HIV testing for HIV prevention: a comparative analysis of policies in Britain, Hungary and Sweden

Growth of vegetative cells and outgrowth of spores of enterotoxigenic psychrotrophic Bacillus cereus in refrigerated minimally processed food products is a public health concern. A study was undertaken to determine the combined effects of pH, nisin, and temperature on growth and survival of 20 strains of B. cereus. The minimum growth temperatures in tryptic soy broth (pH 7.3) and brain heart infusion broth (BHI broth, pH 7.4) were 5 degrees C for two strains and 8 degrees C for five other strains. Vegetative cells of four of eight strains grew at 8 degrees C in BHI broth (pH 6.01 and 6.57) containing 10 micrograms of nisin per ml. At 15 degrees C, all strains grew at pH 5.53 to 6.57; three strains tolerated nisin at 50 micrograms/ml (pH 6.57), whereas two other strains had a maximum tolerance of 10 micrograms of nisin per ml. Tolerance of vegetative cells of B. cereus to nisin increased as the pH of the broth was increased from 5.53 to 6.01 and again to pH 6.57. Outgrowth of spores (six of six strains tested) was inhibited by 5 and 50 micrograms of nisin per ml at 8 and 15 degrees C, respectively. At 15 degrees C, outgrowth of spores of two strains occurred at pH 6.52 in BHI broth containing 10 micrograms of nisin per ml. The effectiveness of nisin in controlling the growth of psychrotrophic strains of B. cereus capable of causing human illness was more pronounced at 8 degrees C than at 15 degrees C and as the pH was decreased from 6.57 to 5.53. Studies to determine the effectiveness of nisin in controlling growth of psychrotrophic B. cereus in nonpasteurized foods held at refrigeration temperatures are warranted.     

Toxin production by Bacillus cereus in dairy products

Spores of a known toxigenic and psychrotrophic dairy isolate of Bacillus cereus (HRM 44) were unable to grow and produce diarrhoeagenic toxin at 6 degrees C in creams and dairy-based products. These findings suggest that the production of B. cereus diarrhoeagenic toxin is unlikely to occur in creams and dairy-based products maintained within the cold chain. Growth and toxin production were readily demonstrated in creams and some desserts stored at 21 degrees C. Growth in creams was associated with obvious spoilage. However, in the flavoured desserts, spoilage was not always obvious before significant growth of B. cereus and toxin production had occurred. Dairy desserts with high sugar content and/or low pH did not support toxin production and these findings are discussed.     

Differential induction of interleukin-12, interleukin-18, and interleukin-1beta converting enzyme mRNA in experimental autoimmune encephalomyelitis of the Lewis rat

Four out of ten Bacillus cereus strains produced spores able to adhere to monolayers of Caco-2 cells (human epithelial cells). One of these strains has been involved in an outbreak of food poisoning where the symptoms were more severe and persisted for longer than a normal B. cereus food poisoning. The hydrophobicity of the spores is a contributing factor for the adhesion to occur. The spores are able to germinate in an environment similar to that of the small intestine and then the vegetative cells can produce the enterotoxin directly at the target place. A concentrated and active form of the enterotoxin will be taken up by the epithelial cells in the small intestine. Spore adhesion could be an important virulence factor for some B. cereus strains.     

Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

Identification of Bacillus cereus by Fourier transform infrared spectroscopy (FTIR)

The objective of this study was to evaluate the potential of Fourier transform infrared spectroscopy (FTIR) for rapid identification of Bacillus cereus isolates. Ten B. cereus group isolates (comprising B. cereus, Bacillus mycoides, and Bacillus thuringiensis strains), five other Bacillus spp., and five non-Bacillus spp. were used. Two types of media, brain heart infusion (BHI) and Trypticase soy agar (TSA), were tested. The results indicated that all B. cereus group isolates produced characteristic absorbance peaks at wave numbers between 1738 and 1740 cm-1. These peaks were not affected by the growth medium. None of the other bacteria tested showed a similar peak after growth on BHI or TSA. Absorbance peaks between 1800 and 1500 cm-1 of members of the B. cereus group had different shapes and sizes, suggesting that FTIR may be useful for rapid identification of species within the B. cereus group.     

