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Ragworts like tansy ragwort (J. vulgaris Gaertn., syn. Senecio jacobaea L.) contain hepatotoxic and cancerogenic pyrrolizidine alkaloids (PA) and their corresponding pyrrolizidine alkaloid N-oxides (PANO). Due to increasing spread of ragworts (Jacobaea spp.) PA/PANO may pose a health risk to animals and humans consuming contaminated feed and food. Therefore, the aim of the present study was to investigate the transfer of individual PA/PANO originating from a well-defined PA/PANO extract into the milk of dairy cows. For this objective, 16 German Holstein cows were assigned to four treatment groups (n = 4) in a 28-day dose-response study. Administration into the reticulorumen was performed daily by gavage after the morning milking. Three groups received different amounts of the J. vulgaris extract resulting in a PA/PANO exposure of 0.47, 0.95, or 1.91 mg PA/PANO/kg body weight/day, respectively. Furthermore, a control group received molasses to account for the sugar content of the used PA/PANO extract. While the composition of the PA/PANO extract was more diverse, the PA/PANO pattern in milk was dominated by the PA in their free base form. It was shown that mainly PA considered stable in the rumen environment were transferred into the milk. The main compounds in milk were jacoline (74.3 ± 2.4% of the PA/PANO sum), jaconine (11.2 ± 1.3%), and jacobine (7.2 ± 0.6%) with concentrations up to 29.7, 4.65 µg/l, or in the highest exposed group, 3.44 µg/l. There was no dose-dependent effect on the total PA/PANO transfer rate into the milk. The average transfer rate was 0.064 ± 0.005% of the administered content. [ABSTRACT FROM AUTHOR]

1,2-Dehydro-pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO) and tropane alkaloids (TA), are toxic plant metabolites. If plant material, containing these toxins, is present in the feed of dairy cows these toxins can be transferred into milk. Here, milk was sampled directly from dairy farms in the German federal states of Bavaria and Schleswig-Holstein in 2020–2022 in order to investigate a possible contamination of milk at the production stage. In total, 228 milk samples were analysed for 54 PA/PANO and two TA by a sensitive LC–ESI-MS/MS method. In addition, a subset of milk samples (n = 85) was independently analysed for TA by a cooperating laboratory for verification. PA/PANO were found in 26 samples (11%) with a low median sum content of the contaminated samples of 0.024 µg/L. The highest level of contamination was 5.6 µg/L. Senecionine-, lycopsamine- and heliotrine-type PA/PANO were detected. In four samples (1.8%), atropine was determined up to 0.066 µg/L. The toxin levels in the milk samples hardly contributed to the total daily exposure. These data are first-time results on contamination rates and levels occurring in milk from individual dairy farms, based on a large sample number. [ABSTRACT FROM AUTHOR]

The increasing spread of ragworts is observed with concern. Ragworts like tansy ragwort (Jacobaea vulgaris Gaertn.) or marsh ragwort (J. aquatica) contain pyrrolizidine alkaloids (PA) which may induce hepatotoxic effects. Grazing animals usually avoid ragworts if their pasture management is appropriate. Preserved feed prepared from ragworts contaminated meadows may, however, lead to a significant exposure to PA. Previous studies on toxicity of PA for dairy cows revealed inconsistent results due to feeding ragwort plant material which was associated with heterogeneous PA exposure and thus failed to conclusively deduce critical PA doses. Therefore, the aim of the present study was to expose dairy cows (n = 4 per group) in a short-term scenario for 28 days with increasing PA doses (PA1: 0.47 mg PA/kg body weight (BW)/day (d); PA2: 0.95 mg PA/kg BW/d; PA3: 1.91 mg PA/kg BW/d) via oral administration by gavage of a defined PA-extract. While group PA3 was dosed with the PA-extract alone, groups PA2 and PA1 received PA-extracts blended in similar volumes with molasses to provide comparable amounts of sugar. Additionally, two control groups were treated either with water (CONWater) or with molasses (CONMolasses) to assess the effects of sugar without PA interference. While clinical traits including dry matter intake, milking performance, rectal body temperature, ruminal activity and body condition score (BCS) were not influenced by PA exposure, activities of enzymes indicative for liver damages, such as gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH), increased significantly over time at an exposure of 1.91 mg total PA/kg BW/d. [ABSTRACT FROM AUTHOR]

