SPR and Double Resonance LPG Biosensors for Helicobacter pylori BabA Antigen Detection.

Given the medical and social significance of Helicobacter pylori infection, timely and reliable diagnosis of the disease is required. The traditional invasive and non-invasive conventional diagnostic techniques have several limitations. Recently, opportunities for new diagnostic methods have appeared based on the recent advance in the study of H. pylori outer membrane proteins and their identified receptors. In the present study we assess the way in which outer membrane protein-cell receptor reactions are applicable in establishing a reliable diagnosis. Herein, as well as in other previous studies of ours, we explore the reliability of the binding reaction between the best characterized H. pylori adhesin BabA and its receptor, the blood antigen Leb. For the purpose we developed surface plasmon resonance (SPR) and double resonance long period grating (DR LPG) biosensors based on the BabA-Leb binding reaction for diagnosing H. pylori infection. In SPR detection, the sensitivity was estimated at 3000 CFU/mL-a much higher sensitivity than that of the RUT test. The DR LPG biosensor proved to be superior in terms of accuracy and sensitivity-concentrations as low as 102 CFU/mL were detected.

Antimicrobial Activity of Arthrospira (Former Spirulina) and Dunaliella Related to Recognized Antimicrobial Bioactive Compounds.

With the increasing rate of the antimicrobial resistance phenomenon, natural products gain our attention as potential drug candidates. Apart from being used as nutraceuticals and for biotechnological purposes, microalgae and phytoplankton have well-recognized antimicrobial compounds and proved anti-infectious potential. In this review, we comprehensively outline the antimicrobial activity of one genus of cyanobacteria (Arthrospira, formerly Spirulina) and of eukaryotic microalgae (Dunaliella). Both, especially Arthrospira, are mostly used as nutraceuticals and as a source of antioxidants for health supplements, cancer therapy and cosmetics. Their diverse bioactive compounds provide other bioactivities and potential for various medical applications. Their antibacterial and antifungal activity vary in a broad range and are strain specific. There are strains of Arthrospira platensis with very potent activity and minimum inhibitory concentrations (MICs) as low as 2-15 µg/mL against bacterial fish pathogens including Bacillus and Vibrio spp. Arthrospira sp. has demonstrated an inhibition zone (IZ) of 50 mm against Staphylococcus aureus. Remarkable is the substantial amount of in vivo studies of Arthrospira showing it to be very promising for preventing vibriosis in shrimp and Helicobacter pylori infection and for wound healing. The innovative laser irradiation of the chlorophyll it releases can cause photodynamic destruction of bacteria. Dunaliella salina has exhibited MIC values lower than 300 µg/mL and an IZ value of 25.4 mm on different bacteria, while Dunaliella tertiolecta has demonstrated MIC values of 25 and 50 µg/mL against some Staphylococcus spp. These values fulfill the criteria for significant antimicrobial activity and sometimes are comparable or exceed the activity of the control antibiotics. The bioactive compounds which are responsible for that action are fatty acids including PUFAs, polysaccharides, glycosides, peptides, neophytadiene, etc. Cyanobacteria, such as Arthrospira, also particularly have antimicrobial flavonoids, terpenes, alkaloids, saponins, quinones and some unique-to-them compounds, such as phycobiliproteins, polyhydroxybutyrate, the peptide microcystin, etc. These metabolites can be optimized by using stress factors in a two-step process of fermentation in closed photobioreactors (PBRs).

Palladium Phthalocyanines Varying in Substituents Position for Photodynamic Inactivation of Flavobacterium hydatis as Sensitive and Resistant Species.

Antimicrobial photodynamic therapy (aPDT) has been considered as a promising methodology to fight the multidrug resistance of pathogenic bacteria. The procedure involves a photoactive compound (photosensitizer), the red or near infrared spectrum for its activation, and an oxygen environment. In general, reactive oxygen species are toxic to biomolecules which feature a mechanism of photodynamic action. The present study evaluates two clinical isolates of Gram-negative Flavobacteriumhydatis (F. hydatis): a multidrug resistant (R) and a sensitive (S) strain. Both occur in farmed fish, leading to the big production losses because of the inefficacy of antibiotics. Palladium phthalocyanines (PdPcs) with methylpyridiloxy groups linked peripherally (pPdPc) or non-peripherally (nPdPc) were studied with full photodynamic inactivation for 5.0 µM nPdPc toward both F. hydatis, R and S strains (6 log), but with a half of this value (3 log) for 5.0 µM pPdPc and only for F. hydatis, S. In addition to the newly synthesized PdPcs as a "positive control" was applied a well-known highly effective zinc phthalocyanine (ZnPcMe). ZnPcMe showed optimal photocytotoxicity for inactivation of both F. hydatis R and S. The present study is encouraging for a further development of aPDT with phthalocyanines as an alternative method to antibiotic medication to keep under control the harmful pathogens in aquacultures' farms.

