Comparison of Antibiotic Resistance, Biofilm Formation and Conjugative Transfer of Staphylococcus and Enterococcus Isolates from International Space Station and Antarctic Research Station Concordia

The International Space Station (ISS) and the Antarctic Research Station Concordia are confined and isolated habitats in extreme and hostile environments. The human and habitat microflora can alter due to the special environmental conditions resulting in microbial contamination and health risk for the crew. In this study, 29 isolates from the ISS and 55 from the Antarctic Research Station Concordia belonging to the genera Staphylococcus and Enterococcus were investigated. Resistance to one or more antibiotics was detected in 75.8 % of the ISS and in 43.6 % of the Concordia strains. The corresponding resistance genes were identified by polymerase chain reaction in 86 % of the resistant ISS strains and in 18.2 % of the resistant Concordia strains. Plasmids are present in 86.2 % of the ISS and in 78.2 % of the Concordia strains. Eight Enterococcus faecalis strains (ISS) harbor plasmids of about 130 kb. Relaxase and/or transfer genes encoded on plasmids from gram-positive bacteria like pIP501, pRE25, pSK41, pGO1 and pT181 were detected in 86.2 % of the ISS and in 52.7 % of the Concordia strains. Most pSK41-homologous transfer genes were detected in ISS isolates belonging to coagulase-negative staphylococci. We demonstrated through mating experiments that Staphylococcus haemolyticus F2 (ISS) and the Concordia strain Staphylococcus hominis subsp. hominis G2 can transfer resistance genes to E. faecalis and Staphylococcus aureus, respectively. Biofilm formation was observed in 83 % of the ISS and in 92.7 % of the Concordia strains. In conclusion, the ISS isolates were shown to encode more resistance genes and possess a higher gene transfer capacity due to the presence of three vir signature genes, virB1, virB4 and virD4 than the Concordia isolates.     

Study of the Biological Dormancy of Aquatic Organisms in Open Space and Space Flight Conditions

Biology Bulletin - In outer space, ultraviolet and cosmic radiation, a wide range of high and low temperatures, altered gravity, electromagnetic fields, the vacuum, and their combinations determine...     

Effects of Simulated Mars Conditions on the Survival and Growth of Escherichia coli and Serratia liquefaciens