Phytochemicals like pyrrolizidine alkaloids (PAs) can affect the health of humans and animals. PAs can occur for example in tea, honey or herbs. Some PAs are known to be cytotoxic, genotoxic, and carcinogenic. Upon intake of high amounts, hepatotoxic and pneumotoxic effects were observed in humans. This study aims to elucidate different toxicokinetic parameters like the uptake of PAs and their metabolism with in vitro models. We examined the transport rates of differently structured PAs (monoester, open-chained diester, cyclic diester) over a model of the intestinal barrier. After passing the intestinal barrier, PAs reach the liver, where they are metabolized into partially instable electrophilic metabolites interacting with nucleophilic centers. We investigated this process by the usage of human liver, intestinal, and lung microsomal preparations for incubation with different PAs. These results are completed with the detection of apoptosis as indicator for bioactivation of the PAs. Our results show a structure-dependent passage of PAs over the intestinal barrier. PAs are structure-dependently metabolized by liver microsomes and, to a smaller extent, by lung microsomes. The detection of apoptosis of A549 cells treated with lasiocarpine and monocrotaline following bioactivation by human liver or lung microsomes underlines this result. Conclusively, our results help to shape the picture of PA toxicokinetics which could further improve the knowledge of molecular processes leading to observed effects of PAs in vivo. [ABSTRACT FROM AUTHOR]

Straw is the main by-product of grain production, used as bedding material and animal feed. If produced or stored under adverse hygienic conditions, straw is prone to the growth of filamentous fungi. Some of them, e.g. Aspergillus, Fusarium and Stachybotrys spp. are well-known mycotoxin producers. Since studies on mycotoxins in straw are scarce, 192 straw samples (wheat n = 80; barley n = 79; triticale n = 12; oat n = 11; rye n = 12) were collected across Germany within the German official feed surveillance and screened for the presence of 21 mycotoxins. The following mycotoxins (positive samples for at least one mycotoxin n = 184) were detected: zearalenone (n = 86, 6.0–785 μg/kg), nivalenol (n = 51, 30–2,600 μg/kg), deoxynivalenol (n = 156, 20–24,000 μg/kg), 15-acetyl-deoxynivalenol (n = 34, 20–2,400 μg/kg), 3-acetyl-deoxynivalenol (n = 16, 40–340 μg/kg), scirpentriol (n = 14, 40–680 μg/kg), T-2 toxin (n = 67, 10–250 μg/kg), HT-2 toxin (n = 92, 20–800 μg/kg), T-2 tetraol (n = 13, 70–480 μg/kg). 15-monoacetoxyscirpenol (30 μg/kg) and T-2 triol (60 μg/kg) were only detected in one barley sample. Macrocyclic trichothecenes (satratoxin G, F, roridin E, and verrucarin J) were also found in only one barley sample (quantified as roridin A equivalent: total 183 μg/kg). The occurrence of stachybotrylactam was monitored for the first time in four samples (n = 4, 0.96–7.4 μg/kg). Fusarenon-X, 4,15-diacetoxyscirpenol, neosolaniol, satratoxin H and roridin-L2 were not detectable in the samples. The results indicate a non-negligible contribution of straw to oral and possibly inhalation exposure to mycotoxins of animals or humans handling contaminated straw. [ABSTRACT FROM AUTHOR]

