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

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

Clostridium strain FAM25158, a unique endospore-forming bacterium related to Clostridium tyrobutyricum and isolated from Emmental cheese shows low tolerance to salt

The genus Clostridium is a large and diverse group of species that can cause food spoilage, including late blowing defect (LBD) in cheese. In this study, we investigated the taxonomic status of strain FAM25158 isolated from Emmental cheese with LBD using a polyphasic taxonomic and comparative genomic approach. A 16S rRNA gene sequence phylogeny suggested affiliation to the Clostridium sensu stricto cluster, with Clostridium tyrobutyricum DSM 2637T being the closest related type strain (99.16% sequence similarity). Average Nucleotide Identity (ANI) analysis revealed that strain FAM25158 is at the species threshold with C. tyrobutyricum, with ANI values ranging from 94.70 to 95.26%, while the digital DNA-DNA hybridization values were below the recommended threshold, suggesting that FAM25158 is significantly different from C. tyrobutyricum at the genomic level. Moreover, comparative genomic analysis between FAM25158 and its four closest C. tyrobutyricum relatives revealed a diversity of metabolic pathways, with FAM25158 differing from other C. tyrobutyricum strains by the presence of genes such as scrA, srcB, and scrK, responsible for sucrose utilization, and the absence of many important functional genes associated with cold and osmolality adaptation, which was further supported by phenotypic analyses. Surprisingly, strain FAM25158 exhibited unique physiologic traits, such as an optimal growth temperature of 30°C, in contrast to its closest relatives, C. tyrobutyricum species with an optimal growth temperature of 37°C. Additionally, the growth of FAM25158 was inhibited at NaCl concentrations higher than 0.5%, a remarkable observation considering its origin from cheese. While the results of this study provide novel information on the genetic content of strain FAM25158, the relationship between its genetic content and the observed phenotype remains a topic requiring further investigation

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

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

Characterization of Clostridium tyrobutyricum Strains Using Three Different Typing Techniques

Clostridium tyrobutyricum is well known as one of the main causative agents of severe cheese spoilage. The metabolism of this anaerobic bacterium during ripening leads to textural and sensory defects in cheese and consequential loss of product value. The potential to induce cheese spoilage, however, may vary among different strains of the same species. Therefore, a better understanding of the intra-species diversity of C. tyrobutyricum may be of practical relevance for the dairy industry. In the present study, we compared the ability of three typing techniques to differentiate 95 C. tyrobutyricum strains on the subspecies level: (1) repetitive element palindromic PCR (rep-PCR) fingerprinting combined with conventional agarose gel electrophoresis, (2) hexaplex-PCR followed by an automated capillary electrophoresis and (3) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) typing. MALDI-TOF MS fingerprinting provided only moderate reproducibility and low discriminatory power. Both PCR-based methods were highly reproducible and discriminative, with hexaplex-PCR fingerprinting being slightly more discriminative than rep-PCR typing. Overall, a high intra-species diversity was observed among the tested strains, indicating that further investigations on the strain level may be of interest

Facilitating Resilience during an African Swine Fever Outbreak in the Austrian Pork Supply Chain through Hybrid Simulation Modelling

This study aimed to simulate the impact of an African swine fever (ASF) outbreak in Austria. ASF is one of the most significant and critical diseases for the global domestic pig population. Hence, the authors evaluated control strategies and identified bottlenecks during an ASF outbreak. A hybrid approach was selected, including discrete-event and agent-based simulation. An extended Susceptible-Exposed-Infectious-Recovered (SEIR) model (within a pig farm) and a standard SEIR model (between pig farms) were used to simulate the chain of infection. A total of 576 scenarios with several parameter variations were calculated to identify the influence of external factors on key performance indicators. The main results show a comparison between two control strategies anchored in law: a standard strategy (SS) and a preventive culling strategy (SC). The calculated scenarios show a difference between these strategies and indicate that with SC during an outbreak, fewer farms would be infected, and fewer pigs would be culled. Furthermore, specific geographical areas were identified, which—due to their density of pigs and farms—would be severely affected in case of an ASF outbreak. The analysis of bottlenecks in rendering plants (RPs) showed an increase in the number of days RPs were overutilized as the transmission rate increased. In addition, SS caused more days of overutilized RPs than SC

Probiotic Supplements Beneficially Affect Tryptophan–Kynurenine Metabolism and Reduce the Incidence of Upper Respiratory Tract Infections in Trained Athletes: A Randomized, Double-Blinded, Placebo-Controlled Trial

