Frequency of SARS-CoV-2 infection in dogs and cats: Results of a retrospective serological survey in Sumadija District, Serbia.

It has long been known that coronaviruses cause various infectious diseases in animals. Although SARS-CoV-2 is genetically related to viruses isolated from Rhinolophus bats, the exact origin, mode of transmission, and how the human species has become the epidemiological reservoir of the virus have not yet been established with certainty. Although the main route of transmission is human-to-human, there are considerable numbers of reported cases of infection in animal species, predominantly among pet animals. The aim of this retrospective study was to assess SARS-CoV-2 seropositivity in dogs and cats during the COVID-19 pandemic in Sumadija District, Serbia. We used serology to identify household contacts of pet animals with infected pet owners and the degree of association. The study presented in this paper is also the first study of this type in Serbia. The results of a retrospective serosurvey, which was conducted in dogs and cats with different exposure risk factors, were analyzed to find the possible modes of transmission between humans and animals. The relative frequency of SARS-CoV-2 infection in dogs was 1.45% bounded with a 95% confidence interval (CI) of 0.0007-7.73%, while in cats, it was 5.56% (95% CI: 0.77-4.13%). The relative frequency of SARS-CoV-2 infection in pet owners was 11% (95% CI: 6.25-18.63%). In pets that were in close contact with COVID-19 positive owners, the seropositivity was found to be 9%. Out of a total of five stray dogs and cats tested, seropositivity was observed in two animals. Detected SARS-CoV-2 infection in pets shows that these animals are susceptible to infection and that the most common means of virus transmission to pets is through contact with diseased owners. However, the presence of infection in stray dogs and cats is not clear and needs further research.

Bacteriophage Cocktail Can Effectively Control Salmonella Biofilm in Poultry Housing.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is the major contaminant of poultry products, and its ability to form biofilms on produced food and poultry farm processing surfaces contributes to Salmonella transmission to humans. Bacteriophages have come under increasing interest for anti-Salmonella biofilm control. In this study, we used the three previously sequenced and described phages UPWr_S1, UPWr_S3, and UPWr_S4 and a phage cocktail, UPWr_S134, containing these three phages to degrade biofilms formed by two S. Enteritidis strains, 327 lux and ATCC 13076, in vitro. It was found that treatment with bacteriophages significantly reduced biofilm on a 96-well microplate (32-69%) and a stainless steel surface (52-98%) formed by S. Enteritidis 327 lux. The reduction of biofilm formed by S. Enteritidis ATCC 13076 in the 96-well microplate and on a stainless steel surface for bacteriophage treatment was in the range of 73-87% and 60-97%, respectively. Under laboratory conditions, an experimental model utilizing poultry drinkers artificially contaminated with S. Enteritidis 327 lux and treated with UPWr_S134 phage cocktail was applied. In in vitro trials, the phage cocktail significantly decreased the number of Salmonella on the surface of poultry drinkers. Moreover, the phage cocktail completely eradicated Salmonella from the abundant bacterial load on poultry drinkers in an experimentally infected chickens. Therefore, the UPWr_S134 phage cocktail is a promising candidate for Salmonella biocontrol at the farm level.

Spatiotemporal Analysis of West Nile Virus Epidemic in South Banat District, Serbia, 2017-2019.

West Nile virus (WNV) is an arthropod-born pathogen, which is transmitted from wild birds through mosquitoes to humans and animals. At the end of the 20th century, the first West Nile fever (WNF) outbreaks among humans in urban environments in Eastern Europe and the United States were reported. The disease continued to spread to other parts of the continents. In Serbia, the largest number of WNV-infected people was recorded in 2018. This research used spatial statistics to identify clusters of WNV infection in humans and animals in South Banat County, Serbia. The occurrence of WNV infection and risk factors were analyzed using a negative binomial regression model. Our research indicated that climatic factors were the main determinant of WNV distribution and were predictors of endemicity. Precipitation and water levels of rivers had an important influence on mosquito abundance and affected the habitats of wild birds, which are important for maintaining the virus in nature. We found that the maximum temperature of the warmest part of the year and the annual temperature range; and hydrographic variables, e.g., the presence of rivers and water streams were the best environmental predictors of WNF outbreaks in South Banat County.

