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The aim of the study was to examine heavy metal tolerance (Cd 2+ , Zn 2+ , Ni 2+ and Cu 2+ ) of single- and mixed-species biofilms (Rhodotorula mucilaginosa and Escherichia coli) and to determine metal removal efficiency (Cd 2+ , Zn 2+ , Ni 2+ , Cu 2+ , Pb 2+ and Hg 2+ ). Metal tolerance was quantified by crystal violet assay and results were confirmed by fluorescence microscopy. Metal removal efficiency was determined by batch biosorption assay. The tolerance of the mixed-species biofilm was higher than the single-species biofilms. Single- and mixed-species biofilms showed the highest sensitivity in the presence of Cu 2+ (E. coli-MIC 4 mg/ml, R. mucilaginosa-MIC 8 mg/ml, R. mucilaginosa/E. coli-MIC 64 mg/ml), while the highest tolerance was observed in the presence of Zn 2+ (E. coli-MIC 80 mg/ml, R. mucilaginosa-MIC 161 mg/ml, R. mucilaginosa-E. coli-MIC 322 mg/ml). The mixed-species biofilm exhibited better efficiency in removal of all tested metals than single-species biofilms. The highest efficiency in Cd 2+ removal was shown by the E. coli biofilm (94.85%) and R. mucilaginosa biofilm (97.85%), individually. The highest efficiency in Cu 2+ (99.88%), Zn 2+ (99.26%) and Pb 2+ (99.52%) removal was shown by the mixed-species biofilm. Metal removal efficiency was in the range of 81.56%-97.85% for the single- and 94.99%-99.88% for the mixed-species biofilm.

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Abstract: In this paper, we investigated the presence of enterobacteria in cheese from Southeastern Serbia which was produced in a traditional way. The samples from different households were collected in spring. Chemical and physical characteristics of samples (water content, fat content, acidity, pH, and sodium chloride content) were examined. In the analyzed samples of cheese, we found one genus of family Enterobacteriaceae, Escherichia sp. The genus Escherichia sp. includes only one species, Escherichia coli. We also determined biochemical characteristics, proteolytic and lipolytic activities, the ability to form a biofilm, sensitivity to streptomycin, chloramphenicol, and tetracycline. According to the rapid latex agglutination test, two isolates belong to serotype E. coli O157. Practical applications: These results contribute to a better understanding of the presence of enterobacteria in unexplored microbiota of cheese from Sokobanja. E. coli is the only member of enterobacteria that appears in the samples collected in spring. Since E. coli is among the indicators of contamination, it does not demonstrate the ability to change the organoleptic properties of cheese. E. coli does not exhibit lipolytic or proteolytic activity, but it demonstrates sensitivity to antibiotics. The bacteria showed no ability to form biofilm. Further research should focus on finding the sources of contamination, as well as on the interaction of E. coli with other members of the cheese microbiota. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The impact of heavy metals, cadmium (Cd2+), zinc (Zn2+) and nickel (Ni2+) on planktonic cells and biofilm of Rhodotorula mucilaginosa and Saccharomyces boulardii was examined. The metal tolerance testing was performed by MBECTM-HTP assay. The minimum inhibitory concentration (MICp) and minimum lethal concentration (MLCp) were determined as well as the minimum biofilm eradication concentration (MBEC). Biofilm was more tolerant on the presence of heavy metals than the planktonic cells. The planktonic cells of R. mucilaginosa were tolerant to high concentrations of Cd2+, Zn2+ and Ni2+, while the planktonic cells of S. boulardii tolerated Zn2+, exclusively. The R. mucilaginosa biofilm was tolerant to all of the tested metal concentrations and the obtained results were confirmed by fluorescence microscopy. S. boulardii did not show ability of biofilm formation. Metal removal efficiency of the R. mucilaginosa planktonic cells and biofilm were also tested. The R. mucilaginosa biofilm showed higher efficiency in metals removing compared to the planktonic cells. Until now, the heavy metal tolerance and the removal efficiency (Cd2+, Zn2+ and Ni2+) analyzes were performed solely on planktonic cells of Rhodotorula species. In this study, we investigated the metal removal efficiency of R. mucilaginosa planktonic cells and biofilm and compared the obtained results. [ABSTRACT FROM AUTHOR]

