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The Bacillus cereus sensu stricto ( s.s .) species comprises strains of biovar Thuringiensis ( Bt ) known for their bioinsecticidal activity, as well as strains with foodborne pathogenic potential. Bt strains are identified (i) based on the production of insecticidal crystal proteins, also known as Bt toxins, or (ii) based on the presence of cry , cyt , and vip genes, which encode Bt toxins. Multiple bioinformatics tools have been developed for the detection of crystal protein-encoding genes based on whole-genome sequencing (WGS) data. However, the performance of these tools is yet to be evaluated using phenotypic data. Thus, the goal of this study was to assess the performance of four bioinformatics tools for the detection of crystal protein-encoding genes. The accuracy of sequence-based identification of Bt was determined in reference to phenotypic microscope-based screening for the production of crystal proteins. A total of 58 diverse B. cereus sensu lato strains isolated from clinical, food, environmental, and commercial biopesticide products underwent WGS. Isolates were examined for crystal protein production using phase contrast microscopy. Crystal protein-encoding genes were detected using BtToxin_Digger, BTyper3, IDOPS (identification of pesticidal sequences), and Cry_processor. Out of 58 isolates, the phenotypic production of crystal proteins was confirmed for 18 isolates. Specificity and sensitivity of Bt identification based on sequences were 0.85 and 0.94 for BtToxin_Digger, 0.97 and 0.89 for BTyper3, 0.95 and 0.94 for IDOPS, and 0.88 and 1.00 for Cry_processor, respectively. Cry_processor predicted crystal protein production with the highest specificity, and BtToxin_Digger and IDOPS predicted crystal protein production with the highest sensitivity. Three out of four tested bioinformatics tools performed well overall, with IDOPS achieving high sensitivity and specificity (>0.90).IMPORTANCEStrains of Bacillus cereus sensu stricto ( s.s .) biovar Thuringiensis ( Bt ) are used as organic biopesticides . Bt is differentiated from the foodborne pathogen Bacillus cereus s.s . by the production of insecticidal crystal proteins. Thus, reliable genomic identification of biovar Thuringiensis is necessary to ensure food safety and facilitate risk assessment. This study assessed the accuracy of whole-genome sequencing (WGS)-based identification of Bt compared to phenotypic microscopy-based screening for crystal protein production. Multiple bioinformatics tools were compared to assess their performance in predicting crystal protein production. Among them, identification of pesticidal sequences performed best overall at WGS-based Bt identification., Competing Interests: The authors declare no conflict of interest.

Bacillus cytotoxicus is a thermotolerant member of the Bacillus cereus group. It has been linked to rare, but at times fatal cases of diarrheal disease and might be missed at routine diagnostic screening temperatures commonly used for the B. cereus group. The pathogen is mostly found on dehydrated foods containing potato starch or insects. How it enters the food chain or whether it persists in food producing environments is largely unknown. Increased consumption of insects and convenience foods in Europe and the lack of information on the persistence of B. cytotoxicus in food environments and its virulence demand for further characterization. In this study, we aimed to obtain a better understanding of i) the food sources of B. cytotoxicus, ii) screening temperatures needed for its isolation from food matrices, iii) cytotoxicity of the organism, and iv) its ecological niche and potential epidemiological links. To this end, 112 food samples were collected, with a focus on foods exhibiting low water activity. The samples were screened for B. cytotoxicus at 42 °C and at 50 °C. Presumptive isolates were characterized by cytK-1 toxin gene PCR for differentiation of B. cytotoxicus from other B. cereus group members. Vero cell cytotoxicity assays were performed, and selected isolates were sequenced. Our results show that screening at 42 °C might be insufficient for detecting B. cytotoxicus in foods that harbor other less thermophilic Bacillus species. When screening at 50 °C, B. cytotoxicus was detected in 23% of the food samples (n = 26 isolates). The highest prevalence was detected in mashed potato products (82%) and potato flakes (67%). In contrast, a wide range of products not containing any potato ingredients did not yield B. cytotoxicus isolates. All B. cytotoxicus isolates exhibited either low or no detectable cytotoxicity. WGS analysis revealed that a highly toxic isolate is closely related to the French outbreak strain NVH 391-98. In addition, we could show that two isolates sampled 5 years apart from the same production facility only differed by seven SNPs, making it likely that B. cytotoxicus is able to persist in production facilities over a long time. Interestingly, the reoccurring strain possessed an additional plasmid and did not show cytotoxic potential when re-isolated after 5 years., Competing Interests: Declaration of competing interest None, (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Staphylococcal enterotoxin C (SEC) can cause staphylococcal food poisoning, one of the most prevalent foodborne intoxications. It is produced by Staphylococcus aureus during growth in the food matrix. While the surrounding bacteria in food matrices usually repress the growth of S.aureus, the organism possesses a remarkable growth advantage under stressful conditions encountered in many foods. Examples for such food matrices are pastry and bakery products with their high sugar content that lowers water availability. While S. aureus can still grow in these challenging environments, it remains unclear how these conditions affect SEC expression. Here, the influence of 30% glucose on sec mRNA in a qPCR assay and SEC protein expression was investigated for the first time in an ELISA. In addition, regulatory knockout mutants Δagr, ΔsarA, and ΔsigB were generated to investigate regulatory gene elements in glucose stress. In five out of seven strains, glucose stress led to a pronounced decrease in sec mRNA transcription and SEC protein levels were substantially lower under glucose stress. It could be shown that key regulatory elements Δagr, ΔsarA, and ΔsigB in strain SAI48 did not contribute to the pronounced downregulation under glucose stress. Based on these findings, glucose effectively lowers SEC synthesis in the food matrix. However, the mechanism by which it acts on toxin expression and regulatory elements in S. aureus remains unclear. Future studies on other regulatory elements and transcriptomics may shed light on the mechanisms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The increasing integration of genomics into routine clinical diagnostics requires reliable computational tools to identify determinants of antimicrobial resistance (AMR) from whole-genome sequencing data. Here, we developed PorinPredict, a bioinformatic tool that predicts defects of the Pseudomonas aeruginosa outer membrane porin OprD, which are strongly associated with reduced carbapenem susceptibility. PorinPredict relies on a database of intact OprD variants and reports inactivating mutations in the coding or promoter region. PorinPredict was validated against 987 carbapenemase-negative P. aeruginosa genomes, of which OprD loss was predicted for 454 out of 522 (87.0%) meropenem-nonsusceptible and 46 out of 465 (9.9%) meropenem-susceptible isolates. OprD loss was also found to be common among carbapenemase-producing isolates, resulting in even further increased MICs. Chromosomal mutations in quinolone resistance-determining regions and OprD loss commonly co-occurred, likely reflecting the restricted use of carbapenems for multidrug-resistant infections as recommended in antimicrobial stewardship programs. In combination with available AMR gene detection tools, PorinPredict provides a robust and standardized approach to link P. aeruginosa phenotypes to genotypes. IMPORTANCE Pseudomonas aeruginosa is a major cause of multidrug-resistant nosocomial infections. The emergence and spread of clones exhibiting resistance to carbapenems, a class of critical last-line antibiotics, is therefore closely monitored. Carbapenem resistance is frequently mediated by chromosomal mutations that lead to a defective outer membrane porin OprD. Here, we determined the genetic diversity of OprD variants across the P. aeruginosa population and developed PorinPredict, a bioinformatic tool that enables the prediction of OprD loss from whole-genome sequencing data. We show a high correlation between predicted OprD loss and meropenem nonsusceptibility irrespective of the presence of carbapenemases, which are a second widespread determinant of carbapenem resistance. Isolates with resistance determinants to other antibiotics were disproportionally affected by OprD loss, possibly due to an increased exposure to carbapenems. Integration of PorinPredict into genomic surveillance platforms will facilitate a better understanding of the clinical impact of OprD modifications and transmission dynamics of resistant clones.