Escherichia coli and Serratia liquefaciens, two bacterial spacecraft contaminants known to replicate under low atmospheric pressures of 2.5 kPa, were tested for growth and survival under simulated Mars conditions. Environmental stresses of high salinity, low temperature, and low pressure were screened alone and in combination for effects on bacterial survival and replication, and then cells were tested in Mars analog soils under simulated Mars conditions. Survival and replication of E. coli and S. liquefaciens cells in liquid medium were evaluated for 7 days under low temperatures (5, 10, 20, or 30°C) with increasing concentrations (0, 5, 10, or 20%) of three salts (MgCl₂, MgSO₄, NaCl) reported to be present on the surface of Mars. Moderate to high growth rates were observed for E. coli and S. liquefaciens at 30 or 20°C and in solutions with 0 or 5% salts. In contrast, cell densities of both species generally did not increase above initial inoculum levels under the highest salt concentrations (10 and 20%) and the four temperatures tested, with the exception that moderately higher cell densities were observed for both species at 10% MgSO₄ maintained at 20 or 30°C. Growth rates of E. coli and S. liquefaciens in low salt concentrations were robust under all pressures (2.5, 10, or 101.3 kPa), exhibiting a general increase of up to 2.5 orders of magnitude above the initial inoculum levels of the assays. Vegetative E. coli cells were maintained in a Mars analog soil for 7 days under simulated Mars conditions that included temperatures between 20 and −50°C for a day/night diurnal period, UVC irradiation (200 to 280 nm) at 3.6 W m⁻² for daytime operations (8 h), pressures held at a constant 0.71 kPa, and a gas composition that included the top five gases found in the martian atmosphere. Cell densities of E. coli failed to increase under simulated Mars conditions, and survival was reduced 1 to 2 orders of magnitude by the interactive effects of desiccation, UV irradiation, high salinity, and low pressure (in decreasing order of importance). Results suggest that E. coli may be able to survive, but not grow, in surficial soils on Mars.The search for extant life on Mars remains a stated goal of NASA''s Mars Exploration Program and Astrobiology Institutes (13, 17). Intrinsic within such a life detection strategy is a requirement to understand how terrestrial life might survive, replicate, and proliferate on Mars. To mitigate the risks of the forward contamination of Mars, the bioloads on spacecrafts targeted for landing must be reduced to low density and diversity (4, 7). Planetary protection guidelines are designed to prevent both the forward contamination of the martian surface and to ensure the scientific integrity of any deployed life detection experiments. To date, 12 spacecraft have landed or crashed onto the Mars surface as a result of U.S., Russian, and European space program missions, but it is currently unknown if terrestrial microorganisms typically found on spacecraft surfaces can grow and replicate under conditions encountered on the surface (44, 45, 48).Despite cleaning and sterilization measures taken to significantly reduce microbial bioloads on spacecraft (26, 56), diverse microbial communities remain at the time of launch (7, 31, 32, 44). The diversity of microorganisms found on spacecraft surfaces are generally characteristic of the clean rooms within which the spacecraft are processed. Spacecraft assembly facilities are oligotrophic extreme environments in which only the most resilient species survive the high-desiccation, low-nutrient conditions, controlled air circulation, and the rigors of bioburden reduction (56, 57). The biological inventory of microorganisms on spacecraft has mostly been limited to isolation and identification using standard culture-based microbiological assays (44, 48, 53). However, culture-based microbiological assays likely underestimate the biological diversity present on spacecraft, as traditional culture techniques fail to capture more than 99.9% of present phylotypes (7). Recently, the simultaneous use of culture-dependent and culture-independent techniques (e.g., Limulus amoebocyte lysate assay [LAL], ATP bioluminescence assay, lipopolysaccharide-based microbial detection, and DNA-based PCR) have identified many nonculturable species (31, 32, 57). Known culturable bacteria recovered from spacecraft surfaces include, but are not limited to, species of Acinetobacter, Bacillus, Corynebacterium, Escherichia, Flavobacterium, Micrococcus, Pseudomonas, Serratia, Staphylococcus, and Streptococcus (44, 53, 57).After launch, spacecraft are exposed to interplanetary conditions of ultralow pressure (3 × 10⁻¹⁰ kPa), extreme desiccating conditions, fluctuating temperatures, solar UV irradiation, and ionizing radiation (22, 44). Furthermore, upon landing, the conditions on the surface of Mars are not much improved over interplanetary space. Diverse biocidal or inhibitory conditions on Mars have been identified in a number of recent publications (8, 21, 22, 35, 36, 38, 44, 48, 59) and include the following (not in order of priority): solar UVC irradiation, low pressure, extreme desiccating conditions, extreme diurnal temperature fluctuations, solar particle events, galactic cosmic rays, UV glow discharge from blowing dust, solar UV-induced volatile oxidants (e.g., O₂⁻, O⁻, H₂O₂, NO_x, O₃), globally distributed oxidizing soils, extremely high salt levels (e.g., MgCl₂, NaCl, FeSO₄, and MgSO₄) in surficial soils at some sites on Mars, high concentrations of heavy metals in martian soils, acidic conditions in martian regolith, high CO₂ concentrations in the global atmosphere, and presence of perchlorates in some regoliths. UV irradiation, especially UVC photons (200 to 280 nm), may be the most biocidal of all factors to microbial survival on the martian surface (34, 37, 39, 47, 50, 52). Microorganisms found on sun-exposed surfaces of spacecraft are killed off within a few tens of minutes of exposure; but if covered by as little as a few hundred micrometers of martian soil, significant protection is provided (11, 34, 47). It is currently unknown if terrestrial microorganisms typically found on spacecraft surfaces can grow and replicate under conditions encountered on the surface of Mars (44, 48).In the studies cited above, most research focused on the survival of dormant spores or vegetative cells under Mars conditions. In contrast, only a few papers have explored the possibility of growth and replication of terrestrial microorganisms under environmental conditions that approach those found in surficial soils of Mars (5, 25, 45, 48). Of these four, 2.5 kPa is the lowest pressure at which replication was observed for a few bacterial species (5, 45, 48).The primary objective of the current study was to expose two non-spore-forming species to environmental stresses present on the surface of Mars to characterize the potential response of the bacteria to martian temperatures, salinities, and pressures. Two bacterial species, Escherichia coli and Serratia liquefaciens, were selected from over 30 prokaryotic species tested in preliminary experiments (5, 45). Their selection was based on their common association with humans, recovery from robotic spacecraft and space-based human life support systems (44, 53), and demonstrated replication at 2.5 kPa of total atmospheric pressure (5, 45). Experiments were conducted on cell suspensions in liquid medium at combinations of low pressure, high salt concentrations, and low temperatures, and then with cells mixed into soils and exposed to simulated Mars conditions. It was predicted for cell suspensions that (i) low temperatures would dramatically retard cell proliferation, (ii) high concentrations of salts would be biocidal on cell suspensions, and (iii) low pressure would have weak to moderate inhibitory effects on cell growth of both species. For cells in soils, growth was not expected under Mars simulations which exposed vegetative cells to low pressure, low temperatures, anaerobic gas composition, and high UVC irradiation similar to the martian surface. Although replication was not predicted, bacterial survival in analog Mars soils under simulated Mars conditions was anticipated.     

