Effect of dairy product environment on the growth of Bacillus cereus.

pH is one of the most important parameters to manage bacterial replication in foodstuffs. In this study, the ability of 2 Bacillus cereus strains, 1 clinical human isolate (GPe2) and 1 isolate from a dairy product (D43), were investigated for in vitro growth at different pH values (from 3.5 to 7.5) at 2 temperatures (15 and 37°C), showing their ability to grow from 5.5 to 7.5 and from 5.0 to 7.5, respectively. The ability of spores of these 2 microorganisms to germinate in different typologies of dairy products (unflavored yogurt, Taleggio cheese, mascarpone cheese, and raw and pasteurized milk) was also investigated by inoculating the spores and maintaining the products at 15°C. No growth was observed in yogurt, likely due to the combined effect of low pH (<5) and the presence of natural microflora. An inhibitory action of the natural microflora on the growth of B. cereus was also hypothesized for Taleggio cheese and raw milk, as these substrates were characterized by a high natural lactic acid bacteria population and permissive pH values (5.8/6.8 in Taleggio cheese, >7 in raw milk). In pasteurized milk and mascarpone cheese, where pH was not restrictive for B. cereus growth and where no significant natural microflora was present, growth occurred rapidly up to loads close to 7 log cfu/g.

Survival and persistence of Bacillus clausii in the human gastrointestinal tract following oral administration as spore-based probiotic formulation.

AIMS: This study aimed to investigate the fate of Bacillus clausii spores orally administered as lyophilized or liquid formulation to healthy volunteers. METHODS AND RESULTS: The study was a randomized, open-label, cross-over trial in which two commercial probiotic formulations containing spores of four antibiotic-resistant B. clausii strains (OC, NR, SIN, T) were given as a single dose administration. Faecal B. clausii units of each strain were counted on selective media and extrapolated for the total weight of evacuated faeces. RAPD-PCR typing was used to confirm B. clausii identification. Bacillus clausii was found alive in faeces for up to 12 days. In some volunteers, the recovered amount of OC, NR or SIN was higher than the number of administered spores. Bioequivalence among the two formulations was demonstrated. CONCLUSIONS: Bacillus clausii spores survive transit through the human gastrointestinal tract. They can undergo germination, outgrowth and multiplication as vegetative forms. Bacillus clausii strains can have different ability to survive in the intestinal environment. Bacillus clausii spores administered as liquid suspension or lyophilized form behave similarly in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes towards a better understanding of the behaviour of B. clausii spores as probiotics.

Effect of microwave radiation on Bacillus subtilis spores.

AIMS: To compare the killing efficacy and the effects exerted by microwaves and conventional heating on structural and molecular components of Bacillus subtilis spores. METHODS AND RESULTS: A microwave waveguide applicator was developed to generate a uniform and measurable distribution of the microwave electric-field amplitude. The applicator enabled the killing efficacy exerted by microwaves on B. subtilis spores to be evaluated in comparison with conventional heating at the same temperature value. The two treatments produced a similar kinetics of spore survival, while remarkably different effects on spore structures were seen. The cortex layer of the spores subjected to conductive heating was 10 times wider than that of the untreated spores; in contrast, the cortex of irradiated spores did not change. In addition, the heated spores were found to release appreciable amounts of dipicolinic acid (DPA) upon treatment, while extracellular DPA was completely undetectable in supernatants of the irradiated spores. These observations suggest that microwave radiation may promote the formation of stable complexes between DPA and other spore components (i.e. calcium ions); thus, making any release of DPA from irradiated spores undetectable. Indeed, while a decrease in measurable DPA concentrations was not produced by microwave radiation on pure DPA solutions, a significant lowering in DPA concentration was detected when this molecule was exposed to microwaves in the presence of either calcium ions or spore suspensions. CONCLUSIONS: Microwaves are as effective as conductive heating in killing B. subtilis spores, but the microwave E-field induces changes in the structural and/or molecular components of spores that differ from those attributable only to heat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the effect of microwaves on B. subtilis spore components.

Rapid determination of vitamin B2 secretion by bacteria growing on solid media.

