[Hyper-Resistance of the Bacillus licheniformis 24 Strain to Oxidative Stress Is Associated with Overexpression of Enzymatic Antioxidant System Genes].

At the International Space Station (ISS), artificial living conditions are created and maintained to satisfy human needs, these conditions are also favorable for the growth of numerous microorganisms, molds and bacteria. Among the microorganisms detected on the ISS are those from the automicroflora of crew members, and a significant number of spore-forming bacteria. In most cases, this group of microorganisms gives rise to strains that are able to colonize, grow and reproduce on interior materials and equipment of stations, and may be involved in biodestructive processes. These bacteria show increased resistance to various stress factors, for example, DNA-damaging and oxidizing agents. The molecular mechanisms of this resistance to stress are poorly understood. As part of the sanitary-microbiological monitoring of the ISS habitat, the Bacillus licheniformis 24 strain was isolated. Here, we demonstrated that this strain has increased resistance to hydrogen peroxide and Paraquat when compared to the "terrestrial" B. licheniformis B-10956 strain. B. licheniformis 24 overexpressed genes encoding enzymes that neutralize reactive oxygen species, such as KatX catalase and the superoxide dismutases SodA and SodF. Apart from this, in comparison with B. licheniformis B-10956, of B. licheniformis 24 cells had lower hydrogen sulfide production that was associated with sharply reduced expression of the cysIJ operon that encodes sulfite reductase. The results indicate that enzymatic antioxidant protective systems make a more significant contribution to the hyper-resistance of Bacillus strains to oxidizing agents than components of non-enzymatic systems, such as hydrogen sulfide.

The Biological Threat: The Threat of Planetary Quarantine Failure as a Result of Outer Space Exploration by Humans.

The paper presents the results of experiments with spore-forming bacteria and microscopic fungi performed in the framework of the Russian Research Program outside the International Space Station. It has been found that microorganisms not only survive in this extreme environment, but also retain reproductive ability. Moreover, most microorganisms exhibit an increase in biochemical activity and resistance to antimicrobial agents, specifically antibiotics. These findings are of obvious interest to the developers of both planetary quarantine methods and biomedical safety systems for manned space exploration missions. In addition, they demonstrate the necessity of experiments on the exposure of bio-objects to simulated environmental factors beyond Earth's magnetosphere.

CLEANING OF METAL SURFACES FROM MICROORGANISMS-BIODESTRUCTORS BY COMBINED USE OF ULTRASOUND GENERATOR <> AND AIR DECONTAMINATION UNIT <>.

The effort was made to assess the feasibility to remove microorganisms from surface of materials with the help of high-intensity ultrasound generator <> and further air decontamination using unit <>. The test data guided construction of a system capable to eliminate completely a mature association of microorganisms from aluminum surfaces. The combined use of 2 units resulted in total decontamination of atmosphere.

Metagenomic Analysis of the Dynamic Changes in the Gut Microbiome of the Participants of the MARS-500 Experiment, Simulating Long Term Space Flight.

A metagenomic analysis of the dynamic changes of the composition of the intestinal microbiome of five participants of the MARS-500 experiment was performed. DNA samples were isolated from the feces of the participants taken just before the experiment, upon 14, 30, 210, 363 and 510 days of isolation in the experimental module, and two weeks upon completion of the experiment. The taxonomic composition of the microbiome was analyzed by pyrosequencing of 16S rRNA gene fragments. Both the taxonomic and functional gene content of the microbiome of one participant were analyzed by whole metagenome sequencing using the SOLiD technique. Each participant had a specific microbiome that could be assigned to one of three recognized enterotypes. Two participants had enterotype I microbiomes characterized by the prevalence of Bacteroides, while the microbiomes of two others, assigned to type II, were dominated by Prevotella. One participant had a microbiome of mixed type. It was found that (1) changes in the taxonimic composition of the microbiomes occurred in the course of the experiment, but the enterotypes remained the same; (2) significant changes in the compositions of the microbiomes occurred just 14-30 days after the beginning of the experiment, presumably indicating the influence of stress factors in the first stage of the experiment; (3) a tendency toward a reversion of the microbiomes to their initial composition was observed two weeks after the end of the experiment, but complete recovery was not achieved. The metagenomic analysis of the microbiome of one of the participants showed that in spite of variations in the taxonomic compositions of microbiomes, the "functional" genetic composition was much more stable for most of the functional gene categories. Probably in the course of the experiment the taxonomic composition of the gut microbiome was adaptively changed to reflect the individual response to the experimental conditions. A new, balanced taxonomic composition of the microbiome was formed to ensure a stable gene content of the community as a whole without negative consequences for the health of the participants.

