Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of bovine paratuberculosis, a chronic granulomatous enteritis leading to economic losses and posing a risk to human health due to its zoonotic potential. The pathogen cannot reliably be detected by standard methods, and immunological procedures during the infection are not well understood. Therefore, the aim of our study was to explore host–pathogen interactions in MAP-infected dairy cows and to improve diagnostic tests. Serum proteomics analysis using quantitative label-free LC-MS/MS revealed 60 differentially abundant proteins in MAP-infected dairy cows compared to healthy controls from the same infected herd and 90 differentially abundant proteins in comparison to another control group from an uninfected herd. Pathway enrichment analysis provided new insights into the immune response to MAP and susceptibility to the infection. Furthermore, we found a higher abundance of Cathepsin S (CTSS) in the serum of MAP-infected dairy cows, which is involved in multiple enriched pathways associated with the immune system. Confirmed with Western blotting, we identified CTSS as a potential biomarker for bovine paratuberculosis. This study enabled a better understanding of procedures in the host–pathogen response to MAP and improved detection of paratuberculosis-diseased cattle.

Horizontal gene transfer by plasmids can confer metabolic capabilities that expand a host cell’s niche. Yet, it is less understood whether the coalescence of specialized catabolic functions, antibiotic resistances and metal resistances on plasmids provides synergistic benefits. In this study, we report whole-genome assembly and phenotypic analysis of five Salmonella enterica strains isolated in the 1980s from milk powder in Munich, Germany. All strains exhibited the unusual phenotype of lactose-fermentation and encoded either of two variants of the lac operon. Surprisingly, all strains encoded the mobilized colistin resistance gene 9 ( mcr-9 ), long before the first report of this gene in the literature. In two cases, the mcr-9 gene and the lac locus were linked within a large gene island that formed an IncHI2A-type plasmid in one strain but was chromosomally integrated in the other strain. In two other strains, the mcr-9 gene was found on a large IncHI1B/IncP-type plasmid, whereas the lac locus was encoded on a separate chromosomally integrated plasmidic island. The mcr-9 sequences were identical and genomic contexts could not explain the wide range of colistin resistances exhibited by the Salmonella strains. Nucleotide variants did explain phenotypic differences in motility and exopolysaccharide production. The observed linkage of mcr-9 to lactose metabolism, an array of heavy-metal detoxification systems, and other antibiotic resistance genes may reflect a coalescence of specialized phenotypes that improve the spread of colistin resistance in dairy facilities, much earlier than previously suspected.

Bacillus cereus causes two types of foodborne disease. During growth of B. cereus in the gastrointestinal tract, virulence factors can be produced, leading to diarrheal illness. Several toxins are suspected as virulence factors, of which the protein toxins Hbl, Nhe and CytK have been studied the most. The emetic syndrome is an intoxication caused by a pre-formed toxin (cereulide). Bacillus anthracis can cause food poisoning by eating undercooked contaminated meet. Bacillus species outside the B. cereus group have also been implicated in foodborne disease. Species such as Bacillus subtilis, Bacillus mojavensis, Bacillus pumilus and Bacillus licheniformis can produce cyclic lipopeptides which show toxic activity, although their link to foodborne disease remains uncertain.

Circular replicase-encoding single-stranded (CRESS) DNA viruses and other circular DNA agents are increasingly found in various samples and animals. A specific class of these agents—termed bovine meat and milk factors (BMMF)—has been supposed to act as a factor in indirect carcinogenesis in humans. Initial observations attributed the BMMF to European cattle breeds and foodstuffs produced thereof. In the present study, blood and fecal samples from African and Asian cattle were examined. BMMF molecules and genomoviruses were detected in all bovids under study. The majority (79%) of the 29 circular elements could be assigned to BMMF groups 1 and 2, whereas CRESS viruses of the family Genomoviridae accounted for the smaller part (21%). Two genomoviruses belong to the genus Gemykibivirus and one to the genus Gemykrogvirus. The remaining three might be considered as novel species within the genus Gemycircularvirus. The majority of all isolated molecules originated from fecal samples, whereas only three derived from blood. The results from this study expand our knowledge on the diversity and presence of circular DNA in different ruminants that serve for food production in many countries over the world.

The presence of thermophilic spore-forming bacteria is challenging in industrial food processing. The presented genome sequences of Aeribacillus pallidus, isolated from raw milk and cocoa powder, provide insights into how to prevent damage to minimally processed foods and products with extended shelf life, such as milk products.

