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There is an urgency to develop novel materials capable of preventing the adherence of microorganisms to their surface. Such materials with antibiofilm properties are needed in hospitals and public places, animal farms and veterinary hospitals, and biofouling prevention. Biofouling is the phenomenon of adhesion and the growth of microorganisms on surfaces in contact with water. It poses a significant problem for filtration processes, water treatment units, bioreactors, and ships performance (marine biofouling). High-pressure techniques such as supercritical solvent impregnation and grafting in the supercritical phase allow for the modification of a solid matrix throughout the whole volume and production of materials of unique properties in an environmentally friendly way. Grafting involves the chemical attachment of an active compound to a solid matrix, ensuring its long-lasting properties. Grafting in supercritical carbon dioxide (scCO2) has been reported as an efficient tool in the production of materials with antibacterial1 , and recently also antibiofilm properties2 . This study aims to investigate the potential of cellulose acetate (CA) grafting with quaternary ammonium compounds (QAC) for acquiring antibiofilm properties. Two QAC were synthesized, and the grafting conditions were investigated. The grafting was performed via hexamethylene diisocyanate (HDI) as a linker1 . Obtained materials were analyzed using FTIR, SEM and Ion microscopy, DSC analyses, contact angle measurements, and standardized microbiological procedures to evaluate antibiofilm properties.

In September 2018, a four-month-old dog with fever and petechial bleeding came to the internal clinic at the Faculty of Veterinary Medicine University of Belgrade. On hematology analysis, thrombocytopenia and mild anemia were observed. Examination of the blood smear revealed platelet inclusions. The commercial serology test was positive for Anaplasma spp. The dog was treated with doxycycline for 14 days, and after 48 hours from the beginning of the treatment, the symptoms subsided. PCR analysis and sequencing confirmed infection with A. platys., U septembru 2018. godine, štene staro četiri meseca, sa znacima groznice i petehijalnim krvarenjem, je došlo u ambulantu Klinike za male životinje Fakulteta veterinarske medicine Univerziteta u Beogradu. Hematološka analiza je pokazala trombocitopeniju i blagu anemiju. Pregledom krvnog razmaza ustanovljene su inkluzije u trombocitima. Komercijalni serološki test je pokazao prisustvo antitela protiv Anaplasma spp. Sprovedena je terapija doksiciklinom u trajanju od 14 dana, a posle 48 sati od početka terapije znaci bolesti su se povukli. PCR analizom i senkvencioniranjem je potvrđeno prisustvo Anaplasma platys.

Simple Summary:& nbsp;West Nile fever is an arthropod-borne viral disease that is transmitted from birds to humans and animals by mosquitoes. Humans may develop a severe disease, which sometimes can be fatal. At the end of the 20th century, the first outbreaks of West Nile fever among humans in urban environments in Eastern Europe and the United States were reported. The epidemics were characterized by the neurological form of the disease with a fatal outcome. Since the first outbreak of the disease in Serbia, the highest number of cases occurred in 2018. West Nile fever spread is driven by location and time, which means nearby locations and periods have similar features. Recognition of patterns of spread of the disease has the potential to facilitate the mosquito control program and disease prevention. This study aimed to examine the geographical and temporal similarities of registered cases during the epidemics in the period 2017-2019 in South Banat District, Serbia. We identified the following factors as crucial for the prediction of possible outbreaks: the presence of virus in natural reservoirs, mosquito abundance; precipitation, high water level of rivers followed by a consequent sudden decrease of precipitation and withdrawal of rivers into the main bed, and favorable temperatures. LT /p> LT br> LT /p> 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 mod

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 β-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.

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).

A novel impregnation process for the fabrication of cotton nanocomposite with strong antimicrobial activity against antibiotics-resistant bacteria and yeast was developed. The impregnation process includes the sol-gel treatment of fabric with (3-aminopropyl)triethoxysilane in the first step, and synthesis of the CuO/Cu2O nanoparticles (NPs) on the fabric surface in the second step. The in situ synthesis of the CuO/Cu2O NPs was based on the adsorption of Cu2+-ions by the introduced amino groups of the sol-gel coating. The adsorbed Cu2+-ions are subsequently reduced in the alkaline solution of NaBH4. X-ray diffraction measurements confirmed the formation of CuO/Cu2O NPs. Scanning electron microscopy and atomic absorption spectrometry analyses indicate that the particle size, agglomeration, and amounts of synthesized NPs were highly affected by the initial concentration of CuSO(4)solution. The toxicity of nanocomposites to human keratinocytes (HaCaT) and antimicrobial activity against Gram-negativeEscherichia coliATCC 25922,E. coliATCC BAA 2469, andKlebsiella pneumoniaeATCC BAA 2146, and Gram-positive bacteriaStaphylococcus aureusATCC 25923,S. aureusATCC 43300 and yeastCandida albicansATCC 24433 strongly depended on the copper content. In addition to excellent antimicrobial activity, controlled release of Cu2+-ions from the fabrics into physiological saline solution was obtained.

