Comparing Spore Resistance of Bacillus Strains Isolated from Hydrothermal Vents and Spacecraft Assembly Facilities to Environmental Stressors and Decontamination Treatments.

Submarine hydrothermal vents are inhabited by a variety of microorganisms capable of tolerating environmental extremes, making them ideal candidates to further expand our knowledge of the limitations for terrestrial life, including their ability to survive the exposure of spaceflight-relevant conditions. The spore resistance of two Bacillus spp. strains, APA and SBP3, isolated from two shallow vents off Panarea Island (Aeolian Islands, Italy), to artificial and environmental stressors (i.e., UVC radiation, X-rays, heat, space vacuum, hydrogen peroxide [H2O2], and low-pressure plasma), was compared with that of two close phylogenetic relatives (Bacillus horneckiae and Bacillus oceanisediminis). Additional comparisons were made with Bacillus sp. isolated from spacecraft assembly facilities (B. horneckiae, Bacillus pumilus SAFR-032, and Bacillus nealsonii) and the biodosimetry strain and space microbiology model organism Bacillus subtilis. Overall, a high degree of spore resistance to stressors was observed for the strains isolated from spacecraft assembly facilities, with an exceptional level of resistance seen by B. pumilus SAFR-032. The environmental isolate SBP3 showed a more robust spore resistance to UVC, X-rays, H2O2, dry heat, and space vacuum than the closely related B. horneckiae. Both strains (SBP3 and APA) were more thermotolerant than their relatives, B. horneckiae and B. oceanisediminis, respectively. SBP3 may have a novel use as a bacterial model organism for future interrogations into the potential of forward contamination in extraterrestrial environments (e.g., icy moons of Jupiter or Saturn), spacecraft sterilization and, broadly, microbial responses to spaceflight-relevant environmental stressors.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy.

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Effects of Low-Temperature Plasma-Sterilization on Mars Analog Soil Samples Mixed with Deinococcus radiodurans.

We used Ar plasma-sterilization at a temperature below 80 °C to examine its effects on the viability of microorganisms when intermixed with tested soil. Due to a relatively low temperature, this method is not thought to affect the properties of a soil, particularly its organic component, to a significant degree. The method has previously been shown to work well on spacecraft parts. The selected microorganism for this test was Deinococcus radiodurans R1, which is known for its remarkable resistance to radiation effects. Our results showed a reduction in microbial counts after applying a low temperature plasma, but not to a degree suitable for a sterilization of the soil. Even an increase of the treatment duration from 1.5 to 45 min did not achieve satisfying results, but only resulted in in a mean cell reduction rate of 75% compared to the untreated control samples.

Improvement of Biological Indicators by Uniformly Distributing Bacillus subtilis Spores in Monolayers To Evaluate Enhanced Spore Decontamination Technologies.

Novel decontamination technologies, including cold low-pressure plasma and blue light (400 nm), are promising alternatives to conventional surface decontamination methods. However, the standardization of the assessment of such sterilization processes remains to be accomplished. Bacterial endospores of the genera Bacillus and Geobacillus are frequently used as biological indicators (BIs) of sterility. Ensuring standardized and reproducible BIs for reliable testing procedures is a significant problem in industrial settings. In this study, an electrically driven spray deposition device was developed, allowing fast, reproducible, and homogeneous preparation of Bacillus subtilis 168 spore monolayers on glass surfaces. A detailed description of the structural design as well as the operating principle of the spraying device is given. The reproducible formation of spore monolayers of up to 5 × 10(7) spores per sample was verified by scanning electron microscopy. Surface inactivation studies revealed that monolayered spores were inactivated by UV-C (254 nm), low-pressure argon plasma (500 W, 10 Pa, 100 standard cubic cm per min), and blue light (400 nm) significantly faster than multilayered spores were. We have thus succeeded in the uniform preparation of reproducible, highly concentrated spore monolayers with the potential to generate BIs for a variety of nonpenetrating surface decontamination techniques.

Utilization of low-pressure plasma to inactivate bacterial spores on stainless steel screws.

A special focus area of planetary protection is the monitoring, control, and reduction of microbial contaminations that are detected on spacecraft components and hardware during and after assembly. In this study, wild-type spores of Bacillus pumilus SAFR-032 (a persistent spacecraft assembly facility isolate) and the laboratory model organism B. subtilis 168 were used to study the effects of low-pressure plasma, with hydrogen alone and in combination with oxygen and evaporated hydrogen peroxide as a process gas, on spore survival, which was determined by a colony formation assay. Spores of B. pumilus SAFR-032 and B. subtilis 168 were deposited with an aseptic technique onto the surface of stainless steel screws to simulate a spore-contaminated spacecraft hardware component, and were subsequently exposed to different plasmas and hydrogen peroxide conditions in a very high frequency capacitively coupled plasma reactor (VHF-CCP) to reduce the spore burden. Spores of the spacecraft isolate B. pumilus SAFR-032 were significantly more resistant to plasma treatment than spores of B. subtilis 168. The use of low-pressure plasma with an additional treatment of evaporated hydrogen peroxide also led to an enhanced spore inactivation that surpassed either single treatment when applied alone, which indicates the potential application of this method as a fast and suitable way to reduce spore-contaminated spacecraft hardware components for planetary protection purposes.