In recent years, a lack of stability of dairy products with extended shelf life (e.g., yoghurt products, UHT desserts) has occurred, with the corresponding products liquefying significantly after days or weeks. This project aimed to identify the enzymes responsible for the liquefaction of the affected products based on differential proteomic analyses. No evidence was found for the presence of starch-degrading bacteria in the affected products. With zymography and proteome analysis, we detected the cause of liquefaction in a pudding by contamination of its aroma component with an engineered amylolytic enzyme, cyclomaltodextrin glucanotransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes. In addition, we detected contamination with Pseudomonas-derived proteolytic ATP-dependent Clp protease in one pudding batch and proteases in technically used amylases, which degraded β-caseins in another batch. Identification of these agents with liquefying properties in dairy products are useful for adjustment of production protocols and/or composition of additives, and thus shelf life extension.

Humans and animals can become asymptomatic carriers of Listeria monocytogenes and introduce the pathogen into their environment with their feces. In turn, this environmental contamination can become the source of food- and feed-borne illnesses in humans and animals, with the food production chain representing a continuum between the farm environment and human populations that are susceptible to listeriosis. Here, we update a review from 2012 and summarize the current knowledge on the asymptomatic carrier statuses in humans and animals. The data on fecal shedding by species with an impact on the food chain are summarized, and the ways by which asymptomatic carriers contribute to the risk of listeriosis in humans and animals are reviewed.

One of the multiple factors determining the onset of the diarrhoeal disease caused by enteropathogenic Bacillus cereus is the ability of the bacteria to actively move towards the site of infection. This ability depends on flagella, but it also varies widely between different strains. To gain more insights into these strain-specific variations, polyclonal rabbit antisera as well as monoclonal antibodies (mAbs) were generated in this study, which detected recombinant and natural B. cereus flagellin proteins in Western blots as well as in enzyme immunoassays (EIAs). Based on mAb 1A11 and HRP-labelled rabbit serum, a highly specific sandwich EIA was developed. Overall, it could be shown that strain-specific swimming motility correlates with the presence of flagella/flagellin titres obtained in EIAs. Interestingly, mAb 1A11, recognizing an epitope in the N-terminal region of the flagellin protein, proved to inhibit bacterial swimming motility, while the rabbit serum rather decreased growth of selected B. cereus strains. Altogether, powerful tools enabling the in-depth characterization of the strain-specific variations in B. cereus swimming motility were developed.

The aim of this study was to examine the prevalence of bacterial contaminants (Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella spp.) in 116 artisanal cheese samples sold in informal markets of Kosovo. The results provide evidence for a presence of the aforementioned contaminants in the artisanal cheese, with levels of positive samples of 64.7%, 39.7% and 3.4% for Escherichia coli, Staphylococcus aureus and Listeria monocytogenes, respectively. Salmonella spp. could not be detected. It also could be shown that the contamination of the cheeses with Escherichia coli and Staphylococcus aureus was higher during the summer season with Staphylococcus aureus counts up to 106 CFU/g, a level sufficient for the potential production of enterotoxins. The number of samples contaminated with Listeria monocytogenes was, however, higher during the winter season. Microbiological analyses relied on the ISO standards methods, an automated Enzyme Linked Fluorescent Assay for detection of Listeria monocytogenes was used in addition. The findings of this study strongly suggest that institutions responsible for public health, need to increase attention and control measures in order to improve the safety of foods sold in informal markets, particularly with regard to artisanal cheese which is a frequently consumed product.

The genes hblC, hblD and hblA encode the components Hbl L2, L1 and B of the pore forming enterotoxin haemolysin BL of Bacillus cereus. Two variants of the operon existand the more common one additionally contains hblB downstream of hblCDA. Up to now, it was completely unclear whether the corresponding protein, Hbl B’, is widely expressed among B. cereus strains and if it has a distinct function. In the present study, it was shown that the hblB gene is indeed expressed and the Hbl B’ protein is secreted by nearly all analysed B. cereus strains. For the latter, a detection system was developed based on monoclonal antibody 11A5. Further, a distinct reduction of cytotoxic and haemolytic activity was observed when recombinant (r)Hbl B’ was applied simultaneously with L2, L1 and B. This effect was due to direct interaction of rHbl B’ with L1. D−6B. cereusAltogether, we present the first simple tool for the detection of Hbl B’ in B. cereus culture supernatants. Moreover, an important regulatory function of Hbl B’ in the mechanism of Hbl was determined, which is best described as an additional control of complex formation, balancing the amounts of Hbl B-L1 complexes and the corresponding free subunits.