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.

A novel impregnation process for the fabrication of cotton nanocomposite with strong antimicrobial activity against antibiotics-resistant bacteria and yeast was developed. The impregnation process includes the sol–gel treatment of fabric with (3-aminopropyl)triethoxysilane in the first step, and synthesis of the CuO/Cu2O nanoparticles (NPs) on the fabric surface in the second step. The in situ synthesis of the CuO/Cu2O NPs was based on the adsorption of Cu2+-ions by the introduced amino groups of the sol–gel coating. The adsorbed Cu2+-ions are subsequently reduced in the alkaline solution of NaBH4. X-ray diffraction measurements confirmed the formation of CuO/Cu2O NPs. Scanning electron microscopy and atomic absorption spectrometry analyses indicate that the particle size, agglomeration, and amounts of synthesized NPs were highly affected by the initial concentration of CuSO4 solution. The toxicity of nanocomposites to human keratinocytes (HaCaT) and antimicrobial activity against Gram-negative Escherichia coli ATCC 25922, E. coli ATCC BAA 2469, and Klebsiella pneumoniae ATCC BAA 2146, and Gram-positive bacteria Staphylococcus aureus ATCC 25923, S. aureus ATCC 43300 and yeast Candida albicans ATCC 24433 strongly depended on the copper content. In addition to excellent antimicrobial activity, controlled release of Cu2+-ions from the fabrics into physiological saline solution was obtained.

Nosema ceranae je микроспоридијa којa паразитира код Европске медоносне пчеле, Apis mellifera, и доминантна је врста рода Nosema у југоисточној Европи. У зависности од степена инфекције и периода године, овај ендопаразит може имати далекосежне последице по пчеле, њихово здравље и продуктивност. Последњих година посебно је истраживан утицај N. ceranae на имунитет пчела а закључци појединих истраживања издвајају ефекат супресије гена значајних за имунитет пчела, односно доказују имуносупресивне последице инфекције врстом N. ceranae. Терапија ноземозе подразумева употребу антибиотика фумагилина, који има доказану ефикасност у сузбијању инфекције али његови споредни ефекти могу представљати проблем за саме пчеле и квалитет пчелињих производа, те је стога је његова примена у многим земљама ограничена. Имајући ово на уму константно се трага за природним и синтетским компонентама које би биле замена за фумагилин. Тестиране су многе супстанце, а међу онима које су показале најбољи ефекат издвајају се екстракти биљака и алги и синтетске витаминско-минералне мешавине. У овом раду је у кавезном експерименту тестирано пет препарата: (1) антибиотик фумагилин, (2) биљни екстракт тимол; и три адитива у исхрани пчела: (3) комерцијални препарат Beewell AminoPlus који представља витаминско-аминокиселинску мешавину, (4) комерцијални препарат Medenko forte који садржи екстракте храстове коре, пелена и жалфије и (5) полисахаридни екстракт гљиве Agaricus blazei. Пчеле су у свим третираним групама трећег дана живота инфициране спорама N. ceranae, а различите групе су третиране препаратима од првог, трећег и шестог дана. Код третираних пчела је праћен ефекат на преживљавање пчела, степен Nosema инфекције, нивое експресије гена значајних за имунитет пчела (абецин, дефензин, хименоптецин, апидецин и вителогенин) и ниво оксидативног стреса праћен кроз активност антиоксидативних ензима (каталазе, супероксид дизмутазе и глутатион С-трансферазе) и концентрације малондиалдехида..., Apis mellifera, and the dominant Nosema species in Southeast Europe. Depending on the infection level it could have significant consequences on bees, their health and productivity. The impact of N. ceranae on honey bees’ immunity has been particularly investigated in recent years. Some conclusions of this research underline N. ceranae-induced suppression of immune-related genes, proving its immunosuppressive impact. Treatment for nosemosis includes the use of the antibiotic fumagillin. Fumagillin has proven effect in Nosema control, but its side effects may pose a problem for the bees and the quality of their products, which is why its application in many countries have been limited. Hence, researchers are constantly looking for natural and synthetic components which could be suitable substitutes for fumagillin. Many products have been tested and plant/algae extracts and synthetic vitamin-mineral complexes showed the best effects. This laboratory (cage) experiment included testing of five products: (1) antibiotic fumagillin (2) a plant extract - thymol, and three dietary supplements: (3) a commercial amino acid and vitamin complex named Beewell AminoPlus, (4) a commercial mixture of oak bark, sage and absinth extracts named Medenko forte, and (5) a polysaccharide extract of Agaricus blazei mushroom. Bees in all treatment groups were infected with N. ceranae spores on day 3, and treated with the listed products from day 1, 3 or 6 after emergence. Bee survival, Nosema loads, levels of immune-related genes (abaecin, apidaecin, hymenoptaecin, defensin and vitellogenin) expression and the level of oxidative stress, assessed through the activity of antioxidative enzymes (catalase, glutathione S-transferase and superoxide dismutase) and the concentration of malondialdehyde, were measured in all experimental groups. The obtained values were compared with those of control (noninfected and infected) groups...