Background: Prolonged intense exercise has been associated with transient suppression of immune function and an increased risk of infections. In this context, the catabolism of amino acid tryptophan via kynurenine may play an important role. The present study examined the effect of a probiotic supplement on the incidence of upper respiratory tract infections (URTI) and the metabolism of aromatic amino acids after exhaustive aerobic exercise in trained athletes during three months of winter training. Methods: Thirty-three highly trained individuals were randomly assigned to probiotic (PRO, n = 17) or placebo (PLA, n = 16) groups using double blind procedures, receiving either 1 × 1010 colony forming units (CFU) of a multi-species probiotic (Bifidobacterium bifidum W23, Bifidobacterium lactis W51, Enterococcus faecium W54, Lactobacillus acidophilus W22, Lactobacillus brevis W63, and Lactococcus lactis W58) or placebo once per day for 12 weeks. The serum concentrations of tryptophan, phenylalanine and their primary catabolites kynurenine and tyrosine, as well as the concentration of the immune activation marker neopterin were determined at baseline and after 12 weeks, both at rest and immediately after exercise. Participants completed a daily diary to identify any infectious symptoms. Results: After 12 weeks of treatment, post-exercise tryptophan levels were lowered by 11% (a significant change) in the PLA group compared to the concentrations measured before the intervention (p = 0.02), but remained unchanged in the PRO group. The ratio of subjects taking the placebo who experienced one or more URTI symptoms was increased 2.2-fold compared to those on probiotics (PLA 0.79, PRO 0.35; p = 0.02). Conclusion: Data indicate reduced exercise-induced tryptophan degradation rates in the PRO group. Daily supplementation with probiotics limited exercise-induced drops in tryptophan levels and reduced the incidence of URTI, however, did not benefit athletic performance

Table_2_Clostridium strain FAM25158, a unique endospore-forming bacterium related to Clostridium tyrobutyricum and isolated from Emmental cheese shows low tolerance to salt.XLSX

The genus Clostridium is a large and diverse group of species that can cause food spoilage, including late blowing defect (LBD) in cheese. In this study, we investigated the taxonomic status of strain FAM25158 isolated from Emmental cheese with LBD using a polyphasic taxonomic and comparative genomic approach. A 16S rRNA gene sequence phylogeny suggested affiliation to the Clostridium sensu stricto cluster, with Clostridium tyrobutyricum DSM 2637T being the closest related type strain (99.16% sequence similarity). Average Nucleotide Identity (ANI) analysis revealed that strain FAM25158 is at the species threshold with C. tyrobutyricum, with ANI values ranging from 94.70 to 95.26%, while the digital DNA-DNA hybridization values were below the recommended threshold, suggesting that FAM25158 is significantly different from C. tyrobutyricum at the genomic level. Moreover, comparative genomic analysis between FAM25158 and its four closest C. tyrobutyricum relatives revealed a diversity of metabolic pathways, with FAM25158 differing from other C. tyrobutyricum strains by the presence of genes such as scrA, srcB, and scrK, responsible for sucrose utilization, and the absence of many important functional genes associated with cold and osmolality adaptation, which was further supported by phenotypic analyses. Surprisingly, strain FAM25158 exhibited unique physiologic traits, such as an optimal growth temperature of 30°C, in contrast to its closest relatives, C. tyrobutyricum species with an optimal growth temperature of 37°C. Additionally, the growth of FAM25158 was inhibited at NaCl concentrations higher than 0.5%, a remarkable observation considering its origin from cheese. While the results of this study provide novel information on the genetic content of strain FAM25158, the relationship between its genetic content and the observed phenotype remains a topic requiring further investigation.</p

Table_1_Clostridium strain FAM25158, a unique endospore-forming bacterium related to Clostridium tyrobutyricum and isolated from Emmental cheese shows low tolerance to salt.DOCX

The genus Clostridium is a large and diverse group of species that can cause food spoilage, including late blowing defect (LBD) in cheese. In this study, we investigated the taxonomic status of strain FAM25158 isolated from Emmental cheese with LBD using a polyphasic taxonomic and comparative genomic approach. A 16S rRNA gene sequence phylogeny suggested affiliation to the Clostridium sensu stricto cluster, with Clostridium tyrobutyricum DSM 2637T being the closest related type strain (99.16% sequence similarity). Average Nucleotide Identity (ANI) analysis revealed that strain FAM25158 is at the species threshold with C. tyrobutyricum, with ANI values ranging from 94.70 to 95.26%, while the digital DNA-DNA hybridization values were below the recommended threshold, suggesting that FAM25158 is significantly different from C. tyrobutyricum at the genomic level. Moreover, comparative genomic analysis between FAM25158 and its four closest C. tyrobutyricum relatives revealed a diversity of metabolic pathways, with FAM25158 differing from other C. tyrobutyricum strains by the presence of genes such as scrA, srcB, and scrK, responsible for sucrose utilization, and the absence of many important functional genes associated with cold and osmolality adaptation, which was further supported by phenotypic analyses. Surprisingly, strain FAM25158 exhibited unique physiologic traits, such as an optimal growth temperature of 30°C, in contrast to its closest relatives, C. tyrobutyricum species with an optimal growth temperature of 37°C. Additionally, the growth of FAM25158 was inhibited at NaCl concentrations higher than 0.5%, a remarkable observation considering its origin from cheese. While the results of this study provide novel information on the genetic content of strain FAM25158, the relationship between its genetic content and the observed phenotype remains a topic requiring further investigation.</p