Dusan Misic

Functionalization of polycaprolactone/hydroxyapatite scaffolds with Usnea lethariiformis extract by using supercritical CO2.

Investigation of an integrated supercritical fluid extraction and supercritical solvent impregnation process for fabrication of microporous polycaprolactone-hydroxyapatite (PCL-HA) scaffolds with antibacterial activity is presented. The HA content and particle size as well as the operating conditions of the integrated process is optimized regarding the amount of impregnated antibacterial agent (Usnea lethariiformis extract) in the PCL-HA matrix, scaffold morphology and antibacterial activity against methicillin resistant Staphylococcus aureus (MRSA) strains. High pressure differential scanning calorimetry (HP-DSC) assay reveals that an increasing amount of HA results in decreasing melting temperature as well as crystallinity at an operating pressure of 17 MPa. The PCL-HA composites with micrometric sizes of the HA particles are convenient for being processed by the integrated process due to the simple preparation, a good interaction between the PCL matrix and filler and the advantageous impact on sorption. The scaffold obtained from PCL-HA with 20% of the HA shows the highest impregnation yield at 17 MPa and 35 °C (5.9%) and subsequently also the best bactericidal effect on the tested MRSA strains at an initial bacterial inoculum of 2 × 10(-4)CFU/mL.

Free-radical scavenging activity and antibacterial impact of Greek oregano isolates obtained by SFE.

The antioxidant and antibacterial properties of Greek oregano extracts obtained by fractional supercritical fluid extraction (SFE) with carbon dioxide were investigated and compared with the properties of essential oil obtained by hydrodistillation. According to DPPH, hydroxyl radical and superoxide anion radical scavenging activity assays, the supercritical extracts expressed stronger antioxidant activity comparing to the essential oil. The most effective was the supercritical extract obtained by fractional extraction at 30 MPa and 100°C after the volatile fraction had been extracted at lower pressure. At the same time this extract showed strong antibacterial activity against staphylococci, including MRSA strain, but did not affect Escherichia coli of normal intestinal flora. The essential oil obtained by hydrodistillation showed stronger antibacterial activity against E. coli, Salmonella and Klebsiella pneumoniae, comparing to the supercritical extracts but at the same affected the normal gut flora.

Estimation of dermatological application of creams with St. John's Wort oil extracts.

Oleum Hyperici, the oil extract of St. John’s Wort (SJW), is one of the oldest folk remedies, traditionally used in the topical treatment of wounds, bruises, ulcers, cuts, burns, hemorrhoids and also as an antiseptic. Considering the advantageous characteristics of emulsion applications, in the present study we have formulated three O/W creams containing 15% (w/v) of SJW oil extract as an active ingredient. The aim was to estimate dermatological application of the prepared creams for the abovementioned indications. The extracts were prepared according to the prescriptions from traditional medicine, however with different vegetable oils used as an extractant, namely: Olive, palm and sunflower oil. The investigated O/W creams demonstrated significant antiinflammatory effects in an in vivo double-blind randomized study, using a sodium lauryl sulphate test. Both skin parameters assessed in the study (electrical capacitance and erythema index), were restored to the baseline value after a seven-day treatment with the tested creams. Almost all investigated SJW oil extracts and corresponding creams displayed the same antimicrobial activity against the most of the investigated microorganisms with obtained minimal inhibitory concentrations values of 1,280 µg/mL, 2,560 µg/mL or >2,560 µg/mL.

European multicenter study on antimicrobial resistance in bacteria isolated from companion animal urinary tract infections

BackgroundThere is a growing concern regarding the increase of antimicrobial resistant bacteria in companion animals. Yet, there are no studies comparing the resistance levels of these organisms in European countries. The aim of this study was to investigate geographical and temporal trends of antimicrobial resistant bacteria causing urinary tract infection (UTI) in companion animals in Europe. The antimicrobial susceptibility of 22 256 bacteria isolated from dogs and cats with UTI was determined. Samples were collected between 2008 and 2013 from 16 laboratories of 14 European countries. The prevalence of antimicrobial resistance of the most common bacteria was determined for each country individually in the years 2012–2013 and temporal trends of bacteria resistance were established by logistic regression.ResultsThe aetiology of uropathogenic bacteria differed between dogs and cats. For all bacterial species, Southern countries generally presented higher levels of antimicrobial resistance compared to Northern countries. Multidrug-resistant Escherichia coli were found to be more prevalent in Southern countries. During the study period, the level of fluoroquinolone-resistant E. coli isolated in Belgium, Denmark, France and the Netherlands decreased significantly. A temporal increase in resistance to amoxicillin-clavulanate and gentamicin was observed among E. coli isolates from the Netherlands and Switzerland, respectively. Other country-specific temporal increases were observed for fluoroquinolone-resistant Proteus spp. isolated from companion animals from Belgium.ConclusionsThis work brings new insights into the current status of antimicrobial resistance in bacteria isolated from companion animals with UTI in Europe and reinforces the need for strategies aiming to reduce resistance.