As bread is a very important staple food, its spoilage threatens global food security. Ropy bread spoilage manifests in sticky and stringy degradation of the crumb, slime formation, discoloration, and an odor reminiscent of rotting fruit. Increasing consumer demand for preservative-free products and global warming may increase the occurrence of ropy spoilage. Bacillus amyloliquefaciens , B. subtilis , B. licheniformis , the B. cereus group, B. pumilus , B. sonorensis , Cytobacillus firmus , Niallia circulans , Paenibacillus polymyxa , and Priestia megaterium were reported to cause ropiness in bread. Process hygiene does not prevent ropy spoilage, as contamination of flour with these Bacillus species is unavoidable due to their occurrence as a part of the endophytic commensal microbiota of wheat and the formation of heat-stable endospores that are not inactivated during processing, baking, or storage. To date, the underlying mechanisms behind ropy bread spoilage remain unclear, high-throughput screening tools to identify rope-forming bacteria are missing, and only a limited number of strategies to reduce rope spoilage were described. This review provides a current overview on (i) routes of entry of Bacillus endospores into bread, (ii) bacterial species implicated in rope spoilage, (iii) factors influencing rope development, and (iv) methods used to assess bacterial rope-forming potential. Finally, we pinpoint key gaps in knowledge and related challenges, as well as future research questions.

Staphylococcal food poisoning is a common food intoxication caused by staphylococcal enterotoxins. While growth of Staphylococcus aureus is not inhibited by the meat-curing agent nitrite, we hypothesize that nitrite has an influence on enterotoxin C (SEC) expression. We investigated the influence of 150 mg/l nitrite on SEC expression at mRNA and protein level in seven strains expressing different SEC variants. Additionally, regulatory knockout mutants (Δagr, ΔsarA, and ΔsigB) of high SEC producing strain SAI48 were investigated at mRNA level. Our findings suggest that nitrite effectively increases sec mRNA transcription, but the effects on SEC protein expression are less pronounced. While Δagr mutants exhibited lower sec mRNA transcription levels than wildtype strains, this response was not stress specific. ΔsigB mutants displayed a nitrite stress-specific response. Whole genome sequencing of the strains revealed a defective agr element in one strain (SAI3). In this strain, sec transcription and SEC protein synthesis was not affected by the mutation. Consequently, additional regulatory networks must be at play in SEC expression. Comparison of our findings about SEC with previous experiments on SEB and SED suggest that each SE can respond differently, and that the same stressor can trigger opposing responses in strains that express multiple toxins., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Plant protection products based on Bacillus thuringiensis have been used to fight agricultural pests for decades and are the world's most frequently applied biopesticide. However, there is growing concern that B. thuringiensis residues in food may occasionally cause diarrheal illness in humans. This has recently sparked a plethora of research activities and vivid discussions across the scientific community, competent authorities, and the public. To support this discussion, we provide a structured overview of the current knowledge on the role of B. thuringiensis as a causative agent of foodborne infections in humans and pinpoint research gaps that need to be addressed for improved risk assessment. We review (i) recent taxonomic changes in the B. cereus group; (ii) the role of B. thuringiensis in transforming agrosystems; and (iii) key considerations for assessing the hazard potential of B. thuringiensis strains detected in foods. We conclude that (i) the taxonomy of the B. cereus group is collapsing, (ii) B. thuringiensis based biopesticides play a key role in realizing the UN's sustainable development goals, and (iii) risk assessment needs to move from taxonomy-driven considerations to strain-specific identification of virulence and pathogenicity traits We also provide an overview of relevant risk-related data for commonly used biopesticide strains., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Enterotoxins (SEs) produced by Staphylococcus aureus are the cause of serious food intoxications. Staphylococcal enterotoxin C (SEC) is one of the main contributors, as it is often highly expressed. S. aureus possesses a competitive growth advantage over accompanying bacterial flora under stress conditions encountered in foods, such as high NaCl concentrations. However, the influence of NaCl as an external stressor on SEC expression is still unclear. We investigated the influence of 4.5% NaCl on sec mRNA and SEC protein levels. A qRT-PCR assay revealed that NaCl stress leads to time-dependently decreased or elevated sec mRNA levels for most strains. SEC protein levels were generally decreased under NaCl stress. Our findings suggest that NaCl stress lowers overall SEC concentration and time-dependently affects sec mRNA levels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Etter, Ukowitz, Eicher, Tasara and Johler.)