Mycobacterium bovis and M. caprae in Bulgaria: insight into transmission and phylogeography gained through whole-genome sequencing.

BACKGROUND: This study aimed to characterize recent Mycobacterium bovis/M. caprae isolates from Bulgaria by whole-genome sequencing (WGS) to gain a first insight into their molecular diversity, transmission, and position within the global phylogeography of this important zoonotic species. RESULTS: The isolates were obtained from cattle in diverse locations of Bulgaria in 2015-2020 and were identified by microbiological and PCR assays. WGS data were used for phylogenetic analysis that also included M. bovis global dataset. Thirty-seven M. bovis/caprae isolates from Bulgaria were studied and 34 of them were SNP genotyped. The isolates were subdivided into 3 major phylogenetic groups. Type Mbovis-13 (Eu2 complex [western Europe and northern Africa]) included one isolate. Mbovis-37 type included 5 isolates outside of known clonal complexes. The Bulgarian M. caprae isolates formed a sub-group within the Mcaprae-27B cluster which also included 22 M. caprae isolates from Poland, Spain, Germany, and the Republic of Congo. The Bulgarian M. caprae isolates share their latest common ancestors with Spanish isolates. The Mbovis-37 group shares a distant common ancestor (pairwise distance 22-29 SNPs) with an isolate from Poland but was very distant (> 200 SNPs) from the rest of the tree. The Mbovis-13 group shares a common ancestor with two human isolates from Germany. Phylogeographically, both M. bovis clades had limited circulation in northeastern Bulgaria while the majority of the studied isolates (M. caprae) were from central and western provinces. A phylogenetic network-based analysis demonstrated that 11 Bulgarian isolates were separated by 1 to 6 SNPs within four clusters, mostly forming pairs of isolates. CONCLUSION: The obtained WGS analysis positioned the Bulgarian isolates within the global phylogeography of M. bovis/M. caprae. Hypothetically, the observed phylogenetic diversity may not have resulted from livestock trade routes, but instead may reflect the deeply rooted M. bovis/M. caprae phylogeography of Europe. A high level of genetic divergence between the majority of the studied isolates suggests limited active transmission of bTB in Bulgaria during the survey period. At the same time, a possibility of the endemic presence of circulating bTB strains in the form of the latent persistent disease cannot be ruled out.

New Potential Pharmacological Targets of Plant-Derived Hydroxyanthraquinones from Rubia spp.

The increased use of polyphenols nowadays poses the need for identification of their new pharmacological targets. Recently, structure similarity-based virtual screening of DrugBank outlined pseudopurpurin, a hydroxyanthraquinone from Rubia cordifolia spp., as similar to gatifloxacin, a synthetic antibacterial agent. This suggested the bacterial DNA gyrase and DNA topoisomerase IV as potential pharmacological targets of pseudopurpurin. In this study, estimation of structural similarity to referent antibacterial agents and molecular docking in the DNA gyrase and DNA topoisomerase IV complexes were performed for a homologous series of four hydroxyanthraquinones. Estimation of shape- and chemical feature-based similarity with (S)-gatifloxacin, a DNA gyrase inhibitor, and (S)-levofloxacin, a DNA topoisomerase IV inhibitor, outlined pseudopurpurin and munjistin as the most similar structures. The docking simulations supported the hypothesis for a plausible antibacterial activity of hydroxyanthraquinones. The predicted docking poses were grouped into 13 binding modes based on spatial similarities in the active site. The simultaneous presence of 1-OH and 3-COOH substituents in the anthraquinone scaffold were emphasized as relevant features for the binding modes' variability and ability of the compounds to strongly bind in the DNA-enzyme complexes. The results reveal new potential pharmacological targets of the studied polyphenols and help in their prioritization as drug candidates and dietary supplements.

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.

