Lukáš Hleba

Antibacterial activity against Clostridium genus and antiradical activity of the essential oils from different origin.

In the present study, the antimicrobial and antiradical activities of 15 essential oils were investigated. The antimicrobial activities were determined by using agar disc diffusion and broth microdilution methods against Clostridium genus and antioxidant properties of essential oils by testing their scavenging effect on DPPH radicals activities. We determined the antibacterial activity of Clostridium butyricum, Clostridium hystoliticum, Clostridium intestinale, Clostridium perfringens and Clostridium ramosum. We obtained the original commercial essential oils samples of Lavandula angustifolia, Carum carvi, Pinus montana, Mentha piperita, Foeniculum vulgare Mill., Pinus sylvestris, Satureia montana, Origanum vulgare L. (2 samples), Pimpinella anisum, Rosmarinus officinalis L., Salvia officinalis L., Abies alba Mill., Chamomilla recutita L. Rausch and Thymus vulgaris L. produced in Slovakia (Calendula a.s., Nova Lubovna, Slovakia). The results of the disk diffusion method showed very high essential oils activity against all tested strains of microorganisms. The best antimicrobial activity against C. butyricum was found at Pimpinella anisum, against C. hystoliticum was found at Pinus sylvestris, against C. intestinale was found at Satureia hortensis L., against C. perfringens was found at Origanum vulgare L. and against C. ramosum was found at Pinus sylvestris. The results of broth microdilution assay showed that none of the essential oils was active against C. hystoliticum. The best antimicrobial activity against C. butyricum was found at Abies alba Mill., against C. intestinale was found at Abies alba Mill., against C. perfringens was found at Satureia montana and against C. ramosum was found at Abius alba and Carum carvi. Antioxidant DPPH radical scavenging activity was determined at several solutions of oil samples (50 μL.mL−1–0.39 μL.mL−1) and the best scavenging effect for the highest concentration (50 μL.mL−1) was observed. The antioxidant properties were different in particular plant species. The highest% of inhibition after 30 min. of reaction was observed at Origanum vulgare (93%), Satureia montana (90.66%) and Lavandula augustifolia (90.22%).

Detection of Listeria monocytogenes in ready-to-eat food by Step One real-time polymerase chain reaction

The aim of this study was to follow contamination of ready-to-eat food with Listeria monocytogenes by using the Step One real time polymerase chain reaction (PCR). We used the PrepSEQ Rapid Spin Sample Preparation Kit for isolation of DNA and MicroSEQ® Listeria monocytogenes Detection Kit for the real-time PCR performance. In 30 samples of ready-to-eat milk and meat products without incubation we detected strains of Listeria monocytogenes in five samples (swabs). Internal positive control (IPC) was positive in all samples. Our results indicated that the real-time PCR assay developed in this study could sensitively detect Listeria monocytogenes in ready-to-eat food without incubation.

In vitro and In vivo antimicrobial activity of propolis on the microbiota from gastrointestinal tract of chickens

The aim of this study was to examine the effect of propolis extracts on the microbial colonization of chicken gastrointestinal tract in vivo. The propolis was administered to both feed mixtures in various amounts except of the control group. The addition of 150 mg propolis to 1 kg of feed was included in the first experimental group, the addition of 450 mg.kg−1 in the second experimental group, the addition of 600 mg.kg−1 the third experimental group and 800 mg kg−1 in the fourth one. The highest count of faecal enterococci was found in the third group (8.6 cfu.g−1) where 600 mg of propolis to 1 kg was added to the feed mixture. The highest count of lactobacilli was detected in the fourth experimental group (8.83 cfu.g−1) where was 800 mg of propolis added to 1 kg of feed mixture and number of Enterobacteriaceae genera count was found in control group (8.73 cfu.g−1). With RTQ PCR detected species from the genus Enterococcus were: E. avium, E. casseliflavus, E cecorum, E. faecalis, E. faecium, E. gallinarum, E. hirae and E. malodoratus and from genus Lactobacillus were: Lactobacillus crispatus, L. acidophilus and L. salivarius. With MALDI TOF MS Biotyper from Enterobacteriaceae genera were identified Citrobacter braakii, Raoultella ornithinolytica, Serratia fonticola, Escherichia coli and Klebsiella oxytoca. Antimicrobial activities In vitro of six species of bacteria isolated from gastrointestinal tract of chickens were also tested. The best antimicrobial effect of Citrobacter braakii on ethanolic propolis extract in all concentrations were found.

