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This paper presents a simple method for the determination of ochratoxins A (OTA) and B (OTB) in pig blood serum. The method includes serum acidification (pH < 1.6) and precipitation of protein with 15% trichloroacetic acid, liquid partitioning with dichloromethane and fluorescence detection. The estimated detection limits were 0.1ng OTA/ml and 0.2ng OTB/ml. The mean recoveries from artificially contaminated samples (n= 6 replicates/mycotoxin) spiked at 0.3, 1 and 3ng OTA and OTB/ml, respectively, were 86.8% (s.d.= 8.4) for OTA and 90.0% (s.d.= 9.8) for OTB. Forty-nine Romanian pig blood serum samples (94% of 52 analysed) were found to be naturally contaminated with OTA in the range 0.1-13.4ng/ml. No sample was found positive for OTB. The method is technically simple, specific, cost effective, suitable for large sample throughput and requires small amount of sample and reagents. It fulfils the criteria for a routine method and could be a suitable tool for surveying OTA in pig herds and in slaughtered pigs. [ABSTRACT FROM AUTHOR]

The causative agents of zoonotic bovine tuberculosis (bTB), Mycobacterium bovis and M. caprae , are members of the M. tuberculosis complex (MTC). Wildlife such as red deer infected with bTB are often without pathological findings, thus meat thereof may be classified as safe for human consumption. The culturing of MTC is time consuming and inappropriate to be applied with fresh meat and food. Therefore, a rapid method "PMA qPCR" to differentiate living and dead cells of MTC was developed in this study. By treating with 50 μM PMA™ dye, dead M. bovis BCG (≤104 cells/ml meat suspension) could be completely discriminated and was not detected by specific MTC PCR. The limit of detection of MTC without treatment with PMA™ dye was 10 cells/ml. All 50 venison samples obtained for field study purposes were negative for MTC. However, 40% were slightly PCR positive for non-TBC mycobacteria. By culturing using selective enrichment, one single colony of M. avium was isolated. This is the first report on the isolation of M. avium from venison. Considering the difficulties of diagnosing mycobacteria in various matrices, the developed PMA qPCR is applicable for the differentiation of dead and living cells of MTC in meat samples. • A sensitive and specific PMA qPCR was developed for discrimination of dead MTC. • The limit of detection of MTC without PMA™ dye was 101 cells/ml meat suspension. • Dead MTC ≤ 104 cells/ml treated with 50 μM PMA were completely discriminated. • The method is applicable for sample types with high risk of MTC contamination. • A non-TBC mycobacterium - M. avium - was isolated in one venison for the first time. [ABSTRACT FROM AUTHOR]

Vacuum packaging and storage conditions at chilled temperatures are commonly used in order to prolong the shelf life of meat. Under these conditions and time-temperature abuse, cold-tolerant (facultatively) anaerobic spoilage microorganisms can continue growing. This study investigated growth of six relevant spoilage microorganisms in vacuum-packed beef (n = 12, 72 subsamples, stored at 10 °C for 28 days) using culture and qPCR methods. Correspondingly, six qPCRs were newly developed/modified (for total bacteria, lactic acid bacteria (LAB), Enterobacterales, total fungi, Kazachstania psychrophila, and cold-tolerant Clostridium spp.). Besides microbial quantification, four spoilage appearances of meat (gas production, spoilage odor, % drip loss, and meat color) were observed. Results obtained from culture and qPCR show that total bacteria, LAB, and Enterobacterales reached their stationary phase at day 7 when spoilage parameters such as gas production were statistically increased and a deviation of odor was detected. Fastidious cold-tolerant Clostridium spp. and K. psychrophila could be detected from day 7. Based on microbiological and sensory analysis results, the maximum shelf life of vacuum-packed beef stored at 10 °C is 7 days. The developed qPCR has the potential to be used as an alternative method to culturing for determination of microbial growth. • Six qPCRs were developed and successfully applied to quantify meat microbiota. • Culture results of total bacteria and LAB correlate with three spoilage parameters. • Cold-tolerant clostridia and yeasts were detected in 17% and 25% of beef samples. • Maximum shelf life of vacuum-packed beef stored at 10 °C was determined to be 7 days. [ABSTRACT FROM AUTHOR]