The First Report of mcr-1-Carrying Escherichia coli Originating from Animals in Serbia.

The aim of this study was continuous monitoring of the presence of mcr-1 to mcr-5 genes in Enterobacterales isolated from cattle, pigs, and domestic poultry at intensive breeding facilities in Northern Vojvodina, Serbia, from 1 January 1 to 1 October 2020. Out of 2167 examined samples, mcr-1 was observed in five E. coli isolates originating from healthy turkeys. Four isolates belonged to the phylogenetic group B1, and one isolate to the phylogenetic group A. Detected E. coli serogenotypes (somatic O and flagellar H antigens) were O8:H25 and O29:H25. Core-genome multi-locus sequence typing (cgMLST) revealed three ST58 isolates clustering together in Clonal Complex (CC) 155 and two singletons of ST641-CC86 and ST410-CC23, respectively. Clonotyping revealed CH4-32 (n = 3), CH6-53 (n = 1) and CH4-24 (n = 1). In all isolates, the mcr-1 gene was located on a large IncX4 replicon type plasmid. Eight virulence-associated genes (VAGs) typical of avian pathogenic E. coli (APEC) (fyuA, fimH, hlyF, iss, ompT, sitA, traT, iroN) were detected in four isolates. These isolates were investigated for susceptibility to four biocides and revealed MIC values of 0.125% for glutardialdehyde, of 0.00003-0.00006% for chlorohexidine, of 4-6% for isopropanol and of 0.001-0.002% for benzalkonium chloride. All obtained MIC values of the tested biocides were comparable to the reference strain, with no indication of possible resistance. This is the first report of mcr-1.1-carrying E. coli from Serbia. Although only samples from turkeys were mcr-positive in this study, continuous monitoring of livestock samples is advised to prevent a spill-over from animals to humans.

The Pheno- and Genotypic Characterization of Porcine Escherichia coli Isolates.

Escherichia (E.) coli is the main causative pathogen of neonatal and post-weaning diarrhea and edema disease in swine production. There is a significant health concern due to an increasing number of human infections associated with food and/or environmental-borne pathogenic and multidrug-resistant E. coli worldwide. Monitoring the presence of pathogenic and antimicrobial-resistant E. coli isolates is essential for sustainable disease management in livestock and human medicine. A total of 102 E. coli isolates of diseased pigs were characterized by antimicrobial and biocide susceptibility testing. Antimicrobial resistance genes, including mobile colistin resistance genes, were analyzed by PCR and DNA sequencing. The quinolone resistance-determining regions of gyrA and parC in ciprofloxacin-resistant isolates were analyzed. Clonal relatedness was investigated by two-locus sequence typing (CH clonotyping). Phylotyping was performed by the Clermont multiplex PCR method. Virulence determinants were analyzed by customized DNA-based microarray technology developed in this study for fast and economic molecular multiplex typing. Thirty-five isolates were selected for whole-genome sequence-based analysis. Most isolates were resistant to ampicillin and tetracycline. Twenty-one isolates displayed an ESBL phenotype and one isolate an AmpC ß-lactamase-producing phenotype. Three isolates had elevated colistin minimal inhibitory concentrations and carried the mcr-1 gene. Thirty-seven isolates displayed a multi-drug resistance phenotype. The most predominant ß-lactamase gene classes were blaTEM-1 (56%) and blaCTX-M-1 (13.71%). Mutations in QRDR were observed in 14 ciprofloxacin-resistant isolates. CH clonotyping divided all isolates into 51 CH clonotypes. The majority of isolates belonged to phylogroup A. Sixty-four isolates could be assigned to defined pathotypes wherefrom UPEC was predominant. WGS revealed that the most predominant sequence type was ST100, followed by ST10. ST131 was detected twice in our analysis. This study highlights the importance of monitoring antimicrobial resistance and virulence properties of porcine E. coli isolates. This can be achieved by applying reliable, fast, economic and easy to perform technologies such as DNA-based microarray typing. The presence of high-risk pathogenic multi-drug resistant zoonotic clones, as well as those that are resistant to critically important antibiotics for humans, can pose a risk to public health. Improved protocols may be developed in swine farms for preventing infections, as well as the maintenance and distribution of the causative isolates.