The fungus Stachybotrys (S.) chartarum was isolated from culinary herbs, damp building materials, and improperly stored animal forage. Two distinct chemotypes of the fungus were described that produced either high-cytotoxic macrocyclic trichothecenes (S type) or low-cytotoxic atranones (A type). Recently, two distinct gene clusters were described that were found to be necessary for the biosynthesis of either macrocyclic trichothecenes (21 SAT (Satratoxin) genes) or atranones (14 ATR (Atranone) genes). In the current study, PCR primers were designed to detect SAT and ATR genes in 19 S. chartarum chemotype S and eight S. chartarum chemotype A strains. Our analysis revealed the existence of three different genotypes: satratoxin-producing strains that harbored all SAT genes but lacked the ATR gene cluster (genotype S), non-satratoxin-producing strains that possessed the ATR genes but lacked SAT genes (genotype A), and a hitherto undescribed hybrid genotype among non-satratoxin-producing strains that harbored all ATR genes and an incomplete set of SAT genes (genotype H). In order to improve the discrimination of genotypes, a triplex PCR assay was developed and applied for the analysis of S. chartarum and S. chlorohalonata cultures. The results show that genes for macrocyclic trichothecenes and atranones are not mutually exclusive in S. chartarum. Correlation of the new genotype-based concept with mycotoxin production data shows also that macrocyclic trichothecenes are exclusively produced by S. chartarum genotype S strains. [ABSTRACT FROM AUTHOR]

The Bacillus (B.) cereus group consists of nine recognized species which are present worldwide. B. cereus play an important role in food-borne diseases by producing different toxins. Yet, only a small percentage of B. cereus strains are able to produce the heat stable cereulide, the causative agent of emetic food poisoning. To minimize the entry of emetic B. cereus into the food chain, food business operators are dependent on efficient and reliable methods enabling the differentiation between emetic and non-emetic strains. Currently, only time-consuming cell bioassays, molecular methods and tandem mass spectrometry are available for this purpose. Thus, the aim of the present study was to establish a fast and reliable method for the differentiation between emetic/non-emetic strains by MALDI-TOF MS. Selected strains/isolates of the B. cereus group as well as other Bacillus spp. (total n = 121) were cultured on sheep blood agar for 48 h before analysis. Subsequently, the cultures were directly analyzed by MALDI-TOF MS without prior extraction steps. The samples were measured in the mass range of m/z 800–1800 Da. Using ClinProTools 3.0 statistical software and Flex analysis software (Bruker Daltonics GmbH, Bremen, Germany), a differentiation between emetic/non-emetic isolates was possible with a rate of correct identification of 99.1% by means of the evaluation of two specific biomarkers (m/z 1171 and 1187 Da). • Fast/reliable MALDI-TOF MS method for differentiation of emetic/non-emetic B. cereus. • Directly analyzed by MALDI-TOF MS without prior extraction steps (direct smear). • Differentiation of emetic/non-emetic B. cereus with 99.1% correct identification. • Biomarkers for differentiation of emetic/non-emetic B. cereus (m/z 1171 and 1187 Da). [ABSTRACT FROM AUTHOR]