Cytotoxic macrocyclic trichothecenes such as satratoxins are produced by chemotype S strains of Stachybotrys chartarum. Diseases such as stachybotryotoxicosis in animals and the sick building syndrome as a multifactorial disease complex in humans have been associated with this mold and its toxins. Less toxic non-chemotype S strains of S. chartarum are morphologically indistinguishable from chemotype S strains, which results in uncertainties in hazard characterization of isolates. To selectively identify macrocyclic trichothecene producing S. chartarum isolates, a set of sat14 gene-specific primers was designed and applied in a loop-mediated isothermal amplification (LAMP) assay using neutral red for visual signal detection. The assay was highly specific for S. chartarum strains of the macrocyclic trichothecene producing chemotype and showed no cross-reaction with non-macrocyclic trichothecene producing S. chartarum strains or 152 strains of 131 other fungal species. The assay's detection limit was 0.635 pg/rxn (picogram per reaction) with a reaction time of 60 min. Its high specificity and sensitivity as well as the cost-saving properties make the new assay an interesting and powerful diagnostic tool for easy and rapid testing. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PA) and their N-oxides (PANO) are a group of toxic secondary plant metabolites occurring predominantly as contaminants in (herbal) teas, honeys and food supplements, as well as in spices and culinary herbs. Depending on the botanical origin of the contaminating plant, the pattern of PA/PANO can strongly vary within a sample. The current study aimed to broaden the existing data on the occurrence of PA/PANO in spices and culinary herbs. For this, 305 authentic samples covering 15 different matrices mainly harvested in 2016 or 2017 and originating from 36 countries were investigated for the presence of 44 PA/PANO. Fifty-eight percent of the samples contained at least one PA/PANO. The average sum content over all samples was 323 µg/kg (median of 0.9 µg/kg, 95% percentile of 665 µg/kg). The highest amount of 24.6 mg/kg was detected in an oregano sample. Additionally, conspicuous analyte patterns were discovered in samples from similar cultivation regions, indicating related botanical sources of PA/PANO contaminations. Particularly, oregano and cumin from Turkey often contained high amounts of PA/PANO. The results were used to assess the acute and chronic health risks related to PA/PANO intake via spices and culinary herbs, indicating a potential health risk in particular for adults and children with high consumption or when considering worst-case contamination scenarios of a sum content of 5500 µg/kg. [ABSTRACT FROM AUTHOR]

The number of natural infections with Mycobacterium caprae in wildlife and in cattle in the Bavarian and Austrian alpine regions has increased over the last decade. Red deer (Cervus elaphus) have been recognized as maintenance reservoir; however, the transmission routes of M. caprae among and from naturally infected red deer are unknown. The unexpected high prevalence in some hot spot regions might suggest an effective indirect transmission of infection. Therefore, this study was undertaken to diagnose the occurrence of M. caprae in faeces and secretions of red deer in their natural habitat. A total of 2,806 red deer hunted in this region during 2014–2016 were included in this study. After pathological examination, organs (lymph nodes, lung, heart), excretions and secretions (faeces, urine, saliva and tonsil swabs) were further investigated by qPCR specific for Mycobacterium tuberculosis complex (MTC), M. bovis and M. caprae. Samples tested positive by qPCR were processed for culturing of mycobacteria. In total, 55 (2.0%) animals were confirmed positive for M. caprae by pathological examination, PCR and culturing of the affected organ material. With the exception of one sample, all of the secretion and excretion samples were negative for mycobacteria of the Mycobacterium tuberculosis complex (MTC). From one red deer, M. caprae could be isolated from the heart sac as well as from the faeces. Whole‐genome sequencing confirmed that both strains were clonally related. This is the first confirmation that M. caprae can be shed with the faeces of a naturally infected red deer. However, further studies focusing on a higher number of infected animals, sample standardization and coordinated multiple sampling are necessary to improve the understanding of transmission routes under natural conditions. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PA) and PA-N-oxides (PANO) are a large group of secondary plant metabolites comprising more than 660 compounds. Exhibiting geno- and hepatotoxic properties, they are responsible for multiple cases of food and feed poisoning over the last 100 years. For food and feed safety reasons, relevant PA/PANO should be monitored extensively in the main sources of PA/PANO intake. In this study, a sensitive analytical method was developed for detecting a broad range of 44 commercially available PA/PANO compounds, and in-house validation procedures were performed for several (herbal) teas. Various extraction solvents and procedures, as well as solid phase extraction materials for sample clean-up and analyte concentration, were tested to establish the methods' efficiency and effectiveness. Chromatographic conditions were optimised to obtain the best possible separation of isomers for the 44 PA/PANO analytes. The final method was proven very sensitive and accurate, with detection limits ranging from 0.1 to 7.0 μg/kg and precisions between 0.7 and 16.1%. For 40 of the analytes, the recovery rates ranged from 60.7 to 128.8%. The applicability and trueness of the method were examined by analysing tea samples from a local supermarket and comparing them to a reference material. At least one PA/PANO analyte was detected in 17 of the 18 samples under investigation, and the sum contents of the samples ranged from 0.1 to 47.9 μg/kg. Knowledge of the PA/PANO composition in a sample can be used to indicate the botanical origin of the impurity and, thus, the geographical region of cultivation. [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 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]