[Biosafety in the study of samples of extraterrestrial origin by an example of preparation for the "Phobos-Grunt" mission].

The necessity of microbial studying the soil from Phobos and terrestrial biological objects, which were for a long time in deep space and may represent a biological hazard has been shown. Developed medical and technical requirements for laboratories comply with Russian regulations and recommendations of international instruments for the ensuring of biosafety on the basis of process (continuous) biorisk management.

[Results of salad machine experiment within the MARS-500 project].

The salad machine experiment was aimed to fulfill performance testing of a prototype of space conveyor-type cylindrical greenhouse PHYTOCYCLE-SL, to study growth and development of plants, and to evaluate microbial contamination of equipment in the closed manned environment. Crops of leaf cabbage Brassica chinensis L., cultivar Vesnianka were raised in the time interval between MARS-500 days 417 and 515. The greenhouse proved it serviceability demanding 17 min/(man x d) in the normal mode. Most likely that the slow growth rate and deviations in plant morphology were caused by the presence of volatile pollutants in the greenhouse compartment Accumulation of micromycetes was observed at the sites of humid surfaces contact with ambient air; reduction of the artificial soil area contacting with air decreased population of micromycetes in 40 times. Cabbage leafs were free of pathogenic microflora. These results of the experiment helped develop recommendations on how to work out some units and systems in projectable greenhouse VITACYCLE-T

[Effect of thermal shock on seed resistance].

The work was performed in order to evaluate possible negative effect of high temperatures on biological properties of seeds in space experiment BIORISK-MSN-2. Laboratory experiments showed that seeds possess high enough resistance to extended exposure to high temperatures. Results of studying effects of positive temperatures in the region of 40 to 100 degrees C on biological properties of seeds of 19 species and varieties of higher plants belonging to three different families lead to the conclusion that heat stability of seeds depends on temperature value, period of exposure, species, variety and individual peculiarities of plants. These data can be useful in future interpretation of results of experiment BIORISK-MSN-2.

[The use of impulse plasma-optical ultraviolet technologies for ensuring microbial safety of the space station environment].

Purpose of the work was to study usability and efficiency of UV decontamination units aboard space station. UV-radiators were high-intensity xenon flashbulbs, as well as standard mercury and amalgam lamps; subjects were reference strains and test-microorganisms isolated from the ISS environment. Quantities of viable airborne and superficial microorganisms were determined before and after UV irradiation. The sanitary-microbiological assessment involved visual and microscopic counting of colony-forming units on material samples (CFU/100 cm2) and Petri dishes after air sampling (CFU/m3 of air). Continuous spectrum of high-intensity pulsed UV was demonstrated to be effective against contamination both by vegetative and high-resistant bacterial spores and molds.

[Radiation sterilization of units of a Mars descent module--a miniature meteorological station].

Subject of the test was a procedure of electron sterilization of Mars descent module units. As a result, data on distribution of absorbed dose field across the surface and by the entire volume of the mockup of a miniature meteorological station (MMS) were obtained In addition, electron sterilization technology was developed and the range of absorbed dose from electron radiation that will sterilize reliably packaged MMS hardware were defined in the interval from 30 to 40 kGy.