Polymeric Microfiltration Membranes Modification by Supercritical Solvent Impregnation-Potential Application in Open Surgical Wound Ventilation.

This study investigated supercritical solvent impregnation of polyamide microfiltration membranes with carvacrol and the potential application of the modified membranes in ventilation of open surgical wounds. The impregnation process was conducted in batch mode at a temperature of 40 °C under pressures of 10, 15, and 20 MPa for contact times from 1 to 6 h. FTIR was applied to confirm the presence of carvacrol on the membrane surface. In the next step, the impact of the modification on the membrane structure was studied using scanning electron and ion beam microscopy and cross-filtration tests. Further, the release of carvacrol in carbon dioxide was determined, and finally, an open thoracic cavity model was applied to evaluate the efficiency of carvacrol-loaded membranes in contamination prevention. Carvacrol loadings of up to 43 wt.% were obtained under the selected operating conditions. The swelling effect was detectable. However, its impact on membrane functionality was minor. An average of 18.3 µg of carvacrol was released from membranes per liter of carbon dioxide for the flow of interest. Membranes with 30-34 wt.% carvacrol were efficient in the open thoracic cavity model applied, reducing the contamination levels by 27% compared to insufflation with standard membranes.

Presence of ß-Lactamase-producing Enterobacterales and Salmonella Isolates in Marine Mammals.

Marine mammals have been described as sentinels of the health of marine ecosystems. Therefore, the aim of this study was to investigate (i) the presence of extended-spectrum ß-lactamase (ESBL)- and AmpC-producing Enterobacterales, which comprise several bacterial families important to the healthcare sector, as well as (ii) the presence of Salmonella in these coastal animals. The antimicrobial resistance pheno- and genotypes, as well as biocide susceptibility of Enterobacterales isolated from stranded marine mammals, were determined prior to their rehabilitation. All E. coli isolates (n = 27) were screened for virulence genes via DNA-based microarray, and twelve selected E. coli isolates were analyzed by whole-genome sequencing. Seventy-one percent of the Enterobacterales isolates exhibited a multidrug-resistant (MDR) pheno- and genotype. The gene blaCMY (n = 51) was the predominant ß-lactamase gene. In addition, blaTEM-1 (n = 38), blaSHV-33 (n = 8), blaCTX-M-15 (n = 7), blaOXA-1 (n = 7), blaSHV-11 (n = 3), and blaDHA-1 (n = 2) were detected. The most prevalent non-ß-lactamase genes were sul2 (n = 38), strA (n = 34), strB (n = 34), and tet(A) (n = 34). Escherichia coli isolates belonging to the pandemic sequence types (STs) ST38, ST167, and ST648 were identified. Among Salmonella isolates (n = 18), S. Havana was the most prevalent serotype. The present study revealed a high prevalence of MDR bacteria and the presence of pandemic high-risk clones, both of which are indicators of anthropogenic antimicrobial pollution, in marine mammals.

Cellulose Acetate Membranes Modification by Aminosilane Grafting in Supercritical Carbon Dioxide towards Antibiofilm Properties.

