Dataset Information

Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores.

ABSTRACT: There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to determine the persistence of Bacillus anthracis and Bacillus subtilis spores on outdoor materials with and without exposure to simulated sunlight, using ultraviolet (UV)-A/B radiation. Spores were inoculated onto glass, wood, concrete, and topsoil and recovered after periods of 2, 14, 28, and 56 days. Recovery and inactivation kinetics for the two species were assessed for each surface material and UV exposure condition. Results suggest that with exposure to UV, decay of spore viability for both Bacillus species occurs in two phases, with an initial rapid decay, followed by a slower inactivation period. The exception was with topsoil, in which there was minimal loss of spore viability in soil over 56 days, with or without UV exposure. The greatest loss in viable spore recovery occurred on glass with UV exposure, with nearly a four log10 reduction after just two days. In most cases, B. subtilis had a slower rate of decay than B. anthracis, although less B. subtilis was recovered initially.

SUBMITTER: Wood JP

PROVIDER: S-EPMC4570822 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores.

Wood Joseph P JP Meyer Kathryn M KM Kelly Thomas J TJ Choi Young W YW Rogers James V JV Riggs Karen B KB Willenberg Zachary J ZJ

PloS one 20150915 9

There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to determine the persistence of Bacillus anthracis and Bacillus subtilis spores on outdoor materials with and without exposure to simulated sunlight, using ultraviolet (UV)-A/B radiation. Spores were inoculated onto glass, wood, concrete, and topsoil and recovered after periods of 2, 14, 28, and 56 days. Recovery and inactivation kinetics f ...[more]

PMID: 26372011

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Project description:Bacterial endospores produced by Bacillus and Clostridium species can remain dormant and highly resistant to environmental insults for long periods, but they can also rapidly germinate in response to a nutrient-rich environment. Multiple proteins involved in sensing and responding to nutrient germinants, initiating solute and water transport, and accomplishing spore wall degradation are associated with the membrane surrounding the spore core. In order to more fully catalog proteins that may be involved in spore germination, as well as to identify protein changes taking place during germination, unbiased proteomic analyses of membrane preparations isolated from dormant and germinated spores of Bacillus anthracis and Bacillus subtilis were undertaken. Membrane-associated proteins were fractionated by SDS-PAGE, gel slices were trypsin digested, and extracted peptides were fractionated by liquid chromatography and analyzed by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. More than 500 proteins were identified from each preparation. Bioinformatic methods were used to characterize proteins with regard to membrane association, cellular function, and conservation across species. Numerous proteins not previously known to be spore associated, 6 in B. subtilis and 68 in B. anthracis, were identified. Relative quantitation based on spectral counting indicated that the majority of spore membrane proteins decrease in abundance during the first 20 min of germination. The spore membranes contained several proteins thought to be involved in the transport of metal ions, a process that plays a major role in spore formation and germination. Analyses of mutant strains lacking these transport proteins implicated YloB in the accumulation of calcium within the developing forespore.IMPORTANCE Bacterial endospores can remain dormant and highly resistant to environmental insults for long periods but can also rapidly germinate in response to a nutrient-rich environment. The persistence and subsequent germination of spores contribute to their colonization of new environments and to the spread of certain diseases. Proteins of Bacillus subtilis and Bacillus anthracis were identified that are associated with the spore membrane, a position that can allow them to contribute to germination. A set of identified proteins that are predicted to carry out ion transport were examined for their contributions to spore formation, stability, and germination. Greater knowledge of spore formation and germination can contribute to the development of better decontamination strategies.

Project description:Bacillus spores incubated on plates for 2 to 98 days at 37°C had identical Ca-dipicolinic acid contents, exhibited identical viability on rich- or poor-medium plates, germinated identically in liquid with all germinants tested, identically returned to vegetative growth in rich or minimal medium, and exhibited essentially identical resistance to dry heat and similar resistance to UV radiation. However, the oldest spores had a lower core water content and significantly higher wet heat and NaOCl resistance. In addition, 47- and 98-day spores had lost >98% of intact 16S and 23S rRNA and 97 to 99% of almost all mRNAs, although minimal amounts of mononucleotides were generated in 91 days. Levels of 3-phosphoglyceric acid (3PGA) also fell 30 to 60% in the oldest spores, but how the 3PGA was lost is not clear. These results indicate that (i) translation of dormant spore mRNA is not essential for completion of spore germination, nor is protein synthesis from any mRNA; (ii) in sporulation for up to 91 days at 37°C, the RNA broken down generates minimal levels of mononucleotides; and (iii) the lengths of time that spores are incubated in sporulation medium should be considered when determining conditions for spore inactivation by wet heat, in particular, in using spores to test for the efficacy of sterilization regimens.IMPORTANCE We show that spores incubated at 37°C on sporulation plates for up to 98 days have lost almost all mRNAs and rRNAs, yet the aged spores germinated and outgrew as well as 2-day spores, and all these spores had identical viability. Thus, it is unlikely that spore mRNA, rRNA, or protein synthesis is important in spore germination. Spores incubated for 47 to 98 days also had much higher wet heat resistance than 2-day spores, suggesting that spore "age" should be considered in generating spores for tests of sterilization assurance. These data are the first to show complete survival of hydrated spores for ∼100 days, complementing published data showing dry-spore survival for years.

Dataset Information

Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores.

Publications

Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores.

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