Comparing the Antimicrobial Actions of Greek Honeys from the Island of Lemnos and Manuka Honey from New Zealand against Clinically Important Bacteria.

Honey is a natural food with a long history as a traditional medicine because of its many biological characteristics, including antimicrobial, antioxidant, anti-tumor and anti-inflammatory properties. In this study, the antimicrobial actions of eight different honeys from Lemnos island (north-eastern Greece) plus manuka honey (from New Zealand, UMF 30+, licensed in many countries as topical medical preparation) were evaluated against 10 clinically relevant bacteria, including five Gram-positive and five Gram-negative. To achieve this, an agar well diffusion assay measured the diameter of inhibition zones (mm) of two selected concentrations for each honey (25% and 12.5% v/v). The minimum inhibitory and bactericidal concentrations (MIC and MBC) of each sample were also calculated and compared against two representative bacterial species (Salmonella Typhimurium and Staphylococcus aureus) using broth microdilution and agar spot methods, respectively. The pH, water activity (aw), 5-hydroxymethylfurfural (HMF) and diastase levels, together with the pollen type and content of each honey, were also determined. Results revealed that all the Lemnos honeys presented antibacterial action, which for some samples was like that of manuka. These all had an acidic pH (3.61 ± 0.04), with a aw ≤ 0.60, while it is worth noting that those found to display the strongest antibacterial actions also presented the lowest HMF content, together with the highest diastase values, both of the latter being used as quality parameters. Pollen composition of the Lemnos honeys was multifloral, underlining the rich plant biodiversity encountered on the island. To summarize, Lemnos honeys could be further exploited as natural antimicrobial systems for use in foods and medicine.

Development of a herbal mouthwash containing a mixture of essential oils and plant extracts and in vitro testing of its antimicrobial efficiency against the planktonic and biofilm-enclosed cariogenic bacterium Streptococcus mutans.

A herbal mouthwash containing essential oils of holy basil and mountain tea, extracts of St John's wort and European goldenrod (Bucovia™) and cetylpyridinium chloride, was developed and in vitro tested for its efficiency against biofilm formation by Streptococcus mutans, together with its eradicating activity against already preformed (48 h with saccharose) streptococcal biofilm. The minimum inhibitory (MIC) and bactericidal concentrations (MBC) of the final formulation, as well as of its individual components, were initially determined. The results revealed that the mouthwash needed to be applied at two-times its MIC (0.63% v.v-1) to completely inhibit biofilm formation by S. mutans, which was otherwise capable of developing a robust biofilm on the tested surface. Once fully developed, the matrix of the biofilm was found to contain a significant amount of exopolysaccharides protecting the cells, being impossible to eradicate even when exposed to pure mouthwash for 15 min, highlighting the great recalcitrance of biofilm-embedded S. mutans.

Exploitation of plant extracts and phytochemicals against resistant Salmonella spp. in biofilms.

Salmonella is one of the most frequent causes of foodborne outbreaks throughout the world. In the last years, the resistance of this and other pathogenic bacteria to antimicrobials has become a prime concern towards their successful control. In addition, the tolerance and virulence of pathogenic bacteria, such as Salmonella, are commonly related to their ability to form biofilms, which are sessile structures encountered on various surfaces and whose development is considered as a universal stress response mechanism. Indeed, the ability of Salmonella to form a biofilm seems to significantly contribute to its persistence in food production areas and clinical settings. Plant extracts and phytochemicals appear as promising sources of novel antimicrobials due to their cost-effectiveness, eco-friendliness, great structural diversity, and lower possibility of antimicrobial resistance development in comparison to synthetic chemicals. Research on these agents mainly attributes their antimicrobial activity to a diverse array of secondary metabolites. Bacterial cells are usually killed by the rupture of their cell envelope and in parallel the disruption of their energy metabolism when treated with such molecules, while their use at sub-inhibitory concentrations may also disrupt intracellular communication. The purpose of this article is to review the current available knowledge related to antimicrobial resistance of Salmonella in biofilms, together with the antibiofilm properties of plant extracts and phytochemicals against these detrimental bacteria towards their future application to control these in food production and clinical environments.