Photoreactivation in the genus Bacillus

Photoreactivation of ultraviolet radiation-induced DNA damage was examined in exponential-phase cells of six mesophilic species of the genus Bacillus. Under the experimental conditions used, it was observed that the laboratory strains B. cereus strain T and B. thuringiensis var. thuringiensis strain NRRL-B4039 exhibited strong photoreactivation (86-fold and 70-fold respectively). Bacillus licheniformis strain ATCC 8480 exhibited moderate (15-fold) photoreactivation. Weak photoreactivation was observed in B. subtilis strain 168 (4-fold) and B. megaterium strain QM B1551 (3.4-fold). Bacillus amyloliquefaciens strain H demonstrated no detectable photoreactivation.     

Identification of contamination sources of Bacillus cereus in pasteurized milk

In order to determine the sources of Bacillus cereus in pasteurized milk, a total of 232 milk samples from various sampling points along milk processing lines and 122 environmental swabs were collected in two dairy plants between March and September, 1996. The incidence of B. cereus vegetative cells in raw milk from the plants was low (< or = 10%). However, the incidence and the average counts of B. cereus spores in the raw milk were very high and similar to those of B. cereus vegetative cells in pasteurized milk or final products after enrichment (> 80% and 1.1 x 10(5) cfu ml(-1), respectively). The incidence and average count of both vegetative cells and spores of B. cereus in environmental swabs was low. Using the microbial identification system (MIDI), a library of B. cereus fatty acid profiles comprising 229 B. cereus isolates from milk samples and environmental swabs was constructed using a critical Euclidian distance of 6.0 units as the cut-off value. Using this library, the relationship between 546 B. cereus isolates from the different sampling points along the milk processing lines and the environmental swabs was determined. Most B. cereus isolates obtained from the pasteurized milk and final products belonged to the same sub-groups as the B. cereus strains germinated from spores in raw milk. Furthermore, specific sub-groups were found in pasteurized milk, different dairy plants and at different sampling times. The results suggested that B. cereus spores in raw milk were the major source of B. cereus in pasteurized milk and that post-pasteurization contamination along the milk processing lines was possibly a minor source of B. cereus in pasteurized milk.     

Analysis of common antigen of flagella in Bacillus cereus and Bacillus thuringiensis

Flagellar antigen of Bacillus cereus H.1 was purified and tested for serodiagnostic antigen by ELISA. The antibody against the flagellar antigen of B. cereus H.1 reacted not only with the homologous specific antigen but also reacted with the flagellar antigens of 23 strains of B. cereus. This common flagellar antigen of B. cereus was found to be due to 61-kDa protein by SDS-PAGE and immunoblot assay. Monoclonal antibody H15A5 against common antigenic epitope of B. cereus also reacted with flagellar antigens of 21 strains of Bacillus thuringiensis by ELISA. This monoclonal antibody reacted with the 61-kDa protein of the flagella of B. cereus H.1 and H.2 and B. thuringiensis Kurstaki HD1, Alesti and Aizawai juroi by immunoblot analysis. These results indicated that the common antigenic epitope of the 61-kDa protein existed in the flagella both of B. cereus and B. thuringiensis.     

Non operative treatment of liver and spleen trauma. Clinical experience

A commercially available ELISA kit was used for the detection of Bacillus diarrhoeal enterotoxin (BDE) in a variety of foods and faeces. The ability of isolates of Bacillus spp., including Bacillus cereus, to produce BDE in Brain Heart Infusion broth containing 0.1% glucose was also checked by use of the kit. Results show that 29 out of 31 B. cereus isolates were enterotoxigenic. Foods positive for performed BDE were always contaminated with > 10(5) B. cereus cfu g-1, but not all foods contaminated with large numbers of B. cereus were positive for BDE. Bacillus sp., other than one isolate which closely resembled B. subtilis, were negative for BDE production. Criteria for the confirmation of Bacillus-mediated diarrhoea should now include reports of symptoms and incubation periods consistent with the diarrhoeal form of food-poisoning by Bacillus spp., together with the results of tests for enterotoxigenicity of the Bacillus isolate, and detection of BDE in either the food and/or faeces.     