The study explores the grafting of cellulose acetate microfiltration membranes with an aminosilane to attain antibiofilm properties. The grafting reaction was performed in the supercritical carbon dioxide used as a transport and reaction medium. The FTIR analyses and dissolution tests confirmed the covalent bonding between the aminosilane and polymer. The membranes' microstructure was investigated using a dual-beam SEM and ion microscopy, and no adverse effects of the processing were found. The modified membranes showed a more hydrophilic nature and larger water permeate flow rate than the neat cellulose acetate membranes. The tests in a cross-filtration unit showed that modified membranes were considerably less blocked after a week of exposure to Staphylococcus aureus and Escherichia coli than the original ones. Microbiological investigations revealed strong antibiofilm properties of the grafted membranes in experiments with Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella Enteritidis.

Application of Supercritical Solvent Impregnation for Production of Zeolite Modified Starch-Chitosan Polymers with Antibacterial Properties.

In the present study, supercritical solvent impregnation (SSI) has been applied to incorporate thymol into bio-composite polymers as a potential active packaging material. Thymol, a natural component with a proven antimicrobial activity, was successfully impregnated into starch-chitosan (SC) and starch-chitosan-zeolite (SCZ) films using supercritical carbon dioxide (scCO2) as a solvent. Experiments were performed at 35 °C, pressures of 15.5 and 30 MPa, and an impregnation time in the range of 4-24 h. The highest impregnation yields of SC films with starch to chitosan mass ratios of 1:1 and 1:2 were 10.80% and 6.48%, respectively. The addition of natural zeolite (15-60%) significantly increased the loading capacity of films enabling thymol incorporation in a quantity of 16.7-27.3%. FTIR and SEM analyses were applied for the characterization of the films. Mechanical properties and water vapor permeability of films before and after the impregnation were tested as well. Thymol release kinetics in deionized water was followed and modeled by the Korsmeyer-Peppas and Weibull model. SCZ films with thymol loading of approximately 24% exhibited strong antibacterial activity against E. coli and methicillin-resistant Staphylococcus (S.) aureus (MRSA).

Broad-Spectrum Cephalosporin-Resistant and/or Fluoroquinolone-Resistant Enterobacterales Associated with Canine and Feline Urogenital Infections.

The aim of the present study was to characterize Enterobacterales resistant to 3rd and 4th generation cephalosporins, carbapenems and/or fluoroquinolones, isolated from dogs and cats with urogenital infections. In total, 36 strains (Escherichia coli (n = 28), Klebsiella pneumoniae (n = 3), Serratia marcescens, Raoultella ornithinolytica, Proteus mirabilis, Citrobacter portucalensis and Enterobacter cloacae (each n = 1)) were included in the present study, 28 from Austria and 8 from Serbia. Isolates were characterized by a polyphasic approach including susceptibility pheno- and genotyping and microarray-based assays. Escherichia (E.) coli isolates were additionally characterized by two-locus (fumC and fimH) sequence phylotyping and multi-locus sequence typing (MLST) of selected isolates. MLST of carbapenem-resistant Enterobacter cloacae isolates was also performed. Among E. coli, the most dominant phylogenetic group was B1 (27.8%), followed by C, (16.6%), A and Clade II (5.5% each), B2 and F (2.77% each). The most predominant ß-lactam resistance genes were blaTEM (70%) and blaCTX-M (38.8%), blaCMY (25%). blaNDM was detected in one carbapenem-resistant Enterobacter cloacae ST114. The most common ST among selected E. coli was 744 (10.7% isolates). The pandemic clones ST131 and ST648 carrying CTX-M-15 were also detected. Remaining STs belonged to 469, 1287, 1463 and 1642. E. coli clonotyping revealed 20 CH types. Based on the presence of certain virulence genes, three isolates were categorized as ExPEC/UPEC. The most prevalent virulence factors were fimH detected in 61%, iucD and iss both in 55%, iroN in 27.8%, papC in 13.8% and sat in 8.3% isolates.

Supercritical Fluid Extraction of Celery and Parsley Fruit-Chemical Composition and Antibacterial Activity.