AIMS: Development of an agar-diffusion assay to measure vitamin B2 in biological samples and application of the method to determine the amount of vitamin B2 secreted by bacteria. METHODS AND RESULTS: A riboflavin-auxotrophic mutant of Bacillus cereus was generated by mini-Tn10 insertion in the ribD gene. ribD mutant sensitivity to exogenous vitamin B2 was investigated by turbidimetric and agar-diffusion assays. In turbidimetric assays, the B. cereus mutant displayed a similar level of sensitivity to vitamin B2 to that of Lactobacillus casei ATCC 7469, the reference organism used for microbiological vitamin B2 quantification. However, only the ribD mutant could be used as an indicator organism in agar-diffusion assays. A total of eight probiotic strains, from five different probiotic formulations, were analysed by the ribD mutant-based assay on agar plates in order to determine their ability to secrete vitamin B2 during growth. CONCLUSION: The agar diffusion method with the ribD mutant of B. cereus is highly reproducible, sensitive, rapid, inexpensive, and can be applied to measure the amount of vitamin B2 in different samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The method developed in this study appears to be a good candidate for the screening of vitamin B2 secretion by bacteria growing on solid media.

Molecular characterization and identification of Bacillus clausii Strains marketed for use in oral bacteriotherapy.

A substantial number of Bacillus species have been marketed for use in oral bacteriotherapy because of their purported ability to prevent or treat various gastrointestinal disorders. Recently, some of the Bacillus strains in Enterogermina, which is made up of aqueous suspensions of viable Bacillus spores, have been partially characterized and aligned with members of the Bacillus alcalophilus subgroup rather than with Bacillus subtilis, as previously reported. With a view toward verifying the original taxonomic position of the Enterogermina strains, we catalogued both phenotypic and genotypic traits exhibited by the four Bacillus strains isolated from the spore mixtures found in original commercial preparations dated 1975 and 1984 and commercial preparations now being propagated industrially. Analyses of physiological and biochemical traits, complete 16S rRNA gene sequences, DNA-DNA reassociation, tRNA intergenic spacer length polymorphism, single-strand conformation polymorphism of PCR-amplified spacer regions of tRNA genes, and randomly amplified polymorphic DNA led to the finding that all of the Enterogermina strains belong to a unique genospecies, which is unequivocally identified as the alkalitolerant species Bacillus clausii. Moreover, we provide evidence that in contrast to several reference strains of B. clausii, the strains constituting Enterogermina are characterized by a notable low level of intraspecific genome diversity and that each strain has remained the same for the last 25 years.

Effect of a novel mucoadhesive polysaccharide obtained from tamarind seeds on the intraocular penetration of gentamicin and ofloxacin in rabbits.

This report describes the efficacy of a novel mucoadhesive polymer, the tamarind seed polysaccharide, as a delivery system for the ocular administration of hydrophilic and hydrophobic antibiotics. Healthy rabbits were subjected to repeated ocular instillations with either conventional gentamicin or ofloxacin or these agents viscosified with the tamarind seed polysaccharide. Administration of viscosified preparations produced antibiotic concentrations both in the aqueous humour and cornea that were significantly higher than those achieved with the drugs alone. The increased drug absorption and the prolonged drug elimination phase obtained with the viscosified formulations indicate the usefulness of the tamarind seed polysaccharide as an ophthalmic delivery system for topical administration of antibiotics.

Characterization of the chemotaxis fli Y and che A genes in Bacillus cereus.

This paper describes the first identification of chemotaxis genes in Bacillus cereus. We sequenced and studied the genomic organization and the expression of the cheA and fliY genes in two different B. cereus strains, ATCC 14579 and ATCC 10987. While cheA encodes a highly conserved protein acting as the main regulator of the chemotactic response in flagellated eubacteria, fliY, which has been previously described only in B. subtilis, is one of the three genes encoding proteins of the flagellar switch complex. Although the sequences and relative position of cheA and fliY were found to be identical in the two B. cereus strains analyzed, the restriction fragment containing both genes was located differently on the physical maps of B. cereus ATCC 14579 and ATCC 10987. Evidence is shown that the genomic organization and the expression of fliY and cheA in B. cereus differ significantly from that described for B. subtilis, which is considered a model microorganism for chemotaxis in gram-positive bacteria.

Control of Candida albicans murine vaginitis by topical administration of polycarbophil-econazole complex.

The complexation of econazole with the mucoadhesive polycarbophil was found to significantly improve the therapeutic benefit of the drug in the topical treatment of experimental vaginal candidiasis in mice, while no difference in the antimycotic activity exerted by econazole and polycarbophil-econazole could be detected in vitro.