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]

Highlights • High number of infection are still caused by foodborne microorganisms. • Increasing number of vulnerable people needs safer food. • emergence and spread of antibiotic resistant bacteria should be controlled. • New methods for effective food preservation are needed. • Magnitudes and sources of risks and ways to effectively control these are needed. [ABSTRACT FROM AUTHOR]

Abstract Psychrophilic and psychrotolerant clostridia (n = 110) were isolated from vacuum-packed meat (beef and lamb), fresh venison and from skin and fecal samples of wild boars. They were identified to species level using MALDI-TOF MS, sequence and phylogeny analysis of the 16S rRNA and species specific multiplex qPCR. The results of all three methods were concordant. The majority of isolates were identified as C. tagluense -like Group I (n = 34) and Group II (n = 42). Thirty-five isolates could be identified to species level as follows: C. estertheticum (n = 15), C. frigoriphilum (n = 13), C. frigidicarnis (n = 1) and C. bowmanii (n = 5). This is the first report of detection and identification of C. frigoriphilum and C. tagluense -like Group II as causative agents of blown pack spoilage of beef. The species specific multiplex qPCR developed in this study could be applied to identify and to quantify the Clostridium species described above in suspicious meat juice samples. Highlights • Investigation of psychrophilic clostridia in meat and in samples of wild boars • Identification of 110 isolates using MALDI-TOF MS and 16S rRNA sequence analysis • Development of a multiplex qPCR for identification of 5 psychrophilic clostridia • First report of C. frigoriphilum and C. tagluense -like as causative agents of BPS in beef [ABSTRACT FROM AUTHOR]

Highlights • High number of infection are still caused by foodborne microorganisms. • Increasing number of vulnerable people needs safer food. • emergence and spread of antibiotic resistant bacteria should be controlled. • New methods for effective food preservation are needed. • Magnitudes and sources of risks and ways to effectively control these are needed. [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]

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]

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]

Stachybotrys ( S.) spp. are omnipresent cellulolytic molds. Some species are highly toxic owing to their ability to synthesize various secondary metabolites such as macrocyclic trichothecenes or hemolysins. The reliable identification of Stachybotrys at species level is currently limited to genome-based identification. This study aimed to establish a fast and reliable MALDI-TOF MS identification method by optimizing the pre-analytical steps for protein extraction for subsequent generation of high-quality fingerprint mass spectra. Eight reference strains of the American Type Culture Collection and the Technical University of Denmark were cultivated in triplicate (biological repetitions) for 2 days in malt extract broth. The mycelia (1.5 ml) were first washed with 75 % ethanol and an additional washing step with dimethyl sulfoxide (10 %) was added to remove unspecific low weight masses. Furthermore, mycelia were broken with roughened glass beads in formic acid (70 %) and acetonitrile. The method was successfully applied to a total of 45 isolates of Stachybotrys originating from three different habitats (indoor, feed, and food samples; n = 15 each): Twenty-seven isolates of S. chartarum and 18 isolates of S. chlorohalonata could be identified by MALDI-TOF MS. The data obtained exactly matched those obtained by genome-based identification. The mean score values for S. chartarum ranged from 2.509 to 2.739 and from 2.148 to 2.622 for S. chlorohalonata with a very good reproducibility: the relative standard deviations were between 0.3 % and 6.8 %. Thus, MALDI-TOF MS proved to be a fast and reliable alternative to identification of Stachybotrys spp. by nucleotide amplification and sequencing. [ABSTRACT FROM AUTHOR]