Supercritical carbon dioxide hops extractswith antimicrobial properties

Abstract Extracts obtained from hops (Humulus lupulus L., Cannabaceae) by supercritical fluid extraction (SFE), SFE followed by isomerization, as well as by conventional technique, were investigated for their chemical composition and antibacterial activity against selected foodborne pathogens and microorganisms capable to cause the food spoilage. The antibacterial activity of the extracts was compared with the antibacterial activity of xanthohumol, compound known for its broad pharmacological properties, isolated from the raw material remained after the SFE. Xanthohumol (XH, 96%) proved to posses the most prominent activity against all the tested strains, with the MIC values ranged between 2.5 and 20 μg mL-1. Supercritical hops extract and potassium isomerized supercritical hops extract showed strong antibacterial activity against the tested strains as well. Escherichia coli was not affected by the extracts, meaning that their oral admission would not cause the same problem as antibiotic application in intestinal flora. The chemical composition of the investigated hops extracts was analysed by GC-MS. Contents of α-acids, β-acids, iso-α-acids and xanthohumol in the samples were determined by HPLC. Graphical Abstract

Usnea barbata CO2-supercritical extract in alkyl polyglucoside-based emulsion system: contribution of Confocal Raman imaging to the formulation development of a natural product

Abstract Topical treatment of skin infections is often limited by drawbacks related to both antimicrobial agents and their vehicles. In addition, considering the growing promotion of natural therapeutic products, our objective was to develop and evaluate naturally-based emulsion system, as prospective topical formulation for skin infections-treatment. Therefore, alkyl polyglucoside surfactants were used for stabilization of a vehicle serving as potential carrier for supercritical CO2-extract of Usnea barbata, lichen with well-documented antimicrobial activity, incorporated using two protocols and three concentrations. Comprehensive physicochemical characterization suggested possible involvement of extract’s particles in stabilization of the investigated system. Raman spectral imaging served as the key method in disclosing extract’s particles potential to participate in the microstructure of the tested emulsion system via three mechanisms: (1) particle–particle aggregation, (2) adsorption at the oil–water interface and (3) hydrophobic particle–surfactant interactions. Stated extract–vehicle interaction proved to be correlated to the preparation procedure and extract concentration on one hand and to affect the physicochemical and biopharmaceutical features of investigated system, on the other hand. Thereafter, formulation with the best preliminary stability and liberation profile was selected for further efficiency and in vivo skin irritation potential evaluation, implying pertinent in vitro antimicrobial activity against G+ bacteria and overall satisfying preliminary safety profile.

Investigation of biofilm formation in vitro ability of Listeria monocytogenes strains isolated from animals.

Listeria monocytogenes is the causative agent of listeriosis in humans and animals and an important food-born pathogen. Control of its presence in food processing plants, particularly on sites where food contamination is expected, is of paramount importance with respect to food safety and protection of human health. Numerous studies demonstrated that this organism can be isolated during several months, even several years, from diverse sites in food processing plants, which is due to its ability to adsorb onto inert surfaces and form a biofilm, alone or in coexistence with other bacterial species. In this study we investigated the ability of 16 animal isolates of L. monocytogenes to form a biofilm on polystyrene microtiter plates. The investigation was performed at three different temperatures 4oC, 25 oC and 37oC that are commonly suitable for growth of Listeria monocytogenes. The research was carried out using three different nutritive media: trypthon-soy broth with yeast extract (TSB-YE), brainheart infusion (BHI) and 1/20 diluted trypthon-soy broth with yeast extract (1/20 TSB-YE). In order to investigate the biofilm formation in vitro an inoculum was prepared from 24-hours-old cultures of isolated strains of L.monocytogenes. The density of the inoculum was 2-10x107 cfu/mL (OD600=0.093 ± 0.009 in TSB-YE). The microtiter plates were incubated at cited temperatures during 48h. Colonization rate of L.monocytogenes strains on polystyrene surface and biofilm formation were monitored using crystal violet stain added to the microtiter plates, as well as using light microscopy. The tested strains demonstrated a diverse ability of biofilm formation, depending on the incubation temperature and nutritive medium. In this paper we presented the results obtained for four strains of Listeria monocytogenes isolated from brain samples of sheep, designated as 785/05, 593/05, 748/05, 1915/04 and one strain isolated from aborted calves fetus, designated as 021/04. The referent strain of L.monocytogenes, 1071 (4b), was used as a control. Strains of L. monocytogenes cultured in nutrient-rich media (e.g. TSB-YE) and at higher temperatures (37oC) exhibited a higher ability of biofilm formation. However, non of the studied strains could be classified as a good biofilm producer, disregard of the incubation temperature and medium used. The highest OD values were obtained at the incubation temperature of 37oC in TSB-YE (OD595=0.346 ± 0.055), when three strains were quantified as moderate and two as weak biofilm producers. The decrease of incubation temperature resulted in decreased OD values, thus four strains were classified as weak biofilm producers at 25oC (OD595=0.289 ± 0.083), and none of the investigated strains was assessed as biofilm producers at 4oC (OD595=0.124 ± 0.011) Light-microscopy examination, confirmed by quantitative values obtained by the crystal violet microtiter test, proved to be a simple and rapid screening method for the quantification of the ability of L.monocytogenes to form biofilms in varying test conditions.