The effects of bee pollen extracts on the broiler chicken's gastrointestinal microflora

The aim of this study was to investigate the effects of bee pollen ethanolic extracts on the in vivo gastrointestinal tract microflora colonization of broiler chickens. A completely randomized experiment based on six treatments (different concentrations of bee pollen - 0, 5, 15, 25, 35 and 45 g kg(-1) diet) was used during 7 weeks. The highest count of faecal Enterococci was found in the experimental group with the addition of 15 g of pollen (8.85 ± 0.87 log CFU g(-1)) per 1 kg of feed mixture. The highest count of Lactobacilli was detected in the experimental group with 35 g of pollen per 1 kg of feed mixture and the highest number of the Enterobacteriaceae genera count was found in the control group (8.43 ± 0.15 log CFU g(-1)). Moreover, the MALDI TOF MS Biotyper identified the following genera: Escherichia coli, Proteus mirabilis, Klebsiella oxytoca, as well as Lactobacillus acidophilus, L. crispatus, L. fermentum and L. salivarius from the Lactobacilli group and Enterococcus avium, E. casseliflavus, E. cecorum, E. faecalis, E. faecium, E. gallinarum, E. hirae and E. malodoratus from the Enterococci group. Additionally, the in vitro antimicrobial activities of pollen against five bacteria species isolated from gastrointestinal tracts of chickens were tested. The best antimicrobial effect of the pollen extract was detected against K. oxytoca.

Determination of wine microbiota using classical method, polymerase chain method and Step One Real-Time PCR during fermentation process

The aim of our study was the identification of grape, must and wine microbiota during the fermentation process using a classical microbiological method and Real-Time PCR. The changes in different groups of microorganisms were monitored in total counts of bacteria, lactobacilli and yeasts. Microbiological parameters were observed during the current collection and processing of grapes in 2009. Samples were taken during the fermentation process in wine enterprises and a private vineyard. During this period 30 samples of wine among Müller Thurgau, Cabernet Sauvignon, Chardonnay, Tramin and Red Bio-wine were examined. Samples were collected from stages of grape-must unfiltered, grape-must filtered, the beginning of fermentation, fermentation, late fermentation and young wine. The highest total counts of bacteria ranged from 0.00 to 176 ± 15 CFU.mL−1 in the wine of Müller Thurgau, the highest number of yeast ranged from 0.00 to 150 ± 9 CFU.mL−1 in the wine of Müller Thurgau and the number of Lactobacillus spp. ranged from 0.00 to 92 ± 5 CFU.mL−1 in the sample of Cabernet Sauvignon wine. The presence and sensitivity of Gram-positive and Gram-negative bacterial species Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus crispatus and Lactobacillus salivarius were detected using Real-Time PCR (RTQ PCR). Susceptibility of Enterococcus faecium varied in different isolates from 1 to 106 CFU.mL−1, the sensitivity of the species Lactobacillus acidophilus in different isolates of the wine samples ranged from 1 to 105 CFU.mL−1. We also monitored representation of species Lactobacillus crispatus, which were captured by RTQ PCR sensitivity and ranged from 1 to 105 CFU.mL−1 . Identification of the species Lactobacillus salivarius in each of isolates by RTQ PCR method showed the presence of these bacteria in the range of 1 to 104 CFU.mL−1.

Phenolic profile and antimicrobial activities to selected microorganisms of some wild medical plant from Slovakia

Abstract Objective To investigate the chemical composition and antimicrobial activity of the methanol extracts of Tussilago farfara (T. farfara), Equisetum arvense, Sambucus nigra (S. nigra) and Aesculus hippocastanum . Methods The antimicrobial activities of the extracts against Enterococcus raffinosus, Escherichia coli, Lactobacillus rhamnosus, Pseudomonas aeruginosa, Serratia rubidaea, Saccharomyces cerevisiae and Staphylococcus epidermis were determined by the microbroth dilution method according to Clinical and Laboratory Standards Institute, while the concentrations of main phenolic acids and flavonoids in the form of trimethylsilyl ethers were analysed using gas chromatography-mass spectrometry. The probit analysis was used for statistical evaluation. Results Of the 4 plant tested, all extracts showed a significant antimicrobial activity against one or more species of examined microorganisms. The most active antimicrobial plant extract was gathered from T. farfara , followed by Aesculus hippocastanum and Equisetum arvense . The extract from S. nigra showed no antimicrobial effects. The flavonoids quercetin and kaempferol, as well as several phenolic acids ( p -hydroxybenzoic acid, gallic acid, ferulic acid and caffeic acid) were identified in all extracts. The highest concentrations of bioactive compounds were detected in the extracts of T. farfara (9 587.6 μg/mg quercetin and 4 875.3 μg/mg caffeic acid) as well as S. nigra (4788.8 μg/mg kaempferol). Conclusions We can state that the methanolic plant extract of T. farfara showed the strongest antimicrobial activity against Saccharomyces cerevisiae as well as other tested microorganisms. At the same time, a good antimicrobial activity was found in the other medical plant extracts as well. No antimicrobial effect of the S. nigra extract was found with respect to the growth of Pseudomonas aeruginosa, Enterococcus raffinosus and Saccharomyces cerevisiae .