Cryptoendolithic microorganisms from Antarctic sandstone of Linnaeus Terrace (Asgard Range): diversity, properties and interactions

Cryptoendolithic microorganisms from stratified communities in Antarctic sandstone were studied for physiological diversity and possible interactions. Cultures of 25 bacteria, five fungi, and two green algae from one boulder grew with a wide variety of organic carbon or nitrogen sources, they exhibited varied exoenzymatic activities and were psychrophilic or psychrotrophic. Many isolates excreted vitamins into the medium and were stimulated by other vitamins. Organic acid excretion and siderophore formation were common, but antibiotic activity was rare. Plasmids were found in 24% of the bacteria, and many of these strains showed resistance to antibiotics and heavy metals. A small plasmid (2.9 kb) from strain AA-341 was electrotransferred into sensitive isolates, thereby rendering these resistant to amplicillin and Cr³⁺ Bacterial cultures in spent algal medium and coculture with algae demonstrated beneficial (rarely inhibitory) interactions. A search for free organic compounds in zones of the sandstone community revealed sugars, sugar alcohols, organic acids and amino acids-in many cases the same compounds that were excreted into the laboratory medium. Data presented here indicate low taxonomic but high physiological diversity among these heterotrophic cryptoendoliths. This physiological diversity, as well as the spatial separation in layers with distinct activities, allows coexistence within the community and contributes to the stability of this ecosystem.     

EXPOSE, an Astrobiological Exposure Facility on the International Space Station - from Proposal to Flight

Following an European Space Agency announcement of opportunity in 1996 for ”Externally mounted payloads for 1st utilization phase” on the International Space Station (ISS), scientists working in the fields of astrobiology proposed experiments aiming at long-term exposure of a variety of chemical compounds and extremely resistant microorganisms to the hostile space environment. The ESA exposure facility EXPOSE was built and an operations´ concept was prepared. The EXPOSE experiments were developed through an intensive pre-flight experiment verification test program. 12 years later, two sets of astrobiological experiments in two EXPOSE facilities have been successfully launched to the ISS for external exposure for up to 1.5 years. EXPOSE-E, now installed at the balcony of the European Columbus module, was launched in February 2008, while EXPOSE-R took off to the ISS in November 2008 and was installed on the external URM-D platform of the Russian Zvezda module in March 2009.     

