Incorporation of Resveratrol-Hydroxypropyl-ß-Cyclodextrin Complexes into Hydrogel Formulation for Wound Treatment.

Resveratrol could be applied in wound healing therapies because of its antioxidant, anti-inflammatory and antibacterial effects. However, the main limitation of resveratrol is its low aqueous solubility. In this study, resveratrol was included in hydroxypropyl-ß-cyclodextrin complexes and further formulated in Pluronic F-127 hydrogels for wound treatment therapy. IR-spectroscopy and XRD analysis confirmed the successful incorporation of resveratrol into complexes. The wound-healing ability of these complexes was estimated by a scratch assay on fibroblasts, which showed a tendency for improvement of the effect of resveratrol after complexation. The antimicrobial activity of resveratrol in aqueous dispersion and in the complexes was evaluated on methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Candida albicans strains. The results revealed a twofold decrease in the MIC and stronger inhibition of the metabolic activity of MRSA after treatment with resveratrol in the complexes compared to the suspended drug. Furthermore, the complexes were included in Pluronic hydrogel, which provided efficient drug release and appropriate viscoelastic properties. The formulated hydrogel showed excellent biocompatibility which was confirmed via skin irritation test on rabbits. In conclusion, Pluronic hydrogel containing resveratrol included in hydroxypropyl-ß-cyclodextrin complexes is a promising topical formulation for further studies directed at wound therapy.

Antimicrobial Resistance and Biofilm Formation of Escherichia coli Isolated from Pig Farms and Surroundings in Bulgaria.

Escherichia coli (E. coli) is a ubiquitous microorganism with pathogenic and saprophytic clones. The objective of this study was to evaluate the presence, virulence, antibiotic resistance and biofilm formation of E. coli in three industrial farms in Bulgaria, as well as their adjacent sites related to the utilization of manure (feces, wastewater in a separator, lagoons, means of transport, and soils). The isolation of single bacterial cultures was performed via standard procedures with modifications, and E. coli isolates were identified via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and polymerase chain reaction (PCR). The disk diffusion method was used to assess antimicrobial resistance, and PCR was used to detect genes for antibiotic resistance (GAR) (qnr, aac(3), ampC, blaSHV/blaTEM and erm) and virulence genes (stx, stx2all, LT, STa, F4 and eae). The protocol of Stepanovic was utilized to measure the biofilm formation of the isolates. A total of 84 isolates from different samples (n = 53) were identified as E. coli. Almost all demonstrated antimicrobial resistance, and most of them demonstrated resistance to multiple antibiotics from different classes. No virulence genes coding the Shiga toxin or enterotoxins or those associated with enteropathogenicity were detected. No GAR from those tested for quinolones, aminoglycosides and macrolides were found. However, all isolates that were resistant to a penicillin-class antibiotic (56) had ß-lactamase-producing plasmid genes. All of them had ampC, and 34 of them had blaTEM. A total of 14 isolates formed strongly adherent biofilms. These results in a country where the use of antibiotics for growth promotion and prophylaxis in farms is highly restricted corroborate that the global implemented policy on antibiotics in human medicine and in animal husbandry needs revision.

Prevalence, Genetic Homogeneity, and Antibiotic Resistance of Pathogenic Yersinia enterocolitica Strains Isolated from Slaughtered Pigs in Bulgaria.

Yersiniosis is the third most commonly reported foodborne zoonosis in the European Union. Here, we evaluated the prevalence of pathogenic Yersinia enterocolitica among healthy pigs (as a major reservoir) in a slaughterhouse in Bulgaria. A total of 790 tonsils and feces from 601 pigs were examined. Isolation and pathogenicity characterization was carried out by the ISO 10273:2003 protocol and Polymerase Chain Reaction (PCR), detecting the 16S rRNA gene, attachment and invasion locus (ail), Yersinia heat-stable enterotoxin (ystA), and Yersinia adhesion (yadA) genes. Genetic diversity was assessed by pulsed-field gel electrophoresis (PFGE), and antimicrobial resistance by the standard disk diffusion method. Of all the pigs tested, 6.7% were positive for Y. enterocolitica. All isolates belonged to Y. enterocolitica bioserotype 4/O:3. ail, and ystA genes were detected in all positive strains (n = 43), while the plasmid Yersinia virulence plasmid (pYV) was detected in 41. High homogeneity was observed among the strains, with all strains susceptible to ceftriaxone, amikacin and ciprofloxacin, and resistant to ampicillin. In conclusion, a low prevalence of Y. enterocolitica 4/O:3 was found in healthy pigs slaughtered in Bulgaria, not underestimating possible contamination of pork as a potential risk to consumer health.

