Use of MALDI-TOF MS to Discriminate between Aflatoxin B1-Producing and Non-Producing Strains of Aspergillus flavus.

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins. One of the producers of AFB1 is Aspergillus flavus. Therefore, its rapid identification plays a key role in various sectors of the food and feed industry. MALDI-TOF mass spectrometry is one of the fastest and most accurate methods today. Therefore, the aim of this research was to develop the rapid identification of producing and non-producing strains of A. flavus based on the entire mass spectrum. To accomplish the main goal a different confirmatory MALDI-TOF MS and TLC procedures such as direct AFB1 identification by scraping from TLC plates, A. flavus mycelium, nutrient media around A. flavus growth, and finally direct AFB1 identification from infected wheat and barley grains had to be conducted. In this experiment, MALDI-TOF mass spectrometry with various modifications was the main supporting technology. All confirmatory methods confirmed the presence of AFB1 in the samples of aflatoxin-producing strains of A. flavus and vice versa; AFB1 was not detected in the case of non-producing strains. Entire mass spectra (from 2 to 20 kDa) of aflatoxin-producing and non-producing A. flavus strains were collected, statistically analyzed and clustered. An in-depth analysis of the obtained entire mass spectra showed differences between AFB1-producing and non-producing strains of A. flavus. Statistical and cluster analysis divided AFB1-producing and non-producing strains of A. flavus into two monasteries. The results indicate that it is possible to distinguish between AFB1 producers and non-producers by comparing the entire mass spectra using MALDI-TOF MS. Finally, we demonstrated that if there are established local AFB1-producing and non-producing strains of A. flavus, the entire mass spectrum database identification of aflatoxigenic A. flavus strains can be even faster and cheaper, without the need to identify the toxin itself.

Escherichia coli as a carrier of tetracyclines and penicillins resistance in wild pheasant (Phasianus colchicus).

Wild animals like pheasant seem to be a good source of information about human activities. Therefore, the wild pheasants and relative stable appendix microcenosis were selected for antibiotic resistance testing. Penicillin resistance by MALDI-TOF Mass Spectrometry and tetracyclines resistance by genetic methods using specific primers were tested. Differences between tetracycline and penicillin resistance were detected. Results showed high prevalence of resistant Escherichia coli isolated from wild pheasant appendix. E. coli isolated from wild pheasant appendix carried plasmids for penicillins and tetracyclines resistance where they were responsible for enzymatic degradation of penicillin and carried genes for regulating efflux pumps for tetracyclines. Results showed that tetracyclines and penicillins resistance is widespread between wild pheasants with a carrier as Escherichia coli isolated from relative stable microcenosis of appendix.

The effect of vacuum packaging, EDTA, oregano and thyme oils on the microbiological quality of chicken's breast.

The effect of ethylenediaminetetraacetate (EDTA), oregano (Origanum vulgare) and thyme (Thymus vulgaris) oils, on the chicken breast fillets was examined in this study. The chicken breast fillets were stored under vacuum packaging (VP), at 4 ± 0.5 °C for a period of 18 days. There were used the following treatments of chicken breast fillets: Air-packaged (AC, control samples), vacuum-packaged (VPC, control samples), VP with EDTA solution 1.50% w/w (VPEC, control samples), VP with oregano oil 0.20% v/w (VP + O) and VP with thyme oil 0.20% v/w, (VP + T). The quality assessment for vacuum packaging of the product in accordance with the terms above and EDTA treatment, oregano and thyme oil was established by microbiological analyzes. The microbiological properties as the total viable counts on Plate Count Agar, after incubation for 2 days at 37 °C and coliform bacteria on Violet Red Bile Glucose agar incubated at 37 °C for 24 h, lactobacilli on Rogosa and Sharpe agar after incubation 48-78 h at 37 °C in an aerobic atmosphere supplemented with carbon dioxide (5% CO2) and Pseudomonas aeruginosa on Pseudomonas Isolation agar (PIA, Oxoid, UK) after incubation at 48 h at 35 °C were monitored. The using of oregano, thyme oil and EDTA with combination of vacuum packaging has significant effects to reduction of all followed groups of microorganisms compared with control group without vacuum packaging and untreated control group. The natural preservatives can be used as alternatives to chemical additives which could extend the meat and meat products shelf life. The knowledge about them can have an important economic feedback by reducing losses attributed to spoilage and by allowing the products to reach distant and new markets. This study shows how using of natural antimicrobials can extend the shelf-life of the meat product.

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%).

Reperfusion after pulmonary embolism - long-term follow-up, risk factors, clinical impact.

BACKGROUND AND AIM: Thromboembolic disease is the third most common cardiovascular disorder and deep vein thrombosis carries the risk of pulmonary embolism (PE). Questions related to reperfusion after PE remain, especially risk factors. Incomplete reperfusion after PE is closely related to the development of chronic thromboembolic pulmonary hypertension. The aim of this study was to determine the relation between reperfusion after PE in the long term over a period of 24 months, laboratory results and clinical risk factors found during the initial PE event. PATIENTS AND METHODS: 85 consecutive patients with a first episode of acute PE, diagnosed at 4 cardiology clinics, were followed up using clinical evaluation, scintigraphy and echocardiography (6, 12 and 24 months after the PE. 35 patients were in the low risk category (41%), 42 (49%) in the intermediate risk group and 8 (9%) in the high risk category. RESULTS: Perfusion defects persisted in 20 patients (26%) after 6 months, in 19 patients (25%) after 12 months and in 14 patients (19%) after 24 months. The incidence was more frequent in older patients, with more serious (higher risk) PE, increased right ventricular internal diameter during the initial episode, and more significant tricuspid insufficiency in the initial echocardiography. Notably, higher hemoglobin levels were also shown as a significant risk factor. The presence of perfusion defects after 24 months correlated with a concurrent higher pulmonary pressure but not with either patient function or adverse events (recurrence of PE, re-hospitalization or bleeding). In 3 cases (4% of patients), long-term echocardiographic evidence of pulmonary hypertension was detected. CONCLUSION: Even after 24 months from acute PE with adequate anticoagulation treatment, incomplete reperfusion was found in 19% of patients with a corresponding risk of chronic thromboembolic pulmonary disease and hypertension.

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 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.