In vitro procjena antimikrobnog potencijala bakterije Streptococcus uberis izolirane iz autohtonog sira jugoistočne Srbije

Streptococcus uberis is an environmental bacterium responsible for bovine mastitis. It is occasionally described as a human pathogen. In our study, the isolation was undertaken of lactic acid bacteria from a local cheese from Southeastern Serbia, produced in a traditional way. S. uberis (7 isolates) and S. agalactiae (1 isolate) were isolated from the cheese samples taken in the summer. The biochemical and physiological characteristics of the isolates were examined. Using tetracycline, chloramphenicol, novobiocin and rifampicin, the antibiotic susceptibility of the isolates was evaluated. The results demonstrated that all the isolates were susceptible to all the tested antibiotics, with a growth inhibition zone from 36-48 mm. Also, the antagonism was examined of S. uberis KGPMF1-7 and S. agalactiae KFPMF8 isolates on the growth of Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella oxytoca KGPMF1, Klebsiella ornithinolytica KGPMF8 and Aeromonas hydrophila, as indicator stains. The results were compared with the activities of chloramphenicol, streptomycin and tetracycline on the tested indicator stains. The strongest antagonism was demonstrated by all Streptococcus isolates on the growth of K. oxytoca KGPMF1 (growth inhibition zone from 12-20 mm) and the A. hydrophila (growth inhibition zone from 13-20 mm). When these results were compared with the results of the sensitivity of tested indicator stains to antibiotics, S. uberis KGPMF1-7 and S. agalactiae KGPMF8 isolates showed a moderate antagonistic effect. Due to the specific way cheese is made in from Sokobanja, these isolates probably originate from cows’ udders.Streptococcus uberis jest okolišna bakterija odgovorna za mastitis kod goveda, a povremeno se opisuje kao patogen u ljudi. U ovom su istraživanju izolirane bakterije mliječne kiseline iz autohtonog sira jugoistočne Srbije, proizvedenog na tradicionalni način. S. uberis (7 izolata) i S. agalactiae (1 izolat) izolirani su iz uzoraka sira prikupljenih tijekom ljeta. Analizirane su biokemijske i fiziološke značajke izolata. Koristeći se tetraciklinom, kloramfenikolom, novobiocinom i rifampicinom, procijenjena je osjetljivost izolata na antibiotike. Rezultati su pokazali da su svi izolati osjetljivi na sve istražene antibiotike, sa zonom inhibicije rasta od 36 do 48 mm. Također, istražen je antagonizam S. uberis KGPMF1-7 i S. agalactiae KFPMF8 izolata na rast Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella oxytoca KGPMF1, Klebsiella ornithinolytica KGPMF8 i Aeromonas hydrophila kao indikatorskih sojeva. Rezultati su uspoređeni s aktivnostima kloramfenikola, streptomicina i tetraciklina na testiranim indikatorskim sojevima. Najjači antagonizam pokazali su svi Streptococcus izolati na rast K. oxytoca KGPMF1 (zona inhibicije rasta od 12 do 20 mm) i A. hydrophila (zona inhibicije rasta od 13 do 20 mm). Usporedbom tih rezultata s rezultatima osjetljivosti istraženih indikatorskih sojeva na antibiotike, za S. uberis KGPMF1-7 i S. agalactiae KGPMF8 izolate utvrđen je umjeren antagonistički učinak. Ti izolati vjerojatno potječu iz vimena krava i u siru iz Sokobanje prisutni su zbog specifičnosti njegove pripreme

Antagonistički potencijal i sposobnost stvaranja biofilma bakterija Enterococcus spp. izoliranih iz sira u Srbiji