Bacillus thuringiensis is a microbial insecticide widely used to control agricultural pests. Although generally regarded as safe, B. thuringiensis is phylogenetically intermingled with the foodborne pathogen B. cereus sensu stricto and has been linked to foodborne outbreaks. Limited data on the pathogenicity potential of B. thuringiensis and the occurrence of biopesticide residues in food compromise a robust consumer risk assessment. In this study, we analyzed whole-genome sequences of 33 B. thuringiensis isolates from biopesticides, food, and human fecal samples linked to outbreaks. All food and outbreak-associated isolates genomically matched (≤ 6 wgSNPs; ≤ 2 cgSNPs) with one of six biopesticide strains, suggesting biopesticide products as their source. Long-read sequencing revealed a more diverse virulence gene profile than previously assumed, including a transposase-mediated disruption of the promoter region of the non-hemolytic enterotoxin gene nhe and a bacteriophage-mediated disruption of the sphingomyelinase gene sph in some biopesticide strains. Furthermore, we provide high-quality genome assemblies of seven widely used B. thuringiensis biopesticide strains, which will facilitate improved microbial source tracking and risk assessment of B. thuringiensis -based biopesticides in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Biggel, Etter, Corti, Brodmann, Stephan, Ehling-Schulz and Johler.)

Staphylococcal enterotoxin C (SEC) is a major cause of staphylococcal food poisoning in humans and plays a role in bovine mastitis. Staphylococcus aureus ( S. aureus ) benefits from a competitive growth advantage under stress conditions encountered in foods such as a low pH. Therefore, understanding the role of stressors such as lactic acid on SEC production is of pivotal relevance to food safety. However, stress-dependent cues and their effects on enterotoxin expression are still poorly understood. In this study, we used human and animal strains harboring different SEC variants in order to evaluate the influence of mild lactic acid stress (pH 6.0) on SEC expression both on transcriptional and translational level. Although only a modest decrease in sec mRNA levels was observed under lactic acid stress, protein levels showed a significant decrease in SEC levels for some strains. These findings indicate that post-transcriptional modifications can act in SEC expression under lactic acid stress.

Bacillus cytotoxicus belongs to the Bacillus cereus group that also comprises the foodborne pathogen Bacillus cereus sensu stricto, Bacillus anthracis causing anthrax, as well as the biopesticide Bacillus thuringiensis . The first B. cytotoxicus was isolated in the context of a severe food poisoning outbreak leading to fatal cases of diarrheal disease. Subsequent characterization of the outbreak strain led to the conclusion that this Bacillus strain was highly cytotoxic and eventually resulted in the description of a novel species, whose name reflects the observed toxicity: B. cytotoxicus . However, only a few isolates of this species have been characterized with regard to their cytotoxic potential and the role of B. cytotoxicus as a causative agent of food poisoning remains largely unclear. Hence, the aim of this study was to gain further insights into the toxicity of B. cytotoxicus . To this end, 19 isolates were obtained from mashed potato powders and characterized by toxin gene profiling and Vero cell cytotoxicity assays. All isolates harbored the cytK1 (cytotoxin K1) gene and species-specific variants of the nhe (non-hemolytic enterotoxin) gene. The isolates exhibited low or no toxicity towards Vero cells. Thus, this study indicates that the cytotoxic potential of B. cytotoxicus may be potentially lower than initially assumed.

Staphylococcal enterotoxins are the most common cause of foodborne intoxications (staphylococcal food poisoning) and cause a wide range of diseases. With at least six variants staphylococcal enterotoxin C (SEC) stands out as particularly diverse amongst the 25 known staphylococcal enterotoxins. Some variants present unique and even host-specific features. Here, we review the role of SEC in human and animal health with a particular focus on its role as a causative agent for foodborne intoxications. We highlight structural features unique to SEC and its variants, particularly, the emetic and superantigen activity, as well as the roles of SEC in mastitis and in dairy products. Information about the genetic organization as well as regulatory mechanisms including the accessory gene regulator and food-related stressors are provided.

Bacillus cytotoxicus CH_213 was isolated from a dehydrated mashed potato product purchased at a Swiss supermarket in 2017. The strain is closely related to strain NVH 391-98, which was linked to a foodborne outbreak of diarrheal syndrome in France in 1998., (Copyright © 2020 Stevens and Johler.)

