Toxins of Foodborne Pathogen Bacillus cereus and the Regulatory Factors Controlling the Biosynthesis of Its Toxins

Bacillus cereus is a versatile organism which causes two distinct types of food poisoning by producing toxins. Toxin formation in B. cereus is very much a complex process involving co-regulation of multiple genes exerting control at transcriptional, translational and post-translational level and such regulations too are often influenced by extrinsic factors. A comprehensive understanding of such factors is very crucial for holistic approaches and strategies in order to minimise food poisoning risk. Hence, this review will focus on the intrinsic and extrinsic factors controlling toxin biosynthesis in B. cereus.

Influence of L-lactate and low glucose concentrations on the metabolism and the toxin formation of Clostridioides difficile

The virulence of Clostridioides difficile (formerly Clostridium difficile) is mainly caused by its two toxins A and B. Their formation is significantly regulated by metabolic processes. Here we investigated the influence of various sugars (glucose, fructose, mannose, trehalose), sugar derivatives (mannitol and xylitol) and L-lactate on toxin synthesis. Fructose, mannose, trehalose, mannitol and xylitol in the growth medium resulted in an up to 2.2-fold increase of secreted toxin. Low glucose concentration of 2 g/L increased the toxin concentration 1.4-fold compared to growth without glucose, while high glucose concentrations in the growth medium (5 and 10 g/L) led to up to 6.6-fold decrease in toxin formation. Transcriptomic and metabolic investigation of the low glucose effect pointed towards an inactive CcpA and Rex regulatory system. L-lactate (500 mg/L) significantly reduced extracellular toxin formation. Transcriptome analyses of the later process revealed the induction of the lactose utilization operon encoding lactate racemase (larA), electron confurcating lactate dehydrogenase (CDIF630erm_01321) and the corresponding electron transfer flavoprotein (etfAB). Metabolome analyses revealed L-lactate consumption and the formation of pyruvate. The involved electron confurcation process might be responsible for the also observed reduction of the NAD+/NADH ratio which in turn is apparently linked to reduced toxin release from the cell.

Enterobacteria in the intestinal microbiocenosis of hivinfected children

Aim. The purpose was to assess the species composition, virulence factors and the nature of the interaction of Enterobacteria with intestinal microsymbionts in HIV-positive children.Material and methods. The composition of intestinal microbiocenosis of 89 HIV-infected children (main group) and 74 children without HIV status (comparison group) was studied. 273 strains of Enterobacteria were isolated and identified to the species. The frequency and level of expression of adhesion, invasion, toxin formation and antagonism factors were studied.Results. In HIV-positive children colonization rate of intestinal mucosa E. coli lac — was 5 times higher, E. coli hly+ 1.5 times higher than in the comparison group. The quantitative level of opportunistic enterobacteria increases with the aggravation of microecological disorders, 98% of enterobacteria forms a many-to-many association. Of these, 86.8% are neutral towards other symbionts. The frequency and level of production of adhesion, invasion and toxin formation factors did not differ in the compared groups.Conclusion. In HIV-infected children is formed enterobacteriales type microbiota. Biological properties of enterobacteria did not depend on the presence of HIV status in children.