Outstanding Antibacterial Activity of Hypericum rochelii-Comparison of the Antimicrobial Effects of Extracts and Fractions from Four Hypericum Species Growing in Bulgaria with a Focus on Prenylated Phloroglucinols.

Microbial infections are by no means a health problem from a past era due to the increasing antimicrobial resistance of infectious strains. Medicine is in constant need of new drugs and, recently, plant products have had a deserved renaissance and garnered scientific recognition. The aim of this work was to assess the antimicrobial activity of ten active ingredients from four Hypericum species growing in Bulgaria, as well as to obtain preliminary data on the phytochemical composition of the most promising samples. Extracts and fractions from H. rochelii Griseb. ex Schenk, H. hirsutum L., H. barbatum Jacq. and H. rumeliacum Boiss. obtained with conventional or supercritical CO2 extraction were tested on a panel of pathogenic microorganisms using broth microdilution, agar plates, dehydrogenase activity and biofilm assays. The panel of samples showed from weak to extraordinary antibacterial effects. Three of them (from H. rochelii and H. hirsutum) had minimum inhibitory concentrations as low as 0.625-78 mg/L and minimum bactericidal concentrations of 19.5-625 mg/L against Staphylococcus aureus and other Gram-positive bacteria. These values placed these samples among the best antibacterial extracts from the Hypericum genus. Some of the agents also demonstrated very high antibiofilm activity against methicillin-resistant S. aureus. Ultra-high-performance liquid chromatography-high-resolution mass spectrometry revealed the three most potent samples as rich sources of biologically active phloroglucinols. They were shown to be good drug or nutraceutical candidates, presumably without some of the side effects of conventional antibiotics.

Advantageous Combinations of Nanoencapsulated Oregano Oil with Selected Antibiotics for Skin Treatment.

The aim of the present study was to evaluate the antimicrobial activity of combinations between encapsulated oregano oil and the most commonly applied antibiotics (ciprofloxacin or gentamicin) against skin infections. In particular, chitosan-alginate nanoparticles loaded with oregano oil and the selected antibiotics were included in methylcellulose hydrogels. Consistency, spreadability, pH of the hydrogel and in vitro release rate of the oil were considered appropriate for topical application. The combination of encapsulated oil and gentamicin in the hydrogel resulted in a synergistic effect against methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus strains. It was expressed in a fourfold reduction in the effective concentration of gentamicin and 98% inhibition of the bacterial metabolic activity. When ciprofloxacin was included in the combination instead of gentamicin, an additive effect with a two-fold decrease in the effective drug concentration and a 96% reduction in the bacterial metabolic activity were observed. Both combinations significantly inhibited the formation of MRSA biofilm by more than 90% when applied. In vivo application of the hydrogel containing the synergistic combination between the encapsulated oil and gentamicin did not induce irritation of the rabbit skin.

Polymeric Microneedles for Transdermal Delivery of Rivastigmine: Design and Application in Skin Mimetic Model.

In the last years, microneedles (MNs) have been considered a valuable, painless, and minimally invasive approach for controlled transdermal drug delivery (TDD). Rivastigmine (RV), a drug administered to patients suffering from dementia, is currently delivered by oral or transdermal routes; however, both present limitations, mainly gastrointestinal adverse symptoms or local skin irritation and drug losses, respectively, for each route. Given this, the objective of the present work was to develop and evaluate the potential of polymeric MNs for RV transdermal delivery in a controlled manner. Polymeric MNs with two needle heights and different compositions were developed with calcein as a fluorescent model molecule. Morphology and mechanical characterisation were accessed. Skin permeation experiments showed the ability of the devices to deliver calcein and confirmed that the arrays were able to efficiently pierce the skin. To obtain a new TDD anti-dementia therapeutic solution, RV was loaded in 800 µm polymeric MNs of alginate and alginate/k-carrageenan MNs. In the presence of RV, the MN's morphology was maintained; however, the presence of RV influenced the compression force. Skin permeation studies revealed that RV-loaded MNs allowed a more efficient controlled release of the drug than the commercial patch. In vivo, skin irritation tests in rabbits revealed that the developed MNs were innocuous upon removal, in contrast with the evidence found for Exelon®, the commercial patch, which caused slight mechanical damage to the skin. The herein-produced MNs demonstrated a more controlled release of the drug, being the more suitable option for the transdermal delivery of RV.

Antimicrobial and Antioxidant Potential of Scenedesmus obliquus Microalgae in the Context of Integral Biorefinery Concept.