U ovoj doktorskoj disertaciji ispitano je prisustvo uzročnika kju groznice - bakterije Coxiella burnetii (C. burnetii) kod nevlasničkih pasa poreklom sa teritorije grada Beograda, primenom molekularnih i seroloških metoda. Molekularni metod je korišćen za otkrivanje uzročnika u reproduktivnim tkivima nevlasničkih pasa, kao i u krpeljima prisutnim na ispitivanim životinjama, dok je imunoenzimskim testom utvrđeno prisustvo specifičnog serološkog odgovora kod ispitivanih životinja. Za otkrivanje prisustva DNK C. burnetii u uzrocima korišćena je lančana reakcija polimeraze (Trans-PCR) kojom se umnožava IS1111 fragment C. burnetii, dok je za otkrivanje specifičnih antitela protiv C. burnetii u serumima ispitivanih pasa korišćen modifikovani komercijalni imunoenzimski test (ELISA). U istraživanju je sakupljeno 316 krpelja sa 51 nevlasničkog psa i identifikovane su tri vrste krpelja: Rhipicephalus sanguineus, Ixodes ricinus i Dermacentor reticulatus. Prisustvo DNK C. burnetii ustanovljeno je u 10,53% (24/228) krpelja vrste R. sanguineus, koji su uzorkovani sa sedam pasa. Reproduktivna tkiva pasa sakupljena su od ukupno 105 pasa - 74 ženke (70,48%) i 31 mužjaka (29,52%). DNK C. burentii je ustanovljena kod 20,95% (22/105) pasa i to u 16,13% (5/31) uzoraka semenika pasa, odnosno u 22,97% (17/74) uzoraka materica i jajnika. Kod 29,52% (31/105) pasa je 5 ustanovljeno prisustvo specifičnih antitela protiv C. burnetii i to 32,26% (10/31) mužjaka i 28,38% (21/74) ispitanih ženki..., In this doctoral dissertation, the presence of the causative agent of Q fever - the bacterium Coxiella burnetii (C. burnetii) in stray dogs has been examined using molecular and serological methods. Molecular method was used for the detection of the agent in the reproductive tissues of stray dogs, as well as in the ticks recovered from the examined animals, while immunoenzyme test was used for the detection of specific serological response in the examined animals. A polymerase chain reaction (Trans-PCR) targeting IS1111 element of C. burnetii was used for the detection of C. burnetii DNA in the samples, while a modified commercial immunoassay (ELISA) was used for the detection of specific antibodies against C. burnetii in the sera of the examined dogs. In this study, 316 ticks were recovered from 51 stray dogs and three tick species were identified: Rhipicephalus sanguineus, Ixodes ricinus and Dermacentor reticulatus. The presence of C. burnetii DNA was established in 10.53% (228/24) R. sanguineus ticks, which originated from seven dogs. Reproductive tissues of dogs were collected from a total of 105 dogs - 74 females (70.48%) and 31 males (29.52%). The presence of C. burentii DNA has been detected in 20.95% (22/105) dogs. C. burnetii DNA was detected in 7 16.13% (5/31) samples of dog testicles and in 22.97% (17/74) samples of uteri and ovaries...