Bacillus thuringiensis CH_48 exhibits extremely high levels of Vero cell cytotoxicity and sphingomyelinase activity., (Copyright © 2020 Stevens and Johler.)

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of severe infections in humans and animals worldwide. Studies elucidating the population structure, staphylococcal cassette chromosome mec types, resistance phenotypes, and virulence gene profiles of animal-associated MRSA are needed to understand spread and transmission., Objectives: The objective of this study was to determine 1) clonal complexes and spa types, 2) resistance phenotypes, and 3) virulence/resistance gene profiles of MRSA isolated from animals in Switzerland., Methods: We analyzed 31 presumptive MRSA isolates collected from clinical infections in horses, dogs, cattle, sheep, and pigs, which had tested positive in the Staphaurex Latex Agglutination Test. The isolates were characterized by spa typing and DNA microarray profiling. In addition, we performed antimicrobial susceptibility testing using the VITEK 2 Compact system., Results: Characterization of the 31 presumptive MRSA isolates revealed 3 methicillin-resistant Staphylococcus pseudintermedius isolates, which were able to grow on MRSA2 Brilliance agar. Of the 28 MRSA isolates, the majority was assigned to CC398 (86%), but CC8 (11%) and CC1 (4%) were also detected. The predominant spa type was t011 (n = 23), followed by t009 (n = 2), t034 (n = 1), t008 (n = 1), and t127 (n = 1)., Conclusions: The results of this study extend the current body of knowledge on the population structure, resistance phenotypes, and virulence and resistance gene profiles of MRSA from livestock and companion animals., Competing Interests: The authors declare no conflicts of interest., (© 2020 The Korean Society of Veterinary Science.)

Staphylococcus aureus is a leading cause for clinical infections and food intoxications, causing over 100,000 yearly cases of bacteremia in the United States and 434 food-borne outbreaks in the European Union. Approximately 30% of the population permanently carry S. aureus asymptomatically in their nasal cavity. The risk of infection and transmission to food items or the environment is higher in individuals that are nasally colonized. In addition, S. aureus can acquire various antimicrobial resistances leading to therapeutic failure, additional medical costs, and fatalities. Methicillin-resistant S. aureus (MRSA) cause a considerable burden of disease in humans and animals. MRSA carriage has been associated with animal and in particular livestock contact. Extensive current data on the virulence gene profiles, as well as data on antimicrobial resistance determinants is crucial in developing effective strategies to mitigate the burden of disease. To this end, we screened the anterior nares of 160 test subjects (87 pupils and 73 members of farmer families) in Switzerland for S. aureus carriage. A total of 73 S. aureus isolates were obtained. Factors such as exposure to farm or companion animals and personal medical history were recorded using a questionnaire. Using a DNA microarray, isolates were assigned to clonal complexes (CCs), and virulence and resistance gene profiles were determined. The collected strains were assigned to 20 CCs, among others CC1, CC7, CC8, CC15, CC30, CC45, CC97, and CC398. Two MRSA strains and one multiresistant isolate carrying genes blaZ/I/R , InuA , aadD , tetK , and fosB were isolated from farmers with intensive exposure to animals. Strains carrying pvl , causing severe skin lesions and necrotizing pneumonia, as well as tetracycline, erythromycin, and kanamycin resistance genes were found in individuals that had taken antibiotics during the last year. A variety of superantigenic toxin genes was detected, including among others, the toxic shock syndrome toxin ( tst1 ), and various enterotoxins ( sea , sec , sel , and the egc cluster). Contact to chickens was identified as a significant factor contributing to S. aureus colonization., (Copyright © 2020 Etter, Corti, Spirig, Cernela, Stephan and Johler.)

Abstract: In contrast to Bacillus cereus, the role of Bacillus thuringiensis in foodborne illness has been controversially discussed. As B. thuringiensis-based biopesticides containing a mixture of crystal toxins and viable spores are widely used, a current European Food Safety Authority opinion underlines the need for additional data to enable risk assessment. However, it is currently poorly understood if B. thuringiensis is able to multiply in food, which is crucial to sound risk assessment. Therefore, the aim of this study was to investigate growth of selected B. thuringiensis strains from food and insecticides in a ratatouille food model. To this end, the growth parameters of three B. thuringiensis strains were determined: insecticide strain ABTS-351 (CH_119, B. thuringiensis serovar kurstaki), insecticide strain ABTS-1857 (CH_121, B. thuringiensis serovar aizawai), and CH_48 (wild-type B. thuringiensis isolated from rosemary), producing extremely high levels of enterotoxins. After an initial drop in colony counts, we observed a statistically significant growth for the tested B. thuringiensis strains between 6 and 24 h at 22, 30, and 37°C, conditions mimicking prolonged holding times. We were also able to show that the enterotoxin overproducer CH_48 can grow up to 108 CFU/g in the ratatouille matrix within 24 h at 37°C. The two midlevel enterotoxin formers ABTS-351 (CH_119) and ABTS-1857 (CH_121) isolated from biopesticides exhibited growth between 6 and 24 h, with one of the strains growing to 107 CFU/g. To our knowledge, this is the first study providing evidence of B. thuringiensis growth in a food model with intact competitive flora. Our findings suggest strain-specific variation and stress the complexity of assessing the risk related to B. thuringiensis in food, indicating that some strains can represent a risk to consumer health when vegetable-based foods are stored under conditions of prolonged temperature abuse., (Copyright ©, International Association for Food Protection.)

