Antibacterial activity of Hungarian varietal honeys against respiratory pathogens as a function of storage time.

Today, antibiotic therapies that previously worked well against certain bacteria due to their natural sensitivity, are becoming less effective. Honey has been proven to inhibit the biofilm formation of some respiratory bacteria, however few data are available on how the storage time affects the antibacterial effect. The activity of black locust, goldenrod, linden and sunflower honeys from three consecutive years (2020, 2021, 2022) was analyzed in 2022 against Gram-negative (Haemophilus influenzae, H. parainfluenzae, Pseudomonas aeruginosa) and Gram-positive (Streptococcus pneumoniae) bacteria using in vitro microbiological methods. After determining the physicochemical parameters of honey, broth microdilution was applied to determine the minimum inhibitory concentration of each honey type against each bacterium, and crystal violet assay was used to test their antibiofilm effect. The possible mechanism of action was explored with membrane degradation test, while structural changes were illustrated with scanning electron microscopy. Honeys stored for one or two years were darker than fresh honeys, while older honeys had significantly lower antibacterial activity. The most remarkable inhibitory effect was exerted by linden and sunflower honeys, and P. aeruginosa proved to be the most resistant bacterium. Based on our results, honey intended for medicinal purposes should be used as fresh as possible during a treatment.

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.

Microbiological Studies on the Influence of Essential Oils from Several Origanum Species on Respiratory Pathogens.

Essential oils (EOs) with established and well-known activities against human pathogens might become new therapeutics in multidrug-resistant bacterial infections, including respiratory tract infections. The aim of this study was to evaluate the antimicrobial activity of EOs obtained from several samples of Origanum vulgare, O. syriacum, and O. majorana cultivated in Poland. EOs were analyzed by GC-MS and tested against four bacterial strains: Staphylococcus aureus (MRSA), Haemophilus influenzae, Haemophilus parainfluenzae, and Pseudomonas aeruginosa. Chemical analyses showed that the Eos were characterized by a high diversity in composition. Based on the chemical data, four chemotypes of Origanum EOs were confirmed. These were carvacrol, terpineol/sabinene hydrate, caryophyllene oxide, and thymol chemotypes. Thin-layer chromatography-bioautography confirmed the presence of biologically active antibacterial components in all tested EOs. The highest number of active spots were found among EOs with cis-sabinene hydrate as the major compound. On the other hand, the largest spots of inhibition were characteristic to EOs of the carvacrol chemotype. Minimal inhibitory concentrations (MICs) were evaluated for the most active EOs: O. vulgare 'Hirtum', O. vulgare 'Margarita', O. vulgare 'Hot & Spicy', O. majorana, and O. syriacum (I) and (II); it was shown that both Haemophilus strains were the most sensitive with an MIC value of 0.15 mg/mL for all EOs. O. majorana EO was also the most active in the MIC assay and had the highest inhibitory rate in the anti-biofilm assay against all strains. The most characteristic components present in this EO were the trans-sabinene hydrate and terpinen-4-ol. The strain with the least sensitivity was the MRSA with an MIC of 0.6 mg/mL for all EOs except for O. majorana, where the MIC value reached 0.3 mg/mL. Scanning electron microscopy performed on the Haemophilus influenzae and Haemophilus parainfluenzae biofilms showed a visible decrease in the appearance of bacterial clusters under the influence of O. majorana EO.

Antibacterial and Antibiofilm Effect of Unifloral Honeys against Bacteria Isolated from Chronic Wound Infections.

Honey is known as an alternative remedy for the treatment of wounds. To evaluate the potential of five Hungarian honey types against wound-associated bacteria, in vitro microbiological assays were conducted on Pseudomonas aeruginosa, Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus (MRSA). Minimum inhibitory concentration (MIC) was determined with the broth macrodilution method, and biofilm degradation capacity was tested with a crystal violet assay. To understand the underlying mechanisms, the effects of honey treatments were assessed on bacterial membrane integrity and quorum sensing (QS). The highest antibacterial activity, indicated by the lowest MIC values, as well as the highest biofilm inhibition rates and membrane disruption, was displayed by chestnut and linden honeys. The most sensitive bacterium was S. epidermidis. Bacterial membrane degradation took place 40 min after treatment with honey solutions of at least a 40% concentration. Each honey sample exhibited anti-QS activity, which was most pronounced in the case of chestnut honey. It was concluded that the antibacterial, biofilm-inhibiting and anti-QS activities of linden and chestnut honeys were superior to those of acacia, goldenrod and milkweed honeys. In addition to the floral source, the antibacterial effect of honey is influenced by the microbial species treated. The use of honey in wound treatment can be justified by its diverse antibacterial mechanisms.

Lutein Isomers: Preparation, Separation, Structure Elucidation, and Occurrence in 20 Medicinal Plants.

