ARTICLE https://doi.org/10.1038/s41467-019-11682-z OPEN Space Station conditions are selective but do not alter microbial characteristics relevant to human health Maximilian Mora1, Lisa Wink1, Ines Kögler1, Alexander Mahnert1, Petra Rettberg 2, Petra Schwendner3, René Demets4, Charles Cockell 3, Tatiana Alekhova5, Andreas Klingl6, Robert Krause1,7, Anna Zolotariof 3, Alina Alexandrova5 & Christine Moissl-Eichinger1,7 1234567890():,; The International Space Station (ISS) is a unique habitat for humans and microorganisms. Here, we report the results of the ISS experiment EXTREMOPHILES, including the analysis of microbial communities from several areas aboard at three time points. We assess microbial diversity, distribution, functional capacity and resistance profile using a combination of cultivation-independent analyses (amplicon and shot-gun sequencing) and cultivation- dependent analyses (physiological and genetic characterization of microbial isolates, anti- biotic resistance tests, co-incubation experiments). We show that the ISS microbial com- munities are highly similar to those present in ground-based confined indoor environments and are subject to fluctuations, although a core microbiome persists over time and locations. The genomic and physiological features selected by ISS conditions do not appear to be directly relevant to human health, although adaptations towards biofilm formation and sur- face interactions were observed. Our results do not raise direct reason for concern with respect to crew health, but indicate a potential threat towards material integrity in moist areas. 1 Medical University of Graz, Department of Internal Medicine, Auenbruggerplatz 15, 8036 Graz, Austria. 2 German Aerospace Center (DLR), Institute of Aerospace Medicine, Radiation Biology Department, Research Group Astrobiology, Linder Höhe, 51147 Cologne, Germany. 3 University of Edinburgh, School of Physics and Astronomy, James Clerk Maxwell Building, Peter Guthrie Tait Road, Edinburgh EH9 3FD, UK. 4 European Space Research and Technology Centre (ESTEC), Keplerlaan 1, 2201 AZ Noordwijk, The Netherlands. 5 Lomonosov Moscow State University, Biological Faculty, ul. Leninskiye Gory, 1, стр. 12, Moscow, Russia. 6 Ludwig Maximilians University of Munich, Plant Development and Electron Microscopy, Department of Biology I, Biocenter, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany. 7 BioTechMed Graz, Mozartgasse 12/II, 8010 Graz, Austria. Correspondence and requests for materials should be addressed to C.M.-E. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:3990 | https://doi.org/10.1038/s41467-019-11682-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11682-z uman space exploration beyond boundaries of Earth and Although an increased risk for the health of the astronauts and HMoon is a declared goal of NASA, ESA, Roscosmos and cosmonauts aboard has often been proposed, infections of crew other space-faring agencies, envisaging a potential human members or health issues related to pathogenic action of micro- Mars mission in the next 20–30 years. Maintenance of crew’s organisms have been reported only rarely24. This may be attri- health during a several hundred days journey in a confined butable to environmental contamination limits (air and surfaces) artificial environment in space is one of the key aspects, which has having rarely been exceeded, at which times effective counter- to be addressed. measures had been implemented25. Moreover, a genomics-based The human immune system was shown to be compromised meta-analysis demonstrated that although pangenomes of Bacil- under space flight conditions, as a significant decrease of lym- lus and Staphylococcus isolated from the ISS differed from Earth- phocytes and also of the activity of innate and adaptive immune based counterparts, these differences did not appear to be health- response was observed1,2. Adding an order of complexity, human threatening26. Thus, a comprehensive understanding of potential health is intertwined with its microbiome, billions of micro- microbial adaptations to the ISS based on complementary geno- organisms thriving on external and internal surfaces of the mic and cultivation approaches is important to better understand human body. Our body’s microbiome is prone to external factors, potential human health implications. including the environmental microbiome, as they are in constant In this study, we report on the realization of the ISS experiment exchange and interaction. EXTREMOPHILES. Our objectives included the analysis of the Several risks with respect to microorganisms and human profile, diversity, dynamics, and functional capacity of the culti- spaceflight have been identified. These include a potentially