This study compares the effect of heavy metals (Hg, Cu, and Pb) on the Rhodotorula mucilaginosa and Saccharomyces boulardii biofilm and planktonic cells. A MBEC-HTP assay was used to test the levels of tolerance to heavy metals. The minimum inhibitory concentration (MICp) and minimum lethal concentration (MLCp) of the R. mucilaginosa and S. boulardii planktonic cells were determined, as well as minimum biofilm eradication concentration (MBEC). Metal removal efficiency was determined by batch biosorption assay. Previous studies had focused on heavy metal tolerance and removal efficiency of planktonic cells from Rhodotorula species only. Hence, our study presents and compares results for metal tolerance and removal efficiency of the R. mucilaginosa planktonic cells and biofilm. Biofilm tolerance was higher than the planktonic cells. The R. mucilaginosa planktonic cells showed the tolerance in the presence of Hg (MICp 0.08 mM), Cu (MICp 6.40 mM), and Pb (MICp 3.51 mM), while the S. boulardii planktonic cells only tolerated Pb (MICp 0.43 mM). The R. mucilaginosa biofilm showed the highest tolerance in the presence of Hg (MBEC >0.31 mM), Cu(MBEC >12.81 mM), Pb (MBEC >7.12 mM), and obtained results were confirmed by fluorescence microscopy. S. boulardii did not show potential in biofilm formation. The R. mucilaginosa biofilm exhibited better efficiency in removal of all tested metals than the planktonic cells. Metal removal efficiency was in the range from 4.79-10.25% for planktonic cells and 91.71-95.39% for biofilm. [ABSTRACT FROM AUTHOR]

The presence, biochemical and physiological characteristics of Lactobacillus and Lactococcus species isolated from traditionally made cheese from Southeastern Serbia (Sokobanja), was investigated. The screening of their antimicrobial potentials, using agar‐well diffusion method, and the ability of biofilm formation, using crystal violet method, was evaluated. The cheese samples, from different households, were obtained during the summer and the autumn. Chemical characteristics of cheese samples were examined. The members of genus Lactobacillus were: Lb. fermentum,Lb. brevis, and Lb. plantarum. The members of genus Lactococcus were: Lc. lactis subsp. lactis and Lc. lactis subsp. lactis biovar. diacetylactis. The antagonism of Lactobacillus and Lactococcus isolates on the growth of some Gram‐negative bacteria, isolated from the same cheese, was examined. The zones of growth inhibition ranged from 10 to 26 mm. The ability of biofilm formation of isolates was strains‐specific. Lactobacillus (KGPMF 28, KGPMF 29, KGPMF 35) and Lactococcus (KGPMF 23 and KGPMF 26) isolates demonstrated the potential for further investigation in natural preservation of food. Practical applications: Lactic acid bacteria, which can be found in traditional dairy products, presents unique and autochthonous microflora responsible for fermentation and quality of dairy products. These bacteria often have a great potential in natural food preservation, but their number and diversity are depending of season, vegetation, and the nutrition of animals. In this paper, the seasonal differences in bacterial community (especially community of lactobacilli and lactococci) between cheese samples, were investigated. In addition, the antagonism against some enterobacteria, which can affect the hygienic safety of cheese, was screened. Also, it was showed that some isolates had the ability of biofilm formation. Further investigation need to include the potential of isolates to be uses like probiotic or starter cultures. [ABSTRACT FROM AUTHOR]

