Slaviša Stanković

Antimicrobial Activity of Serbian Propolis Evaluated by Means of MIC, HPTLC, Bioautography and Chemometrics

New information has come to light about the biological activity of propolis and the quality of natural products which requires a rapid and reliable assessment method such as High Performance Thin-Layer Chromatography (HPTLC) fingerprinting. This study investigates chromatographic and chemometric approaches for determining the antimicrobial activity of propolis of Serbian origin against various bacterial species. A linear multivariate calibration technique, using Partial Least Squares, was used to extract the relevant information from the chromatographic fingerprints, i.e. to indicate peaks which represent phenolic compounds that are potentially responsible for the antimicrobial capacity of the samples. In addition, direct bioautography was performed to localize the antibacterial activity on chromatograms. The biological activity of the propolis samples against various bacterial species was determined by a minimum inhibitory concentration assay, confirming their affiliation with the European poplar type of propolis and revealing the existence of two types (blue and orange) according to botanical origin. The strongest antibacterial activity was exhibited by sample 26 against Staphylococcus aureus, with a MIC value of 0.5 mg/mL, and Listeria monocytogenes, with a MIC as low as 0.1 mg/mL, which was also the lowest effective concentration observed in our study. Generally, the orange type of propolis shows higher antimicrobial activity compared to the blue type. PLS modelling was performed on the HPTLC data set and the resulting models might qualitatively indicate compounds that play an important role in the activity exhibited by the propolis samples. The most relevant peaks influencing the antimicrobial activity of propolis against all bacterial strains were phenolic compounds at RF values of 0.37, 0.40, 0.45, 0.51, 0.60 and 0.70. The knowledge gained through this study could be important for attributing the antimicrobial activity of propolis to specific chemical compounds, as well as the verification of HPTLC fingerprinting as a reliable method for the identification of compounds that are potentially responsible for antimicrobial activity. This is the first report on the activity of Serbian propolis as determined by several combined methods, including the modelling of antimicrobial activity by HPTLC fingerprinting.

Molecular characterization of Pseudomonas syringae isolates from fruit trees and raspberry in Serbia

Infection of fruit trees by Pseudomonas syringae is a potentially serious problem that may limit the establishment and sustained productivity of pome and stone fruit orchards in Serbia. To estimate possible diversity of Pseudomonas syringae fruit trees strains, we collected a set of strains in several areas of Serbia. The samples were taken from infected orchards with raspberry, plum, cherry, sour cherry, peach, pear and apple trees. Genetic diversity of P. syringae strains isolated from fruit trees was determined by using SpeI macrorestriction analysis of genomic DNAs by pulsed-field gel electrophoresis (PFGE) and REP-PCR. Molecular analysis showed that most of isolates had unique profiles, with the exception of isolates from plum and cherry that displayed profiles identical to each other and similar to P. syringae pv. morsprunorum. The study presented here clearly demonstrates the discriminative power of molecular techniques in enabling a detailed analysis of the genetic variations between strains of P. syringae from different pome and stone fruit hosts in Serbia.

Licheniocin 50.2 and Bacteriocins from Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 Inhibit Biofilms of Coagulase Negative Staphylococci and Listeria monocytogenes Clinical Isolates.

Background Coagulase negative staphylococci (CoNS) and Listeria monocytogenes have important roles in pathogenesis of various genital tract infections and fatal foetomaternal infections, respectively. The aim of our study was to investigate the inhibitory effects of two novel bacteriocins on biofilms of CoNS and L. monocytogenes genital isolates. Methods The effects of licheniocin 50.2 from Bacillus licheniformis VPS50.2 and crude extract of bacteriocins produced by Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 (BGBU1-4 crude extract) were evaluated on biofilm formation and formed biofilms of eight CoNS (four S. epidermidis, two S. hominis, one S. lugdunensis and one S. haemolyticus) and 12 L. monocytogenes genital isolates. Results Licheniocin 50.2 and BGBU1-4 crude extract inhibited the growth of both CoNS and L. monocytogenes isolates, with MIC values in the range between 200–400 AU/ml for licheniocin 50.2 and 400–3200 AU/ml for BGBU1-4 crude extract. Subinhibitory concentrations (1/2 × and 1/4 × MIC) of licheniocin 50.2 inhibited biofilm formation by all CoNS isolates (p < 0.05, respectively), while BGBU1-4 crude extract inhibited biofilm formation by all L. monocytogenes isolates (p < 0.01 and p < 0.05, respectively). Both bacteriocins in concentrations of 100 AU/mL and 200 AU/mL reduced the amount of 24 h old CoNS and L. monocytogenes biofilms (p < 0.05, p < 0.01, p < 0.001). Conclusions This study suggests that novel bacteriocins have potential to be used for genital application, to prevent biofilm formation and/or to eradicate formed biofilms, and consequently reduce genital and neonatal infections by CoNS and L. monocytogenes.