Data on the occurrence, population structure and toxinogenic potential of Bacillus cereus sensu lato isolated from flour is essential to enable improved risk assessment. We aimed to provide data on the occurrence of B. cereus sensu lato in flour products at retail level. In addition, we screened the isolates for Bacillus thuringiensis and Bacillus cytotoxicus and determined population structure and toxin gene profiles. We screened 89 flour products for presence of B. cereus sensu lato, resulting in 75 positive samples (84%). We were able to show that the population structure of members of the B. cereus group isolated from flour is highly diverse. Isolates were assigned to panC types II (4%), III (21%), IV (39%) and V (36%). Production of parasporal crystals characteristic for Bacillus thuringiensis was detected in seven isolates assigned to panC type III, IV and V. No B. cytotoxicus were detected. Two of the isolates harbored ces encoding cereulide, which causes the emetic syndrome. Various enterotoxin genes were found, with all isolates harboring nhe, 75% of isolates harboring hbl and 51% of the isolates harboring cytK-2. Our findings suggest that toxinogenic B. cereus sensu lato are common in flour products at retail level., (© FEMS 2019.)

Many parts of pork meat processing are currently not used for human consumption in Switzerland, although they are of great nutritional value. Therefore, data on the occurrence of pathogenic organisms on byproducts is extremely scarce and the prevalence and population structure of Staphylococcus aureus on meat processing sidestreams is unknown. Hence, abattoir byproducts of pork origin including ear, forefoot, heart, intestine, liver, rib bone, sternum, bladder, stomach, hind foot and tongue originating from six abattoirs were screened for S. aureus. The obtained isolates were investigated by spa typing and DNA microarray analysis to reveal their genomic profile and population structure. The prevalence of S. aureus was generally low with a mean of 8%. In total, 40 S. aureus strains were detected and assigned to 12 spa types (t015, t1491, t1778, t091, t337, t899, t2922, t7439, t1333, t208, t4049, t034) and seven clonal complexes (CC1, CC7, CC9, CC30, CC45, CC49, CC398). Detected enterotoxin genes included sea, seb, sec, seh, sel and egc encoded toxin genes seg, sei, sem, sen, seo, and seu. None of the isolates harbored genes conferring methicillin resistance, but blaZ/I/R genes causing penicillin resistance were frequently found. In addition, strains from CC398 exhibited tetM and tetK, conferring tetracycline resistance. Similarity calculations based on microarray profiles revealed no association of clonal complexes with particular body parts, but revealed a certain correspondence of clonal complex and originating abattoir., Competing Interests: The authors have declared that no competing interests exist.

Prevention, prediction, control, and handling of bacterial foodborne diseases - an ongoing, serious, and costly concern worldwide - are continually facing a wide array of difficulties. Not the least due to that food matrices, highly variable and complex, can impact virulence expression in diverse and unpredictable ways. This review aims to present a comprehensive overview of challenges related to the presence of enterotoxigenic Staphylococcus aureus in the food production chain. It focuses on characteristics, expression, and regulation of the highly stable staphylococcal enterotoxins and in particular staphylococcal enterotoxin A (SEA). Together with the robustness of the pathogen under diverse environmental conditions and the range of possible entry routes into the food chain, this poses some of the biggest challenges in the control of SFP. Furthermore, the emergence of new enterotoxins, found to be connected with SFP, brings new questions around their regulatory mechanisms and expression in different food environments. The appearance of increasing amounts of antibiotic resistant strains found in food is also highlighted. Finally, potentials and limitations of implementing existing risk assessment models are discussed. Various quantitative microbial risk assessment approaches have attempted to quantify the growth of the bacterium and production of disease causing levels of toxin under various food chain and domestic food handling scenarios. This requires employment of predictive modeling tools, quantifying the spatiotemporal population dynamics of S. aureus in response to intrinsic and extrinsic food properties. In this context, the armory of predictive modeling employs both kinetic and probabilistic models to estimate the levels that potentiate toxin production, the time needed to reach that levels, and overall, the likelihood of toxin production. Following risk assessment, the main challenge to mitigate the risk of S. aureus intoxication is first to prevent growth of the organism and then to hamper the production of enterotoxins, or at least prevent the accumulation of high levels (e.g., >10-20 ng) in food. The necessity for continued studies indeed becomes apparent based on the challenges to understand, control, and predict enterotoxin production in relation to the food environment. Different types of food, preservatives, processing, and packaging conditions; regulatory networks; and different staphylococcal enterotoxin-producing S. aureus strains need to be further explored to obtain more complete knowledge about the virulence of this intriguing pathogen.

The Bacillus cereus group comprises genetical closely related species with variable toxigenic characteristics. However, detection and differentiation of the B. cereus group species in routine diagnostics can be difficult, expensive and laborious since current species designation is linked to specific phenotypic characteristic or the presence of species-specific genes. Especially the differentiation of Bacillus cereus and Bacillus thuringiensis , the identification of psychrotolerant Bacillus mycoides and Bacillus weihenstephanensis , as well as the identification of emetic B. cereus and Bacillus cytotoxicu s, which are both producing highly potent toxins, is of high importance in food microbiology. Thus, we investigated the use of a machine learning approach, based on artificial neural network (ANN) assisted Fourier transform infrared (FTIR) spectroscopy, for discrimination of B. cereus group members. The deep learning tool box of Matlab was employed to construct a one-level ANN, allowing the discrimination of the aforementioned B. cereus group members. This model resulted in 100% correct identification for the training set and 99.5% correct identification overall. The established ANN was applied to investigate the composition of B. cereus group members in soil, as a natural habitat of B. cereus , and in food samples originating from foodborne outbreaks. These analyses revealed a high complexity of B. cereus group populations, not only in soil samples but also in the samples from the foodborne outbreaks, highlighting the importance of taking multiple isolates from samples implicated in food poisonings. Notable, in contrast to the soil samples, no bacteria belonging to the psychrotolerant B. cereus group members were detected in the food samples linked to foodborne outbreaks, while the overall abundancy of B. thuringiensis did not significantly differ between the sample categories. None of the isolates was classified as B. cytotoxicus , fostering the hypothesis that the latter species is linked to very specific ecological niches. Overall, our work shows that machine learning assisted (FTIR) spectroscopy is suitable for identification of B. cereus group members in routine diagnostics and outbreak investigations. In addition, it is a promising tool to explore the natural habitats of B. cereus group, such as soil.

