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Effect of cultivation pH on the surface hydrophobicity of Bacillus subtilis spores.

ABSTRACT: Bacillus subtilis spores are often used as biological indicators (BI) to monitor decontamination processes with gaseous hydrogen peroxide. Results in practical inactivation validation tests, however, vary considerably with no available explanation so far. This study reports on the effect of cultivation pH on spore surface hydrophobicity. Surface hydrophobicity is suspected to have an impact on the decontamination of technical surfaces such as packaging material when gaseous, condensing hydrogen peroxide is applied. It is the aim of this study to examine the impact of different cultivation pH levels on surface hydrophobicity and resistance of B. subtilis spores. Submersed cultivation of B. subtilis in bioreactors at controlled conditions with different static pH levels led to contact angles ranged between 50° and 80°, which was analyzed with water on a homogeneous layer of spores on a filter sheet. Resistance of spores was also affected by the cultivation pH. The results show that the culturing conditions during BI production should be controlled to obtain BI with specified characteristics in inactivation validation tests.

SUBMITTER: Eschlbeck E

PROVIDER: S-EPMC5532177 | biostudies-other | 2017 Dec

REPOSITORIES: biostudies-other

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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.

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Effect of cultivation pH on the surface hydrophobicity of Bacillus subtilis spores.

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