Novel antilisterial bacteriocin licheniocin 50.2 from Bacillus licheniformis VPS50.2 isolated from soil sample.

To isolate and characterize bacteriocin, licheniocin 50.2, from soil bacteria identified as Bacillus licheniformis.

The Profile and Antimicrobial Activity of Bacillus Lipopeptide Extracts of Five Potential Biocontrol Strains

In this study the efficacy of two different methods for extracting lipopeptides produced by five Bacillus strains-ethyl acetate extraction, and acid precipitation followed by methanol extraction—was investigated using mass spectrometry. High performance thin layer chromatography (HPTLC) was also used for the simultaneous separation of complex mixtures of lipopeptide extracts and for the determination of antimicrobial activity of their components. The mass spectra clearly showed well-resolved groups of peaks corresponding to different lipopeptide families (kurstakins, iturins, surfactins, and fengycins). The ethyl acetate extracts produced the most favorable results. The extracts of SS-12.6, SS-13.1, and SS-38.4 showed the highest inhibition zones. An iturin analog is responsible for the inhibition of Xanthomonas arboricola and Pseudomonas syringae phytopathogenic strains. HPTLC bioautography effectively identified the active compounds from a mixture of lipopeptide extracts, proving in situ its potential for use in direct detection and determination of antimicrobials. In the test of potential synergism among individual extracts used in different mixtures, stronger antimicrobial effects were not observed. Biochemical and phylogenetic analysis clustered isolates SS-12.6, SS-13.1, SS-27.2, and SS-38.4 together with Bacillus amyloliquefaciens, while SS-10.7 was more closely related to Bacillus pumilus.

Biogenesis of secondary mycogenic minerals related to wall paintings deterioration process

Present study addresses potential of fungal strains, isolated from deteriorated mural paintings and surrounding air environment of the Church of the Holy Ascension in Veliki Krčimir (Serbia), to precipitate mycogenic minerals, when cultivated on agarized B4 medium. Utilizing culture-based isolation methods, 38 filamentous fungi were obtained in total, 23 from mural paintings and 15 from air, respectively, mainly ascomycetes, while Bjerkandera adusta and Thanatephorus cucumeris were only basidiomycetes. A total of 31 of 38 fungal isolates, more than 80%, were able to form minerals of different morphologies and variable size, determined via SEM-EDS and XRPD, to be either calcite or calcite and weddellite association. Among screened fungi, all Penicillium, Chaetomium and Cladosporium isolates, as well as most of the Aspergillus isolates (8/11) precipitated minerals, whereas cultures of Bionectria, Bjerkandera, and Seimatosporium isolates lacked any observable crystal forms. With the exception of two Alternaria alternata strains, no apparent disparity in potential to precipitate minerals in general, or form particular crystal phase was documented among the air and mural paintings isolates. Possible mechanisms of fungal mineralization of calcite and weddellite are further proposed. In addition to providing experimental evidence for fungal induced precipitation of oxalate and carbonate minerals, presented data suggest that fungal activity could be an important factor in a weathering process affecting cultural heritage exhibited and stored in inadequate conditions. Implementation of B4 plate assay for screening of mineralization potential of the isolated fungi could be used to assess biodegradative risk mycobiota pose to the mural paintings, so appropriate conservation measures may be utilized.

Chemical Defence in a Millipede: Evaluation and Characterization of Antimicrobial Activity of the Defensive Secretion from Pachyiulus hungaricus (Karsch, 1881) (Diplopoda, Julida, Julidae)

The chemical defence of the millipede Pachyiulus hungaricus is reported in the present paper, in which a chemical characterization is given and antimicrobial activity is determined. In total, independently of sex, 44 compounds were identified. All compounds belong to two groups: quinones and pentyl and hexyl esters of long-chain fatty acids. The relative abundances of quinones and non-quinones were 94.7% vs. 5.3% (males) and 87.3% vs. 12.7% (females), respectively. The two dominant quinones in both sexes were 2-methyl-1,4,-benzoquinone and 2-methoxy-3-methyl-1,4-benzoquinone. Antibacterial and antifungal activity of the defensive secretion was evaluated in vitro against seven bacterial strains and eight fungal species. With the aid of a dilution technique, the antimicrobial potential of the secretion and high sensitivity of all tested strains were confirmed. The lowest minimum concentrations of these compounds (0.20–0.25 mg/mL) were sufficient for inhibition of Aeromonas hydrophila, Listeria monocytogenes and Methicillin resistant Staphylococcus aureus (MRSA). The growth of eight tested fungal species was inhibited by slightly lower concentrations of the secretion, with Fusarium equiseti as the most sensitive fungus and Aspergillus flavus as the most resistant. Values of MIC and MFC in the employed microdilution assay ranged from 0.10 to above 0.35 mg/mL. The given extract contains antimicrobial components potentially useful as therapeutic agents in the pharmaceutical and agricultural industries.