In this paper, the presence, biochemical and physiological characteristics of the members of genus Enterococcus, isolated from traditionally made Serbian cheese, were investigated. The members were identified as: Enterococcus faecium (8 isolates), E. faecalis (14 isolates), E. hirae (4 isolates) and E. durans (4 isolates), using the biochemical tests and MALDI-TOF mass spectrometry. All the tested isolates showed good acidification ability in pure and enriched milk. The antagonism of enterococci on the growth of Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella oxytoca KGPMF1, Klebsiella ornithinolytica KGPMF8 and Aeromonas hydrophila, as well as their ability to form biofilms, were examined. The tested isolates showed moderate inhibitory activity (10-22 mm) on the growth of the indicator strains. Among all the isolates, only E. hirae KGPMF9 and E. faecium KGPMF14 showed the ability of biofilm formation. The results provide a basis for further research into the possible practical application of the isolated enterococci.U ovom je radu istražena prisutnost te biokemijske i fiziološke značajke bakterija iz roda Enterococcus, izoliranih iz domaćeg sira u Srbiji, proizvedenoga na tradicionalan način. Biokemijskim testom i masenom spektrometrijom MALDI-TOF dokazani su Enterococcus faecium (8 izolata), E. faecalis (14 izolata), E. hirae (4 izolata) i E. durans (4 izolata). Svi su analizirani izolati pokazali dobru sposobnost acidifikacije u sirovu i obogaćenom mlijeku. Istraženi su antagonizam enterokoka na rast bakterija Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella oxytoca KGPMF1, Klebsiella ornithinolytica KGPMF8 i Aeromonas hydrophila te njihova sposobnost stvaranja biofilma. Analizirani izolati pokazali su umjerenu inhibicijsku aktivnost (10 - 22 mm) u rastu navedenih sojeva. Među izolatima samo su E. hirae KGPMF9 i E. faecium KGPMF14 pokazali sposobnost stvaranja biofilma. Rezultati ove studije daju temelj za daljnja istraživanja mogućnosti praktične primjene izoliranih enterokoka

The influence of environmental factors on the planktonic growth and biofilm formation of Escherichia coli

In this study, the effects of environmental factors (different media, temperature, pH, salt and sugar concentrations) on the planktonic growth, biofilm formation and formed biofilm of Escherichia coli KGPMF 16 and Escherichia coli KGPMF 17 were investigated. Tested bacteria were isolated from traditionally made cheese produced in Southeastern Serbia (Sokobanja region). The influence on planktonic growth, biofilm formation and formed biofilm was determined using spectrophotometric method. The limiting factors for the planktonic growth and biofilm formation were temperature of 4 °C and all tested concentrations of salt. The growth of tested bacteria was higher in media enriched with lactose than in media containing glucose. TSB was more congenial media to the planktonic growth of bacteria than MHB broth. None of the tested bacteria demonstrated the ability to form biofilm at 4 °C and 44 °C. Only E. coli KGPMF 17 showed ability to form biofilm in TSB at 37 °C. Different concentrations of salt, glucose and lactose exhibited inhibitory effect on biofilm formation, but all tested concentrations of lactose showed stimulating effect on formed biofilm of E. coli KGPMF 17. These results contribute to better understanding of the effects of environmental factors on the development of E. coli in cheese

Probiotički potencijal i procjena sigurnosnog aspekta sojeva Enterococcus sp. izoliranih iz tradicionalno rađenog sira u Srbiji

The purpose of this research paper was the evaluation of the safety aspects and probiotic potential of six isolates from the genus Enterococcus isolated from traditionally made cheese. The isolates represented part of the unexplored microflora of the cheese. The tolerance of Enterococcus isolates to different gastrointestinal conditions (low pH, the presence of pepsin, pancreatin and bile salts) were investigated. Using the microdilution method, sensitivity to clinically relevant antimicrobial agents (tetracycline, ampicillin, gentamicin, vancomycin and polymyxin B) was evaluated. The adhesion ability of autochthonous Enterococcus isolates to solvents, as well as the ability of auto- aggregation and co-aggregation between them and Escherichia coli clinical isolates, was investigated. The results indicated that isolates showed tolerance to the simulated gastrointestinal condition in a high percentage. Isolates were sensitive to all the tested antibiotics, especially to ampicillin, with MIC values obtained from 0.19-2.5 μg/mL. The isolates showed the ability of growth in medium with phenol and showed no ability to synthesize histamine and tyramine. The highest percentage of adhesion was detected with chloroform, and the lowest with xylene. The isolates showed moderate auto-aggregation ability, while a different degree of co-aggregation with E. coli was observed. The results indicated that the potential application of investigated Enterococcus isolates is selective and limited.Cilj ovoga rada bio je procjena sigurnosnog aspekta i probiotičkog potencijala šest izolata roda Enterococcus izoliranih iz tradicionalno rađenog sira. Izolati su predstavljali dio neistražene mikroflore sira. Analizirana je tolerancija izolata Enterococcus na različite gastrointestinalne uvjete (nizak pH, prisutnost pepsina, pankreatina i soli žučnih kiselina). Mikrodilucijskom metodom procijenjena je osjetljivost na klinički relevantne antimikrobne agense (tetraciklin, ampicilin, gentamicin, vankomicin i polimiksin B). Istraživana je i adhezivna sposobnost autohtonih izolata Enterococcus na otapala, kao i sposobnost autoagregacije i koagregacije između njih i kliničkih izolata Escherichia coli. Izolati su pokazali toleranciju na simulirane gastrointestinalne uvjete u velikom postotku. Utvrđena je osjetljivost na sve testirane antibiotike, posebno na ampicilin, s MIC vrijednostima od 0,19 do 2,5 μg/mL. Izolati su pokazali sposobnost rasta na hranjivoj podlozi s fenolom, a nisu pokazali sposobnost sintetizacije histamina i tiramina. Otkriveno je da je najveći postotak adhezije s kloroformom, a najmanji sa ksilenom. Izolati su pokazali umjerenu sposobnost autoagregacije, a opažen je i različit stupanj koagregacije s E. coli. Rezultati pokazuju da je potencijalna primjena istraživanih izolata bakterije Enterococcus selektivna i ograničena

