RESUMO
AIMS: To determine the mechanism of autoclave killing of Geobacillus stearothermophilus spores used in biological indicators (BIs) for steam autoclave sterilization, and rates of loss of spore viability and a spore enzyme used in BIs. METHODS AND RESULTS: Spore viability, dipicolinic acid (DPA) release, nucleic acid staining, α-glucosidase activity, protein structure and mutagenesis were measured during autoclaving of G. stearothermophilus spores. Loss of DPA and increases in spore core nucleic acid staining were slower than loss of spore viability. Spore core α-glucosidase was also lost more slowly than spore viability, although soluble α-glucosidase in spore preparations was lost more rapidly. However, spores exposed to an effective autoclave sterilization lost all viability and α-glucosidase activity. Apparently killed autoclaved spores were not recovered by artificial germination in supportive media, much spore protein was denatured during autoclaving, and partially killed autoclave-treated spore preparations did not acquire mutations. CONCLUSIONS: These results indicate that autoclave-killed spores cannot be revived, spore killing by autoclaving is likely by protein damage, and spore core α-glucosidase activity is lost more slowly than spore viability. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides insight into the mechanism of autoclave killing of spores of an organism used in BIs, and that a spore enzyme in a BI is more stable to autoclaving than spore viability.
Assuntos
Geobacillus stearothermophilus , Vapor , Esterilização , Proteínas de Bactérias/química , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/genética , Mutação , Ácidos Picolínicos/análise , Esporos Bacterianos/químicaRESUMO
In this proof-of-concept study, we describe the use of the main red beet pigment betanin for the quantification of calcium dipicolinate in bacterial spores, including Bacillus anthracis. In the presence of europium(III) ions, betanin is converted to a water-soluble, non-luminescent orange 1â¶1 complex with a stability constant of 1.4 × 10(5) L mol(-1). The addition of calcium dipicolinate, largely found in bacterial spores, changes the color of the aqueous solution of [Eu(Bn)(+)] from orange to magenta. The limit of detection (LOD) of calcium dipicolinate is around 2.0 × 10(-6) mol L(-1) and the LOD determined for both spores, B. cereus and B. anthracis, is (1.1 ± 0.3)× 10(6) spores mL(-1). This simple, green, fast and low cost colorimetric assay was selective for calcium dipicolinate when compared to several analogous compounds. The importance of this work relies on the potential use of betalains, raw natural pigments, as colorimetric sensors for biological applications.
Assuntos
Bacillus anthracis/fisiologia , Beta vulgaris/química , Colorimetria/instrumentação , Ácidos Picolínicos/análise , Raízes de Plantas/química , Esporos Bacterianos/química , Limite de DetecçãoRESUMO
Two decades of scientific ocean drilling have demonstrated widespread microbial life in deep sub-seafloor sediment, and surprisingly high microbial-cell numbers. Despite the ubiquity of life in the deep biosphere, the large community sizes and the low energy fluxes in this vast buried ecosystem are not yet understood. It is not known whether organisms of the deep biosphere are specifically adapted to extremely low energy fluxes or whether most of the observed cells are in a dormant, spore-like state. Here we apply a new approach--the D:L-amino-acid model--to quantify the distributions and turnover times of living microbial biomass, endospores and microbial necromass, as well as to determine their role in the sub-seafloor carbon budget. The approach combines sensitive analyses of unique bacterial markers (muramic acid and D-amino acids) and the bacterial endospore marker, dipicolinic acid, with racemization dynamics of stereo-isomeric amino acids. Endospores are as abundant as vegetative cells and microbial activity is extremely low, leading to microbial biomass turnover times of hundreds to thousands of years. We infer from model calculations that biomass production is sustained by organic carbon deposited from the surface photosynthetic world millions of years ago and that microbial necromass is recycled over timescales of hundreds of thousands of years.
Assuntos
Organismos Aquáticos/isolamento & purificação , Archaea/crescimento & desenvolvimento , Bactérias/crescimento & desenvolvimento , Biomassa , Sedimentos Geológicos/microbiologia , Água do Mar/microbiologia , Altitude , Aminoácidos/análise , Aminoácidos/química , Aminoácidos/metabolismo , Organismos Aquáticos/química , Organismos Aquáticos/crescimento & desenvolvimento , Archaea/química , Archaea/citologia , Archaea/isolamento & purificação , Bactérias/química , Bactérias/citologia , Bactérias/isolamento & purificação , Biomarcadores/análise , Carbono/metabolismo , Parede Celular/química , Ácidos Murâmicos/análise , Oceanos e Mares , Oxirredução , Peru , Fotossíntese , Ácidos Picolínicos/análise , Esporos Bacterianos/química , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/isolamento & purificação , Fatores de TempoRESUMO
AIMS: The aim of this work was to detect Bacillus thuringiensis endospore production during fermentation under conditions hindering endospore detection, i.e. in a complex undefined industrial medium with a high content of solids in suspension. METHODS AND RESULTS: Bacterial endospore production was measured using the photoluminescence of dipicolinate (DPA) with Tb3+. The high temperature and pressure of a conventional autoclave was used to release DPA from the endospores. The endospore was obtained from B. thuringiensis var. kurstaki HD-73 fermentations in industrial-type media with 25.1 and 54.1 g l(-1) glucose, 4.4 and 35.3 g l(-1) soybean meal, 5.8 g l(-1) yeast extract, 9.2 g l(-1) corn steep solids and mineral salts. CONCLUSIONS: In this study, we successfully determined the DPA concentrations during the culture of B. thuringiensis in high-concentration soybean meal media. A good correlation was found between microscope endospore counting and DPA measurements in the cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of synergy between Cry protein and endospore in B. thuringiensis bioinsecticides formulation, it is important to be able to determine endospore development during B. thuringiensis industrial-type fermentation, in order to ascertain the beginning of sporulation.