Catalytic activity of thermolysin under extremes of pressure and temperature: modulation by metal ions

The catalytic activity of thermolysin (TL), a Zn-dependent neutral protease from Bacillus thermoproteolyticus, has been studied over a wide interval of pressures (1 bar to 4 kbar) and temperatures (20 degreesC to 80 degreesC) by monitoring hydrolysis of a low-molecular-mass substrate, 3-(2-furylacryloyl)-glycyl-L-leucine amide. This reaction shows a very large negative value for the activation volume and, because of that, simultaneous increase in temperature and pressure leads to a significant (up to 40-fold) acceleration of the reaction. At pressures higher than 2-2.5 kbar, the reaction rate starts to decrease due to disactivation of TL. This disactivation is explained in part by pressure-promoted dissociation of zinc ion from the active site and can be inhibited by adding exogenous zinc. Thus, this thermostable protease does not specifically show a higher stability at high pressure in comparison with small mesophilic proteases.     

Sensitivity of aerobic spore-forming species of the genus Bacillus to the pteridine compound O129

Discs containing the pteridine compound O129 at various concentrations may be useful in differentiating the following closely related species belonging to the genus Bacillus, viz. B. subtilis, B. licheniformis and B. pumilus; B. polymyxa and B. macerans; together with B. cereus and its subspecies B. cereus var. mycoides. At concentrations of 10 micrograms O129, the ATCC type strain of B. subtilis was resistant whereas B. licheniformis and B. pumilus were sensitive. However, B. subtilis was sensitive to O129 at 150 micrograms. Similar reactions differentiated the ATCC type strains of B. polymyxa and B. macerans. The ATCC type strain of B. cereus was resistant to O129 at both 150 micrograms and 10 micrograms, but its subspecies B. cereus var. mycoides (ATCC 28) was partially sensitive at 150 micrograms concentration. When clinical isolates of B. cereus var. mycoides were subsequently tested, this partial sensitivity was found to be a variable characteristic.     

Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues

To identify the critical sites for protein thermostabilization by proline substitution, the gene for oligo-1,6- glucosidase from a thermophilic Bacillus coagulans strain, ATCC 7050, was cloned as a 2.4-kb DNA fragment and sequenced. In spite of a big difference in their thermostabilities, B. coagulans oligo-1,6-glucosidase had a large number of points in its primary structure identical to respective points in the same enzymes from a mesophilic Bacillus cereus strain, ATCC 7064 (57%), and an obligately thermophilic Bacillus thermoglucosidasius strain, KP1006 (59%). The number of prolines (19 for B. cereus oligo-1,6-glucosidase, 24 for B. coagulans enzyme, and 32 for B. thermoglucosidasius enzyme) was observed to increase with the rise in thermostabilities of the oligo-1,6-glucosidases. Classification of proline residues in light of the amino acid sequence alignment and the protein structure revealed by X-ray crystallographic analysis also supported this tendency. Judging from proline residues occurring in B. coagulans oligo-1,6-glucosidase and the structural requirement for proline substitution (second site of the beta turn and first turn of the alpha helix) (K. Watanabe, T. Masuda, H. Ohashi, H. Mihara, and Y. Suzuki, Eur. J. Biochem. 226:277-283, 1994), the critical sites for thermostabilization were found to be Lys-121, Glu-290, Lys-457, and Glu-487 in B. cereus oligo-1,6-glucosidase. With regard to protein evolution, the oligo-1,6-glucosidases very likely follow the neutral theory. The adaptive mutations of the oligo-1,6-glucosidases that appear to increase thermostability are consistent with the substitution of proline residues for neutrally occurring residues. It is concluded that proline substitution is an important factor for the selection of thermostability in oligo-1,6-glucosidases.     

