Cold-tolerant microorganisms causing spoilage of vacuum-packed beef under time-temperature abuse determined by culture and qPCR.

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.

Rapid and selective detection of macrocyclic trichothecene producing Stachybotrys chartarum strains by loop-mediated isothermal amplification (LAMP).

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.

Differentiation of S. chartarum (Ehrenb.) S. Hughes Chemotypes A and S via FT-IR Spectroscopy.

Stachybotrys (S.) chartarum is a cellulolytic mould with the ability to produce highly cytotoxic macrocyclic trichothecenes. Two chemotypes are defined according to their ability to produce either atranones or satratoxins. S. chartarum has been well known as the causative agent of the lethal disease stachybotryotoxicosis in horses. Further investigations revealed that this disease is strictly correlated with the presence of macrocyclic trichothecenes. Furthermore, their occurrence in water-damaged buildings has been linked to adverse health effects such as the sick building syndrome. As the chemotypes cannot be characterized via phenotypic criteria, different methods such as PCR, MALDI-TOF MS, LC-MS/MS, thin-layer chromatography and cytotoxicity assays have been used so far. Fourier-transform-infrared spectroscopy (FT-IR) is commonly used for the differentiation of bacteria and yeasts, but this technique is also applicable to filamentous fungi. Hence, this study aimed at evaluating to which extent a reliable differentiation of S. chartarum chemotypes A and S is possible. Besides, another objective was to verify if the recently introduced third genotype of S. chartarum can be identified. Therefore, 28 strains including the two chemotypes and the third genotype H were cultivated on malt extract agar (MEA) and potato dextrose agar in three biological replicates. Each sample was applied to FT-IR measurements on day 7, 14 and 21 of cultivation. In this study, we achieved a distinction of the chemotypes A and S via FT-IR spectroscopy after incubation for 7 days on MEA. In terms of genotype differentiation, the PCR detecting satratoxin- and atranone-gene clusters remained the only applicable method.

Development of a sensitive analytical method for determining 44 pyrrolizidine alkaloids in teas and herbal teas via LC-ESI-MS/MS.

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.

Differentiation of live and dead Mycobacterium tuberculosis complex in meat samples using PMA qPCR.

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.

Identification of cereulide producing Bacillus cereus by MALDI-TOF MS.

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).

Endogenous microbial contamination of melons (Cucumis melo) from international trade: an underestimated risk for the consumer?

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.

Hepatitis E Virus in Wild Boar in Northwest Poland: Sensitivity of Methods of Detection.

In northwest Poland, 163 blood and 53 fecal samples of wild boars were collected in winter 2012/13 and 2013/14. All blood samples were tested for the presence of hepatitis E virus (HEV) ribonucleic acid (RNA) by two reverse transcription-polymerase chain reaction (RT-PCR) based methods and by anti-HEV IgG enzyme-linked immunosorbent assay (ELISA). About 17.2% of blood samples were seropositive. One-step nested RT-PCR turned out to be too insensitive (11.6% were positive). Therefore a two-step nested RT-PCR was applied where 25.8% of the blood samples were tested positive for HEV RNA. About 50.0% of blood samples positive in ELISA were also positive in two-step nested RT-PCR. The prevalence of HEV RNA in feces was 9.4%. Based on the results of blood (ELISA, PCR) and fecal (PCR) tests, the overall prevalence of HEV in wild boars in northwest Poland was 36.8%. There was no correlation between the ELISA results and the presence of HEV RNA in plasma or in feces. According to the sequencing results of 348 bp PCR products of HEV, there were four different subtypes identified. Reports on the prevalence of HEV in wild boar populations are varying due to different sensitivities of the detection methods. However, this study reveals based on a highly sensitive method that HEV is widely spread in wild boar populations in the northwestern region of Poland and posing a potential risk to the consumer of game meat.

Identification of Stachybotrys spp. by MALDI-TOF mass spectrometry.

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.

Spoilage of vacuum-packed beef by the yeast Kazachstania psychrophila.

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.

Listeria monocytogenes in Different Specimens from Healthy Red Deer and Wild Boars.

In the past, Listeria monocytogenes has been isolated from game feces and meat. However, less information is available on the occurrence of L. monocytogenes in other specimens originating from game animals. Hence, the aim of this study was to get an overview of the occurrence and distribution of L. monocytogenes in game animals by characterization of isolates from different matrices. For that purpose, samples were collected from red deer (Cervus elaphus), wild boars (Sus scrofa), and feed during the hunting season 2011-2012 in three different regions of Germany and Austria. Six samples from each animal were examined: tonsils, content of the rumen or the stomach, liver, intestinal lymph nodes, cecum content, and feces. Nineteen of 45 red deer and 12 of 49 wild boars were found to be positive for L. monocytogenes as well as 4 of 22 pooled feed samples. L. monocytogenes was isolated most frequently from the rumen of red deer (14 of 19) and the tonsils of wild boars (7 of 12). Serotypes 1/2a, 1/2b, 4a, and 4b were detected in samples of game animals and feed, and serotypes 1/2a and 4b were the most prevalent serotypes. The presence of L. monocytogenes serotype 4a had not yet been described in red deer. This might be due to the fact that it was only isolated from the content of rumen and that no other study has yet examined ruminal content. Pulsed-field gel electrophoresis showed a wide variety of strains. Some strains occurred in both species and feed samples, but one strain was dominant in one region. The results show that red deer and wild boars can be carriers of L. monocytogenes in different matrices, although the feces samples can be negative.