[Results of the experiment with extended exposure of microorganisms in open space].

Purpose of the experiment was to define the hypothesized margins of phenotype adaptation and genotype modifications of bacteria-fungi associations that form the typical microbiota on space vehicle materials. The fundamental questions to answer were whether the selected test-cultures would remain vital after extended (comparable to the Mars mission duration) exposure in open space as part of microbesstructural material model systems, and how the space factors would affect microorganisms in the context of eco-safety of space vehicles and planetary quarantine precautions. The first extended (1.5 years) exposure of the model systems outside of the International space station proved survivability of bacterial and microfungal spores in open space. Ultrastructures of eukaryotic and prokaryotic organisms were found altered, and so were their biological properties.

[Opportunistic pathogenic and toxic micro-fungi among synthetic polymer destructors].

Analysis of species diversity of the micro-fungi typically detected at the sites of biodamage of synthetic polymers on space vehicles exhibited the presence of a broad variety of opportunistic pathogens and toxic species. Thus, 78 species of micromycetes of 300 polymer destructing fungi are associated with biological risk levels BSL-1 and BSL-2 (low and moderate levels, respectively). As many as 56 species are able to produce toxic compounds.

[Main results of the Biorisk experiment on the International Space Station].

To get better appreciation of the margins of phenotypic adaptation and genotypic changes in bacteria-fungi associations within the typical microbiota residing on structural materials of space-flown equipment, developed were a program and hardware for a series of experiments under the general name BIORISK. Protocol of each experimental cycle is based on the well-proven method of exposure of "passive" samples of materials (Biorisk-KM), microorganisms-materials systems inside the ISS service module (Biorisk-MSV), and microorganisms-materials systems on the outside of the ISS SM (Biorisk-MSN). Each six months the samples are returned to the laboratory in conjunction with crew rotation. Already the first in-hand data from the experiment point to the dramatic effect of space flight on growth, reproduction, and biological properties of test microbes and fungi. Thus, the activity of enzymes that characterize the pathogenic potential (RNA-ase and DNA-ase), and resistance of microorganisms to aseptic agents were found increased.

[Studies on the growth and reproduction of bacterial communities on structural materials of the international space station].

Probability of microbial growth and reproduction on the ISS interior and equipment materials varying in chemical composition was studied with the strains of Bacillus subtilis, Staphylococcus epidermidis, Staphylococcus saprophyticus, Pseudomonas putida etc. sampled from the ISS environment. Controls were ground reference strains of same bacterial species. Based on our results, some of the microorganisms are able to survive and proliferate on structural materials; the ability was greater in space isolates as compared with their ground analogs. The greatest ability to grow and proliferate on materials was demonstrated by Bacillus subtilis.

[Air purification aboard space vehicles]. / Obezzarazhivanie vozdushnoi sredy na bortu kosmicheskikh letatel'nykh apparatov.

Air purification unit POTOK 150MK has been designed to clean air of space vehicles from microbial contaminant. Comparison of the data of analysis of air samples collected in different periods of operation of the space station MIR and ISS reconfirmed efficiency of the unit.

[Penicillium expansum, a resident fungal strain of the orbital complex Mir, producing xanthocillin X and questiomycin A]. / Grib Penicillium expansum--shtamm-rezident orbital'nogo komplexa "MIR"--produtsent ksantotsillina X i kvestiomitsina A.

It was demonstrated that the fungus Penicillium expansum 2-7, a resident strain of the orbital complex Mir, which became dominating at the end of a long-term space flight, formed biologically active secondary metabolites (antibiotics). Using physicochemical methods, these metabolites were identified as xanthocyllin X and questiomycin A. Time courses of their biosyntheses during growth and development of the producer culture were studied. Addition of zinc to the culture medium affected both the growth of the culture and the biosyntheses of the antibiotics. The concentrations of zinc in the medium, optimum for xanthocyllin X and questiomycin A production, amount to 0.3 and 3.0 mg/l, respectively.

