Marek Selwet

Different antibacterial activity of novel theophylline-based ionic liquids – Growth kinetic and cytotoxicity studies

The aim of this study was to investigate novel theophylline-based ionic liquids and their cytotoxic effects towards model Gram-positive and Gram-negative bacteria (Bacillus cereus and Escherichia coli, respectively). Growth kinetics, respiratory rates and dehydrogenase activities were studied in the presence of ionic liquids at concentrations ranging from 10 to 1000mg/L. Additionally, the influence of ionic liquids on bacterial cells associated with specific interactions based on the structure of cell wall was evaluated. This effect was assessed by viability tests and scanning electron microscope observations. The obtained results confirmed that ionic liquids exhibit different levels of toxicity in relation to Gram-positive and Gram-negative bacteria. Those effects are associated with the chemical structure of the cationic species of the ionic liquids and their critical micelle concentration value. It was established that the presence of an alkyl or allyl group increased the toxicity, whereas the presence of an aryl group in the cation decreased the toxic effect of ILs. Results presented in this study also revealed unexpected effects of self-aggregation of E. coli cells. Overall, it was established that the studied ILs exhibited higher toxicity towards Gram-positive bacteria due to different interactions between the ILs and the cell membranes. These findings may be of importance for the design of ILs with targeted antimicrobial properties.

Impact of Globulins Derived from Genetically Modified and Conventional Soybean on Swine Lymphocyte Proliferation in in vitro Cultures

Impact of Globulins Derived from Genetically Modified and Conventional Soybean on Swine Lymphocyte Proliferation in in vitro Cultures The majority of the global feed market is dominated by the Roundup Ready 40-3-2 transgenic soybean varieties developed and marketed by Monsanto Company, which are characterized by tolerance to glyphosate, the active ingredient of the Roundup herbicide. It should be remembered, however, that soybean is one of the major allergens which may affect animal health. The aim of the study was to compare allergenic properties of globulins derived from genetically modified (GM) soybean imported from the USA and conventional soybean developed in Poland. Analyses were performed by measuring porcine lymphocyte proliferation in in vitro cultures. It turned out that both genetically modified and conventional soybean proteins caused immune response at the level of negative control. A slight increase in relation to the negative control was observed in the case of 7S and 11S fractions derived from the GM meal and 7S fraction isolated from Nawiko meal. Wpływ globulin soi genetycznie modyfikowanej i konwencjonalnej na proliferację limfocytów świńskich w kulturach in vitro Większość globalnego rynku zdominowała soja transgeniczna Roundup Ready 40-3-2, wprowadzona przez firmę Monsanto, cechująca się tolerancją na glifosat, składnik aktywny herbicydu Roundup. Uprawia się również w niewielkich ilościach soję konwencjonalną. Należy jednak pamiętać, że soja należy do jednego z głównych alergenów, który może mieć wpływ na zdrowotność zwierząt. Celem pracy było porównanie właściwości alergennych globulin soi modyfikowanej genetycznie i konwencjonalnej wyhodowanej w Polsce. Analizy dokonano mierząc proliferację limfocytów świńskich w kulturach in vitro. Okazało się, iż soja modyfikowana genetycznie oraz konwencjonalna wywołują odpowiedź immunologiczną na poziomie kontroli negatywnej. Nieznaczny wzrost w stosunku do kontroli negatywnej obserwowano w przypadku frakcji 7S i 11S pochodzących ze śruty GM oraz frakcji 7S izolowanej ze śruty Nawiko.

Interaction of Gram-Positive and Gram-Negative Bacteria with Ceramic Nanomaterials Obtained by Combustion Synthesis – Adsorption and Cytotoxicity Studies

This paper presents the interactions of Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas putida) bacteria with ceramic materials obtained by combustion synthesis. These studies were conducted based on an analysis of the adsorption of bacteria onto aggregates of ceramic materials in an aqueous suspension. The materials used in the studies were of a nanostructured nature and consisted mainly of carbides: silicon carbide (SiC) in the form of nanofibers (NFs) and nanorods (NRs), titanium carbide, and graphite, which can also be formed by combustion synthesis. Micrometric SiC was used as a reference material. Gram-positive bacteria adsorbed more strongly to these materials. It seems that both the point of zero charge value and the texture of the ceramic material affected the bacterial adsorption process. Additionally, the viability of bacteria adsorbed onto aggregates of the materials decreased. Generally, P. putida cells were more sensitive to the nanomaterials than S. aureus cells. The maximum loss of viability was noted in the case of bacteria adsorbed onto NRSiC and NFSiC aggregates.