Bacillus cytotoxicus is a member of the Bacillus cereus group linked to fatal cases of diarrheal disease. Information on B. cytotoxicus is very limited; in particular comprehensive genomic data is lacking. Thus, we applied a genomic approach to characterize B. cytotoxicus and decipher its population structure. To this end, complete genomes of ten B. cytotoxicus were sequenced and compared to the four publicly available full B. cytotoxicus genomes and genomes of other B. cereus group members. Average nucleotide identity, core genome, and pan genome clustering resulted in clear distinction of B. cytotoxicus strains from other strains of the B. cereus group. Genomic content analyses showed that a hydroxyphenylalanine operon is present in B. cytotoxicus, but absent in all other members of the B. cereus group. It enables degradation of aromatic compounds to succinate and pyruvate and was likely acquired from another Bacillus species. It allows for utilization of tyrosine and might have given a B. cytotoxicus ancestor an evolutionary advantage resulting in species differentiation. Plasmid content showed that B. cytotoxicus is flexible in exchanging genes, allowing for quick adaptation to the environment. Genome-based phylogenetic analyses divided the B. cytotoxicus strains into four clades that also differed in virulence gene content.

Mashed potato powder as well as powdered infant formula (PIF) are frequently contaminated with Bacillus cereus sensu lato (B. cereus s.l.), mainly with its spores. These products have also been implicated in foodborne illnesses. Here, we characterized B. cereus s.l. isolates originating from powdered products based on sporulation assays, toxin gene profiling, and panC typing combined with a SplitsTree analysis. Furthermore, cytotoxicity assays with B. cytotoxicus isolates were performed. 78% of PIF tested positive for B. cereus s.l., whereas 92% of all mashed potato powders were positive. In total, 43 isolates were further characterized. The nhe and cytK2 genes were most frequently detected. Moreover, a cereulide-producer was detected from PIF. Most isolates were assigned to panC group III, but members of group II, IV, V, and VII could also be found. Nine B. cytotoxicus were isolated out of nine mashed potato powders. All panC group VII isolates were positive for cytK1. Cytotoxicity assays of these nine isolates revealed one highly cytotoxic strain, while all other isolates exhibited no detectable cytotoxicity, underpinning that cytotoxicity of a certain B. cereus group strain cannot be deduced from the sole presence or absence of toxin genes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

While the relevance of Bacillus (B.) cereus as a major cause of gastroenteritis is undisputed, the role of the closely related B. thuringiensis in foodborne disease is unclear. B. thuringiensis strains frequently harbor enterotoxin genes. However, the organism has only very rarely been associated with foodborne outbreaks, possibly due to the fact that during outbreak investigations, B. cereus is routinely not differentiated from B. thuringiensis . A recent EFSA scientific opinion stresses the urgent need for further data allowing for improved risk assessment, in particular as B. thuringiensis is a commonly used biopesticide. Therefore, the aim of this study was to gain further insights into the hazardous potential of B. thuringiensis . To this end, 39 B. thuringiensis isolates obtained from commercially used biopesticides, various food sources, as well as from foodborne outbreaks were characterized by panC typing, panC -based SplitsTree analysis, toxin gene profiling, FTIR spectroscopic analysis, a cytotoxicity assay screening for enterotoxic activity, and a sphingomyelinase assay. The majority of the tested B. thuringiensis isolates exhibited low (23%, n = 9) or mid level enterotoxicity (74%, n = 29), and produced either no (59%, n = 23) or low levels (33%, n = 13) of sphingomyelinase, which is reported to act synergistically with enterotoxins Nhe and Hbl. One strain isolated from rosemary was however classified as highly enterotoxic surpassing the cytotoxic activity of the high-level reference strain by a factor of 1.5. This strain also produced vast amounts of sphingomyelinase. Combining all results obtained in this study into a fingerprint pattern, several enterotoxic biopesticide strains were indistinguishable from those of isolates from foods or collected in association with outbreaks. Our study shows that many B. thuringiensis biopesticide strains exhibit mid-level cytotoxicity in a Vero cell assay and that some of these strains cannot be differentiated from isolates collected from foods or in association with outbreaks. Thus, we demonstrate that the use of B. thuringiensis strains as biopesticides can represent a food safety risk, underpinning the importance of assessing the hazardous potential of each strain and formulation used.