Improvement of the Antimicrobial Activity of Oregano Oil by Encapsulation in Chitosan-Alginate Nanoparticles.

Oregano oil (OrO) possesses well-pronounced antimicrobial properties but its application is limited due to low water solubility and possible instability. The aim of this study was to evaluate the possibility to incorporate OrO in an aqueous dispersion of chitosan-alginate nanoparticles and how this will affect its antimicrobial activity. The encapsulation of OrO was performed by emulsification and consequent electrostatic gelation of both polysaccharides. OrO-loaded nanoparticles (OrO-NP) have small size (320 nm) and negative charge (-25 mV). The data from FTIR spectroscopy and XRD analyses reveal successful encapsulation of the oil into the nanoparticles. The results of thermogravimetry suggest improved thermal stability of the encapsulated oil. The minimal inhibitory concentrations of OrO-NP determined on a panel of Gram-positive and Gram-negative pathogens (ISO 20776-1:2006) are 4-32-fold lower than those of OrO. OrO-NP inhibit the respiratory activity of the bacteria (MTT assay) to a lower extent than OrO; however, the minimal bactericidal concentrations still remain significantly lower. OrO-NP exhibit significantly lower in vitro cytotoxicity than pure OrO on the HaCaT cell line as determined by ISO 10993-5:2009. The irritation test (ISO 10993-10) shows no signs of irritation or edema on the application site. In conclusion, the nanodelivery system of oregano oil possesses strong antimicrobial activity and is promising for development of food additives.

Junipers of Various Origins as Potential Sources of the Anticancer Drug Precursor Podophyllotoxin.

Juniper representatives are natural sources of plenty of bioactive metabolites and have been used since ancient times as folk remedies against tapeworms, warts, cancer, etc. The antiproliferative activities of junipers are attributed to podophyllotoxin (PPT), which is a precursor for the synthesis of efficient anticancer drugs. However, the natural sources of PPT, Sinopodophyllum hexandrum (Royle) T. S. Ying and Podophyllum peltatum L., are already endangered species because of their intensive industrial exploitation. Therefore, identification of other sources of PPT is necessary. This study is a broad comparative investigation of junipers, for which original sources have been accessed from different continents of the world. The present research is aimed at the identification of species, producing PPT and other lignans at concentrations that are sufficient for the high antiproliferative activity of the corresponding extracts. Cytotoxic juniper leaf extracts demonstrated a broad spectrum of activity on a panel of cancer cell lines. The antiproliferative properties of junipers were attributed to the combined activity of great diversity of lignans (podophyllotoxin, deoxypodophyllotoxin, ß-peltatin, yatein, matairesinol, anhydropodorhizol, etc.), detected by UHPLC-HRMS and LC-ESI-MS/MS in the corresponding extracts. Several species of the genus Juniperus L. were outlined as perspective sources of drug precursors with potential pharmaceutical applications.

In Vitro Antineoplastic and Antiviral Activity and In Vivo Toxicity of Geum urbanum L. Extracts.

This study evaluated the in vitro antineoplastic and antiviral potential and in vivo toxicity of twelve extracts with different polarity obtained from the herbaceous perennial plant Geum urbanum L. (Rosaceae). In vitro cytotoxicity was determined by ISO 10993-5/2009 on bladder cancer, (T-24 and BC-3C), liver carcinoma (HEP-G2) and normal embryonic kidney (HEK-293) cell lines. The antineoplastic activity was elucidated through assays of cell clonogenicity, apoptosis induction, nuclear factor kappa B p65 (NFκB p65) activation and total glutathione levels. Neutral red uptake study was applied for antiviral activity. The most promising G. urbanum extract was analyzed by UHPLC-HRMS. The acute in vivo toxicity analysis was carried out following OEDC 423. The ethyl acetate extract of aerial parts (EtOAc-AP) exhibited the strongest antineoplastic activity on bladder cancer cell lines (IC50 = 21.33-25.28 µg/mL) by inducing apoptosis and inhibiting NFκB p65 and cell clonogenicity. EtOAc and n-butanol extracts showed moderate antiviral activity against human adenovirus type 5 and human simplex virus type I. Seventy four secondary metabolites (gallic and ellagic acid derivatives, phenolic acids, flavonoids, etc.) were identified in EtOAc-AP by UHPLC-HRMS. This extract induced no signs of acute toxicity in liver and kidney specimens of H-albino mice in doses up to 210 mg/kg. In conclusion, our study contributes substantially to the detailed pharmacological characterization of G. urbanum, thus helping the development of health-promoting phytopreparations.