Characterization of fungi isolated from the equipment used in the International Space Station or Space Shuttle

As a part of a series of studies regarding the microbial biota in manned space environments, fungi were isolated from six pieces of equipment recovered from the Japanese Experimental Module “KIBO” of the International Space Station and from a space shuttle. Thirty‐seven strains of fungi were isolated, identified and investigated with regard to morphological phenotypes and antifungal susceptibilities. The variety of fungi isolated in this study was similar to that of several previous reports. The dominant species belonged to the genera Penicillium, Aspergillus and Cladosporium, which are potential causative agents of allergy and opportunistic infections. The morphological phenotypes and antifungal susceptibilities of the strains isolated from space environments were not significantly different from those of reference strains on Earth.     

Evaluation of a Treadmill with Vibration Isolation and Stabilization (TVIS) for use on the International Space Station

A treadmill with vibration isolation and stabilization designed for the International Space Station (ISS) was evaluated during Shuttle mission STS-81. Three crew members ran and walked on the device, which floats freely in zero gravity. For the majority of the more than 2 hours of locomotion studied, the treadmill showed peak to peak linear and angular displacements of less than 2.5 cm and 2.5 degrees, respectively. Vibration transmitted to the vehicle was within the microgravity allocation limits that are defined for the ISS. Refinements to the treadmill and harness system are discussed. This approach to treadmill design offers the possibility of generating 1G-like loads on the lower extremities while preserving the microgravity environment of the ISS for structural safety and vibration free experimental conditions.     

The significance of the study about the biological effects of solar ultraviolet radiation using the Exposed Facility on the International Space Station.

It is believed that ultraviolet (UV) radiation from the sun participated in events related to the chemical evolution and birth of life on the primitive Earth. Although UV radiation would be also a driving force for the biological evolution of life on Earth, life space of the primitive living organisms would be limited in the UV-shielded place such as in the water at an early stage of the evolution of life. After the formation of stratospheric ozone layer through the production of oxygen by photoautotroph, living organisms were able to expand their domain from water to land. As a result, now, many kinds of living organisms containing human beings are flourishing on the ground. In the near future, increased transmission of harmful solar UV radiation may reach the Earth's surface due to stratospheric ozone layer depletion. In order to learn more about the biological effects of solar UV radiation with or without interruption by the ozone layer, the utilization of an Exposed Facility on the International Space Station is required. Experiments proposed for this facility would provide a tool for the scientific investigation of processes involved in the birth and evolution of life on Earth, and could also demonstrate the importance of protecting the Earth's future environment from future ozone layer depletion.     

Development and verification of hardware for life science experiments in the Japanese Experiment Module "Kibo" on the International Space Station

Japan Aerospace Exploration Agency (JAXA) has developed a cell biology experiment facility (CBEF) and a clean bench (CB) as a common hardware in which life science experiments in the Japanese Experiment Module (JEM known as "Kibo") of the International Space Station (ISS) can be performed. The CBEF, a CO2 incubator with a turntable that provides variable gravity levels, is the basic hardware required to carry out the biological experiments using microorganisms, cells, tissues, small animals, plants, etc. The CB provides a closed aseptic operation area for life science and biotechnology experiments in Kibo. A phase contrast and fluorescence microscope is installed inside CB. The biological experiment units (BEU) are designed to run individual experiments using the CBEF and the CB. A plant experiment unit (PEU) and two cell experiment units (CEU type1 and type2) for the BEU have been developed.     

Inertial shear force and the impact on facilities for the International Space Station

Inertial shear force is a surface force that is generated in centrifuges especially with attached samples on flat surfaces and plays a significant role in gravitational and space research. The magnitude of this force is proportional to the radius of the centrifuge and surface area of the sample compartment. In gravitational research we want to study the impact of weight onto a system. However, the force of inertial shear is perpendicular to the gravity vector, hence, results may be obscured or even misinterpreted by this artifact.     