Small-scale photobioreactors (PBRs) in the inoculum stage were designed with internal (red or green) and external white LED light as an initial step of a larger-scale installation aimed at fulfilling the integral biorefinery concept for maximum utilization of microalgal biomass in a multifunctional laboratory. The specific growth rate of Scenedesmus obliquus (Turpin) Kützing biomass for given cultural conditions was analyzed by using MAPLE software. For the determination of total polyphenols, flavonoids, chlorophyll "a" and "b", carotenoids and lipids, UHPLC-HRMS, ISO-20776/1, ISO-10993-5 and CUPRAC tests were carried out. Under red light growing, a higher content of polyphenols was found, while the green light favoured the flavonoid accumulation in the biomass. Chlorophylls, carotenoids and lipids were in the same order of magnitude in both samples. The dichloromethane extracts obtained from the biomass of each PBR synergistically potentiated at low concentrations (0.01-0.05 mg/mL) the antibacterial activity of penicillin, fluoroquinolones or oregano essential oil against the selected food-borne pathogens (Staphylococcus aureus, Escherichia coli and Salmonella typhimurium) without showing any in vitro cytotoxicity. Both extracts exhibited good cupric ion-reducing antioxidant capacity at concentrations above 0.042-0.08 mg/mL. The UHPLC-HRMS analysis revealed that both extracts contained long chain fatty acids and carotenoids thus explaining their antibacterial and antioxidant potential. The applied engineering approach showed a great potential to modify microalgae metabolism for the synthesis of target compounds by S. obliquus with capacity for the development of health-promoting nutraceuticals for poultry farming.

Prevalence of Antibiotic-Resistant Escherichia coli Isolated from Swine Faeces and Lagoons in Bulgaria.

Antimicrobial resistance (AMR) is a worldwide health problem affecting humans, animals, and the environment within the framework of the "One Health" concept. The aim of our study was to evaluate the prevalence of pathogenic strains of the species Escherichia coli (E. coli), their AMR profile, and biofilm-forming potential. The isolated strains from three swine faeces and free lagoons (ISO 16654:2001/Amd 1:2017) were confirmed using Phoenix M50 and 16S rDNA PCR. The antibiotic sensitivity to 34 clinically applied antibiotics was determined by Phoenix M50 and the disc diffusion method, according to the protocols of the CLSI and EUCAST. We confirmed the presence of 16 E. coli isolates, of which 87.5% were multi-drug-resistant and 31.25% performed strong biofilms. The possibility for the carrying and transmission of antibiotic-resistance genes to quinolones (qnr), aminoglycosides (aac(3)), ß-lactamase-producing plasmid genes ampC, and blaSHV/blaTEM was investigated. We confirmed the carrying of blaSHV/blaTEM in one and ampC in seven isolates. The strains were negative for the virulence genes (ETEC (LT, STa, and F4), EPEC (eae), and STEC/VTEC (stx and stx2all)). The results should contribute to the development of effective measures for limitation and control on the use of antibiotics, which is a key point in the WHO action plan.

Cytotoxicity and Microbicidal Activity of Commonly Used Organic Solvents: A Comparative Study and Application to a Standardized Extract from Vaccinium macrocarpon.

The cytotoxicity and microbicidal capacity of seven organic solvents commonly applied for studying plant extracts and bioactive compounds were systematically investigated based on international standards. Four cell lines of normal (CCL-1, HaCaT) or tumor (A-375, A-431) tissue origin, seven bacterial and one fungal strain were used. The impact of the least toxic solvents in the determination of in vitro cytotoxicity was evaluated using a standardized extract from Vaccinium macrocarpon containing 54.2% v/v proanthocyanidins (CystiCran®). The solvents ethanol, methoxyethanol and polyethylene glycol were the least cytotoxic to all cell lines, with a maximum tolerated concentration (MTC) between 1 and 2% v/v. Ethanol, methanol and polyethylene glycol were mostly suitable for antimicrobial susceptibility testing, with minimum inhibitory concentrations (MICs) ≥ 25% v/v. The MTC values of the solvents dimethyl sulfoxide, dimethoxyethane and dimethylformamide varied from 0.03% to 1.09% v/v. The MICs of dimethyl sulfoxide, methoxyethanol and dimethoxyethane were in the range of 3.125-25% v/v. The cytotoxic effects of CystiCran® on eukaryotic cell lines were directly proportional to the superimposed effect of the solvents used. The results of this study can be useful for selecting the appropriate solvents for in vitro estimation of the cytotoxic and growth inhibitory effects of bioactive molecules in eukaryotic and prokaryotic cells.