was registered for cinnamic acid with inhibition effects between 48% and 91%. The active phenolics can be natural interventions against food-contaminating yeasts in future preservative developments.

Plant Phenolics and Phenolic-Enriched Extracts as Antimicrobial Agents against Food-Contaminating Microorganisms.

Phenolic compounds and extracts with bioactive properties can be obtained from many kinds of plant materials. These natural substances have gained attention in the food research as possible growth inhibitors of foodborne pathogenic and spoilage bacteria. Many phenolic-enriched plant extracts and individual phenolics have promising anti-quorum sensing potential as well and can suppress the biofilm formation and toxin production of food-related pathogens. Various studies have shown that plant phenolics can substitute or support the activity of synthetic food preservatives and disinfectants, which, by the way, can provoke serious concerns in consumers. In this review, we will provide a brief insight into the bioactive properties, i.e., the antimicrobial, anti-quorum sensing, anti-biofilm and anti-enterotoxin activities, of plant phenolic extracts and compounds, with special attention to pathogen microorganisms that have food relation. Carbohydrase aided applications to improve the antimicrobial properties of phenolic extracts are also discussed.

Anti-Biofilm Effect of Selected Essential Oils and Main Components on Mono- and Polymicrobic Bacterial Cultures.

Biofilms are surface-associated microbial communities resistant to sanitizers and antimicrobials. Various interactions that can contribute to increased resistance occur between the populations in biofilms. These relationships are the focus of a range of studies dealing with biofilm-associated infections and food spoilage. The present study investigated the effects of cinnamon (Cinnamomum zeylanicum), marjoram (Origanum majorana), and thyme (Thymus vulgaris) essential oils (EOs) and their main components, i.e., trans-cinnamaldehyde, terpinen-4-ol, and thymol, respectively, on single- and dual-species biofilms of Escherichia coli, Listeria monocytogenes, Pseudomonas putida, and Staphylococcus aureus. In dual-species biofilms, L. monocytogenes was paired with each of the other three bacteria. Minimum inhibitory concentration (MIC) values for the individual bacteria ranged between 0.25 and 20 mg/mL, and trans-cinnamaldehyde and cinnamon showed the highest growth inhibitory effect. Single-species biofilms of L. monocytogenes, P. putida, and S. aureus were inhibited by the tested EOs and their components at sub-lethal concentrations. Scanning electron microscopy images showed that the three-dimensional structure of mature biofilms embedded in the exopolysaccharide matrix disappeared or was limited to micro-colonies with a simplified structure. In most dual-species biofilms, to eliminate living cells from the matrix, concentrations exceeding the MIC determined for individual bacteria were required.

Anti-listerial effect of selected essential oils and thymol.

The anti-listerial effect of marjoram, thyme essential oils (EOs) and thymol on Listeria monocytogenes inoculated chicken breast fillets was investigated. Before inoculation the fillets were pretreated by washing or not under running tap water. Inoculated samples were kept at 6 °C for 24 h to allow the growth of L. monocytogenes. After this, the fillets were put in marinating solutions containing salt (5%) and EOs or thymol in MIC/2 concentration established in vitro. Total germ count (TGC) and L. monocytogenes count was monitored on the meat surface and in the marinating solutions following 24 and 48 h storage at 6 °C. Thyme and thymol reduced significantly Listeria cell count (1-3 log CFU) in both samples. They also gave good flavour to the fried meat. The doses of EOs used were optimal for antimicrobial efficiency and had a pleasant flavour effect. Washing was not efficient in reducing total germ count.