Melilotus albus Medic. and Dorycnium herbaceum Vill. (Fabaceae) acetone, ethyl acetate, and ethanol extracts were investigated for their in vitro antimicrobial, antibiofilm, and anti- oxidant activity with quantification of phenolic compound contents. In general, D. herba- ceum extracts showed better antibacterial and antioxidant activity than M. albus extracts. Bacteria Bacillus subtilis , Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa , and Proteus mirabilis were the most susceptible with the minimum inhibitory concentrations (MICs), determined by microdilution method, between 1.25 e 10 mg/mL. Antifungal activity was lower with the detectable MICs at 10 mg/mL and 20 mg/mL. The plant extracts, using the crystal violet assay, inhibit P. aeruginosa biofilm formation in concentration range from 5 mg/mL to 20 mg/mL whereas the effect on mature bacterial biofilm was lower. The antioxidant activity was evaluated using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging and reducing power model systems. The intensity of DPPH radicals scavenging activity, expressed as half maximal effective concentration (EC50 ) values, was from 84.33 μg/mL to > 1000 μg/mL. The extracts demonstrated reduced power in a concentration- dependent manner, with ethanol extract as the most active. The total phenols, flavonoids, and proanthocyanidins were determined spectrophotometrically while total extractable tannins were obtained by precipitation method. The phenolic compounds showed differences in their total contents depending on solvents polarities and plant species. Although the plants M. albus and D. herbaceum have not yet been fully explored, these results contribute better understanding of their biotic properties and potential application as antimicrobial and antioxidant agents. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Trapa natans L. (water chestnut or water caltrop) is a widespread aquatic plant, which has been cultivated for food and traditional medicine since ancient times. Pharmacological studies showed that water chestnut exhibits the wide range of biological activities, such as antimicrobial, antioxidative, analgesic, anti-inflammatory, as well as antiulcer. Aim of the study Evaluation of anti-virulence potential and toxicity of T. natans methanol (TnM), acetone (TnA) and ethyl acetate (TnEA) leaf extracts. Materials and methods The anti-quorum sensing activity of Tn extracts was addressed by measuring their effects on biofilm formation, swarming motility and pyocyanin and elastase production in Pseudomonas aeruginosa . Specific P. aeruginosa biosensors were used to identify which of the signaling pathways were affected. The lethal and developmental toxicity of extracts were addressed in vivo using the zebrafish ( Danio rerio ) model system. The phenolic composition of T. natans leafs extracts was analyzed by a linear ion trap-OrbiTrap hybrid mass spectrometer (LTQ OrbiTrapMS) and UHPLC system configured with a diode array detector (DAD) hyphenated with the triple quadrupole mass spectrometer. Results Subinhibitory concentrations of Tn leaf extracts (0.2 MIC) inhibited pyocyanin and elastase production up to 50% and 60%, respectively, and reduced swarming zones, comparing to non-treated P. aeruginosa . TnA inhibited biofilm formation by 15%, TnM showed a stimulatory effect on biofilm formation up to 20%, while TnEA showed no effect. The bioactive concentrations of TnM and TnA were not toxic in the zebrafish model system. Twenty-two phenolic compounds were tentatively identified in TnM, where thirteen of them were identified in T. natans for the first time. Tn extracts, as well as their major components, ellagic and ferulic acids, demonstrated the ability to interfere with P. aeruginosa Las and PQS signaling pathways. Conclusions This study demonstrates anti-virulence potential of Tn leaf extracts against medically important pathogen P. aeruginosa and confirms the ethnopharmacological application of this plant against microbial infections. [ABSTRACT FROM AUTHOR]

The spectroscopically predicted structure of the obtained copper(II)-complex with S-propyl derivative of thiosalicylic acid was confirmed by X-ray structural study. The binuclear copper(II)-complex with S-propyl derivative of thiosalicylic acid crystallized in two polymorphic forms with main structural difference in the orientation of phenyl rings relative to corresponding carboxylate groups. The antibacterial activity was tested determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) by using microdilution method. The influence on bacterial biofilm formation was determined by tissue culture plate method. In general, the copper(II)-complexes manifested a selective and moderate activity. The most sensitive bacteria to the effects of Cu(II)-complexes was a clinical isolate of Pseudomonas aeruginosa . For this bacteria MIC and biofilm inhibitory concentration (BIC) values for all tested complexes were in the range or better than the positive control, doxycycline. Also, for the established biofilm of clinical isolate Staphylococcus aureus , BIC values for the copper(II)-complex with S-ethyl derivative of thiosalicylic acid,[Cu 2 (S-et-thiosal) 4 (H 2 O) 2 ] ( C3) and copper(II)-complex with S-butyl derivative of thiosalicylic acid, [Cu 2 (S-bu-thiosal) 4 (H 2 O) 2 ] ( C5) were in range or better than the positive control. All the complexes acted better against Gram-positive bacteria ( Staphylococcus aureus and Staphylococcus aureus ATCC 25923) than Gram-negative bacteria ( Proteus mirabilis ATCC 12453, Pseudomonas aeruginosa , and P. aeruginosa ATCC 27855). The complexes showed weak antioxidative properties tested by two methods (1,1-diphenyl-2-picrylhydrazyl (DPPH) and reducing power assay). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]