Can Plants Grow on Mars and the Moon: A Growth Experiment on Mars and Moon Soil Simulants

When humans will settle on the moon or Mars they will have to eat there. Food may be flown in. An alternative could be to cultivate plants at the site itself, preferably in native soils. We report on the first large-scale controlled experiment to investigate the possibility of growing plants in Mars and moon soil simulants. The results show that plants are able to germinate and grow on both Martian and moon soil simulant for a period of 50 days without any addition of nutrients. Growth and flowering on Mars regolith simulant was much better than on moon regolith simulant and even slightly better than on our control nutrient poor river soil. Reflexed stonecrop (a wild plant); the crops tomato, wheat, and cress; and the green manure species field mustard performed particularly well. The latter three flowered, and cress and field mustard also produced seeds. Our results show that in principle it is possible to grow crops and other plant species in Martian and Lunar soil simulants. However, many questions remain about the simulants'' water carrying capacity and other physical characteristics and also whether the simulants are representative of the real soils.     

Phototropism of Arabidopsis thaliana in microgravity and fractional gravity on the International Space Station

While there is a great deal of knowledge regarding plant growth and development in microgravity aboard orbiting spacecraft, there is little information available about these parameters in reduced or fractional gravity conditions (less than the nominal 1g on Earth). Thus, in these experiments using the European Modular Cultivation System on the International Space Station, we studied the interaction between phototropism and gravitropism in the WT and mutants of phytochrome A and B of Arabidopis thaliana. Fractional gravity and the 1 g control were provided by centrifuges in the spaceflight hardware, and unidirectional red and blue illumination followed a white light growth period in the time line of the space experiments. The existence of red-light-based positive phototropism in hypocotyls of seedlings that is mediated by phytochrome was confirmed in these microgravity experiments. Fractional gravity studies showed an attenuation of red-light-based phototropism in both roots and hypocotyls of seedlings occurring due to gravitational accelerations ranging from 0.l to 0.3 g. In contrast, blue-light negative phototropism in roots, which was enhanced in microgravity compared with the 1g control, showed a significant attenuation at 0.3 g. In addition, our studies suggest that the well-known red-light enhancement of blue-light-induced phototropism in hypocotyls is likely due to an indirect effect by the attenuation of gravitropism. However, red-light enhancement of root blue-light-based phototropism may occur via a more direct effect on the phototropism system itself, most likely through the phytochrome photoreceptors. To our knowledge, these experiments represent the first to examine the behavior of flowering plants in fractional or reduced gravity conditions.     

Gravisensitivity and automorphogenesis of lentil seedling roots grown on board the International Space Station

The GRAVI-1 experiment was brought on board the International Space Station by Discovery (December 2006) and carried out in January 2007 in the European Modular Cultivation System facility. For the first run of this experiment, lentil seedlings were hydrated and grown in microgravity for 15 h and then subjected for 13 h 40 min to centrifugal accelerations ranging from 0.29 x 10(-2) g to 0.99 x 10(-2) g. During the second run, seedlings were grown either for 30 h 30 min in microgravity (this sample was the control) or for 21 h 30 min and then subjected to centrifugal accelerations ranging from 1.2 x 10(-2) g to 2.0 x 10(-2) g for 9 h. In both cases, root orientation and root curvature were followed by time-lapse photography. Still images were downlinked in near real time to ground Norwegian User Support and Operations Center during the experiment. The position of the root tip and the root curvature were analyzed as a function of time. It has been shown that in microgravity, the embryonic root curved strongly away from the cotyledons (automorphogenesis) and then straightened out slowly from 17 to 30 h following hydration (autotropism). Because of the autotropic straightening of roots in microgravity, their tip was oriented at an angle close to the optimal angle of curvature (120 degrees -135 degrees ) for a period of 2 h during centrifugation. Moreover, it has been demonstrated that lentil roots grown in microgravity before stimulation were more sensitive than roots grown in 1 g. In these conditions, the threshold acceleration perceived by these organs was found to be between 0 and 2.0 x 10(-3) g and estimated punctually at 1.4 x 10(-5) g by using the hyperbolic model for fitting the experimental data and by assuming that autotropism had no or little impact on the gravitropic response. Gravisensing by statoliths should be possible at such a low level of acceleration because the actomyosin system could provide the necessary work to overcome the activation energy for gravisensing.     

