Quirks of dye nomenclature. 3. Trypan blue

Trypan blue is colorant from the 19^th century that has an association with Africa as a chemotherapeutic agent against protozoan (Trypanosomal) infections, which cause sleeping sickness. The dye still is used for staining biopsies, living cells and organisms, and it also has been used as a colorant for textiles.     

Modelling the number of viable vegetative cells of Bacillus cereus passing through the stomach

Aims:  Model the number of viable vegetative cells of B. cereus surviving the gastric passage after experiments in simulated gastric conditions.
Materials and Methods:  The inactivation of stationary and exponential phase vegetative cells of twelve different strains of Bacillus cereus , both mesophilic and psychrotrophic strains isolated from food and faeces from healthy and ill individuals, in simulated gastric conditions was determined using decimal reduction times at low pH ( D _pH). Subsequently inactivation rates were calculated. Inclusion of the inactivation rates into models describing the course of the gastric pH after the consumption of meal of solid food and the transfer of food from the stomach to the small intestine resulted in numbers of viable Bacillus cereus vegetative cells able to pass the stomach.
Conclusions:  According to the model, 3–26% of the ingested vegetative cells from Bacillus cereus may survive the gastric passage, dependent on the growth phase of the vegetative cells, the type of strains, and the age of the consumer.
Significance and Impact of the Study:  Vegetative cells of Bacillus cereus may be involved in the onset of diarrhoeal disease to a greater extent than expected since up to 26% of the ingested cells survive simulated gastric conditions.     

Bacillus cereus can attack the cell membranes of the alga Chara corallina by means of HlyII

We studied the influence of Bacillus cereus bacteria on cells of the freshwater alga Chara corallina. These bacteria and recombinant Bacillus subtilis strains are capable of producing the secreted toxin HlyII, which changes the electrophysiological parameters of the algal electrically excitable plasma membrane by forming pores. Cooperative incubation of bacterial cells, which carry active hlyII gene, and Chara corallina cells caused a decrease in the resting potential (V(m)) and plasma membrane resistance (R(m)) of algal cells. The efficiency of each strain was commensurable with its ability to produce HlyII. Purified hemolysin II caused a similar effect on V(m) and R(m) of intact and perfused cells. This protein changed the kinetics and magnitude of transient voltage-dependent calcium and calcium-activated chloride currents owing to the formation of additional Ca(2+)-permeable pores in algal cell membrane. Occurrence of the cellulose cell wall with pores 2.1 to 4.6nm in diameter suggests that HlyII molecules reach the plasma membrane surface strictly as monomers.     

Heat resistance of Bacillus cereus emetic toxin, cereulide

Aims: The study describes the effects of heating temperature and exposure time on the thermal stability of cereulide under different conditions (pH, presence/absence of oil phase and cereulide concentration). Methods and Results: Cereulide heat inactivation was investigated at 100, 121 and 150°C under different alkaline pH values (8.6–10.6) and in the presence of oil phase (0.6–1.4%). Three different cereulide concentrations (0.5, 5 and 6 μg ml^?1) were used. Cereulide detection was performed with computer‐aided semen analyzer and with HPLC–MS. Highly alkaline pH was needed to achieve inactivation. At lower cereulide concentrations less drastic conditions were needed. Removal of alkaline buffer after the heat treatment resulted in the recovery of toxic activity. Conclusions: Heat stability of cereulide has been proved to be remarkable, even at highly alkaline pH values, at all temperatures tested. The loss of activity appeared to be reversible. Significance and Impact of the Study: The study demonstrates the inability of any heat treatment used in the food industry to inactivate cereulide. Food safety has to rely on prevention and cold chain maintenance. Cleaning practices also need to be adapted as cereulide may remain in its active form upon sterilization of used material.     

蜡样芽胞杆菌在双歧杆菌四联活菌片治疗小鼠腹泻中的作用研究

目的研究双歧杆菌四联活菌片中的蜡样芽胞杆菌对小鼠腹泻治疗作用的影响。方法将SPF小鼠采用抗生素联合伤寒沙门菌和志贺菌诱导建立腹泻模型,取112只造模成功后的小鼠随机分为7组,其中三组分别喂养3种不同剂量的四联活菌片,三组分别喂养3种不同剂量的不含蜡样芽胞杆菌的三联活菌片,余下一组为对照组灌胃生理盐水。比较各组小鼠腹泻的治愈时间,测定粪便菌群、结肠组织耗氧能力及氧化还原电势、肠内sIgA含量及脾脏指数变化。结果相同剂量条件下,含蜡样芽胞杆菌的四联活菌片治愈腹泻时间比三联活菌片缩短48~72h。治疗48h后,四联活菌片组肠内sIgA水平明显增加,与三联活菌片组相比差异有统计学意义(t=5.5783,P=0.0002);治疗结束后,四联活菌片组的脾脏指数也显著高于三联活菌片组(t=18.8648,P=0.0001)。结论含蜡样芽胞杆菌的四联活菌片对小鼠腹泻的治疗及肠道菌群的恢复作用比三联活菌片效果更佳,能显著促进腹泻小鼠肠黏膜局部免疫力的提高。     