Lutein and its cis-isomers occur in a lot of plants, including a variety of flowers. In this study, lutein isomers were produced via iodine-catalyzed isomerization, and four cis-isomers (9Z-, 9'Z-, 13Z-, and 13Z') were isolated by means of column chromatography and semipreparative HPLC. The structures of the 9'Z- and 13'Z-isomers were elucidated via NMR measurements. These compounds were used as standards for the HPLC-DAD-MS determination of the carotenoid composition of the flowers of 20 plant species, in which lutein and its geometrical isomers are the main components. The flowers showed great variation in their cis- and trans-lutein content, and also in the presence or absence of other carotenoids, such as violaxanthin, neoxanthin, ß-cryptoxanthin, and ß-carotene. Some of the investigated flowers were found to be rich sources of lutein without zeaxanthin.

Immortelle (Helichrysum italicum (Roth) G. Don) Essential Oil Showed Antibacterial and Biofilm Inhibitory Activity against Respiratory Tract Pathogens.

The biofilm formation of bacteria in different parts of the human body can influence the success of antibiotic therapy. Essential oils (EOs) and their components are becoming increasingly popular in point of view of medicinal applications, because of their antibacterial efficacy. The immortelle EO has been used traditionally as an expectorant; however, there are no studies summarizing its antibacterial effect against respiratory tract bacteria. Our aim was to investigate the antibacterial and biofilm inhibitory activity of immortelle (Helichrysum italicum) EO against respiratory tract pathogens such as Haemophilus influenzae, H. parainfluenzae, Pseudomonas aeruginosa and Streptococcus pneumoniae. In order to prove the antibacterial effect of the immortelle EO, broth microdilution and biofilm inhibition tests, and membrane damage assay were investigated. Scanning electron microscopy was used to identify the structural modifications in bacterial cells. Our results showed that immortelle EO has antibacterial and anti-biofilm effects against respiratory tract bacteria used in this study. H. parainfluenzae was the most sensitive to each treatment, however, P. aeruginosa was the most resistant bacteria. In conclusion, the studied EO may have a role in the treatment of respiratory tract infections due to their antibacterial and anti-biofilm activity.

Preparation, characterisation and microbiological examination of Pickering nano-emulsions containing essential oils, and their effect on Streptococcus mutans biofilm treatment.

Essential oils (EOs) are commonly applied in mouth care products like mouthwashes, mostly as an ethanolic solution or by usage of surfactants as solubilising agents. In this study, we present a formulation for preparation of Pickering nano-emulsions (PnE) of EOs as a novel form for application of EOs in mouth care. For the preparation of PnE, we have synthesised surface-modified silica nanoparticles with a mean diameter of 20 nm, as well as we have examined the effect of EOs concentration on PnE droplet size and stability. In vitro study of their effect on the Streptococcus mutans biofilm as the main pathogen of dental health problems has been performed. We have found that EOs in the PnE form has the highest effectiveness against biofilm formation. Diffusion through the biofilm model membrane was studied to explain this observation. We have found that PnEs have a better performance in the transportation of EOs trough model membrane than the ethanolic solutions and conventional emulsions (CEs).

Antibacterial activity evaluation of selected essential oils in liquid and vapor phase on respiratory tract pathogens.

BACKGROUND: The increasing number of multidrug-resistant bacteria and the fact of antibiotic resistance is leading to a continuous need for discovering alternative treatments against infections, e.g. in the case of respiratory tract diseases. Essential oils (EOs), because of their volatility, can easily reach both the upper and lower parts of the respiratory tract via inhalation. Therefore, the aim of the present study was the antibacterial evaluation of clove, cinnamon bark, eucalyptus, thyme, scots pine, peppermint, and citronella EOs against respiratory tract pathogens such as Streptococcus pneumoniae, S. mutans, S. pyogenes, Haemophilus influenzae, H. parainfluenzae, and Moraxella catarrhalis. Furthermore, we wanted to compare the antibacterial effect of these EOs in two different test systems to provide data for the development of an appropriate product formulation. METHODS: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined with in vitro vapor phase test (VPT) and broth macrodilution test (BDT). The chemical and percentage compositions of the EOs were determined by GC-MS and GC-FID analysis. RESULTS: Among the EOs, thyme was the most effective against S. mutans (MIC: 0.04 mg/mL in BDT, but cinnamon bark and clove oils also presented high inhibition in liquid medium with MIC values of 0.06 mg/mL and 0.1 mg/mL against S. pneumoniae and S. pyogenes, respectively. M. catarrhalis was the most sensitive to thyme EO (MIC: 0.09 mg/mL). Cinnamon bark EO was the most effective against Haemophilus spp. (MIC: 0.06 mg/mL). In the VPT, cinnamon bark was the most effective oil against all investigated pathogens with MIC values in the range of 15.62-90 µl/L. Surprisingly, the eucalyptus and scots pine showed weak activity against the test bacteria in both test systems. CONCLUSIONS: The EO of thyme, clove and cinnamon bark may provide promising antibacterial activity against respiratory tract pathogens either in liquid medium or in vapor phase. However, their effect is lower than that of the reference antibiotics. The combination of EOs and antibiotics may be beneficial in the alternative treatment of respiratory tract diseases. In vivo studies are necessary to calculate the effective dose of EOs in patients and determine their possible side effects and toxicity.