The impact of pH on Clostridioides difficile sporulation and physiology

ABSTRACTClostridioides difficile is a pathogenic bacterium that infects the human colon to cause diarrheal disease. Growth of the bacterium is known to be dependent on certain bile acids, oxygen levels and nutrient availability in the intestine, but how the environmental pH can influence C. difficile is mostly unknown. Previous studies indicated that C. difficile modulates the intestinal pH, and prospective cohort studies have found a strong association between a more alkaline fecal pH and C. difficile infection. Based on these data we hypothesized that C. difficile physiology can be affected by various pH conditions. In this study, we investigated the impact of a range of pH conditions on C. difficile to assess potential effects on growth, sporulation, motility and toxin production in the strains 630∆erm and R20291. We observed pH-dependent differences in sporulation rate, spore morphology and viability. Sporulation frequency was lowest under acidic conditions, and differences in cell morphology were apparent at low pH. In alkaline environments, C. difficile sporulation was greater for strain 630∆erm, whereas R20291 produced relatively high levels of spores in a broad range of pH conditions. Rapid changes in pH during exponential growth impacted sporulation similarly among the strains. Furthermore, we observed an increase in C. difficile motility with increases in pH, and strain-dependent differences in toxin formation under acidic conditions. The data demonstrate that pH is an important parameter that affects C. difficile physiology and may reveal relevant insights into the growth and dissemination of this pathogen.IMPORTANCEClostridioides difficile is an anaerobic bacterium that causes gastrointestinal disease. C. difficile forms dormant spores, which can survive harsh environmental conditions, allowing their spread to new hosts. In this study, we determine how intestinally relevant pH conditions impact C. difficile physiology in the two divergent strains, 630∆erm and R20291. Our data demonstrate that low pH conditions reduce C. difficile growth, sporulation, and motility. However, toxin formation and spore morphology are differentially impacted in the strains at low pH. In addition, we observed that alkaline environments reduced C. difficile growth, but increased cell motility. When pH was adjusted rapidly during growth, we observed similar impacts on both strains. This study provides new insights into the phenotypic diversity of C. difficile grown under the diverse pH conditions present in the intestinal tract, and demonstrates similarities and differences in the pH responses of different C. difficile isolates.

MYCOTOXINS AND TOXIN-PRODUCING MICROSCOPIC FUNGI IN COTTON-SEED CAKE FROM TAJIKISTAN

The article presents the results of complex study of cotton-seed cake from the Republic of Tajikistan, including the determination of content of mycotoxins, the analysis of exposure to microscopic fungi and the assessment of the potential of their toxin formation. The ability to produce mycotoxins was established for 11 species of micromycetes belonging to the genus Aspergillus, Penicillium, Fusarium, Scop-ulariopsis, and Trichothecium. Alternariol, zearalenone, fu-monisins, aflatoxin B1, sterigmatocystin, ochratoxin A, citrinin and emodin were found in the composition of the cotton-seed cake. Prospects for further research and the practical significance of the results are discussed.

Suppression of cellular immunity factors by toxic fraction of supernatant of broth culture Pasteurella haemolytica

In the etiology of many infectious animal diseases, Pasteurella haemolytica belongs to a specific place. An important factor in the pathogenicity of this microorganism, like the Pasteurella multocida serotype D, is the thermostable exotoxin. It can be obtained from bacterial-purified culture fluid. Although the study of toxin formation among microorganisms is quite popular, however, the features of toxin formation in hemolytic pasteurals, depending on the virulence of the pathogen, the nature of the toxic effects of these objects in vivo remain unclear. Materials for research were 16 isolates of Pasteurella haemolytica, isolated from pathological and biological material obtained during the outbreaks of respiratory pathology in farm animals. Initially, the nature of the research concerned the establishment of the potential for toxin formation in the isolates obtained. The method provided for a comparative analysis of the DNA nucleotide sequences of each of the investigated isolates P. haemolytica and information obtained from the international database. Another part of the work concerned the actual allocation of the major groups of toxic components of Pasteurella haemolytica by extraction to determine their biochemical nature. Exotoxin isolation was carried out from the Pasteurella spp. The components of the sediment and supernatant were separated by ion exchange chromatography on TSK gels. In order to detect the harmful effects of toxin hemolytic pasteurals on the body, they used the method of determining the opsonic index (the ratio of the phagocytic number in the mixture without the products of toxic fractions to the mixture with the toxin-containing fraction). It has been established that an important factor of the pathogenetic effect in Pasteurella haemolytica is the toxic fraction. Electrophoregram analysis of the results of DNA amplification in a comparative aspect with the data of standard samples helped to determine the presence of elements of the genome, which indicate the potential for toxin formation in isolated hemolytic pasteurized isolates from the test material. Toxic fractions isolated from Pasteurella haemolytica broth culture supernatant are substances of protein-carbohydrate nature. The isolated peak toxicogenic fractions of dialysate of a bacterial culture sieve contained protein and carbohydrates within the limits of 12.5–20 μg / ml and 0–20 μg/ml, respectively. In the dialysate of the broth culture supernatant, where 5 groups of toxigenic fractions were identified, the content of protein and carbohydrates in them varied, respectively, in the range from 20 to 95 μg/ml and from 3.3 to 26.62 μg/ml. At reproduction of opsono-phagocytic reaction with participation of toxigenic fractions of hemolytic pasteurel, a sufficiently expressed immunosuppressive effect of these complexes on the body of warm-blooded substances with an opsonic index of 3 ± 0.03 was established. During further research it is planned to determine the dermal necrotic and lethal effects of the isolated toxicogenic fractions of hemolytic pasteuride on the body of warm-blooded ones.