Bacillus cereus pore-forming toxins hemolysin II and cytotoxin K: polymorphism and distribution of genes among representatives of the cereus group

Abstract-Phylogenetic interrelation between 40 strains of the Bacillus cereus group has been established using BcREP fingerprinting. The PCR method has shown that the frequency of occurrence of the genes of cytotoxin K (cytK) and hemolysin II (hlyII) is 61% and 56%, respectively, and the gene of the hemolysin II regulator (hlyIIR) occurs together with hlyII. Comparison of the results of fingerprinting, PCR, and RFLP of the toxin genes showed that bacteria with the hlyII+ and cytK+ genotypes did not form separate clusters. However, microorganisms with the similar fingerprints were shown to have toxin genes of the same type. The proposed variant of RFLP analysis made it possible to clearly distinguish between the cytK1 and cytK2 genes. Twenty-three strains having the cytK genes carried no cytK1 dangerous for mammals. Additionally, the entire collection of microorganisms was tested for the ability to grow at 4 degrees C. This property was revealed for five strains, which should most likely be classified as B. weihenstephanensis. Two of the five psychrotolerant microorganisms carried the hemolysin II gene variant of the same type according to RFLP. None of the five strains had the cytK gene. These strains did not form close groups upon clustering by the applied method of Bc-REP fingerprints.     

The toxicological spectrum of the Bacillus cereus toxin cereulide points towards niche-specific specialisation

Most microbes share their environmental niches with very different forms of life thereby engaging in specialised relationships to enable their persistence. The bacterium Bacillus cereus occurs ubiquitously in the environment with certain strain backgrounds causing foodborne and opportunistic infections in humans. The emetic lineage of B. cereus is capable of producing the toxin cereulide, which evokes emetic illnesses. Although food products favouring the accumulation of cereulide are known, the ecological role of cereulide and the environmental niche of emetic B. cereus remain elusive. To better understand the ecology of cereulide-producing B. cereus, we systematically assayed the toxicological spectrum of cereulide on a variety of organisms belonging to different kingdoms. As cereulide is a potassium ionophore, we further tested the effect of environmental potassium levels on the action of cereulide. We found that adverse effects of cereulide exposure are species-specific, which can be exacerbated with increased environmental potassium. Additionally, we demonstrate that cereulide is produced within an insect cadaver indicating its potential ecological function for a saprophytic lifestyle. Collectively, distinct cereulide susceptibilities of other organisms may reflect its role in enabling competitive niche specialization of emetic B . cereus.     

Heat-stable toxin production by strains of Bacillus cereus, Bacillus firmus, Bacillus megaterium, Bacillus simplex and Bacillus licheniformis

Strains of Bacillus cereus can produce a heat-stable toxin (cereulide). In this study, 101 Bacillus strains representing 7 Bacillus species were tested for production of heat-stable toxins. Strains of B. megaterium, B. firmus and B. simplex were found to produce novel heat-stable toxins, which showed varying levels of toxicity. B. cereus strains (18 out of 54) were positive for toxin production. Thirteen were of serovar H1, and it was of interest that some were of clinical origin. Two were of serovars 17B and 20, which are not usually implicated in the emetic syndrome. Partial purification of the novel B. megaterium, B. simplex and B. firmus toxins showed they had similar physical characteristics to the B. cereus emetic toxin, cereulide.     

Antifungal activity displayed by cereulide, the emetic toxin produced by Bacillus cereus

In this study, the fungistatic activity of Bacillus cereus cereulide-producing strains was demonstrated against nine fungal species. The role of cereulide was confirmed using plasmid-cured derivatives and ces knockout mutants. The fungistatic spectra of cereulide and valinomycin, a chemically related cyclododecadepsipeptide, were also compared and found to be similar but distinct.     