Optimization of Silver Nanoparticle Synthesis by Banana Peel Extract Using Statistical Experimental Design, and Testing of their Antibacterial and Antioxidant Properties.

BACKGROUND: In this study, silver nanoparticles (AgNPs) were synthesized using Banana Peel Extract (BPE), and characterized using UV- Vis absorbance spectroscopy, X-Ray Powder Diffraction (XRD), Atomic Force Microscopy (AFM), and Fourier Transform Infrared Spectroscopy (FTIR). UV-Vis absorbance spectroscopy showed the characteristic plasmon resonance of AgNPs at 433 nm. The synthesized AgNPs were tested for their antibacterial and antioxidant properties. METHODS: Nanoparticle size (between 5 and 9 nm) was measured using AFM, whereas their crystallinity was shown by XRD. FTIR identified the ligands that surround the nanoparticle surface. The synthesis conditions were optimised using Central Composite Design (CCD) under Response Surface Methodology (RSM). Silver nitrate (AgNO3) and BPE concentrations (0.25-2.25 mM, 0.2-1.96 % v/v respectively), incubation period (24-120 h) and pH level (2.3-10.1) were chosen as the four independent factors. The fitting parameters (i.e. the wavelength at peak maximum, the peak area, and the peak width) of a Voigt function of the UV- Vis spectra were chosen as the responses. The antibacterial properties of the AgNPs were tested against Escherichia coli and Staphylococcus aureus using the tube dilution test. The synthesized nanoparticles were tested for total phenolic composition (TPC) using the Folin - Ciocalteau method, whereas their radical scavenging activity using the 1,1-diphenyl-2- picrylhydrazyl (DPPH) free radical assay. RESULTS: An optimum combination of all independent factors was identified (BPE concentration 1.7 % v/v, AgNO3 concentration 1.75 mM, incubation period 48 h, pH level 4.3), giving minimum peak wavelength and peak width. The nanoparticles inhibited the growth of E. coli, whereas S. aureus growth was not affected. However, no superiority of AgNPs compared to AgNO3 used for their fabrication (1.75 mM), with respect to antibacterial action, could be here demonstrated. AgNPs were found to present moderate antioxidant activity (44.71± 3.01%), as measured using DPPH assay, while the BPE (used for their fabrication) presented alone (100%) an antioxidant action equal to 86±1%, something expected due to its higher total phenolic content (TPC) compared to that of nanoparticles. CONCLUSION: Altogether, the results of this study highlight the potential of an eco-friendly method to synthesize nanoparticles and its promising optimization through statistical experimental design. Future research on the potential influence of other synthesis parameters on nanoparticles yield and properties could further promote their useful biological activities towards their successful application in the food industry and other settings.

Disinfection efficiencies of sage and spearmint essential oils against planktonic and biofilm Staphylococcus aureus cells in comparison with sodium hypochlorite.

Staphylococcus aureus causes human infections and foodborne intoxications. This study explored the potential antibacterial actions of sage and spearmint essential oils (EOs) against both its planktonic and biofilm cells, in comparison with sodium hypochlorite (NaOCl), a commonly applied chemical sanitizer. Initially, the minimum inhibitory and bactericidal concentrations (MICs, MBCs) of each plant mixture were determined against planktonic cultures, following growth at 30°C for 24h. Stationary phase planktonic bacteria were then individually exposed for 6min to either each EO (applied at 1-2×MBC; 2.5-5%), or NaOCl (250-450ppm). These were also left to form biofilms on 96-well polystyrene microplates, at 30°C for 96h, with medium renewal at 48h, in the presence of 10 different concentrations of each EO, expanding from sub- to super-inhibitory for planktonic growth, and the minimum biofilm inhibitory concentrations (MBICs; >90% inhibition) of each plant mixture were calculated. Formed biofilms were finally exposed for 6min to either each EO (applied at 2-6×MBC; 5-15%), or NaOCl (7500-25,000ppm; applied either alone or in combination with each EO at 5%). Results showed that both EOs presented MIC and MBC equal to 1.25 and 2.5%, respectively. As expected, their application at their MIC and above significantly inhibited biofilm formation, while spearmint EO was still able to cause this at ½ of its MIC, with MBICs equal to 1.25 and 0.63% for sage and spearmint EOs, respectively. Alarmingly, the application of both EOs at 1/8 to 1/16 of their MIC further increased biofilm formation. Regarding biofilm disinfection experiments, the individual application of each EO against the pre-established sessile communities resulted in log decrease ranges of 0.8-3logCFU/cm2, while in the case of NaOCl application (either alone or combined with each EO), the observed reductions never exceeded 1.7logCFU/cm2. These last results highlight the great antimicrobial recalcitrance of biofilm communities, found here to be ca. 100 times more resistant to NaOCl compared to planktonic ones, and stress the urgent need for further research on alternative, adequate and safe disinfection strategies to control them in food processing and other environments.