Alkaline-fermented foods: a review with emphasis on pidan fermentation

Alkaline-fermented foods constitute a group of less-known food products that are widely consumed in Southeast Asia and African countries. They can be made from different raw ingredients. For instance, Japanese natto, Thai thua-nao, and kinema are made from cooked soybeans, dawadawa from African locust beans, ogiri from melon seeds, ugba from African oil beans, kawal from fresh legale leaves, owoh from cotton seeds, and pidan from fresh poultry eggs. In alkaline-fermented foods, the protein of the raw materials is broken down into amino acids and peptides; ammonia is released during the fermentation, raising the pH of the final products and giving the food a strong ammoniacal smell. Most alkaline fermentations are achieved spontaneously by mixed bacteria cultures, principally dominated by Bacillus subtilis. In other cases, pure cultures can be used. For example, Japanese natto is inoculated with a pure culture of B. subtilis var natto. Pidan is a special example of alkaline fermentation. Instead of using microorganisms, pidan is made using an alkali-treated fermentation. Sodium hydroxide (NaOH) is produced from the reaction of sodium carbonate (Na2CO3), water (H2O), and calcium oxide (CaO) of pickle or coating mud. NaOH penetrates into the eggs, causing the physicochemical changes, color changes, and gelation. The appearance of pidan differs from fresh eggs in that the white becomes a semitransparent tea-brown color, and the yolk is solid or semisolid with a dark-green color. The nutritional value of pidan is slightly decreased compared with fresh eggs, but pidan has an extremely long shelf life and a pleasant, fragrant taste that is preferred by most people in Southeast Asian countries. In a small-scale laboratory study conducted by the authors, B. subtilis was not found in pidan. Four Staphylococcus spp. (S. cohnii, S. epidermidis, S. haemolyticus, and S. warneri) and two strains of Bacillus spp. (B. cereus and B. macerans) were isolated from pidan. Staphylococcus spp. did not contribute to the fermentation and were considered contaminants.     

A small, thermostable, and monofunctional chorismate mutase from the archaeon Methanococcus jannaschii

The gene for chorismate mutase (CM) from the archaeon Methanococcus jannaschii, an extreme thermophile, was subcloned and expressed in Escherichia coli. This gene, which belongs to the aroQ class of CMs, encodes a monofunctional enzyme (AroQf) able to complement the CM deficiency of an E. coli mutant strain. The purified protein follows Michaelis-Menten kinetics (kcat = 5.7 s-1 and Km = 41 microM at 30 degreesC) and displays pH-independent activity in the range of pH 5-9. Its activation parameters [Delta H = 16.2 kcal/mol, Delta S = -1. 7 cal/(mol.K)] are similar to those of another well characterized AroQ class CM, the mesophilic AroQp domain from E. coli. Like AroQp, the thermophilic CM is an alpha-helical dimer, but approximately 5 kcal/mol more stable than its mesophilic counterpart as judged from equilibrium denaturation studies. The possible origins of the thermostability of M. jannaschii AroQf, the smallest natural CM characterized to date, are discussed in light of available sequence and tertiary structural information.     

Lipoprotein profile determinants in children and adolescents from a lipid consultation clinic. The impact of diet, body composition and physical activity]

The production of extracellular beta-amylase by some Bacillus cereus, Bacillus megaterium and Bacillus polymyxa [corrected] strains was investigated, and the maximal yields of the enzyme were 3.6; 9.3 and 20.4 U/mL of the culture fluid, respectively (U, 1 mumol of maltose equivalent per min at 30 degrees C). Several cultivation media were used for beta-amylase production. Bacillus cereus and some strains of Bacillus megaterium gave good yields of beta-amylase only in medium with the addition of nutrient broth. However, beta-amylase produced during growth in protein rich medium (nutrient broth) was highly unstable, probably due to inactivation by proteolytic enzymes co-existing in the culture fluid. Bacillus polymyxa [corrected] strains can produce good yields of beta-amylase on a semi-synthetic medium consisting of inorganic salts, potato starch and inexpensive soybean extract instead of costly peptone and meat extract. The most potential beta-amylase producer was the strain Bacillus polymyxa [corrected] NCIB 8524. The tested Bacillus megaterium and Bacillus polymyxa [corrected] strains were apparently differentiated by temperature cultivation (30 and 37 degrees C) suitable for beta-amylase amylase yield.     

The unusually slow unfolding rate causes the high stability of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus: equilibrium and kinetic studies of guanidine hydrochloride-induced unfolding and refolding