Transformation of Bacillus cereus vegetative cells by electroporation

Transformation of untreated vegetative cells of Bacillus cereus 569 with plasmid pC194 (1.8 megadaltons) by high-voltage electroporation resulted in a maximum of 2 x 10(-5) transformants per viable cell. Transformation of a 130-megadalton plasmid occurred at a comparable frequency. The method was simple, rapid, and yielded transformant colonies in 14 to 24 h. Transformation was obtained with unpurified total plasmid DNA.     

Helix 4 mutants of the Bacillus thuringiensis insecticidal toxin Cry1Aa display altered pore-forming abilities

The role played by alpha-helix 4 of the Bacillus thuringiensis toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis. The majority of the resulting mutant proteins were considerably less toxic to Manduca sexta larvae than Cry1Aa. Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix. Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation. For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to N-methyl-D-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation. These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores. They provide further evidence that alpha-helix 4 plays a crucial role in the mechanism of pore formation.     

Bacillus cereus is common in the environment but emetic toxin producing isolates are rare

AIMS: To determine the incidence of emetic toxin producing Bacillus cereus in soil, animal faeces and selected vegetable produce to compare the results with the previously reported high incidence in rice paddy fields. To examine whether the emetic toxin has antibiotic activity. METHODS AND RESULTS: The incidence of emetic toxin producing B. cereus was evaluated by plating on selective agar 271 samples of soils, animal faeces, raw and processed vegetables. Overall, 45.8% of samples were positive for B. cereus. One hundred and seventy-seven B. cereus isolates were recovered at 30 degrees C with the grand mean spore count being 2.6 +/- 1.7 log(10) CFU g(-1) and 148 B. cereus isolates were recovered at 7 degrees C with the grand mean spore count being 2.2 +/- 1.2 log(10) CFU g(-1) of the 177 B. cereus isolated at 30 degrees C, only 3 were positive for emetic toxin production at a titre of 1/64, 1/32, 1/16, respectively. Also, 1 of 148 B. cereus isolated at 7 degrees C was positive for emetic toxin production to a titre of 1/128. All positive isolates came from washed or unwashed potato skins, one was psychrotrophic as determined by PCR and growth at 7 degrees C on subculture. The emetic toxin was not shown to have any antibiotic effects in growth inhibition studies. CONCLUSIONS: While B. cereus was a common isolate, the incidence of the emetic strain was rare. This is in contrast to previous findings of the high incidence in rice paddy fields and the processing environment, which may suggest rice is a selective area for growth of the emetic strain of B. cereus. SIGNIFICANCE AND IMPACT OF STUDY: The finding that a psychrotrophic isolate of B. cereus can produce emetic toxin is the first ever such observation and suggests the possibility that psychrotrophic isolates could grow in refrigerated fresh foods and cause emesis. The incidence of emetic B. cereus strains in rice paddy fields now requires further study for comparison with the low incidence found in other soils. The emetic toxin failed to inhibit the growth of other bacterial, fungal and yeast species. Whether the toxin (which is similar in structure to the antibiotic valinomycin) plays a competitive role in the environment therefore remains unclear.     

Fluorescence of Trypan blue in frozen-dried embryos of the rat

Freeze-drying and fluorescence microscopy techniques were combined to create a sensitive method for the visualization of the teratogenic dye, Trypan blue, in both protein-bound and free forms. In the development and initial application of this method, visceral yolk sacs of several gestational ages as well as normal appearing, 12-day embryos obtained from dye-injected rats were utilized. Observations on paraffinized sections of the yolk sac placentae demonstrated that only the protein-bound form of the dye exists in the yolk sac cavity whereas both forms of the dye exist in supranuclear regions of cells of the visceral endoderm. Paraffin sections of the normal appearing, 12-day embryos displayed the protein-bound form of dye within lumina of mid- and hind-gut, and both forms of dye in the primitive mucosa of mid- and hind-gut. The advantages of the method are derived not only from the use of fluorescence microscopy but also from the avoidance of solvents that are employed in more routine microtechniques.     

A novel dodecadepsipeptide, cereulide, is an emetic toxin of Bacillus cereus

Abstract A vacuole-formation substance, cereulide of Bacillus cereus , is an emetic toxin in animals. Both oral administration and intraperitoneal injection of cereulide caused dose-dependent emesis in Suncus murinus , a new animal model of emesis. Vagotomy or a 5-HT₃ receptor antagonist completely abolished this emetic effect. Therefore, cereulide causes emesis through the 5-HT₃ receptor and stimulation of the vagus afferent. We also found that our purified cereulide caused swelling of mitochondria of HEp-2 cells.