Yersinia artesiana sp. nov., Yersinia proxima sp. nov., Yersinia alsatica sp. nov., Yersina vastinensis sp. nov., Yersinia thracica sp. nov. and Yersinia occitanica sp. nov., isolated from humans and animals.

Thirty-three Yersinia strains previously characterized by the French Yersinia National Reference Laboratory (YNRL) and isolated from humans and animals were suspected to belong to six novel species by a recently described core genome multilocus sequence typing scheme. These strains and five additional strains from the YNRL were characterized using a polyphasic taxonomic approach including a phylogenetic analysis based on 500 core genes, determination of average nucleotide identity (ANI), determination of DNA G+C content and identification of phenotypic features. Phylogenetic analysis confirmed that the 38 studied strains formed six well-demarcated clades. ANI values between these clades and their closest relatives were <94.7 % and ANI values within each putative novel species were >97.5 %. Distinctive biochemical characteristics were identified in five out of the six novel species. All of these data demonstrated that the 38 strains belong to six novel species of the genus Yersinia: Yersinia artesiana sp. nov., type strain IP42281T (=CIP 111845T=DSM 110725T); Yersinia proxima sp. nov., type strain IP37424T (=CIP 111847T=DSM 110727T); Yersinia alsatica sp. nov., type strain IP38850T (=CIP 111848T=DSM 110726T); Yersinia vastinensis sp. nov., type strain IP38594T (=CIP 111844T=DSM 110738T); Yersinia thracica sp. nov., type strain IP34646T (=CIP 111842T=DSM 110736T); and Yersinia occitanica sp. nov., type strain IP35638T (=CIP 111843T=DSM 110739T).

Migratory birds along the Mediterranean - Black Sea Flyway as carriers of zoonotic pathogens.

At the crossroad between Europe, Asia, and Africa, Bulgaria is part of the Mediterranean - Black Sea Flyway (MBSF) used by millions of migratory birds. In this study, bird species migrating through Bulgaria were investigated as carriers of zoonotic pathogens. In total, 706 birds belonging to 46 species were checked for the presence of various bacterial pathogens (Campylobacter, Yersinia, Salmonella, Listeria, Escherichia coli, Staphylococcus aureus, Francisella tularensis, Coxiella burnetii, Borrelia burgdorferi, and Brucella spp.). From 673 birds we investigated fecal samples, from the remaining 33, blood samples. We detected Campylobacter 16S rDNA gene in 1.3% of birds, but none were of pathogenic Campylobacter jejuni and Campylobacter coli species. Escherichia coli 16S rDNA gene was found in 8.8% of the birds. Out of 34 birds that transported Yersinia enterocolitica strains (5.05%), only 1 carried a pathogenic isolate. Three birds (0.4%) were carriers of nonpathogenic Salmonella strains. Four avian samples (0.6%) were positive for Listeria monocytogenes and 1 (0.15%) was positive for Brucella spp. None of the birds tested carried the tick-borne pathogens C. burnetii or B. burgdorferi sensu lato. Antibiotic-resistant strains were detected, suggesting that migratory birds could be reservoirs and spreaders of bacterial pathogens as well as antibiotic resistance genes.

HPLC-UV and LC-MS Analyses of Acylquinic Acids in Geigeria alata (DC) Oliv. & Hiern. and their Contribution to Antioxidant and Antimicrobial Capacity.