Review of the knowledge of microbial contamination of the Russian manned spacecraft.

The 15-year experience of orbital station Mir service demonstrated that specifically modified space vehicle environments allows for the consideration of spaceship habitats as a certain ecological niche of microbial community development and functioning, which was formed from the organisms of different physiological and taxonomical groups. The base unit of the orbital station (OS) Mir was launched on February 20, 1986, and on March 13 the first crew arrived to it. From that moment a unique microbiocenosis started forming in the closed environment of the space station, and vital activity of the microorganisms continued for the next 15 years in a specifically changed environment, in conditions of continuous influence of a set of factors intrinsic to space flight. A total of 234 species of bacteria and fungi were found onboard orbital station Mir, among which microorganisms capable of resident colonization of the environment of space objects as a unique anthropotechnological niche were revealed. In such conditions the evolution of microflora is followed by the rise of medical and technical risks that can affect both sanitary-microbiological conditions of the environment and the safety and reliability characteristics of space equipment. The latter is caused by progressing biological damage to the structural materials. The microbial loading dynamic does not have linearly progressing character, but it is a wavy process of alternation of the microflora activation and stabilization phases; on this background there is a change of the dominating species by quantity and prevalence. The accumulated data is evidence of the necessity of the constant control of the microbial environmental factors to maintain their sanitary and microbiological optimum condition and to prevent the processes of constructional materials biodestruction.

[The concept of microbiological safety of a piloted Martian expedition]. / Kontseptsiia obespecheniia mikrobiologicheskoi bezopasnosti pilotiruemoi marsianskoi ékspeditsii.

It is the peculiar evolution of the microbial association aboard long-operating space vehicles that lends additional medical, technical and technological risks that may impact crew safety and orbital systems performance. Based on the experience of the Russian space stations, a concept of microbiological safety of a piloted expedition to Mars has been proposed comprising preventive measures, methods, means and technologies to control the microbiological environment in transport vehicles, lander and Martian habitation module.

[Basic patterns of microflora development in the environment of orbital complex Mir]. / Osnovnye zakonomernosti formirovaniia mikroflory sredy obitaniia orbital'nogo kompleksa "Mir".

During the multi-year operation of orbital complex Mir a systematic survey of development and behavior of microflora was performed in order to assess the associated risks to the piloted vehicle. Microorganisms isolated from the Mir environment represented 234 species of which 108 were bacteria and 126 microscopic fungi. Bacteria included opportunistic pathogens causing opportunistic infections. There were also fungi that were pathogenic for humans leading to mycosis and mycotoxication. The largest species diversity was characteristic of fungi, so-called technophils damaging polymers and corroding metals. Staphylococcus, Micrococcus, Corynebacterium Bacillus dominated among the bacteria and Penicullium, Aspergillus and Cladosporium were most common, omnipresent and numerous among the fungi. Microbial contamination of Mir undulated so that activation periods alternated period of stabilization and other species prevailed in number and spread. Evaluation of microflora in this environment was conducive of medical and technological risks.

[Comparative evaluation of microorganisms biological characteristics isolated in the orbital complex "Mir" on different phases of its operation]. / Sravnitel'naia otsenka biologicheskikh svoistv mikroorganizmov, vydelennykh v orbital'nom komplekse "Mir" v razlichnye sroki ékspluatatsii.

Colonizing and bio-destructive activities and pathogenetic potential of microorganisms isolated from the MIR environment at different phases of its operations were tested and evaluated in comparison with standard "museum" cultures of analogous species. Comparison of bacterial and fungal species revealed certain cultural, biochemical, and morphological distinctions of the "flight" strains. However, to assess the character and the risk associated with peculiar evolution of microflora in long-term space missions, more careful investigations should be followed with the use of the strain genetic labeling and other modern techniques.