To elucidate the energetic features of the anomalously high-level stabilization of a hyperthermophile pyrrolidone carboxyl peptidase (PfPCP) from a hyperthermophilic archaeon, Pyrococcus furiosus, equilibrium and kinetic studies of the guanidine hydrochloride (GuHCl)-induced unfolding and refolding were carried out with CD measurements at 220 nm in comparison with those from the mesophile homologue (BaPCP) from Bacillus amyloliquefaciens. The mutant protein of PfPCP substituted with Ser at both Cys142 and Cys188 (PfC142/188S) was used. The GuHCl unfolding for PfC142/188S and BaPCP was reversible. It was difficult to obtain the equilibrated unfolding curve of the hyperthermophile proteins at temperatures below 50 degreesC and pH 7, because of the remarkably slow rate of the unfolding. The unfolding for PfC142/188S attained equilibrium after 7 days at 60 degreesC, resulting in the coincidence between the unfolding and refolding curves. The Gibbs energy change of unfolding, DeltaGH2O (56.6 kJ/mol), for PfC142/188S at 60 degreesC and pH 7 was dramatically higher than that (7.6 kJ/mol) for BaPCP at 40 degreesC and pH 7. The unfolding and refolding kinetics for PfC142/188S and BaPCP at both 25 and 60 degreesC at pH 7 were approximated as a single exponential. The rate constant in water (kuH2O) of the unfolding reaction for PfC142/188S (1.6 x 10(-)15 s-1) at 25 degreesC and pH 7 was drastically reduced by 7 orders of magnitude compared to that (1.5 x 10(-)8 s-1) for BaPCP, whereas the refolding rates (krH2O) in water for PfC142/188S (9.3 x 10(-)2 s-1) and BaPCP (3.6 x 10(-)1 s-1) at 25 degreesC and pH 7 were similar. These results indicate that the greater stability of the hyperthermophile PCP was characterized by the drastically slow unfolding rate.     

Screening and characterization of microorganisms with glutaryl-7ADCA acylase activity

A screening of microorganisms producing glutaryl-7ADCA acylase, an enzyme able to hydrolyse glutaric acid selectively from glutaryl-3-deacetoxy-7-aminocephalosporanic acid (glutaryl-7ADCA), has been carried out in soil samples. Five microorganisms expressing acylase activity were isolated and classified as Bacillus cereus, Achromobacter xylosooxidans, Bacillus sp., Pseudomonas sp. and Pseudomonas paucimobilis. The screening was carried out by preparing enrichment cultures containing glutaryl-7-ADCA or cephalosporin C as the selective carbon source. Four model compounds (adipoyl-, glutamyl- and glutaryl-p-nitroanilide and glutarylcoumarin), mimicking the glutaryl-7ADCA beta-lactam moiety, were synthesized as substrates suitable for the rapid screening of the microorganisms (2500) isolated from the enrichment cultures. A total of 300 strains were active on the model substrates and only 5 displayed acylase activity on glutaryl-7ADCA. The fermentation parameters, such as pH and inducer concentration, for the optimal acylase expression and acylase specificity towards the model substrates were different for each strain.     

Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant?

Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain. The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B. More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease. To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2. We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B. Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases. All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper. One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC. Mutation of the CPC motif resulted in a nonfunctional protein, which demonstrates that this motif is essential for copper transport by ATP7B. Of the two putative ATP7B normal variants tested, one resulted in a nonfunctional protein, which suggests that it is a disease-causing mutation.     

Identification of lactic acid bacteria from chili bo, a Malaysian food ingredient

Ninety-two strains of lactic acid bacteria (LAB) were isolated from a Malaysian food ingredient, chili bo, stored for up to 25 days at 28 degreesC with no benzoic acid (product A) or with 7,000 mg of benzoic acid kg-1 (product B). The strains were divided into eight groups by traditional phenotypic tests. A total of 43 strains were selected for comparison of their sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) whole-cell protein patterns with a SDS-PAGE database of LAB. Isolates from product A were identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus farciminis, Pediococcus acidilactici, Enterococcus faecalis, and Weissella confusa. Five strains belonging to clusters which could not be allocated to existing species by SDS-PAGE were further identified by 16S rRNA sequence comparison. One strain was distantly related to the Lactobacillus casei/Pediococcus group. Two strains were related to Weissella at the genus or species level. Two other strains did not belong to any previously described 16S rRNA group of LAB and occupied an intermediate position between the L. casei/Pediococcus group and the Weissella group and species of Carnobacterium. The latter two strains belong to the cluster of LAB that predominated in product B. The incidence of new species and subspecies of LAB in chili bo indicate the high probability of isolation of new LAB from certain Southeast Asian foods. None of the isolates exhibited bacteriocin activity against L. plantarum ATCC 14917 and LMG 17682.