Bovine paratuberculosis is a serious chronic disease of the gastrointestinal tract that causes economic losses and dramatically affects animal health in livestock. The underlying infectious agent, Mycobacterium avium subspecies paratuberculosis (MAP), cannot reliably be detected by standard diagnostic tests due to the long asymptomatic disease stage. The aim of this study was to detect proteomic changes in peripheral blood mononuclear cells (PBMC) from cows of the same herd with different MAP infection status after co-incubation with viable MAP in vitro using label-free LC-MS/MS. In our proteomic discovery experiment, we detected 2631 differentially regulated proteins between cows with negative MAP infection status (so-called MAP-resistant cows) and cows with positive MAP infection status (so-called persistently MAP-infected cows). In MAP-resistant cows, we detected enriched immune-related signaling pathways for TLR2 and MHC class II component proteins, among others, indicating a successful defensive immune response of the cows to MAP. In contrast, persistently MAP-infected cows were not directly enriched in immune-related signaling pathways associated with ITGA2B and KCNMA1, among others. The introduction of these distinct immune responses contributes to a better understanding of the bovine immune response and mechanisms of susceptibility to MAP.

In recent years, a variety of circular replicase-encoding single-stranded (CRESS) DNA viruses and unclassified virus-like DNA elements have been discovered in a broad range of animal species and environmental samples. Key questions to be answered concern their presence in the human diet and their potential impact on disease emergence. Especially DNA elements termed bovine meat and milk factors (BMMF) are suspected to act as co-factors in the development of colon and breast cancer. To expand our knowledge on the occurrence of these potential pathogens in human nutrition, a total of 73 sheep and 40 goat milk samples were assayed by combining rolling circle amplification (RCA), PCR and Sanger sequencing. The present study further includes retail milk from the aforementioned species. We recovered 15 single stranded (ss) circular genomes. Of those, nine belong to the family Genomoviridae and six are members of the unclassified group of BMMF. Thus, dairy sheep and goats add to dispersal of CRESS viruses and circular ssDNA elements, which enter the food chain via milk. The presence of these entities is therefore more widespread in Bovidae than initially assumed and seems to be part of the common human nutrition.

Bacillus cereus sensu stricto is an important pathogen causing food poisoning, as well as extraintestinal diseases [...].

Isolation and characterization of circular replicase-encoding single-stranded (ss) DNA from animal, plant and environmental samples are rapidly evolving in virology. We detected 21 circular DNA elements, including one genomoviral sequence, in individual milk samples from domesticated Asian water buffaloes (Bubalus arnee f. bubalis). Most of the obtained genomes are related to Sphinx 1.76 and Sphinx 2.36 sequences and share a high degree of similarity to recently published circular DNAs-named BMMF (bovine meat and milk factors)-that have been isolated from commercial milk, as well as from bovine serum. Characteristic features such as rep genes, tandem repeats and inverted repeats were detected. These BMMF have recently been found to be present in taurine-type dairy cattle breeds descending from the aurochs (Bos primigenius). Importantly, the occurrence of BMMF has been linked to the higher incidence of colorectal and breast cancer in North America and Western Europe compared with Asia. This is the first report of circular ssDNA detected in milk from the domesticated form of the wild Asian water buffalo (B. arnee) belonging to the subfamily Bovinae. This novelty should be taken into account in view of the above-mentioned cancer hypothesis.

Mycobacterium avium subspecies paratuberculosis (MAP) are detectable viable in milk and other dairy products. The molecular mechanisms allowing the adaptation of MAP in these products are still poorly understood. To obtain information about respective adaptation of MAP in milk, we differentially analyzed the proteomes of MAP cultivated for 48 h in either milk at 37 °C or 4 °C or Middlebrook 7H9 broth as a control. From a total of 2197 MAP proteins identified, 242 proteins were at least fivefold higher in abundance in milk. MAP responded to the nutritional shortage in milk with upregulation of 32% of proteins with function in metabolism and 17% in fatty acid metabolism/synthesis. Additionally, MAP upregulated clusters of 19% proteins with roles in stress responses and immune evasion, 19% in transcription/translation, and 13% in bacterial cell wall synthesis. Dut, MmpL4_1, and RecA were only detected in MAP incubated in milk, pointing to very important roles of these proteins for MAP coping with a stressful environment. Dut is essential and plays an exclusive role for growth, MmpL4_1 for virulence through secretion of specific lipids, and RecA for SOS response of mycobacteria. Further, 35 candidates with stable expression in all conditions were detected, which could serve as targets for detection. Data are available via ProteomeXchange with identifier PXD027444.