Investigation of antibacterial activity of supercritical extracts of plants, as well as of extracts obtained by other technological processes on some bacteria isolated from animals

The multiresistance of bacteria to antibiotics, as well as the lack of new antibiotics on the market encouraged the research of antibacterial activity of non-antibiotic substances including plant extracts. During the previous decades, it has been proven that extracts of certain plants have a strong antibacterial activity, but their clinical use was limited due to the presence of organic solvents. However, plant extracts obtained by the process of supercritical fluid extraction contain no traces of solvents, and the latest researches have established that they do have antibacterial effects on some gram-positive bacteria. This comparative study included extracts of Common Mullein, Angelica and Echinacea obtained by means of supercritical fluid extraction, Soxlet extraction and ultrasound-assisted extraction. The study of their antibacterial activity was performed on some strains of Staphylococcus, Enterobacter cloacae and E. coli isolated from clinical material of human and animal origin. A referential strain of S. aureus ATCC 25923 was included in the research. In the study broth macrodilution method was applied by which the MIC values of extracts were determined. The Angelica extract obtained by ultrasound-assisted extraction had the strongest antibacterial activity, i.e. the lowest MIC value of 40 μg/mL for S. epidermidis strain. The Angelica extract obtained by supercritical fluid extraction also showed substantial antibacterial activity to all Staphylococcus strains included in this study, with the MIC values of 320 to 640 μg/mL. The extracts of Echinacea and Common Mullein obtained by supercritical fluid extraction, as well as of Echinacea extract obtained by Soxlet extraction showed no antibacterial activity since the MIC values of these extracts were 2560 μg/mL or >2560 μg/mL for all bacterial strains icluded in the study.

Clonal persistence of Salmonella enterica serovars Montevideo, Tennessee, and Infantis in feed factories

INTRODUCTION Novel molecular techniques applied in biotechnology research have provided sound evidence on clonal persistence of distinct serovars of Salmonella in feed factory environments, over long periods of time (months, even years), which can be responsible for repeated in-house contamination of final products. In this study, we examined the possibility of clonal persistence of isolates of three Salmonella serovars that have been repeatedly identified in animal feed samples from three feed factories throughout a two-year period. METHODOLOGY The isolates Salmonella enterica serovars Tennessee (n = 7), Montevideo (n = 8), and Infantis (n = 4) were tested for genetic diversity using pulsed-field gel electrophoresis (PFGE) and multicellular behavior patterns by applying the Congo red agar test. RESULTS SpeI and XbaI macro-restriction profiles indicated that isolates S. Montevideo and S. Infantis were identical, whereas isolates of S. Tennessee demonstrated greater genetic diversity, although the genetic differences did not exceed 10%. All Salmonella serovars demonstrated the ability to produce predominant matrix compounds essential for biofilm formation, curli fimbriae and cellulose. CONCLUSIONS The identification of identical clones of S. Montevideo and S. Infantis, as well as the minor genetic diversity of S. Tennessee, which have been repeatedly isolated from animal feed in three production plants throughout a two-year period, indirectly suggests the possibility of their persistence in feed factory environments. Their ability to express the key biofilm matrix components further supports this hypothesis.

Investigation of the presence of extended spectrum beta-lactamases (ESBL) in multiresistant strains of E. Coli and salmonella species originated from domestic animals

Bacterial strains which possess genes to produce ESBL most often are multiresistant and also carry genes responsible for the resistance to most other antibiotics, including aminoglycosides, sulfamethoxazole+trimethoprim and fluoroquinolones. Therefore, practically the biggest contemporary clinical problem are infections of humans and animals caused by ESBL-producing strains of E. coli, Kleibsiella, Enterobacter, Proteus, Serratia, Citrobacter, Salmonella and Shigella species. The investigation of the ESBL presence was completed on multiresistant E. coli and Salmonella strains originating from dogs, cats, cattle, sheep, goats, pigs and poultry. The investigated strains were isolated from ear, skin, vaginal, faecal, urine, egg and eggshell swabs, from healthy and diseased individual animals of various ages and breed categories. The sum of 112 E. coli and 45 Salmonella strains was investigated. All strains resistant to 3 or more antibiotics were categorized as multiresistant, which led to a conclusion that 35 E. coli and 6 Salmonella strains out of all investigated were multiresistant to antibiotics. The largest number of multiresistant E. coli strains was discovered in cattle - 12 in total, and the minimal number in goats and sheep, with two strains each. All multiresistant Salmonella strains belonged to the Salmonella Enteritidis species (S. enterica subsp. enterica serovar Enteritidis). The sum of multiresistant Salmonella strains compared to all investigated strains was relatively low (13.3%), but the resistance prevalence for some antibiotics in these strains was extremely high, for ampicillin and amoxicillin with clavulanic acid as high as 100%, and for tetracycline 83.3%. For the control in this investigation were used ESBL positive E. coli strains originated in human urine specimens. No presence of positive ESBL strains was established. However, when the screening investigation was performed, almost all the strains were suspect, thus a confirmatory test had to be performed for all strains.