INTRODUCTION: Geigeria alata is a traditional plant used in Sudanese folk medicine for treatment of diabetes, cough, epilepsy and intestinal complaints. OBJECTIVE: To analyze phenolic acids in Geigeria alata roots and leaves and to evaluate their antioxidant and antimicrobial activities. METHODOLOGY: Phenolic acids in the aqueous-methanol extracts were identified by LC-MS. Major compounds were isolated using low-pressure liquid chromatography. The quantitative analysis of phenolic acids was performed by a validated HPLC-UV method with limits of detection ranging from 0.04 to 0.57 µg/mL. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazine-6-sulphonic acid) (ABTS) and ferric reducing antioxidant power (FRAP) methods were used for antioxidant activity evaluation. In addition, the minimal inhibitory concentration and the minimal bactericidal concentration against a panel of pathogenic bacteria and fungi were determined by the broth microdilution test. RESULTS: For the first time protocatechuic, caffeic, p-coumaroylquinic, caffeoylsinapoylquinic, caffeoylferuloylquinic, three feruloylquinic, six caffeoylquinic acids, and a caffeic acid hexoside were detected in Geigeria alata roots by LC-MS. HPLC-UV analyses showed that 3,5-dicaffeoylquinic acid (25.96 ± 2.08 mg/g dry weight (DW)) was the most abundant phenolic acid in roots, while 4,5-dicaffeoylquinic acid (8.99 ± 0.56 mg/g DW) was the main compound present in leaves. 3,5-Dicaffeoylquinic acid demonstrated stronger radical scavenging activity and reducing power compared with the crude extracts and the positive control 5-caffeoylquinic acid. 3,4,5-Tricaffeoylquinic acid revealed the highest antibacterial potential against the penicillin sensitive and resistant Staphylococcus aureus strains, as well as methicillin-resistant S. aureus. CONCLUSION: The caffeoylquinic acids content of up to 6.22% in Geigeria alata roots establishes this species as a new source rich in these bioactive molecules. Copyright © 2016 John Wiley & Sons, Ltd.

Biopolymeric Nanogel as a Drug Delivery System for Doxorubicin-Improved Drug Stability and Enhanced Antineoplastic Activity in Skin Cancer Cells.

In this study, doxorubicin was loaded in a chitosan-albumin nanogel with the aim of improving its stability and exploring the potential of the system in the treatment of skin cancer. Infrared spectroscopy and X-ray diffraction confirmed the encapsulation of the drug. Transmission electron microscopy revealed the spherical shape of the nanogel particles. The drug-loaded nanogel was characterized with a small diameter of 29 nm, narrow polydispersity (0.223) and positive zeta potential (+34 mV). The exposure of encapsulated doxorubicin to light (including UV irradiation and daylight) did not provoke any degradation, whereas the nonencapsulated drug was significantly degraded. In vitro studies on keratinocytes (HaCaT) and epidermoid squamous skin carcinoma cells (A-431) disclosed that the encapsulated doxorubicin was more cytotoxic on both cell lines than the pure drug was. More importantly, the cytotoxic concentration of encapsulated doxorubicin in carcinoma cells was approximately two times lower than that in keratinocytes, indicating that it would not affect them. Thus, the loading of doxorubicin into the developed chitosan-albumin nanogel definitely stabilized the drug against photodegradation and increased its antineoplastic effect on the skin cancer cell line.

Polycarbonate-Based Copolymer Micelles as Biodegradable Carriers of Anticancer Podophyllotoxin or Juniper Extracts.

Podophyllotoxin (PPT) is used in the industrial production of efficient anticancer, antiviral and other drugs. Sinopodophyllum hexandrum or Podophyllum peltatum are natural sources of PPT, but at present they are considered as endangered species. Their PPT content is variable, depending on the growing conditions. Searching for new sources of PPT, some representatives of the genus Juniperus were found to exhibit efficient PPT biosynthesis. However, PPT is highly toxic and poorly soluble in water compound, which limits its clinical applications. In this connection, amphiphilic polymer micelles are considered to be suitable PPT carriers, aimed at increase in water solubility and decrease in toxicity. The present research deals with the evaluation of MPEG-polycarbonate block copolymer micelles loaded with PPT or juniper extracts. The active component-loaded polymer nanocarriers were characterized by dynamic and electrophoretic light scattering, as well as by transmission electron microscopy. The active component loading efficiency and loading capacity were also determined. Highly efficient antiproliferative activity of the loaded micelles was determined in a panel of cancer cell lines. The obtained amphiphilic nanocarriers, loaded with PPT-containing bioactive components, have application in future in vivo preclinical trials of their pharmacokinetics and pharmacodynamics as potential therapeutical agents in the prospective nanomedicine.

Pathogenic Potential of Opportunistic Gram-Negative Bacteria Isolated from the Cloacal Microbiota of Free-Living Reptile Hosts Originating from Bulgaria.