Oxidative stress in bacteria (Pseudomonas putida) exposed to nanostructures of silicon carbide

Silicon carbide (SiC) nanostructures produced by combustion synthesis can cause oxidative stress in the bacterium Pseudomonas putida. The results of this study showed that SiC nanostructures damaged the cell membrane, which can lead to oxidative stress in living cells and to the loss of cell viability. As a reference, micrometric SiC was also used, which did not exhibit toxicity toward cells. Oxidative stress was studied by analyzing the activity of peroxidases, and the expression of the glucose-6-phosphate dehydrogenase gene (zwf1) using real-time PCR and northern blot techniques. Damage to nucleic acid was studied by isolating and hydrolyzing plasmids with the formamidopyrimidine [fapy]-DNA glycosylase (also known as 8-oxoguanine DNA glycosylase) (Fpg), which is able to detect damaged DNA. The level of viable microbial cells was investigated by propidium iodide and acridine orange staining.

Bacterial Communities from the Arsenic Mine in Złoty Stok, Sudety Mountains, Poland

Investigations of bacterial communities and characterization of mineralogy of the environment in the Złoty Stok As-Au deposit were carried out. PXRD analysis revealed the presence of picropharmacolite as the most common secondary arsenic mineral in the mine. Total DNA was extracted from slime streams or slime biofilms samples to investigate the bacterial communities. PCR amplification of 16S rDNA was performed followed by subcloning of its products. Over 170 clones were analyzed by means of RFLP method. Eight group of clones representing different restriction patterns were identified. The nucleotide sequences of their inserts suggest that bacteria present in the mine environment belong to: Flavobacteria, Sphingobacteriia, Bacteroides, Proteobacteria, Mollicutes and Firmicutes. The metagenomic approach allows to demonstrate a higher diversity of microbiota than classical microbiological studies of cultivable isolates.

IMPACT OF PROBIOTIC ON THE NUMBER OF LACTIC ACID RODS FORMING HYDROGEN PEROXIDE ISOLATED FROM PORKERS AND ON CHANGES IN DRUG RESISTANCE OF SELECTED ESCHERICHIA COLI ISOLATES

Abstract The presented investigations were conducted on a group of 60 porkers of crossbreed Polish Landrace x Large White Polish. The animals were divided into two equal experimental groups. The control group (K) was fed diets without supplementation with probiotics, group (P) - diets with the addition of probiotic (0.2 kg t-1 feed). The aim of the study was to determine the effect of probiotic preparation on total numberof lactic acid rods from the Lactobacillus genus and those forming hydrogen oxide. The second part of experiment concerned the influence of probiotic preparation on the number, haemolytic ability and changes in drug resistance of Escherichia coli isolated from animal faeces. The significantly highest number of Lactobacillus sp. were determined in the saliva of porkers fed diets with the addition of probiotic, while the lowest in the control group. Lactobacillus sp. rods capable of forming hydrogen peroxide were isolated from 17 animals in group K and from three animals in group P. E. coli was determined in each examined sample of faeces. In groups K and P, counts of these bacteria were similar and did not differ statistically. High numbers of haemolytic isolates (haemolysis β) were found in faeces of animals fed diets with the addition of probiotic. Number and proportions of resistant isolates in groups K and P were different. Gentamicin was characterised by exceptionally high in vitro effectiveness. The used probiotic increased drug resistance of E. coli and increased frequency of incidence of haemolysis β.