The Effect of Spaceflight on Growth of Ulocladium chartarum Colonies on the International Space Station

The objectives of this 14 days experiment were to investigate the effect of spaceflight on the growth of Ulocladium chartarum, to study the viability of the aerial and submerged mycelium and to put in evidence changes at the cellular level. U. chartarum was chosen for the spaceflight experiment because it is well known to be involved in biodeterioration of organic and inorganic substrates covered with organic deposits and expected to be a possible contaminant in Spaceships. Colonies grown on the International Space Station (ISS) and on Earth were analysed post-flight. This study clearly indicates that U. chartarum is able to grow under spaceflight conditions developing, as a response, a complex colony morphotype never mentioned previously. We observed that spaceflight reduced the rate of growth of aerial mycelium, but stimulated the growth of submerged mycelium and of new microcolonies. In Spaceships and Space Stations U. chartarum and other fungal species could find a favourable environment to grow invasively unnoticed in the depth of surfaces containing very small amount of substrate, posing a risk factor for biodegradation of structural components, as well as a direct threat for crew health. The colony growth cycle of U. chartarum provides a useful eukaryotic system for the study of fungal growth under spaceflight conditions.     

Effects of a Closed Space Environment on Gene Expression in Hair Follicles of Astronauts in the International Space Station

Adaptation to the space environment can sometimes pose physiological problems to International Space Station (ISS) astronauts after their return to earth. Therefore, it is important to develop healthcare technologies for astronauts. In this study, we examined the feasibility of using hair follicles, a readily obtained sample, to assess gene expression changes in response to spaceflight adaptation. In order to investigate the gene expression changes in human hair follicles during spaceflight, hair follicles of 10 astronauts were analyzed by microarray and real time qPCR analyses. We found that spaceflight alters human hair follicle gene expression. The degree of changes in gene expression was found to vary among individuals. In some astronauts, genes related to hair growth such as FGF18, ANGPTL7 and COMP were upregulated during flight, suggesting that spaceflight inhibits cell proliferation in hair follicles.     

Shotgun metagenomic analysis of kombucha mutualistic community exposed to Mars-like environment outside the International Space Station

Kombucha is a multispecies microbial ecosystem mainly composed of acetic acid bacteria and osmophilic acid-tolerant yeasts, which is used to produce a probiotic drink. Furthermore, Kombucha Mutualistic Community (KMC) has been recently proposed to be used during long space missions as both a living functional fermented product to improve astronauts' health and an efficient source of bacterial nanocellulose. In this study, we compared KMC structure and functions before and after samples were exposed to the space/Mars-like environment outside the International Space Station in order to investigate the changes related to their re-adaptation to Earth-like conditions by shotgun metagenomics, using both diversity and functional analyses of Community Ecology and Complex Networks approach. Our study revealed that the long-term exposure to space/Mars-like conditions on low Earth orbit may disorganize the KMC to such extent that it will not restore the initial community structure; however, KMC core microorganisms of the community were maintained. Nonetheless, there were no significant differences in the community functions, meaning that the KMC communities are ecologically resilient. Therefore, despite the extremely harsh conditions, key KMC species revived and provided the community with the genetic background needed to survive long periods of time under extraterrestrial conditions.     

Development and Improvement of Countermeasures against Negative Effects of Weightlessness in Long-Term Space Flights on Board International Space Station

Human Physiology - The paper describes the history of development and improvement of the means of the Russian system of countermeasures against the negative effects of space flights over the past...