Extracts of Agrimonia eupatoria L. as sources of biologically active compounds and evaluation of their antioxidant, antimicrobial, and antibiofilm activities

In this study, we determined the concentration of total phenols, flavonoids, tannins, and proanthocyanidins in the water, diethyl ether, acetone, and ethanol extracts of Agrimonia eupatoria L. We also investigated the antioxidant activity of these extracts using two methods [2,2-diphenyl-1-picrylhydrazyl (DPPH) and reducing power] and their in vitro antimicrobial (antibacterial and antifungal) activity on some selected species of bacteria and fungi. In addition, the effects of the acetone and water extracts on the inhibition of biofilm formation of Proteus mirabilis and Pseudomonas aeruginosa were investigated using the crystal violet method. The concentration of total phenols was measured according to the Folin–Ciocalteu method and the values obtained ranged from 19.61 mgGA/g to 220.31 mgGA/g. The concentration of flavonoids was examined by the aluminum chloride method and the values obtained ranged from 20.58 mgRU/g to 97.06 mgRU/g. The total tannins concentration was measured by the polyvinylpolypyrrolidone method and the values obtained ranged from 3.06 mgGA/g to 207.27 mgGA/g. The concentration of proanthocyanidins was determined by the butanol–HCl method and the values obtained ranged from 4.15 CChE/g to 103.72 CChE/g. Among the various extracts studied, the acetone extract exhibited good antioxidant activity (97.13%, as determined by the DPPH method). The acetone extract was active in the absorbance value range from 2.2665 to 0.2495 (as determined by the reducing power method). The strongest antimicrobial activity was detected on G+ bacteria, especially on probiotic species, and the acetone extract demonstrated the highest activity. Biofilm inhibitory concentration required to reduce biofilm coverage by 50% values for acetone extract was 4315 μg/mL for P. mirabilis and 4469.5 μg/mL for P. aeruginosa. The results provide a basis for further research of this plant species

In vitro interaction between Agrimonia eupatoria L.: Extracts and antibiotic

Synergistic activity between water, acetone, ethanol and diethyl ether extract of Agrimonia eupatoria L. and commonly used antibiotic (ampicillin) were evaluated. Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Pseudomonas aeruginosa were used. Interaction between plant extracts and antibiotic were tested by checkerboard method and expressed as fractional inhibitory concentration (FIC) index showed indifferent, additive and synergistic effects. Synergism was observed against E. coli for every combination of agents. FICI values were ranged from 0.03 to 0.29. Inhibitory concentration (IC50) was evaluated for every combination of tested extracts and antibiotic and the best combinations for every tested bacteria were combination of diethyl ether extract + ampicillin and combination of acetone extract + ampicillin

Effect of water activity on the radial growth of fungi isolated from dry-cured sheep ham, in vitro (Serbia)