U ovoj doktorskoj disertaciji je ispitana mogućnost primene simpleks i multipleks PCR protokola sa prethodno opisanim prajmerima za preciznu identifikaciju najčešće izolovanih serovarijeteta Salmonella vrsta na teritoriji Vojvodine. Takođe je ispitana efikasnost primene metode sekvenciranja gena za 16Ѕ rRNK u preciznoj identifikaciji salmonela. Ispitano je 107 izolata Salmonella enterica ssp. enterica identifikovanih klasičnom serotipizacijom kao: Infantis (52), Enteritidis (33), Tenessee (5), Mbandaka (4), Montevideo (3), Havana (2), Lille (2), Senftenberg (2), Typhimurium (1), Agona (1), Derby (1) i Livingstone (1). Korišćena je tripleks PCR reakcija u detekciji bcfC, steB i sdf lokusa, optimizovana tokom preliminarnih ispitivanja. Primenom tripleks PCR, samo S. Enteritidis je mogla precizno da bude razlikovana od drugih serovarijeteta, dok su ostali serovarijeteti na osnovu rezultata tripleks PCR svrstavani u dve grupe (Grupa 1 i Grupa2). S. Infantis, najzastupljeniji serovarijetet među ispitanim izolatima nije mogao biti precizno identifikovan jer se primenom tripleks PCR nije razlikovao od ostalih članova Grupe 2. Za njegovu konačnu identifikaciju je primenjen simpleks PCR za umnožavanje segmenta fljB koji je karakterističan samo za S. Infantis. Sekvenciranje gena za 16Ѕ rRNK je primenjeno u identifikaciji izolata devet različitih serovarijeteta. Poredjenjem rezultata dobijenih u klasičnoj serotipizaciji i u PCR, uočeno je slaganje u 91 slučaju od ukupno 107 (85%). Od 33 izolata S. Enteritidis njih 31 (94%) je dao očekivan PCR profil. U slučaju S. Infantis PCR je potvrdila identifikaciju u samo 75% (39 od 52) slučajeva. Ostali ispitani serovarijeteti dali su očekivane PCR profile. Sekvenciranje gena za 16Ѕ rRNK je bilo pouzdano samo u određivanju pripadnosti ispitanih izolata rodu Salmonella., Aim of this doctoral thesis was to test the applicability of published primers for the identification of serovars isolated most frequently in the north part of our country using multiplex and simplex PCR reactions. The efficacy of sequencing of gene for 16S ribosomal RNA for identification of Salmonella strains was also checked. The study was conducted on 107 serotyped Salmonella enterica ssp. enterica isolates. Strains belonged to the following serovars: Infantis (52), Enteritidis (33), Tenessee (5), Mbandaka (4), Montevideo (3), Havana (2), Lille (2), Senftenberg (2), Typhimurium (1), Agona (1), Derby (1) i Livingstone (1). A triplex PCR protocol (bcfC, steB, sdf) optimized during preliminary investigations was used. This reaction separated S. Enteritidis from other strains, while the remaining serovars divided in two distinct groups (Group 1 and Group 2). As the used PCR protocol could not differentiate S. Infantis, the most numerous serovar among isolates, from other members of the Group 2, its identification was completed using a simplex PCR reaction targeting fljB specific for S. Infantis. Comparison of the results of serotyping and PCR protocols revealed a concordance in 91 cases from the total of 107 (85%). From 33 strains serotyped as S. Enteritidis 31 (94%) had the predicted PCR profile. Regarding S. Infantis, identification based on genomic markers confirmed this serovar only in 75% of cases (39 from 52). Other serovars showed their typical PCR profiles. Sequencing of gene for 16S ribosomal RNA could reliably determine only that tested isolates are the members of the genus Salmonella

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-mecRI-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.

Two new nitrato complexes of zinc and nickel with 1,4,8,11-tetrakis(2-pyridylmethyl)-1,4,8,11-tetraazacyclotetradecane (tpmc), have been synthesized and characterized. The IR spectral peaks showed that the coordinated and ionic nitrate ions are in agreement with the formula proposed by elemental analysis. Conductometric titrations predicted methanol to be a convenient solvent for synthesis and revealed the stoichiometry of the complexes, while molar electrical conductivities indicated a 1 : 3 complex electrolyte type for the zinc complex, and a 1 : 2 complex electrolyte type for the nickel complex. The optimized complex structure was obtained by molecular modeling and density functional theory calculations. The biological activity of the novel complexes was examined by screening eight different bacterial strains and two cancer cell lines. The zinc complex showed better antimicrobial activity against the bacterial strains, while the complexes did not show significance antiproliferative activity toward cancer cells MCF-7 and MDA-MB-231.