Viable Campylobacter jejuni on Eggshells and Its Potential to Cross-contaminate Egg White and Yolk When Using a Manual Separation Technique, Determined by Culture and Propidium Monoazide (PMA) qPCR.

Manual separation of egg yolk from egg white using the eggshell is common practice in private households. For this, the egg is cracked and both components are separated by passing the egg yolk back and forth between the two halves of the eggshell, allowing the egg white to drip down while the egg yolk remains in the shell. During this process, the egg content naturally gets in contact with the outside of the eggshell, which might lead to a cross-contamination with its microorganisms, thus was correspondingly assessed in this study. Campylobacter jejuni is one of the most important zoonotic pathogens that can be found on eggshells. Therefore, this bacterium was used to artificially contaminate the eggshells (n = 22) with concentrations of 3.1 ± 0.6 log10 cfu/g. After separating the egg yolk from the egg white, cross-contamination was determined using culture and qPCR. Altogether, cross-contaminations with C. jejuni were found in 15 egg white (68%) and in three egg yolk (14%) samples. Afterward, 90 eggs from 30 egg packs from different producers in and around Munich (Germany) were obtained for field study purposes. To address the problem of culturing due to a possible viable but nonculturable (VBNC) status of C. jejuni, a method to differentiate viable and dead C. jejuni on eggshell using 10 µM propidium monoazide (PMA) and qPCR was developed. As a result, seven egg packs (23%) were positive for C. jejuni. Of these, only one (3%) was contaminated with viable cells, but still in a concentration of 3.3 log10 cells/g shell. According to these results and considering that eggshells might also be naturally contaminated with other pathogens, the authors recommend avoiding the manual separation technique of egg white and yolk by the eggshell. Especially if raw egg white or yolk is used for preparation of not sufficiently heated foods, where contaminating pathogens are not inactivated during processing, this technique might be a safety hazard for the consumer.

New route for fast detection of antibodies against zoonotic pathogens in sera of slaughtered pigs by means of flow-through chemiluminescence immunochips.

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.

Kazachstania psychrophila sp. nov., a novel psychrophilic yeast isolated from vacuum-packed beef.

Five novel ascosporogenous yeast strains (H382, H396, H409, H433(T) and H441) were found through a survey of vacuum-packed beef microbiota. Sequence analysis of ITS domain and LSU rRNA genes showed that the new strains represent a distinct lineage within the genus Kazachstania, closely related to Kazachstania lodderae (97.0 % identity) and Kazachstania ichnusensis (96.1 % identity). The main difference of strains H382, H396, H409, H433(T) and H441 to strains of known Kazachstania species is the maximum growth temperature, which is below 20 °C for the new strains, whereas related species grow at 25 °C. Furthermore, the strains differed from known Kazachstania species in assimilation and fermentation patterns of carbon sources. Based on these characteristics, the five strains are considered to represent a novel species of the genus Kazachstania for which the name Kazachstania psychrophila sp. nov. is proposed. The type strain is H433(T) (DSM 26230(T)=CBS 12689(T)). The Mycobank number of the type strain is MB 803980.

Symbiotic Husbandry of Chickens and Pigs Does Not Increase Pathogen Transmission Risk.