Biodegradative potential of fungal isolates from sacral ambient: In vitro study as risk assessment implication for the conservation of wall paintings

The principal purpose of the study was to evaluate in vitro the potential ability of fungal isolates obtained from the painted layer of frescoes and surrounding air to induce symptoms of fresco deterioration, associated with their growth and metabolism, so that the risk of such deterioration can be precisely assessed and appropriate conservation treatments formulated. Biodegradative properties of the tested microfungi were qualitatively characterized through the use of a set of special agar plates: CaCO3 glucose agar (calcite dissolution), casein nutrient agar (casein hydrolysis), Czapek-Dox minimal medium (pigment secretion); and Czapek-Dox minimal broth (acid and alkali production). Most of the tested isolates (71.05%) demonstrated at least one of the degradative properties, with Penicillium bilaiae as the most potent, since it tested positive in all four. The remaining isolates (28.95%) showed no deterioration capabilities and were hence considered unlikely to partake in the complex process of fungal deterioration of murals via the tested mechanisms. The obtained results clearly indicate that utilization of fast and simple plate assays can provide insight into the biodegradative potential of deteriogenic fungi and allow for their separation from allochthonous transients, a prerequisite for precise assessment of the amount of risk posed by a thriving mycobiota to mural paintings.

Molecular assessment of genetic diversity of Xanthomonas arboricola pv. juglandis strains from Serbia by various DNA fingerprinting techniques

The purpose of the present study was to investigate the genetic diversity of X. arboricola pv. juglandis strains in Serbia. This bacterium is the causal agent of walnut blight and is also associated with apical necrosis of immature walnut fruits. Although walnut blight is long known and widespread in Serbia, a systematic strain diversity study of Xanthomonas arboricola pv. juglandis strains from different regions in Serbia has not been performed. The objectives of this work were to examine the molecular diversity and its possible biological significance of 59 isolates of X. arboricola pv. juglandis collected from different geographic locations in Serbia. Genomic variability was assessed by using repetitive PCR, SpeI macrorestriction analysis of genomic DNAs by pulsed-field gel electrophoresis (PFGE) and partial sequencing of the gyrB gene. Molecular analyses showed substantial genetic diversity among strains and existence of diverse populations of X. arboricola pv. juglandis in Serbia.

Further insight into the bioactivity of the freshwater sponge Ochridaspongia rotunda

Abstract Context: Bioprospection has become a dynamic scientific field that explores novel possibilities for the implementation of natural products in medicine and pharmacy. Compared to marine species from all kingdoms, freshwater species have been highly neglected. Objective: This work focuses on the screening of acetylcholinesterase inhibitory (AChE) and mutagenic activities of the acetone extract (obtained by maceration) of the freshwater sponge Ochridaspongia rotunda Arndt (Malawispongiidae) in vitro. Materials and methods: AChE inhibitory activity was evaluated both in liquid (five different concentrations of the extract, from 1 to 100 μg/mL) and in solid (seven different concentrations of the extract, from 0.5 to 10.0 μg) by methods well described in literature, while mutagenicity was estimated using the Ames test (four different concentrations of the extract, from 0.106 to 1.328 mg/plate). Results: Ochridaspongia rotunda acetone extract exhibited promising AChE inhibitory activity in a dose-dependent manner both in liquid (IC50 23.07 μg/mL) and in solid (1.50 μg). Furthermore, the Ames test revealed no sign of mutagenicity at any concentration tested. Its FTIR spectrum coupled with the positive Liebermann?Burchard, Salkowski and Zak color reactions (tests) indicated the presence of sterol compounds. Discussion and conclusion: The screened extract may inspire a search for novel anticholinesterase therapeutic agent(s) potentially used in the treatment of Alzheimers disease. Further research will be directed toward its detailed chemical analysis along with addressing the issue of a real producer of the natural product(s) responsible for the AChE activity observed.