Bacillus cereus Hemolysin BL is a tripartite toxin responsible for a diarrheal type of food poisoning. Open questions remain regarding its mode of action, including the extent to which complex formation prior to cell binding contributes to pore-forming activity, how these complexes are composed, and the properties of the pores formed in the target cell membrane. Distinct complexes of up to 600 kDa were found on native gels, whose structure and size were primarily defined by Hbl B. Hbl L1 and L2 were also identified in these complexes using Western blotting and an LC-MS approach. LC-MS also revealed that many other proteins secreted by B. cereus exist in complexes. Further, a decrease of toxic activity at temperatures ≥60 °C was shown, which was unexpectedly restored at higher temperatures. This could be attributed to a release of Hbl B monomers from tight complexation, resulting in enhanced cell binding. In contrast, Hbl L1 was rather susceptible to heat, while heat treatment of Hbl L2 seemed not to be crucial. Furthermore, Hbl-induced pores had a rather small single-channel conductance of around 200 pS and a probable channel diameter of at least 1 nm on planar lipid bilayers. These were highly instable and had a limited lifetime, and were also slightly cation-selective. Altogether, this study provides astonishing new insights into the complex mechanism of Hbl pore formation, as well as the properties of the pores.

Inflammasomes are important for host defence against pathogens and homeostasis with commensal microbes. Here, we show non-haemolytic enterotoxin (NHE) from the neglected human foodborne pathogen Bacillus cereus is an activator of the NLRP3 inflammasome and pyroptosis. NHE is a non-redundant toxin to haemolysin BL (HBL) despite having a similar mechanism of action. Via a putative transmembrane region, subunit C of NHE initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, thus forming a tripartite lytic pore that is permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting a versatile functional repertoire in different host species. These data indicate that NHE and HBL operate synergistically to induce inflammation and show that multiple virulence factors from the same pathogen with conserved function and mechanism of action can be exploited for sensing by a single inflammasome.

Despite its benefits as biological insecticide, Bacillus thuringiensis bears enterotoxins, which can be responsible for a diarrhoeal type of food poisoning. Thus, all 24 isolates from foodstuffs, animals, soil and commercially used biopesticides tested in this study showed the genetic prerequisites necessary to provoke the disease. Moreover, though highly strain-specific, various isolates were able to germinate and also to actively move, which are further requirements for the onset of the disease. Most importantly, all isolates could grow under simulated intestinal conditions and produce significant amounts of enterotoxins. Cytotoxicity assays classified 14 isolates as highly, eight as medium and only two as low toxic. Additionally, growth inhibition by essential oils (EOs) was investigated as preventive measure against putatively enteropathogenic B. thuringiensis. Cinnamon Chinese cassia showed the highest antimicrobial activity, followed by citral, oregano and winter savory. In all tests, high strain-specific variations appeared and must be taken into account when evaluating the hazardous potential of B. thuringiensis and using EOs as antimicrobials. Altogether, the present study shows a non-negligible pathogenic potential of B. thuringiensis, independently from the origin of isolation. Generally, biopesticide strains were indistinguishable from other isolates. Thus, the use of these pesticides might indeed increase the risk for consumers' health. Until complete information about the safety of the applied strains and formulations is available, consumers or manufacturers might benefit from the antimicrobial activity of EOs to reduce the level of contamination.

The ubiquitous soil bacterium Bacillus cereus presents major challenges to food safety. It is responsible for two types of food poisoning, the emetic form due to food intoxication and the diarrheal form emerging from food infections with enteropathogenic strains, also known as toxico-infections, which are the subject of this review. The diarrheal type of food poisoning emerges after production of enterotoxins by viable bacteria in the human intestine. Basically, the manifestation of the disease is, however, the result of a multifactorial process, including B. cereus prevalence and survival in different foods, survival of the stomach passage, spore germination, motility, adhesion, and finally enterotoxin production in the intestine. Moreover, all of these processes are influenced by the consumed foodstuffs as well as the intestinal microbiota which have, therefore, to be considered for a reliable prediction of the hazardous potential of contaminated foods. Current knowledge regarding these single aspects is summarized in this review aiming for risk-oriented diagnostics for enteropathogenic B. cereus.

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/nonemetic 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).