Heated food is known to be often contaminated with B. cereus , leading to cases of diarrhoeal or emetic diseases. Battalion kitchens or army catering facilities present a food safety risk, as temperature abuse and long storage time can result in serious public health problems affecting a high number of served people. In contrast to civil catering facilities, no microbiological monitoring systems are currently implemented in Swiss military kitchens. In this study toxin gene profiles and cytotoxicity levels of 21 isolates of B. cereus originating from six different food categories were determined. Nearly all isolates (95%) harbored the nhe gene, whereas no hbl could be detected. Seven isolates displayed the cytK2 gene and one cereulide-producer was isolated out of vegetables. While most isolates displayed low cytotoxicity, highly cytotoxic strains were detected, with three isolates even exceeding the cytotoxicity level of the reference strain for high-level toxin production, underpinning that cytotoxicity cannot be deduced only from presence or absence of toxin genes. These findings further underline the importance of rapid cooling of foods or maintenance over 65°C before serving. This is especially important in mass catering facilities, such as military kitchens, in which food is often prepared a long time in advance., Competing Interests: Conflict of interest: the authors declare no potential conflict of interest.

Streptococcus agalactiae is a leading cause of morbidity and mortality among neonates and causes severe infections in pregnant women and nonpregnant predisposed adults, in addition to various animal species worldwide. Still, information on the population structure of S. agalactiae and the geographical distribution of different clones is limited. Further data are urgently needed to identify particularly successful clones and obtain insights into possible routes of transmission within one host species and across species borders. We aimed to determine the population structure and virulence gene profiles of S. agalactiae strains from a diverse set of sources and geographical origins. To this end, 373 S. agalactiae isolates obtained from humans and animals from five different continents were typed by DNA microarray profiling. A total of 242 different S. agalactiae strains were identified and further analyzed. Particularly successful clonal lineages, hybridization patterns, and strains were identified that were spread across different continents and/or were present in more than one host species. In particular, several strains were detected in both humans and cattle, and several canine strains were also detected in samples from human, bovine, and porcine hosts. The findings of our study suggest that although S. agalactiae is well adapted to various hosts including humans, cattle, dogs, rodents, and fish, interspecies transmission is possible and occurs between humans and cows, dogs, and rabbits. The virulence and resistance gene profiles presented enable new insights into interspecies transmission and make a crucial contribution to the identification of suitable targets for therapeutic agents and vaccines.

Surface carbohydrate moieties are essential for bacterial communication, phage-bacteria and host-pathogen interaction. Most Staphylococcus aureus produce polyribitolphosphate type Wall teichoic acids (WTAs) substituted with α- and/or β-O-linked N-acetyl-glucosamine (α-/β-O-GlcNAc) residues. GlcNAc modifications have attracted particular interest, as they were shown to govern staphylococcal adhesion to host cells, to promote phage susceptibility conferring beta-lactam resistance and are an important target for antimicrobial agents and vaccines. However, there is a lack of rapid, reliable, and convenient methods to detect and quantify these sugar residues. Whole cell Fourier transform infrared (FTIR) spectroscopy could meet these demands and was employed to analyse WTAs and WTA glycosylation in S. aureus. Using S. aureus mutants, we found that a complete loss of WTA expression resulted in strong FTIR spectral perturbations mainly related to carbohydrates and phosphorus-containing molecules. We could demonstrate that α- or β-O-GlcNAc WTA substituents can be clearly differentiated by chemometrically assisted FTIR spectroscopy. Our results suggest that whole cell FTIR spectroscopy represents a powerful and reliable method for large scale analysis of WTA glycosylation, thus opening up a complete new range of options for deciphering the staphylococcal pathogenesis related glycocode.

Staphylococcal food poisoning (SFP) is the most prevalent cause of food-borne intoxications worldwide. Consumption of enterotoxins preformed in food causes violent vomiting and can be fatal in children and the elderly. While being repressed by competing bacteria in most matrices, Staphylococcus aureus benefits from crucial competitive advantages in foods with high osmolarity or low pH. During recent years, the long-standing belief in the feasibility of assessing SFP risk based on colony-forming units of S. aureus present in food products has been disproven. Instead, researchers and food business operators are acutely aware of the imminent threat arising from unforeseeable enterotoxin production under stress conditions. This paradigm shift led to a variety of new publications enabling an improved understanding of enterotoxin expression under stress conditions encountered in food. The wealth of data provided by these studies is extremely diverse, as it is based on different methodological approaches, staphylococcal strains, stressors, and enterotoxins. Therefore, in this review, we aggregated and critically evaluated the complex findings of these studies, to provide readers with a current overview of the state of research in the field., Competing Interests: The authors declare no conflict of interest.

Staphylococcal food poisoning (SFP) is a common cause of foodborne illness worldwide, and enterotoxin D (SED) is one of the most frequent Staphylococcus aureus enterotoxins associated with it. It has been reported that the expression and formation of SED in S. aureus is regulated by the quorum sensing Agr system. In this study, the effect of agr deletion on sed expression in S. aureus grown on boiled ham was investigated. Growth, sed mRNA and SED protein levels in an S. aureus wild type strain and its isogenic Δ agr mutant were monitored for 14 days at 22 °C. The results showed that although deletion of the agr gene did not affect the growth rate or maximum cell density of S. aureus on boiled ham, it had a pronounced effect on SED formation during the first 5 days of incubation. The SED concentration was not reflected in the amount of preceding sed transcripts, suggesting that sed transcription levels may not always reflect SED formation. The expression of RNAIII transcript, the regulatory signal of the Agr system, was also monitored. Similar transcription patterns were observed for RNAIII and sed . Surprisingly, in the Δ agr mutant, sed expression was comparable to that in the wild type strain, and was thus unaffected by deletion of the Agr system. These results demonstrate that the Agr system appears to only partially affect SED formation, even in a real food environment.