Reptiles are known to be asymptomatic carriers of various zoonotic pathogens. A number of Gram-negative opportunistic commensals are causative agents of bacterial infections in immunocompromised or stressed hosts and are disseminated by reptiles, whose epidemiological role should not be neglected. Since most studies have focused on exotic species, in captivity or as pet animals, the role of wild populations as a potential source of pathogens still remains understudied. In the present study, we isolated a variety of Gram-negative bacteria from the cloacal microbiota of free-living lizard and tortoise hosts (Reptilia: Sauria and Testudines) from the Bulgarian herpetofauna. We evaluated their pathogenic potential according to their antibiotic susceptibility patterns, biofilm-forming capacity, and extracellular production of some enzymes considered to play roles as virulence factors. To our knowledge, the phenotypic manifestation of virulence factors/enzymatic activity and biofilm formation in wild reptile microbiota has not yet been widely investigated. All isolates were found to be capable of forming biofilms to some extent and 29.6% of them could be categorized as strong producers. Two strains proved to be excellent producers. The majority of the isolated strains showed extracellular production of at least one exoenzyme. The most pronounced pathogenicity could be attributed to the newly isolated Pseudomonas aeruginosa strain due to its multiresistance, excellent biofilm formation, and expression of exoenzymes.

Insight into the influence of natural deep eutectic solvents on the extraction of phenolic compounds from poplar type propolis: Composition and in vitro biological activity.

Natural deep eutectic solvents (NADESs) have been considered promising to replace traditional volatile and toxic organic solvents for the extraction of biologically active substances from natural sources. This work applied an efficient and ethanol-exclusion strategy for extraction of phenolic compounds from poplar type propolis using five known NADESs (lactic acid:1,2-propanediol 1:1, lactic acid:fructose 5:1, choline chloride:1,2-propanediol 1:3, choline chloride:1,2-propanediol:water 1:1:1 and betaine:malic acid:water 1:1:6). The selected NADESs' extractability was evaluated by measuring the concentrations of total phenolics and total flavones and flavonols in the propolis extracts obtained, which qualitative chemical composition was further determined in detail by gas chromatography-mass spectrometry (GC-MS) analysis. It demonstrated that the chemical profiles of NADES and 70% ethanolic propolis extracts are similar. To expand the knowledge about the role of the applied solvents in the poplar propolis extraction process, the in vitro antimicrobial, cytotoxic and genotoxic activity of both NADESs and liquid NADES extracts were evaluated. The results revealed that the use of the selected NADESs as an extraction media for phenolic compounds from poplar propolis not only delivered a good extraction yield in some cases, but generally led to the preservation of propolis extracts' biological activity and even to the enhancement of their antimicrobial effect in comparison with the hydroethanolic one. Besides, the tested NADESs except for lactic acid:fructose and betaine:malic acid:water exerted low to negligible toxicity against normal cells treated and apart from lactic acid:fructose the remaining solvents demonstrated concentration-dependent moderate to subtle genotoxicity. There is a probability that not the supramolecular structure of the NADESs, but their components, played a key role for the observed biological effects. The present study has demonstrated an alternative approach for extracting the biologically active complex from poplar type propolis using NADESs, which could be useful for further pharmaceutical and cosmeceutical applications.

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.

Functional Hydrogels for Delivery of the Proteolytic Enzyme Serratiopeptidase.

Hydrogels are superior wound dressings because they can provide protection and hydration of the wound, as well as the controlled release of therapeutic substances to aid tissue regeneration and the healing process. Hydrogels obtained from natural precursors are preferred because of their low cost, biocompatibility, and biodegradability. We describe the synthesis of novel functional hydrogels based on two natural products-citric acid (CA) and pentane-1,2,5-triol (PT, a product from lignocellulose processing) and poly(ethylene glycol) (PEG-600)-via an environment friendly approach. The hydrogels were prepared via monomer crosslinking through a polycondensation reaction at an elevated temperature in the absence of any solvent. The reagents were blended at three different compositions with molar ratios of hydroxyl (from PT and PEG) to carboxyl (from CA) groups of 1:1, 1:1.4, and 1.4:1, respectively. The effect of the composition on the physicomechanical properties of materials was investigated. All hydrogels exhibited pH-sensitive behavior, while the swelling degree and elastic modulus were dependent on the composition of the polymer network. The proteolytic enzyme serratiopeptidase (SER) was loaded into a hydrogel via physical absorption as a model drug. The release profile of SER and the effects of the enzyme on healthy skin cells were assessed. The results showed that the hydrogel carrier could provide the complete release of the loaded enzyme.

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.

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.