A survey of the psychrotolerant yeast microbiota of vacuum-packed beef was conducted between 2010 and 2012. Chilled vacuum-packed beef (n = 50) sampled from 15 different producers was found to have a mean psychrotolerant yeast count of 3.76 log cfu per cm 2 . During this assessment, a recently described yeast named Kazachstania psychrophila was shown to be associated with this product. In order to gain basic knowledge about the spoilage potential of K. psychrophila in vacuum-packed beef, challenge studies were performed and the survival of three different K. psychrophila strains was analyzed during storage of artificially contaminated beef. Beef samples were inoculated with the yeasts at a contamination level of 2 log cfu per cm 2 . Survival and growth of K. psychrophila strains was monitored on malt extract agar at regular intervals over 84 days. Kazachstania levels rapidly increased about 5 log units within 16 days under chill conditions (4 °C). Gas bubbles were observed after 16 days, while discoloration and production of off-flavors became evident after 42 days in inoculated samples. This study demonstrates for the first time, that the psychrotolerant yeast K. psychrophila is a dominant spoilage microorganism of vacuum-packed beef products stored at low temperatures, causing sensory defects which result in reduced shelf life, and consequently in considerable economic losses. [ABSTRACT FROM AUTHOR]

Foodborne diseases are of major concern for public health. Here we assess the microbiological quality and safety of marinated pork steaks (n = 300) and marinades (n = 30) which were used for the production of marinated steaks by analyzing quantitative microbiological parameters and foodborne pathogens. Salmonella spp. and Listeria monocytogenes were isolated from about 2%, Staphylococcus aureus from 8% and Bacillus cereus from 21% of the steaks. One steak was MRSA-positive and one contained EHEC/STEC. B. cereus was the only pathogen detected in the marinades. Similar toxin patterns of B. cereus strains from meat and marinades suggested that a contamination of meat with B. cereus occurred via marinades. [ABSTRACT FROM AUTHOR]

The research on fast screening methods for antibodies against zoonotic pathogens in slaughter animals is important for food safety in farming and meat-processing industries. As a proof-of-concept study, antibodies against the emerging zoonotic pathogen hepatitis E virus (HEV) and enteropathogenic Yersinia spp. were analyzed in parallel using immobilized recombinant antigens (rAgs) of HEV genotypes 1 and 3 and Yersinia outer protein D (YopD) on a flow-through chemiluminescence immunochip. These rAgs are usually part of commercially available line immunoassays (LIAs) used for human diagnostics. In this study, sera from slaughtered pigs were tested on the microarray analysis platform MCR 3 to detect anti-HEV and anti-Yersinia IgG. The new method was characterized regarding signal reproducibility and specificity. The analytical performance was compared with in-house enzyme-linked immunosorbent assay (ELISA) and a LIA based on recomLine HEV (Mikrogen) or the ELISA test kit pigtype Yersinia Ab (Qiagen), respectively. The immunochip revealed the highest analytical sensitivity and was processed in 9 min automatically on the MCR 3. A comparative screening of swine serum samples from Bavarian slaughterhouses regarding anti-HEV and anti-Yersinia IgG seroprevalence was conducted. By using the LIA, 78% of the sera were tested positive for HEV antibodies. The immunochip and the ELISA identified anti-HEV IgG in 96% and 93% of the tested samples using the O2C-gt1 and O2C-gt3 rAg, respectively. The screening for anti-Yersinia IgG resulted in 86% positive findings using the immunochip and 57% and 48% for the ELISA methods, respectively, indicating a higher detection capability of the new method. Serum samples of slaughtered pigs could be analyzed faster and in an automated way on the microarray analysis platform MCR 3 which shows the great potential of the new immunochip assay format for multiplexed serum screening purposes. [ABSTRACT FROM AUTHOR]