The genus Stachybotrys produces a broad diversity of secondary metabolites, including macrocyclic trichothecenes, atranones, and phenylspirodrimanes. Although the class of the phenylspirodrimanes is the major one and consists of a multitude of metabolites bearing various structural modifications, few investigations have been carried out. Thus, the presented study deals with the quantitative determination of several secondary metabolites produced by distinct Stachybotrys species for comparison of their metabolite profiles. For that purpose, 15 of the primarily produced secondary metabolites were isolated from fungal cultures and structurally characterized in order to be used as analytical standards for the development of an LC-MS/MS multimethod. The developed method was applied to the analysis of micro-scale extracts from 5 different Stachybotrys strains, which were cultured on different media. In that process, spontaneous dialdehyde/lactone isomerization was observed for some of the isolated secondary metabolites, and novel stachybotrychromenes were quantitatively investigated for the first time. The metabolite profiles of Stachybotrys species are considerably influenced by time of growth and substrate availability, as well as the individual biosynthetic potential of the respective species. Regarding the reported adverse effects associated with Stachybotrys growth in building environments, combinatory effects of the investigated secondary metabolites should be addressed and the role of the phenylspirodrimanes re-evaluated in future research. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Fruits and vegetables have increasingly been related to foodborne outbreaks. Besides surface contamination, a possible internalization of microorganisms into edible parts of plants during growth has already been observed. To examine an actual risk for the consumer, microbial contamination of the rind and pulp of 147 muskmelons from international trade was assessed using cultural and biochemical methods, polymerase chain reaction and matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry. RESULTS: One hundred percent of the rind samples [3.69–8.92 log colony forming units (CFU) g−1] and 89.8% of the pulp samples (maximum load 3.66 log CFU g−1) were microbiologically contaminated. Among the 432 pulp isolates, opportunistic and potentially pathogenic bacteria were identified, mainly Staphylococcus spp. (48.9%), Clostridium spp. (42.9%) and Enterobacteriaceae (27.9%). Salmonella spp., Escherichia coli and isolates of the Bacillus cereus group were found on the rind (1.4%, 0.7% and 42.9%, respectively) and in the pulp (0.7%, 1.4% and 4.7%). Clostridium perfringens was isolated from the rind of seven melons. CONCLUSION: The present study revealed a regularly occurring internal contamination of melons. Possible health risks for consumers because of an occurrence of microorganisms in melon pulp should be considered in future food safety assessments. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The contamination of honey with hepatotoxic pyrrolizidine alkaloids (PAs) is a well-known hazard for food safety. While management strategies and controls of the honey industry aim to reduce the PA levels, uncertainties remain with regard to the safety of regionally produced and marketed honey. In addition, a previous study showed large differences of results obtained after various periods of storage and apparent differences between the analytical results of different laboratories. Therefore, this study aimed at examining these uncertainties by monitoring the impact of storage on the PA and PA N-oxide (PANO) content of two freshly harvested honeys and on possible demixing effects caused by pollen settling. Additionally, three analytical approaches - target analysis with matrix-matched calibration or standard addition and a sum parameter method - were applied for a comparative analysis of 20 honeys harvested in summer 2016. All samples originated from Schleswig-Holstein in Northern Germany where the PA plant Jacobaea vulgaris is currently observed on a massive scale. The results of the time series analyses showed that PANO levels markedly decreased within a few weeks and practically reached the LOD 16 weeks after harvest. Tertiary PAs, by contrast, remained stable and did not increase as a consequence of PANO decrease. The experiments on a putative demixing, which may result in a heterogeneous distribution of PAs/PANOs, revealed that there was no such effect during storage of up to 12 weeks. A comparison of the PA/PANO levels obtained by different analytical approaches showed that in some cases the sum parameter method yielded much higher levels than the target approaches, whereas in other cases, the target analysis with standard addition found higher levels than the other two methods. In summary, the results of this study highlight uncertainties regarding the validity and comparability of analytical results and consequently regarding health risk assessment. [ABSTRACT FROM AUTHOR]

1,2-Dehydro-pyrrolizidine alkaloids (PA) and their N-oxides (PANO) exhibit acute and chronic toxic effects on the liver and other organs and therefore are a hazard for animal and human health. In certain regions of Germany, an increasing spread of Senecio spp. (ragwort) on grassland and farmland areas has been observed during the last years leading to a PA/PANO-contamination of feed and food of animal and plant origin. This project was carried out to elucidate whether the process of grass pellet production applying hot air drying influences the content of PA and PANO. Samples of hay (n = 22) and grass pellets (n = 28) originated from naturally infested grassland (around 10% and 30% dominance of Senecio aquaticus) and from a trial plot with around 50% dominance. Grass pellets were prepared from grass originating from exactly the same plots as the hay samples. The samples were analysed by liquid chromatography-tandem mass spectrometry for PA/PANO typically produced by this weed. The results of the study revealed that PA/PANO levels (predominantly sum of senecionine, seneciphylline, erucifoline and their N-oxides) in hay ranged between 2.1 and 12.6 mg kg−1 dry matter in samples with 10% and 30% dominance of S. aquaticus, respectively. Samples from the trial plot (50% dominance) had levels of up to 52.9 mg kg−1. Notably, the hot air drying process during the production of grass pellets did not lead to a reduction of PA/PANO levels. Instead, the levels in grass pellets with 10% and 30% S. aquaticus ranged from 3.1 to 55.1 mg kg−1. Grass pellets from the trial plot contained up to 96.8 mg kg−1. In conclusion, hot air drying and grass pellet production did not affect PA/PANO contents in plant material and therefore, heat-dried products cannot be regarded as safe in view of the toxic potential of 1,2-dehydro-pyrrolizidine alkaloids. [ABSTRACT FROM AUTHOR]

