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.

Carbapenemase Producing Klebsiella pneumoniae (KPC): What Is the Best MALDI-TOF MS Detection Method.

Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria is a group of highly dangerous antibiotic resistant Gram-negative Enterobacteriaceae. They cause infections associated with significant morbidity and mortality. Therefore, the rapid detection of KPC-producing bacteria plays a key role in clinical microbiology. Matrix assisted laser desorption/ionization time-of- flight (MALDI-TOF) is a rapidly evolving technology that finds application in various clinical, scientific, and industrial disciplines. In the present study, we demonstrated three different procedures of carbapenemase-producing K. pneumoniae (KPC) detection. The most basic model of MALDI-TOF instrument MS Microflex LT was used, operating in the linear ion-positive mode, commonly used in modern clinical laboratories. The first procedure was based on indirect monitoring of carbapenemase production with direct detection of hydrolyzed carbapenem antibiotic degradation products in the mass spectrum. The second procedure was based on direct detection of blaKPC accompanying peak with an 11,109 Da in the mass spectrum of carbapenemase-producing K. pneumoniae (KPC), which represents the cleaved protein (pKpQIL_p019) expressed by pKpQIL plasmid. In addition, several unique peaks were detected in the carbapenemase-producing K. pneumoniae (KPC) mass spectrum. The third procedure was the identification of carbapenemase-producing K. pneumoniae (KPC) based on the protein fingerprint using local database created from the whole mass spectra. By comparing detection procedures, we determined that the third procedure was very fast and relatively easy. However, it requires previous verification of carbapenemase-producing K. pneumoniae (KPC) using other methods as genetic blaKPC identification, detection of carbapenem degradation products, and accompanying peak with 11,109 Da, which represents cleaved pKpQIL_p019 protein expressed by pKpQIL plasmid. Detection of carbapenemase-producing K. pneumoniae using MALDI-TOF provides fast and accurate results that may help to reduce morbidity and mortality in hospital setting when applied in diagnostic situations.

Antifungal and Antitoxigenic Effects of Selected Essential Oils in Vapors on Green Coffee Beans with Impact on Consumer Acceptability.

The main objective of this study is to evaluate the effect of selected essential oils thyme chemotype linalool (Thymus zygis L.), thyme chemotype tymol (Thymus vulgaris L.), eucalyptus (Eucalyptus globulus Labill.), lavender (Lavandula angustifolia Mill.), mint (Mentha piperita L.), almond (Prunbus dulcis Mill.), cinnamon bark (Cinnamomum zeylanicum Nees), litsea (Litsea cubeba Lour. Pers), lemongrass (Cympogon citrati L. Stapf), and ginger (Zingiber officinalis Rosc.) in the vapor phase on growth, sporulation, and mycotoxins production of two Aspergillus strains (Aspergillus parasiticus CGC34 and Aspergillus ochraceus CGC87), important postharvest pathogens of green and roasted coffee beans. Moreover, the effect of the essential oils (EOs) on the sensory profile of the coffee samples treated with EOs was evaluated. The major components of tested EOs were determined by gas chromatography and mass spectrometry (GC-MS) and gas chromatography with flame ionization detector (GC-FID). The results showed that almond, cinnamon bark, lemongrass, and litsea EOs are able to significantly inhibit the growth, sporulation, and mycotoxins production by toxigenic fungi. Sensory evaluation of coffee beans treated with EOs before and after roasting showed that some EOs (except lemongrass and litsea) do not adversely affect the taste and aroma of coffee beverages. Thus, application of the vapors of almond and cinnamon EOs appears to be an effective way that could serve to protect coffee during its transport and storage from toxigenic fungi.

Antifungal and synergistic activities of some selected essential oils on the growth of significant indoor fungi of the genus Aspergillus.

The study aimed to assess the antifungal activity of twenty-five essential oils (EOs) and the potential synergistic activity of the most effective EOs against significant indoor fungi of the genus Aspergillus [A. fumigatus (KBio-122), A. flavus (KBio-134), A. terreus (KBio-145) and A. niger (KBio-202)]. The chemical composition of all EOs was evaluated by the gas chromatography coupled with mass spectrometry (GC/MS) and gas chromatography with flame ionization detector (GC-FID) analysis. The antifungal susceptibility of EOs was evaluated by using the broth microdilution method. The most effective EOs were selected to determine the minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) at a concentration range from 256 to 0.125 µg/mL. For the synergistic activities, the most effective EOs were tested using the chessboard pattern. The most sensitive strain to treatments with essential oils alone and in the combination of EOs was A. flavus (KBio-134). The chessboard assay showed that combinations of lemongrass and thyme EOs proved the most potent synergistic antifungal activity (FICI = 0.1875) against A. fumigatus (KBio-122). The synergy displayed by a combination of some EOs may be used to control fungal growth or increasing resistance to available synthetic antifungals, consequently permitting the reduction of their most active doses.

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 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.

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.