Detection of foodborne pathogenic and spoilage bacteria by RNA-DNA hybridization is an alternative to traditional microbiological procedures. To achieve high sensitivity with RNA-DNA-based methods, efficient bacterial lysis and release of nucleic acids from bacteria are needed. Here we report the specific detection of the hygiene indicator microorganism Escherichia coli in meat by use of electrochemical biochips. We improved RNA isolation from bacteria in meat juice from pork and beef. Samples, either naturally or artificially contaminated by E. coli. were enriched by incubation in full or minimal medium. A combined treatment of the samples with lysozyme, proteinase K, and sonication resulted in efficient cell disruption and high total RNA yields. Together with optimization of enrichment time, this ensures high sensitivity of electrochemical measurements on biochips. A short enrichment period and the triple-lysis regimen in combination with electrochemical biochip measurement were tested with 25 meat samples. The lower limit of detection of the biochip was approximately 2,000 CFU of E. coli per ml. The entire analysis procedure (5 h of enrichment, triple lysis, and biochip detection) has a lower limit of detection of 1 CFU of E. coli per ml within a total time needed for analysis of 7 h. [ABSTRACT FROM AUTHOR]

Abstract: Electrochemical biochips are an emerging tool for point-of-care diagnostic systems in medicine, food and environmental monitoring. In the current study, a thermostable reporter enzyme, esterase 2 (EST2) from Alicyclobacillus acidocaldarius, is used for specific and sensitive detection of bacteria by one-step rRNA/DNA hybridization between a bacterium-specific capture oligodeoxynucleotide (ODN), bacterial 16S rRNA and an uniform EST2–ODN reporter conjugate. The detection limit corresponds to approximately 500colony forming units (cfu) Escherichia coli. Beside high sensitivity, the application of electrochemical biochips allows discrimination of two Gram-negative and two Gram-positive bacteria demonstrating the specificity and the potential for parallel detection of microorganisms. The feasibility of identification of foodborne bacteria was studied with meat juice contaminated with E. coli. This detection system has the capability to be applied for monitoring of bacterial food contamination. [Copyright &y& Elsevier]

Sixty vacuum-packed beef samples retailed in Germany were investigated for the occurrence of cold-tolerant Clostridium spp. After a storage period at 4 °C for eight weeks, meat juice from all samples was processed for culturing, DNA extraction and SYBR green qPCR for Clostridium species. After that, a previously developed multiplex qPCR, sequence analysis of the 16S rRNA gene, and MALDI-TOF MS were applied in order to identify Clostridium spp. found in samples. Subsequently, 23 samples were found positive for C. frigoriphilum (n = 19), C. estertheticum (n = 2), C. tagluense (n = 1) and C. lacusfryxellense / C. frigoris (n = 1). By using a new multiplex qPCR and a new RFLP method developed in this study, a further 15 meat juice samples were revealed to be contaminated with C. algidicarnis. With some samples being co-contaminated with two different species, 53% (n = 32) of all investigated vacuum-packed beef samples were found to be positive for cold-tolerant clostridia. This is the first report of detection and identification of C. algidicarnis in meat samples in Germany and Central Europe. • High incidence (53%) of psychrophilic clostridia in vacuum-packed fresh beef • 60% of identified processing plants potentially contaminated with said clostridia • Multiplex qPCR developed to identify C. putrefaciens & C. algidicarnis in meat juice • RFLP developed to identify C. putrefaciens & C. algidicarnis in meat juice • First report of detection of C. algidicarnis in meat sold in Germany [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]

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]

A correction to an article on the identification of Stachybotrys spp. by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry," by Sebastian Ulrich and others, that was published in a 2016 issue is presented.