Comparison of the performance of multiple whole-genome sequence-based tools for the identification of Bacillus cereus sensu stricto biovar Thuringiensis.

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.

Stress Lowers Staphylococcal Enterotoxin C Production Independently of Agr, SarA, and SigB.

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.

PorinPredict: In Silico Identification of OprD Loss from WGS Data for Improved Genotype-Phenotype Predictions of P. aeruginosa Carbapenem Resistance.

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.

Ropiness in Bread-A Re-Emerging Spoilage Phenomenon.

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.

Nitrite stress increases staphylococcal enterotoxin C transcription and triggers the SigB regulon.

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.

Mild NaCl Stress Influences Staphylococcal Enterotoxin C Transcription in a Time-Dependent Manner and Reduces Protein Expression.

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.

Recent paradigm shifts in the perception of the role of Bacillus thuringiensis in foodborne disease.

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.

Mild Lactic Acid Stress Causes Strain-Dependent Reduction in SEC Protein Levels.

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.

Further Insights into the Toxicity of Bacillus cytotoxicus Based on Toxin Gene Profiling and Vero Cell Cytotoxicity Assays.

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.

Whole Genome Sequencing Reveals Biopesticidal Origin of Bacillus thuringiensis in Foods.

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.

Staphylococcal Enterotoxin C-An Update on SEC Variants, Their Structure and Properties, and Their Role in Foodborne Intoxications.

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.

Draft Genome Sequence of CH_213, a Highly Cytotoxic Bacillus cytotoxicus Strain Isolated from Mashed Potatoes.

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.

Draft Genome Sequence of CH_48, a Highly Cytotoxic Bacillus thuringiensis Strain Isolated from Rosemary at the Retail Level.

Bacillus thuringiensis CH_48 exhibits extremely high levels of Vero cell cytotoxicity and sphingomyelinase activity.

DNA microarray-based characterization and antimicrobial resistance phenotypes of clinical MRSA strains from animal hosts.

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.

Staphylococcus aureus Population Structure and Genomic Profiles in Asymptomatic Carriers in Switzerland.

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.

Temperature-Dependent Growth Characteristics of Bacillus thuringiensis in a Ratatouille Food Model.

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.

Population structure and toxin gene profiles of Bacillus cereus sensu lato isolated from flour products.

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.

Microarray based genetic profiling of Staphylococcus aureus isolated from abattoir byproducts of pork origin.

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.

The Role of Regulatory Mechanisms and Environmental Parameters in Staphylococcal Food Poisoning and Resulting Challenges to Risk Assessment.

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.

Detection and Identification of Bacillus cereus, Bacillus cytotoxicus, Bacillus thuringiensis, Bacillus mycoides and Bacillus weihenstephanensis via Machine Learning Based FTIR Spectroscopy.

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 cytotoxicus, 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.