Supercritical fluid extraction as an environmentally friendly technology was applied to isolate biologically active extracts from celery and parsley fruits for potential applications in the food industry. The extractions were performed under mild temperature conditions of 39.85 °C and at pressures of 10 and 30 MPa. The extracts were analyzed regarding their chemical composition, antibacterial activity, and cytotoxic effect. Sedanolide was the dominant component of the celery fruit extracts, comprising more than 70% of the obtained fraction, while the content of apiole in the parsley fruit SC CO2 extracts exceeded 85%. The celery fruit extracts showed strong and moderately strong antibacterial activity against tested Staphylococcus aureus, Bacillus (B.) cereus, B. subtilis, B. circulans, Listeria (L.) greyi, L. seeligeri and L. welshimeri, with minimal inhibitory concentration (MIC) values between 160 and 640 µg/mL, and weak activity against the selected Salmonella isolates with a MIC of 2560 µg/mL. The parsley extract obtained at 10 MPa showed strong and moderately strong antibacterial effects against Bacillus strains with obtained MICs of 160-640 µg/mL, and weak activity against Staphylococcus, Listeria, and Salmonella with a MIC of 2560 µg/mL. Cytotoxicity investigation showed that the extracts with proven antibacterial activity had no cytotoxic effect on rabbit kidney cells at concentrations of up to 640 µg/mL.

Characterization of mecC gene-carrying coagulase-negative Staphylococcus spp. isolated from various animals.

The presence of the methicillin resistance gene mecC in coagulase-negative Staphylococcus spp. (CoNS) is scarce. The aim of this study was to characterize mecC-positive CoNS isolated from various wild and domestic animals. The presence of the mecC gene was screened in 4299 samples from wild animals and domestic animals. Fifteen coagulase-negative staphylococci, that displayed a cefoxitin-resistant phenotype, were tested mecC-positive by PCR. Antimicrobial susceptibility testing was performed for all isolates. The 15 isolates were genotyped by sequencing of the entire class E mec gene complex (blaZ-mecC-mecR1-mecI), the ccrA and ccrB recombinase genes and other determinants within the type XI SCCmec element. DNA microarray analysis was performed and five selected isolates were additionally whole genome sequenced and analyzed. S. stepanovicii (n = 3), S. caprae (n = 1), S. warneri (n = 1), S. xylosus (n = 1) and S. sciuri (n = 9) were detected. All but the S. sciuri isolates were found to be susceptible to all non-beta lactams. The entire class E mec gene complex was detected in all isolates but ccrA and ccrB genes were not identified in S. stepanovicii and S. xylosus. The genes erm(B) and fexA (n = 4, each) were the most predominant non-beta lactam resistance genes detected in the S. sciuri isolates. Even though the presence of the mecC gene among CoNS is a rare observation, this study further expands our knowledge by showing that the mecC gene, including its allotypes, are present in more staphylococcal species from different animal species than has been previously described.

Genetic Profiling and Comparison of Human and Animal Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates from Serbia.

The aim of this study was to characterize a collection of methicillin-resistant Staphylococcus aureus (MRSA) isolates of human and animal origin from Serbia. In total, 36 MRSA isolates-30 obtained from humans and six from companion animals-were investigated by PCR for the presence of antibiotic and biocide resistance determinants and virulence genes (PVL-Panton⁻Valentine leukocidin, ETs-exfoliative toxins, TSST-toxic shock syndrome toxin, SEs-staphylococcal enterotoxins, and MSCRAMMs-microbial surface components recognizing adhesive matrix molecules and biofilm). Isolates were analyzed by staphylococcal cassette chromosome mec (SCCmec), spa, and dru typing, as well as by multiple locus variable number of tandem repeat analyses (MLVA), multilocus sequence typing (MLST), and subsequently, eBURST. The majority of human MRSA isolates were resistant to gentamicin, erythromycin, clindamycin, and ciprofloxacin. Different antibiotic resistance genes were detected: aac-aphD, ant(6')-Ia, erm(A), erm(B), erm(C), tet(K), tet(M), fexA, and catpC221. All isolates were susceptible to teicoplanin and linezolid. SCCmec type III was prevalent in human isolates, while SCCmec elements in animals were mostly nontypeable. t037 was the predominant spa type in human and t242 in animal MRSA isolates. The prevalent dru type was dt11c in human and dt10a in animal MRSA isolates. MRSA isolates exhibited 27 different MLVA types. ST239 was predominant in human, while ST5 was prevalent in canine MRSA isolates. PVL was found in two, while tsst-1 was detected in three human isolates. Human-associated clones belonging to ST5, ST45, and ST239 MRSA clones were discovered in companion animals, which suggests anthropozoonotic transmission.