A symbiotic or mixed animal husbandry (e.g., pigs and chickens) is considered to have a positive effect for animal welfare and sustainable agriculture. On the other hand, a risk of infection and transmission of microorganisms, especially of zoonotic pathogens, between animal species may potentially occur and thus might increase the risk of foodborne illnesses for consumers. To prove these assumptions, two groups of animals and their environmental (soil) samples were investigated in this study. Animals were kept in a free-range system. In the first group, pigs and chickens were reared together (pasture 1), while the other group contained only pigs (pasture 2). During a one-year study, fecal swab samples of 240 pigs and 120 chickens, as well as 120 ground samples, were investigated for the presence of Campylobacter spp., Salmonella spp. and E. coli. Altogether, 438 E. coli and 201 Campylobacter spp. strains were isolated and identified by MALDI-TOF MS. Salmonella spp. was not isolated from any of the sample types. The prevalences of Campylobacter coli and C. jejuni in pigs were 26.7% and 3.3% in pasture 1 and 30.0% and 6.7% in pasture 2, while the prevalences of C. coli and C. jejuni in chickens from pasture 1 were 9.2% and 78.3%, respectively. No correlation between the rearing type (mixed vs. pigs alone) and the prevalence of Campylobacter spp. was observed. All swab samples were positive for E. coli, while the average prevalences in soil samples were 78.3% and 51.7% in pasture 1 and 2, respectively. Results of similarity analysis of the MALDI-TOF MS spectra (for C. coli, C. jejuni and E. coli) and FT-IR spectra (for E. coli) of the same bacterial species showed no recognizable correlations, no matter if strains were isolated from chickens, pig or soil samples or isolated at different sampling periods. The results of the study indicate that the symbiotic husbandry of pigs and chickens neither results in an increased risk of a transmission of Campylobacter spp. or E. coli, nor in a risk of bacterial alteration, as shown by MALDI-TOF MS and FT-IR spectra. In conclusion, the benefits of keeping pigs and chickens together are not diminished by the possible transmission of pathogens.

A European-wide dataset to uncover adaptive traits of Listeria monocytogenes to diverse ecological niches.

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes listeriosis, a serious foodborne illness. In the nature-to-human transmission route, Lm can prosper in various ecological niches. Soil and decaying organic matter are its primary reservoirs. Certain clonal complexes (CCs) are over-represented in food production and represent a challenge to food safety. To gain new understanding of Lm adaptation mechanisms in food, the genetic background of strains found in animals and environment should be investigated in comparison to that of food strains. Twenty-one partners, including food, environment, veterinary and public health laboratories, constructed a dataset of 1484 genomes originating from Lm strains collected in 19 European countries. This dataset encompasses a large number of CCs occurring worldwide, covers many diverse habitats and is balanced between ecological compartments and geographic regions. The dataset presented here will contribute to improve our understanding of Lm ecology and should aid in the surveillance of Lm. This dataset provides a basis for the discovery of the genetic traits underlying Lm adaptation to different ecological niches.

High incidence of cold-tolerant Clostridium frigoriphilum and C. algidicarnis in vacuum-packed beef on retail sale in Germany.

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.

Phenotypic and Genetic Comparison of a Plant-Internalized and an Animal-Isolated Salmonella Choleraesuis Strain.

Contamination of fresh produce with human pathogens poses an important risk for consumers, especially after raw consumption. Moreover, if microorganisms are internalized, no removal by means of further hygienic measures would be possible. Human pathogenic bacteria identified in these food items are mostly of human or animal origin and an adaptation to this new niche and particularly for internalization would be presumed. This study compares a plant-internalized and an animal-borne Salmonella enterica subsp. enterica serovar Choleraesuis aiming at the identification of adaptation of the plant-internalized strain to its original environment. For this purpose, a phenotypical characterization by means of growth curves under conditions resembling the indigenous environment from the plant-internalized strain and further analyses using Pulsed-field gel electrophoresis and Matrix-assisted laser desorption ionization time of flight spectrometry were assessed. Furthermore, comparative genomic analyses by means of single nucleotide polymorphisms and identification of present/absent genes were performed. Although some phenotypical and genetic differences could be found, no signs of a specific adaptation for colonization and internalization in plants could be clearly identified. This could suggest that any Salmonella strain could directly settle in this niche without any evolutionary process being necessary. Further comparative analysis including internalized strains would be necessary to assess this question. However, these kinds of strains are not easily available.

Distribution of T-2 toxin and HT-2 toxin during experimental feeding of yellow mealworm (Tenebrio molitor).

Within the European Union (EU), edible insects need to be approved as "Novel Food" according to Regulation (EU) 2015/2283 and must comply with the requirements of European food law with regard to microbiological and chemical food safety. Substrates used for feeding insects are susceptible to the growth of Fusarium spp. and consequently to contamination with trichothecene mycotoxins. Therefore, the current study aimed to investigate the influence of T-2 and HT-2 toxins on the larval life cycle of yellow mealworm (Tenebrio molitor (L.)) and to study the transfer of T-2, HT-2, T-2 triol and T-2 tetraol in the larvae. In a 4-week feeding study, T. molitor larvae were kept either on naturally (oat flakes moulded with Fusarium sporotrichioides) or artificially contaminated oat flakes, each at two levels (approximately 100 and 250 µg/kg total T-2 and HT-2). Weight gain and survival rates were monitored, and mycotoxins in the feeding substrates, larvae and residues were determined using LC-MS/MS. Larval development varied between the diets and was 44% higher for larvae fed artificially contaminated diets. However, the artificially contaminated diets had a 16% lower survival rate. No trichothecenes were detected in the surviving larvae after harvest, but T-2 and HT-2 were found both in the dead larvae and in the residues of naturally and artificially contaminated diets.