We present the draft genome sequence of an atypical tetracycline-susceptible livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) strain. It contains 2,817,340 bp and 2,858 coding sequences, including 6 rRNA operons, 56 tRNAs, and 4 noncoding RNA (ncRNA) genes. The strain harbors a tet (M) gene, but 15 point mutations in amino acids are present that likely impair the functionality of TetM., (Copyright © 2017 Stevens et al.)

Staphylococcal enterotoxin B (SEB) causes staphylococcal food poisoning and is produced in up to ten times higher quantities than other major enterotoxins. While Staphylococcus aureus growth is often repressed by competing flora, the organism exhibits a decisive growth advantage under some stress conditions. So far, data on the influence of food-related stressors and regulatory mutations on seb expression is limited and largely based on laboratory strains, which were later reported to harbor mutations. Therefore, the aim of this study was to investigate the influence of stress and regulatory mutations on seb promoter activity. To this end, transcriptional fusions were created in two strains, USA300 and HG003, carrying different seb upstream sequences fused to a blaZ reporter. NaCl, nitrite, and glucose stress led to significantly decreased seb promoter activity, while lactic acid stress resulted in significantly increased seb promoter activity. Loss of agr decreased seb promoter activity and loss of sigB increased promoter activity, with the magnitude of change depending on the strain. These results demonstrate that mild stress conditions encountered during food production and preservation can induce significant changes in seb promoter activity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Staphylococcus aureus frequently isolated from milk products in sub-Saharan Africa (SSA) is a major pathogen responsible for food intoxication, human and animal diseases. SSA hospital-derived strains are well studied but data on the population structure of foodborne S. aureus required to identify possible staphylococcal food poisoning sources is lacking. Therefore, the aim was to assess the population genetic structure, virulence and antibiotic resistance genes associated with milk-derived S. aureus isolates from Côte d'Ivoire, Kenya and Somalia through spa-typing, MLST, and DNA microarray analysis. Seventy milk S. aureus isolates from the three countries were assigned to 27 spa (7 new) and 23 (12 new) MLST sequence types. Milk-associated S. aureus of the three countries is genetically diverse comprising human and livestock-associated clonal complexes (CCs) predominated by the CC5 (n = 10) and CC30 (n = 9) isolates. Panton-Valentine leukocidin, toxic shock syndrome toxin and enterotoxin encoding genes were predominantly observed among human-associated CCs. Penicillin, fosfomycin and tetracycline, but not methicillin resistance genes were frequently detected. Our findings indicate that milk-associated S. aureus in SSA originates from human and animal sources alike highlighting the need for an overarching One Health approach to reduce S. aureus disease burdens through improving production processes, animal care and hygienic measures., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of nosocomial infections and a major public health concern worldwide. During the last decade, MRSA of CC398 have emerged as important colonizers of livestock. These strains also represent an increasing cause of human infections. A recent study reporting a new dominant spa type among MRSA from Finish fattening pigs (CC398/t2741) identified a strain lacking both the global virulence regulator gene locus agr and the adhesion gene fnbB. The aim of this study was to characterize this agr/fnbB-negative livestock-associated MRSA strain in terms of growth, hemolysis and adhesive capacity, and to provide data on its genomic background. To this end, growth curves and hemolysis patterns were generated and adhesion assays on human keratinocyte and porcine nasal mucosa cell lines were performed. Whole genome sequencing was used to determine the nature and extent of the relevant deletions in the livestock strains. For comparison, an agr-positive, fnbB-negative CC398/t2741 strain from the same pig herd, an agr/fnbB- positive CC398/t034 strain from another pig herd and one human MRSA strain and its isogenic Δagr knockout mutant were used. The agr-negative strains adhered significantly better to human and porcine host cells than the agr-positive control strains. For the agr-positive porcine MRSA strains, cytotoxic effects on porcine mucosal cells were observed. The strong adhesive capacity of the naturally agr-negative livestock-associated MRSA, in combination with diminished cytotoxic effects, is likely favorable for inducing persistent colonization in pigs. Independently of the host cell type, similar adhesive capacities of the naturally agr-negative livestock-associated MRSA and the human MRSA strain were shown. Our results indicate that loss of agr in the livestock-associated MRSA strain investigated in this study may have increased its potential to be transmitted to and amongst humans., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Staphylococcus aureus 1608 is a strain that caused a lethal mastitis in cows. Here, the draft genome sequence of the strain is presented., (Copyright © 2017 Stevens et al.)

Ingestion of staphylococcal enterotoxins preformed by Staphylococcus aureus in food leads to staphylococcal food poisoning, the most prevalent foodborne intoxication worldwide. There are five major staphylococcal enterotoxins: SEA, SEB, SEC, SED, and SEE. While variants of these toxins have been described and were linked to specific hosts or levels or enterotoxin production, data on sequence variation is still limited. In this study, we aim to extend the knowledge on promoter and gene variants of the major enterotoxins SEB, SEC, and SED. To this end, we determined seb, sec, and sed promoter and gene sequences of a well-characterized set of enterotoxigenic Staphylococcus aureus strains originating from foodborne outbreaks, human infections, human nasal colonization, rabbits, and cattle. New nucleotide sequence variants were detected for all three enterotoxins and a novel amino acid sequence variant of SED was detected in a strain associated with human nasal colonization. While the seb promoter and gene sequences exhibited a high degree of variability, the sec and sed promoter and gene were more conserved. Interestingly, a truncated variant of sed was detected in all tested sed harboring rabbit strains. The generated data represents a further step towards improved understanding of strain-specific differences in enterotoxin expression and host-specific variation in enterotoxin sequences.