Antifungal activity of essential oils against selected terverticillate penicillia

The aim of this study was to screen 15 essential oils of selected plant species, viz. Lavandula angustifolia, Carum carvi, Pinus mungo var. pulmilio, Mentha piperita, Chamomilla recutita L., Pinus sylvestris, Satureia hortensis L., Origanum vulgare L., Pimpinella anisum, Rosmarinus officinalis L., Salvia officinalis L., Abietis albia etheroleum, Chamomilla recutita L. Rausch, Thymus vulgaris L., Origanum vulgare L. for antifungal activity against five Penicillium species: Penicillium brevicompactum, Penicillium citrinum, Penicillium crustosum, Penicillium expansum and Penicillium griseofulvum. The method used for screening included the disc diffusion method. The study points out the wide spectrum of antifungal activity of essential oils against Penicillium fungi. There were five essential oils of the 15 mentioned above which showed a hopeful antifungal activity: Pimpinella anisum, Chamomilla recutita L., Thymus vulgaris, Origanum vulgare L. The most hopeful antifungal activity and killing effect against all tested penicillia was found to be Origanum vulgare L. and Pimpinella anisum. The lowest level of antifungal activity was demonstrated by the oils Pinus mungo var. pulmilio, Salvia officinalis L., Abietis albia etheroleum, Chamomilla recutita L. Rausch, Rosmarinus officinalis.

The StepOne real-time polymerase chain reaction detection of Salmonella sp., Salmonella enterica ser. typhimurium and enteritidis in milk and meat

The aim of this study was to follow contamination of ready to eat milk and meat products with Salmonella spp. by using the StepOne real-time polymerase chain reaction (PCR). Classical microbiological methods for detection of foodborne bacteria involve the use of pre-enrichment and/or specific enrichment, following isolation of bacteria in solid media and the final confirmation by biochemical and/or serological tests. We used the PrepSEQ Rapid Spin Sample Preparation Kit for isolation of DNA and MicroSEQ® Salmonella spp. Detection Kit for pursuance of the real-time PCR (Applied Biosystems). In samples without incubation we detected strain of Salmonella sp. in 5 out of 25 samples (swabs), as well as in the internal positive control (IPC), which was positive in all samples. This StepOne real-time PCR assay is extremely useful for any laboratory equipped by real-time PCR. It is a fast, reproducible, simple, specific and sensitive way to detect nucleic acids, which could be used in clinical diagnostic tests in the future. Our results indicated that real-time PCR assay developed in this study could sensitively detect Salmonella spp. in ready-to-eat food. This could prevent infection caused by Salmonella, and also could benefit food manufacturing companies by extending their products shelf-life as well as saving the cost of warehousing their food products while awaiting pathogen testing results.

Resistance in bacteria and indirect beta-lactamase detection in E. coli isolated from Culex pipiens detected by matrix-assisted laser desorption ionization time of flight mass spectrometry.

ABSTRACT The aim of this study was detections of antibiotic resistance and resistance mechanism in bacteria isolated from mosquitos (Culex pipiens) living near humans. Therefore, antibiotic resistance in bacteria isolated from Culex pipiens was investigated by disk diffusion test and MIC E-test in this study. MALDI-TOF mass spectrometry was used for detection of resistant mechanism. In this study, hydrolytic breakdown products after a few hours of incubation of the bacteria isolated from Culex pipiens were detected. Results show that enzymatic destruction of ampicillin by beta-lactamases is able to be detected by MALDI-TOF mass spectrometry from wild strains of potential pathogens. The MALDI-TOF mass spectrometry is useful method for routine detection of beta-lactamases resistant mechanism, but overnight incubation of pure culture is necessary. The results are important for proper and fast intervention to limit the spread of beta-lactamase-producing wild bacteria and provide information for appropriate initial therapy of the infections caused by these microbes.

Chemical Composition of the Essential oil of Bougainvillea spectabilis from Montenegro

The composition of the essential oil produced from the aerial parts of Bougainvillea spectabilis was analyzed by GC and GC-MS. Among the thirty nine compounds constituting 97.2 % of the essential oil, the main components were characterized as methyl salicylate (21.8 %), terpinolene (8.2 %) and α-(E)-ionone (9.8 %).