In the Western Balkans, traditional dry-cured sheep ham called Pastrma or Stelja is produced. Dry-cured sheep ham from Sjenica is produced in a very complex manner, and a prerequisite for its production is the sanitary safety of raw materials in accordance with veterinary and sanitary regulations. Isolation and preliminary identification of fungi from sheep ham were carried out in this study, as well as in vitro testing of the effects of water activity (aw) on the growth of isolated fungi. Fungi were isolated from 9 samples of dry-cured sheep meat taken from three households in the area of Sjenica in two productive years (2015 and 2016). Species of genus Penicillium were isolated as dominant in all the investigated samples. Water activity was tested on MY50GF agar from the series of malt extract yeast extract glucose fructose agar. Water activity was set to values of 0.87, 0.89, and 0.97. The results of the research showed that the growth of fungal colonies is under the direct influence of water activity. Fungi grew fastest at water activity of 0.97, and the highest growth of all tested species was recorded after 3 (A. niger), 7 (P. patulum), and 10 days (A. nidulans)

Antimicrobial, antioxidant and DNA-binding studies of palladium(II) complexes with different chelate ligands containing nitrogen donor atoms

The antimicrobial and antioxidant activities, as well as the DNA-binding of four square-planar Pd(II) complexes, [Pd(terpy)Cl]+ (C1), [Pd(en)Cl2] (C2), [Pd(DMEAImiPr)Cl2] (C3) and [Pd(dach)Cl2] (C4) (terpy = 2,2′:6′,2′′- -terpyridine, en = ethylenediamine, dach = trans-1,2-diaminocyclohexane and DMEAImiPr = N2-((1,3-dihydro-1,3-diisopropyl-4,5-dimethyl)-2H-imidazol-2- ylidene)-N1,N1-dimethyl-1,2-ethanediamine are reported. The antimicrobial activities of the Pd(II) complexes with the appropriate ligands were tested using the microdilution method against 18 strains of microorganisms, whereby the minimal inhibitory concentration (MIC) and the minimal microbicidal concentration (MMC) were determined. The antibiofilm activity of [Pd(terpy)Cl]+ and the corresponding ligand were determined on a formed biofilm. The intensity of antimicrobial activity varied depending on the type of microorganism and the tested compound. The C1 complex with the corresponding ligand demonstrated significantly greater overall antimicrobial activity than C2, C3 and C4. The antibacterial activity of the C1 complex was better than its antifungal activity that was overall greater than that of the positive control, fluconazole. The greatest sensitivity for C1 and L1 was with Penicillium italicum (MIC < 0.49 μg mL-1) among the fungi, and with Proteus mirabilis ATCC 12453 (MIC = 0.98 μg mL-1) among the tested bacteria. The tested compounds show low and moderate antibiofilm activity. The complexes showed weak antioxidant properties when tested using the DPPH (1,1-diphenyl-2- -picrylhydrazyl) method. The interaction of the metal complexes C1–C4 with calf thymus DNA (CT-DNA) was further examined by absorption (UV–Vis) and emission spectral studies (EthBr displacement studies). Overall, the investigated complexes exhibited good DNA interaction ability. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 172011 and Grant no. 173032

Spider mites and predatory mites (Acari: Tetranychidae, Phytoseiidae) on stone fruit trees (Prunus spp.) in Serbia

The results of a research conducted from 1980 to 2016 of leaf mites of stone fruit trees (Prunus spp.) in Serbia are presented. The species complex of 9 spider mite species and 16 phytoseiid species was recognized on 10 Prunus species. A total of 17 mite species were found on cherry plum (Prunus cerasifera Ehrh.) and blackthorn (Prunus spinosa L.), 16 species were found on sweet cherry (Prunus avium L.), and 13 species were found on plum (Prunus domestica L.). The highest coefficient of constancy among the spider mites was found for Bryobia rubrioculus (Scheuten) (27.9%), and among the phytoseiids for Euseius finlandicus (Oudemans) (58.7%). Very significant positive values of the association index were found for the pairs: Typhlodromus (Typhlodromus) pyri Scheuten-Bryobia angustisetis Jakobashvili, Neoseiulella aceri (Collyer)-Panonychus ulmi (Koch), N. aceri-Bryobia sp., Phytoseius corniger Wainstein-T.(T.) pyri, N. aceri-P. echinus, P. macropilis -Paraseiulus soleiger (Ribaga). Significant positive values were noted for the the pairs P. corniger-Bryobia sp., P. echinus-Bryobia sp., and K. aberrans-P. echinus. Very significant negative index values were detected for the pairs E. finlandicus-K. aberrans and E. finlandicus-P. macropilis, and a significant negative index of association for the pair A. andersoni-A. viennensis