Lunasin – a bioactive peptide from triticale(X Triticosecale Wittmack ) seeds, inhibits proliferationof cancer HeLa and SK-OV-3 cells

The presented study demonstrates the influence of lunasin, a bioactive peptide isolated from seeds of winter triticale, on the proliferation of neoplastic cells. In order to conduct investigations, 2S fraction low-molecular-weight albumin proteins were extracted. SDS-PAGE electrophoresis separation of extracts indicated the presence of a 5 kDa peptide in the crude extract, which corresponded to the weight of lunasin. The next stage of protein purification involved ion-exchange column chromatography in an FPLC system on DEAE Sepharose Fast Flow. The separation of biomolecules by means of 2D electrophoresis confirmed that a peptide with an isoelectric point was present in the area, with a pH of about 5.5 and molecular mass of about 5 kDa. The presence of lunasin in the purified samples was confirmed by Western blot. The lunasin peptide affected the proliferation of cervical cancer (HeLa) and ovarian cancer (SK-OV-3) cell lines. The average decrease in the HeLa cell proliferation rate was 17%. The SK-OV-3 cell line was more susceptible to inhibition by the protein fraction containing lunasin than HeLa cells, with an average decrease in the proliferation rate of 48%.

Monitoring of Virulence Genes, Drug-Resistance in Campylobacter coli Isolated from Golden Retrievers

The investigation was performed on 75 of Golden Retriever puppies. Faecal samples were collected on the 42 day of the puppies life (con-trol). Probiotic preparation was administered on 43 day of the puppies life and 10 days after the application of the probiotic, faecal samples were collected again (on 53 day of puppies life). All isolates of Campylobacter coli isolated prior to the administration of the probiotic were found to contain the cadF gene responsible for adhesion, as well as, the flaA gene influencing motility of the examined bacteria. Significant differences (P < 0.05) were recorded only in the case of enrofloxacin.

Synthesis of SiC/Ag/Cellulose Nanocomposite and Its Antibacterial Activity by Reactive Oxygen Species Generation

We describe the synthesis of nanocomposites, based on nanofibers of silicon carbide, silver nanoparticles, and cellulose. Silver nanoparticle synthesis was achieved with chemical reduction using hydrazine by adding two different surfactants to obtain a nanocomposite with silver nanoparticles of different diameters. Determination of antibacterial activity was based on respiration tests. Enzymatic analysis indicates oxidative stress, and viability testing was conducted using an epifluorescence microscope. Strong bactericidal activity of nanocomposites was found against bacteria Escherichia coli and Bacillus cereus, which were used in the study as typical Gram-negative and Gram-positive bacteria, respectively. It is assumed that reactive oxygen species generation was responsible for the observed antibacterial effect of the investigated materials. Due to the properties of silicon carbide nanofiber, the obtained nanocomposite may have potential use in technology related to water and air purification. Cellulose addition prevented silver nanoparticle release and probably enhanced bacterial adsorption onto aggregates of the nanocomposite material.

Impact of the Priobiotic on the Presence of Selected Virulence Genes and Drug-Resistance Among Campylobacter Coli Isolated from Piglets

Abstract The investigations comprised 100 piglets of crossbreed Polish Landrace x Large White Polish breed. Faeces samples were collected on the 2nd d of piglets’ life (control). On the 5th d of life of the piglets, probiotic paste was applied and 7 d later, faecal samples were collected again. The material included 100 isolates of Campylobacter sp. obtained from healthy piglets. All isolates were assigned to the Campylobacter coli species. The occurrence of virulence genes was determined by the PCR method. Drugresistance of the obtained isolates was determined using diffusion tests and E-test strips. All isolates deriving from the control group piglets were found to contain the cadF gene responsible for adhesion, as well as, gene flaA influencing motility of the examined bacteria. In piglets fed diets supplemented with probiotics, the cadF gene occurred in 100% isolates and gene flaA - in 99% isolates. Campylobacter coli isolates obtained from piglets from the control group exhibited the highest resistance with respect to ciprofloxacin and enrofloxacin. The similar results were recorded in the case of isolates obtained after the probiotic application. The majority of the isolates generated α type haemolysis (91%-92%). No significant differences were recorded in the capability of generating haemolysis between isolates obtained before probiotic administration and the isolates obtained after the application of the experimental probiotic.