vable and noncultivable microbiome aboard. Moreover, we increased infection risk, as it has been shown that microgravity assessed their genomic and physiological adaptation toward ISS affects the virulence of certain microorganisms, such as Salmo- conditions and refuted the hypothesis, that, as indicated by pre- nella typhimurium3, Listeria monocytogenes, and Enterococcus vious literature reports, ISS microorganisms possess a higher faecalis4. Another stressor for the indigenous microorganisms is extremo-tolerance and antibiotics-resistance potential compared the strict maintenance regime, which could result in an increase with ground controls. Moreover, we observed surface-microbe of antimicrobial resistances, as recently shown for highly-main- interaction with regard to material integrity, exhibited by selected, tained, confined built environments5. Some microorganisms freshly isolated ISS strains. might even pose a risk to the material integrity of a spacecraft: So- called technophilic microorganisms, in particular fungi, are able to corrode alloys and polymers used in spacecraft assembly6. Results Technophilic microorganisms caused major problems on the Study set-up and sampling overview. In-flight sampling on former Russian space station MIR, partaking in damage to board the ISS was performed April to June 2017 under the ESA structural materials as well as malfunctioning of various space operation named EXTREMOPHILES. All samples (n = 24, plus systems and equipment7,8. Specifically, Bacillus, Penicillium, and controls) were taken during three sampling sessions. With a span Aspergillus species were associated with the progressive destruc- of 72 days between session A and B they were conceptualized for tion of a window in MIRs descent module9, and mold on wiring time-course sampling (same sampling locations). For compara- connectors was associated with electrical outages10. tive purposes, an ISS-relevant clean room and a therein housed The majority of information with respect to environmental cargo-spacecraft were sampled, namely clean room S5C at the microbiome composition and dynamics aboard manned space- Centre Spatial Guayanais near Kourou in French Guiana with craft is retrieved from ground-based simulation studies, such as an ATV spacecraft. the Mars50011 and the HI-SEAS (http://hi-seas.org/) experiments. However, the International Space Station (ISS) has, like no other currently available testbed for long-term manned space missions, ISS microbiome is dominated by human-associated micro- the scientific benefit of providing real spaceflight conditions, organisms. The microbial community composition was assessed including microgravity and an elevated background radiation. by amplicon sequence analysis of wipe samples obtained from The ISS orbits Earth ∼400 km above ground and is meanwhile sessions A–C and the Kourou clean room. Archaeal and bacterial constantly inhabited for more than 18 years. Except for cargo RSVs (>3.500) were retrieved from ISS samples with the universal exchange and the arrival of new crew members roughly every approach (Fig. 1). The signatures belonged to 377 genera, with 6 months, the ISS is completely sealed off from any biological Streptococcus, Corynebacterium, Lactobacillus, Acinetobacter, ecosystem12. Staphylococcus as dominant taxa (Fig. 1, Supplementary Fig. 1, Analyses of the ISS microbiome have already been performed, Supplementary Data 1). Firmicutes, Proteobacteria, Actino- including microbial analyses of ISS debris and dust13–16, the study bacteria, and Bacteroidetes were found to be the predominant of the astronauts’ microbiome17, the characterization of bacterial bacterial phyla (all samples), whereas archaeal signatures (Woe- and fungal isolates from the ISS18,19, and the (molecular) micro- searchaeota, Thaumarchaeota, and Euryarchaeota) were fre- bial analysis of swab and wipe samples taken inside the ISS20.A quently detected in air (Cupola air: 14.1% and Columbus air study investigating the growth behavior of nonpathogenic (ter- session B: 3.6% of all sequences) and on various surfaces (Fig. 1). restrial) bacteria aboard the ISS found no changes in most bac- By the Archaea-targeting approach, mostly gut associated teria, given that they have enough nutrients21. Other publications Methanobrevibacter sequences (surface Cupola, Waste and focused on the detection of antimicrobial resistance genes aboard Hygiene Compartment), Woesearchaeota (ATU; hand grips the ISS and evaluated the potential risk these genes might repre- Columbus), and unclassified Archaea (ATU, dining table) were sent in a closed spacecraft environment22. Singh et al. assessed the detected. To a lesser extent, we also found signatures from succession and persistence