Hepatotoxic 1,2-dehydro-pyrrolizidine alkaloids (PA) and their N -oxides (PANO) in feed are a potential threat for animal and human health. However, their risk assessment in preserved animal forage is difficult due to data gaps regarding their occurrence in field samples and contradictory results regarding their behavior during the ensilage process. In this study, 115 samples of grass silage originating from different districts in Bavaria (Germany) were analyzed for their PA and PANO contents. A sensitive LC–ESI-MS/MS method for the detection of 10 PA and 4 PANO was developed including a clean-up of the aqueous acidic extract by cation-exchange cartridges. The mean recoveries were between 70% for seneciphylline- N -oxide and 104% for senecionine- N -oxide. The limits of detection ranged between 0.14 μg senkirkine/kg dry matter (DM) and 1.3 μg retrorsine- N -oxide or monocrotaline- N -oxide/kg DM. Eighteen percent of the samples contained one or more of the analyzed compounds. The highest observed value, calculated as the sum of seneciphylline and senecionine, was 30 μg/kg DM. Other samples contained senecionine- N -oxide, seneciphylline- N -oxide, lycopsamine, intermedine, echimidine, and heliotrine in lower amounts. An accompanying laboratory scale ensilage trial showed a compound-dependent transformation of PANO during the fermentation process while PA-amounts remained stable or even increased. Impacts on safety of ensiled animal feed are discussed with regard to animal health. [ABSTRACT FROM AUTHOR]

Honey was previously considered to be one of the main food sources of human pyrrolizidine alkaloid (PA) exposure in Europe. However, comprehensive analyses of honey and tea sampled in the Berlin retail market revealed unexpected high PA amounts in teas. This study comprised the analysis of 87 honey as well as 274 tea samples including black, green, rooibos, melissa, peppermint, chamomile, fennel, nettle, and mixed herbal tea or fruit tea. Total PA concentrations in tea ranged from < LOD to 5647 µg kg−1, while a mean value of about 10 µg kg−1 was found in honey samples. Additionally, herbal drugs were investigated to identify the source of PA in teas. Results suggest that PA in tea samples are most likely a contamination caused by co-harvesting of PA-producing plants. In some cases such as fennel, anise or caraway, it cannot be excluded that these plants are able to produce PA themselves. [ABSTRACT FROM AUTHOR]

A sensitive LC-MS/MS method for the simultaneous determination of type A, B and D trichothecenes in cereals is presented. The limits of detection ranged between 0.1 and 0.7 µg kg-1 for all analytes. The method was applied to 289 representatively drawn samples of wheat, rye and oat products. Ninety-four percent of the wheat samples (n = 130), 95% of the rye samples (n = 61) and 100% of the oat samples (n = 98) were contaminated with the type A trichothecenes T-2 and HT-2 toxin. Median levels of T-2/HT-2 (sum of the toxins) were 0.91, 0.53 and 8.2 µg kg-1, respectively. Highest levels were found in wheat bran (24 µg kg-1), rye kernels (3.1 µg kg-1) and oat flakes (85 µg kg-1). All wheat and rye samples and 75% of the oat samples were contaminated with the type B trichothecene deoxynivalenol. Median levels of this toxin were 23, 15 and 0.53 µg kg-1, respectively. Highest levels were found in wheat bran (1160 µg kg-1), rye kernels (288 µg kg-1) and oat flakes (55 µg kg-1). The type B trichothecene nivalenol was detected in 67% of the wheat samples, in 3% of the rye samples and in 24% of the oat samples with highest levels in wheat bran (96 µg kg-1), rye kernels (1.8 µg kg-1) and in oat flakes (17 µg kg-1), respectively. Levels of other type A and B trichothecenes played a minor role, although the rates of contamination were often high. Neither macrocyclic type D trichothecenes (satratoxin G and H, verrucarin A, roridin A) nor diacetylverrucarol and verrucarol (type A trichothecenes), were detected in any of the samples. [ABSTRACT FROM AUTHOR]