Enterotoxin production and antimicrobial susceptibility in Staphylococci isolated from traditional raw milk cheeses in Serbia.

This study was undertaken to determine the prevalence of coagulase positive staphylococci (CPS) by examining a total of 71 raw milk cheeses. Additionally, enterotoxigenicity, antimicrobial susceptibility and the presence of mecA and mecC genes in the staphylococcal isolates were investigated. The isolation and enumeration procedure of CPS followed the International Organization for Standardization (ISO) standard. The presumptive staphylococci were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the VITEK MS system. VIDAS® Staph enterotoxin II assay was used for the detection of classical enterotoxins. Antimicrobial susceptibility testing (AST) was accomplished performing the disk diffusion method. All suspected methicillin resistant staphylococci were investigated for the presence of mecA and mecC genes by PCR assay. A high prevalence (87.32%) of CPS was detected in the cheeses at contamination levels up to 5.58 log CFU g-1. Among 47 staphylococcal isolates screened for enterotoxin production, only one isolate, identified as S. hyicus, was confirmed as being enterotoxigenic. Resistance to penicillin (63.70%) was the most common resistance among the tested Staphylococcus aureus isolates. The dominant phenotypic resistance patterns in coagulase negative staphylococci (CNS) were resistance to ofloxacin and fusidic acid. All CNS isolates were susceptible to the clinically important antibiotics clindamycin, chloramphenicol, gentamicin, linezolid, rifampicin and trimethoprim-sulfamethoxazole. The mecA and mecC genes were not detected. To the best of our knowledge, this is the first study concerning evaluation of the presence of methicillin resistant staphylococci (MRS) in dairy products in Serbia.

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

BACKGROUND: There 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. RESULTS: The 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. CONCLUSIONS: This 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.

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.

Circulating immune complexes of calves with bronchopneumonia modulate the function of peripheral blood leukocytes: In vitro evaluation.

In this work we studied if circulating immune complexes (CIC) of calves with bronchopneumonia have the capacity to modulate function of peripheral blood leukocytes of healthy cattle. CIC of three month old calves (6 healthy and 6 diseased) were isolated by PEG precipitation. Peripheral blood mononuclear cells (MNCs) and granulocytes from healthy calves and cows were the CIC responder cells in in vitro tests. The most remarkable increase of adhesiveness to polystyrene and ROS synthesis (assessed by NBT test) was detected in cows' granulocytes stimulated with CIC of diseased calves. Results of MTT test showed that CIC of both healthy and diseased calves reduced granulocytes' viability. The strongest effect of inhibition of cows' granulocytes resulted from CIC of diseased calves. CIC only moderately reduced spontaneous viability of calves' MNCs. Again, the strongest effect of CIC isolated from diseased calves was observed. In contrast to the low impact of CIC on non-stimulated cells, their inhibitory effect on viability of mitogen stimulated MNCs was very strong. With CFSE assay we showed that both types of CIC stimulated spontaneous, but inhibited mitogen induced proliferation of calves' MNCs. Propidium iodide staining reviled that CIC increased apoptosis/necrosis of both non-stimulated and mitogen stimulated MNCs. CIC of both healthy and diseased calves modulated the function of peripheral blood MNCs and granulocytes, but a stronger effect of CIC of diseased calves was shown. The age of the donors (calves or cows) of the responder cells, and the activation state of these cells, were also of influence.

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

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.

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.