Effect of Equilibrated pH and Indigenous Spoilage Microorganisms on the Inhibition of Proteolytic Clostridium botulinum Toxin Production in Experimental Meals under Temperature Abuse

ABSTRACT Clostridium botulinum is a foreseeable biological hazard in prepared refrigerated meals that needs to be addressed in food safety plans. The objective of this study was to evaluate the effect of product composition and storage temperature on the inhibition of botulinum toxin formation in nine experimental meals (meat, vegetable, or carbohydrate based). Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin in samples stored at 25°C for up to 96 h for phase 1, or at 25°C for 12 h and then transferred to 12.5°C for up to 12 and 6 weeks in phases 1 and 2, respectively. For phase 1, none of the treatments (equilibrated pH 5.8) supported toxin production when stored at 25°C for 48 h, but toxin production was observed in all treatments at 72 h. For the remaining experiments with storage at 12.5°C, toxin production was dependent on equilibrated pH, storage time, and growth of indigenous spoilage microorganisms. In phase 1, no gross spoilage and no botulinum toxin was detected for any treatment (pH ≤5.8) stored at 12.5°C for 12 weeks. In phase 2, gross spoilage varied by commodity, with the brussels sprouts meal with pH 6.5 showing the most rapid spoilage within 2 weeks and botulinum toxin detected at 5 and 6 weeks for the control and cultured celery juice treatments, respectively. In contrast, spoilage microbes decreased the pH of a pH 5.9 beef treatment by 1.0 unit, potentially inhibiting C. botulinum through 6 weeks at 12.5°C. None of the other treatments with pH 5.8 or below supported toxin production or spoilage. This study provides validation for preventive controls in refrigerated meals. These include equilibrated product pH and storage temperature and time to inhibit toxin formation by proteolytic C. botulinum, but the impact of indigenous microflora on safety and interpretation of challenge studies is also highlighted.

Effect of Cultured Celery Juice, Temperature, and Product Composition on the Inhibition of Proteolytic Clostridium botulinum Toxin Production

ABSTRACTClostridium botulinum may be of concern in prepared refrigerated meals, for which strict cold chain management cannot be guaranteed. This study evaluated the effect of temperature, product composition, and cultured celery juice powder (CCJP) as a source of nitrite on the inhibition of botulinum toxin formation in two experimental (meat- and vegetable-based) prepared meals. Data obtained from the challenge study were compared with a published mathematical model to determine whether the model is fail-safe with regard to the tested meals. Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin at appropriate intervals in samples stored at 10, 15, or 20°C for up to 8 weeks. None of the treatments stored at 10°C for 8 weeks supported toxin production by proteolytic C. botulinum. The addition of CCJP delayed toxin production by 1 and 3 weeks in cauliflower potatoes and in Dijon pork, respectively, stored at 15°C. Toxin production was delayed by 1 week at 20°C when CCJP was added to the cauliflower potatoes. This study found that the predictive model was fail-safe but was overly conservative for the experimental meals described. Finally, this study confirms that product composition, the addition of nitrite via CCJP, storage time, and temperature play important roles in the inhibition of toxin formation by proteolytic C. botulinum.