There is a lack of information on the effect of swine caecal microbiota on fumonisin metabolism. In this in vitro study, the biotransformation of fumonisin B1 (FB1) by the gut microbiota of adult, healthy pigs was examined. Suspensions of caecal contents and McDougall buffer solution were incubated anaerobically with pure FB1 for 0, 12, 24, 48 and 72 h. After 48 h, the conversion of FB1 to partially hydrolysed FB1 (46%) was nearly equal to the percentage ratio of FB1, while by 72 h it was 49%. In vitro, the conversion of fumonisin B1 to aminopentol was less than 1%. The results show that the caecal microbiota are capable of transforming fumonisin B1 to the above metabolites. Further studies on FB1 metabolism in the small intestine are clearly justified. [ABSTRACT FROM AUTHOR]

1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites occurring as food contaminants that can cause severe liver damage upon metabolic activation in hepatocytes. However, it is yet unknown how these contaminants enter the cells. The role of hepatic transporters is only at the beginning of being recognized as a key determinant of PA toxicity. Therefore, this study concentrated on assessing the general mode of action of PA transport in the human hepatoma cell line HepaRG using seven structurally different PAs. Furthermore, several hepatic uptake and efflux transporters were targeted with pharmacological inhibitors to identify their role in the uptake of the PAs retrorsine and senecionine and in the disposition of their N-oxides (PANO). For this purpose, PA and PANO content was measured in the supernatant using LC-MS/MS. Also, PA-mediated cytotoxicity was analyzed after transport inhibition. It was found that PAs are taken up into HepaRG cells in a predominantly active and structure-dependent manner. This pattern correlates with other experimental endpoints such as cytotoxicity. Pharmacological inhibition of the influx transporters Na+/taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter 1 (SLC22A1) led to a reduced uptake of retrorsine and senecionine into HepaRG cells, emphasizing the relevance of these transporters for PA toxicokinetics. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PA) and PA N-oxides (PANO) are secondary plant metabolites exhibiting genotoxic and carcinogenic properties. Apart from the roots and leaves, PA/PANO are particularly present in pollen and nectar. Therefore, the spread of Jacobaea vulgaris in certain regions of northern Germany has an impact on the safety of honey produced in that region. In this study, raw honey samples (n = 437) were collected from usually three individual beehives per site (n = 73) in the district of Ostholstein and analyzed for 25 PA/PANO. The results reveal mean levels of 8.4, 1.5, and 72.6 µg/kg and maximum levels of 111, 59.4, and 3313 µg/kg, depending on the season (summer 2015 and spring/summer 2016, respectively). As far as individual data are concerned, sites near areas with J. vulgaris growth did not necessarily result in high PA/PANO values. Furthermore, intra-site investigations revealed remarkable differences in PA/PANO levels of raw honey collected by different bee colonies at the same site. Consumption of these regionally produced honeys entails an increased exposure to PA/PANO, especially in children and high consumers. Margin of exposure values of <10,000 and an exceedance of the health-based guidance value highlight that regionally produced and marketed honey must be considered with care for a proper risk assessment and risk management. [ABSTRACT FROM AUTHOR]

The original version of this paper was published with error. The supplementary materials originally provided were not captured during article production. [ABSTRACT FROM AUTHOR]