Hydrosol of Thymbra capitata Is a Highly Efficient Biocide against Salmonella enterica Serovar Typhimurium Biofilms.

UNLABELLED: Salmonella is recognized as one of the most significant enteric foodborne bacterial pathogens. In recent years, the resistance of pathogens to biocides and other environmental stresses, especially when they are embedded in biofilm structures, has led to the search for and development of novel antimicrobial strategies capable of displaying both high efficiency and safety. In this direction, the aims of the present work were to evaluate the antimicrobial activity of hydrosol of the Mediterranean spice Thymbra capitata against both planktonic and biofilm cells of Salmonella enterica serovar Typhimurium and to compare its action with that of benzalkonium chloride (BC), a commonly used industrial biocide. In order to achieve this, the disinfectant activity following 6-min treatments was comparatively evaluated for both disinfectants by calculating the concentrations needed to achieve the same log reductions against both types of cells. Their bactericidal effect against biofilm cells was also comparatively determined by in situ and real-time visualization of cell inactivation through the use of time-lapse confocal laser scanning microscopy (CLSM). Interestingly, results revealed that hydrosol was almost equally effective against biofilms and planktonic cells, whereas a 200-times-higher concentration of BC was needed to achieve the same effect against biofilm compared to planktonic cells. Similarly, time-lapse CLSM revealed the significant advantage of the hydrosol to easily penetrate within the biofilm structure and quickly kill the cells, despite the three-dimensional (3D) structure of Salmonella biofilm. IMPORTANCE: The results of this paper highlight the significant antimicrobial action of a natural compound, hydrosol of Thymbra capitata, against both planktonic and biofilm cells of a common foodborne pathogen. Hydrosol has numerous advantages as a disinfectant of food-contact surfaces. It is an aqueous solution which can easily be rinsed out from surfaces, it does not have the strong smell of the essential oil (EO) and it is a byproduct of the EO distillation procedure without any industrial application until now. Consequently, hydrosol obviously could be of great value to combat biofilms and thus to improve product safety not only for the food industries but probably also for many other industries which experience biofilm-related problems.

Acquired acid adaptation of Listeria monocytogenes during its planktonic growth enhances subsequent survival of its sessile population to disinfection with natural organic compounds.

The abilities of Listeria monocytogenes to adapt to stress and also to attach to surfaces are believed to contribute to its persistence in food processing environments. In this study, the possible influence of acid adaptation of L. monocytogenes cells during their planktonic growth on their subsequent resistance against 3 different natural disinfection treatments (0.05% lactic acid, 1% essential oil or 100% hydrosol of S. thymbra) upon their attachment to stainless steel (SS) was evaluated by simultaneously using the bead vortexing technique and a conductance method. Prior to disinfection, both nonadapted and acid-adapted stationary phase bacteria were left to attach to SS coupons statically incubated for 10days into Brain Heart Infusion (BHI) broth under 3 different temperatures (5, 16 or 30°C). Results revealed that both essential oil and hydrosol presented sufficient bactericidal activity against all formed sessile populations, always resulting in counts following disinfection below the plate counting detection limit. However, the conductance method, able to detect metabolically active sessile bacteria unable to be recovered by the bead vortexing, revealed the positive influence of previously acquired acid adaptation on disinfection resistance of attached cells against these plant extracts. A similar effect of acid adaptation was also evident for lactic acid disinfection.