The occurrence of several mycotoxins, including ergot alkaloids, alternariol (AOH), deoxynivalenol (DON), and zearalenone (ZEA) in beer ( n = 44) from the German market was studied by using enzyme immunoassay methods. Detection limits in beer were 2.1 μg/L (DON), 0.14 μg/L (ZEA), 0.06 μg/L (ergometrine equivalents), and 0.18 μg/L (AOH). DON was detected in 75% of the samples (2.2–20 μg/L, median 3.7 μg/L). All samples were positive for ZEA (0.35–2.0 μg/L, median 0.88 μg/L) and AOH (0.23–1.6 μg/L, median 0.45 μg/L). Most samples (93%) were positive for ergot alkaloids (0.07–0.47 μg/L, median 0.15 μg/L). Correlating toxin levels in beer with European Union tolerable daily intake (TDI) levels for DON (1 μg/kg b.w.), ZEA (0.25 μg/kg b.w.), and ergot alkaloids (0.6 μg/kg b.w.), beer does not represent a major source of intake of these toxins. No TDI data are available for AOH, but considering toxin levels in other foods, beer does not seem to be a major source of intake of this toxin either. Nevertheless, the frequency of their occurrence warrants further study of ergot alkaloids and AOH in raw materials used for beer brewing. [ABSTRACT FROM AUTHOR]

The increasing antimicrobial resistance (AMR) among pathogenic and opportunistic pathogenic microorganisms is one of the main global public health problems. The consumption of food contaminated with such bacteria (ARB), especially of raw products, might result in the direct acquisition of ARB and in a spread of resistant bacteria along the food chain. The aim of the study was to characterize the antimicrobial susceptibility of potentially extended spectrum β-lactamase (ESBL) producing or AmpC resistant Enterobacteriaceae isolated from the surface of 147 muskmelons from wholesale and retail. A phenotypic analysis was carried out by using minimum inhibitory concentration (MIC) test strips for ESBL detection and MIC susceptibility plates against 14 antimicrobials. Furthermore, ESBL genes, sul -genes and plasmid-mediated AmpC resistance were analyzed by real-time PCR. Additionally, a further insight in the AmpC resistance of isolates of the Enterobacter cloacae complex (ECC) was obtained by analyzing the sequence of the ampC regulatory region (n = 15). A total of 73 potentially resistant Enterobacteriaceae were isolated from 56 muskmelons. Of these, 15 isolates of the ECC were suspicious for ESBL/AmpC resistance, and eleven thereof were positive for the AmpC family EBC. Phenotypic analysis showed diminished susceptibility against "critically" and "highly important" antimicrobials, according to the WHO classification. Furthermore, divergence in the ampC regulatory region was detected between the 15 isolates. These findings highlight the important role that raw produce might play in the transmission of antimicrobial resistances along the food chain. • A total of 15 AMR ECC strains were isolated from 15 of the 147 analyzed muskmelons. • Eleven ECC isolates were additionally positive for AmpC EBC plasmid. • All ECC isolates were negative for ESBL-/ sul -genes. • Analyzed ampR gene of ECC isolates showed heterogeneity even within same species. [ABSTRACT FROM AUTHOR]

Properly handled fish is usually marketed as “fresh fish” until day 10 after fishing. About 40% of the total fishery that is used for direct human consumption is marketed in fresh form stored at temperatures up to +2 °C. Currently, there are no validated methods available for controlling the recommended period of storage. Apart from being a potential source for food fraud, spoiled fish represents a major source of foodborne illnesses and intoxications. In this study, a rapid MALDI-TOF mass spectrometry based screening method was developed using the vitreous fluid of fish eyes as specimen for the examination of different days of storage. The vitreous fluid was collected from n = 100 freshly fished brown trouts at day 0, 3, 7, 9, and 11 post mortem (n = 20 brown trouts each day of examination). The samples were immediately measured by MALDI-TOF mass spectrometry in linear positive mode (mass range m/z 2000–20,000 Da). For quality assurance the experiment was repeated with a set of brown trouts (n = 100) originating from the same fish farm and with brown trouts (n = 100) originating from a different fish farm. For specificity testing rainbow trouts (n = 10) were examined accordingly. All obtained mass spectra were processed by means of MALDI Biotyper OC 3.1 and ClinProTools 3.0 software. The MALDI Biotyper approach showed limited applicability for the identification of the time of storage. However, it was suitable to reliably discriminate between the closely related species brown and rainbow trout. Processing by ClinProTools revealed four crucial mass peaks ( m/z 2594 Da, m/z 4857 Da, m/z 4879 Da, m/z 4899 Da) which enabled a reliable differentiation between day 0 and 3, 7, 9, 11 (rate of correct identification > 90%) as well as the differentiation between day 3 and 7, 9, 11 (rate of correct identification > 72%). However, this approach showed limited applicability within the end of the tested period of storage when comparing between day 7, 9, or 11. [ABSTRACT FROM AUTHOR]

The consumption of edible insects (entomophagy) will gain greater significance facing the increasing global population, which is suggested to reach 9 billion people in 2050 (FAO., 2009). Due to their high amount of proteins, fatty acids, vitamins, and minerals insects represent a valuable source of essential nutrients. While the consumption of insects is very common in many countries of Africa and Asia, there is a far smaller acceptance for entomophagy in Western cultures. Though, products such as noodles or burger paddies made from insect meal have a better compliance and can already be purchased in some countries of the European Union. This processing step however involves the risk of adulteration, because there is no more possibility to authenticate the insects once they are ground. The aim of this study was to investigate whether edible insects could be measured and distinguished by MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry). Therefore, different kinds of edible insects (buffalo worms, mealworms, crickets and grasshoppers) were purchased via online shops and ground subsequently. The insect powder was extracted by vigorously shaking in diluted formic acid and measured by MALDI-TOF MS. The measurement provided reproducible as well as specific mass spectra and enabled a precise differentiation of the different species. [ABSTRACT FROM AUTHOR]

Culturing methods are conventionally applied to investigate the contamination of food with several microorganisms after heat processing. However, with these methods, it is not possible to evaluate whether heat-treated meat products, such as cooked sausages, contained parts of spoiled meat. Therefore, two specific multiplex qPCRs were developed in this study in order to determine the microbiological quality of the raw materials used for these products. The PCR targets focused on four bacterial groups often found on meat (family Enterobacteriaceae , genus Pseudomonas , genus Staphylococcus and species Brochothrix thermosphacta). Specificity as well as sensitivity of the developed multiplex qPCRs, validated by using 68 microbial species, were 100%. The applicability of both multiplex qPCRs compared to culturing methods was performed using 96 meat samples (fresh and naturally spoiled) and 12 inhouse-made "Lyoner" sausages containing variable ratios of spoiled meat (0%, 5%, 12% and 25%; n = 3 for each group). Both methods showed similar results by evaluating the ∆log 10 cfu/g, the relative accuracy and the t -test analysis (p > 0.05). Comparing qPCR results of the different sausage groups, a significant difference between sausages containing fresh meat and sausages containing spoiled meat (12% and 25%) was found only for Pseudomonas and B. thermosphacta in both raw and cooked sausages. The statistical difference between 5% vs. 12% and 25% spoiled meat in cooked sausages, was also found only for these two bacterial groups. The developed multiplex qPCRs were further applied to 30 commercially available "Bologna-type" sausages. The results showed a total of 14 sausages considered to be suspicious for Food Fraud. While the role of Staphylococcus spp. in meat spoilage remains unclear, Pseudomonas , Enterobacteriaceae and B. thermosphacta could together be used as an indicator for "spoiled meat" used in sausages. The developed qPCR systems in this study allow the detection of four relevant bacterial groups in the heated Bologna-type sausages and provide information about the hygienic quality of raw materials used. This method could thus be helpful for screening food suspected of Food Fraud. • Two qPCRs successfully used to quantify 4 bacterial groups in heated meat products. • Specificity and sensitivity of qPCRs for each target bacterial group were 100%. • Pseudomonas and Enterobacteriaceae always found in high amounts in spoiled meat. • Brochothrix thermosphacta and Staphylococcus found in variable amounts in spoiled meat. • 14 of 30 investigated sausages from retail found to be suspicious for Food Fraud. [ABSTRACT FROM AUTHOR]