Microbial Detection and Quantification of Low-Biomass Water Samples Using an International Space Station Smart Sample Concentrator.

The pressing need to safeguard the health of astronauts aboard the International Space Station (ISS) necessitates constant and rigorous microbial monitoring. Recognizing the shortcomings of traditional culture-based methods, NASA is deliberating the incorporation of molecular-based techniques. The challenge, however, lies in developing and validating effective methods for concentrating samples to facilitate this transition. This study is dedicated to investigating the potential of an ISS Smart Sample Concentrator (iSSC) as an innovative concentration method. First, the iSSC system and its components were tested and optimized for microgravity, including various testing environments: a drop tower, parabolic flight, and the ISS itself. Upon confirming the system's compatibility with microgravity, we further evaluated its proficiency and reliability in concentrating large volumes (i.e., 1 L) of water samples inoculated with different microbes. The samples carried 102 to 105 colony-forming units (CFUs) of Sphingomonas paucimobilis, Ralstonia pickettii, or Cupriavidus basilensis per liter, aligning with NASA's acceptable limit of 5 × 104 CFU/L. The average retrieved volume post-concentration was ≈450 µL, yielding samples that were ≈2200 times more concentrated for subsequent quantitative PCR (qPCR) and CFU analysis. The average microbial percent recovery, as assessed with CFU counts, demonstrated consistency for C. basilensis and R. pickettii at around 50% and 45%, respectively. For S. paucimobilis, the efficiency oscillated between 40% and 80%. Interestingly, when we examined microbial recovery using qPCR, the results showed more variability across all tested species. The significance of these findings lies not merely in the successful validation of the iSSC but also in the system's proven consistency, as evidenced by its alignment with previous validation-phase results. In conclusion, conducted research underscored the potential of the iSSC in monitoring microbial contamination in potable water aboard the ISS, heralding a paradigm shift from culture-based to molecular-based monitoring methods.

Microbial applications for sustainable space exploration beyond low Earth orbit.

With the construction of the International Space Station, humans have been continuously living and working in space for 22 years. Microbial studies in space and other extreme environments on Earth have shown the ability for bacteria and fungi to adapt and change compared to "normal" conditions. Some of these changes, like biofilm formation, can impact astronaut health and spacecraft integrity in a negative way, while others, such as a propensity for plastic degradation, can promote self-sufficiency and sustainability in space. With the next era of space exploration upon us, which will see crewed missions to the Moon and Mars in the next 10 years, incorporating microbiology research into planning, decision-making, and mission design will be paramount to ensuring success of these long-duration missions. These can include astronaut microbiome studies to protect against infections, immune system dysfunction and bone deterioration, or biological in situ resource utilization (bISRU) studies that incorporate microbes to act as radiation shields, create electricity and establish robust plant habitats for fresh food and recycling of waste. In this review, information will be presented on the beneficial use of microbes in bioregenerative life support systems, their applicability to bISRU, and their capability to be genetically engineered for biotechnological space applications. In addition, we discuss the negative effect microbes and microbial communities may have on long-duration space travel and provide mitigation strategies to reduce their impact. Utilizing the benefits of microbes, while understanding their limitations, will help us explore deeper into space and develop sustainable human habitats on the Moon, Mars and beyond.

Microbiome-liver crosstalk: A multihit therapeutic target for liver disease.

Liver disease has become a leading cause of death, particularly in the West, where it is attributed to more than two million deaths annually. The correlation between gut microbiota and liver disease is still not fully understood. However, it is well known that gut dysbiosis accompanied by a leaky gut causes an increase in lipopolysaccharides in circulation, which in turn evoke massive hepatic inflammation promoting liver cirrhosis. Microbial dysbiosis also leads to poor bile acid metabolism and low short-chain fatty acids, all of which exacerbate the inflammatory response of liver cells. Gut microbial homeostasis is maintained through intricate processes that ensure that commensal microbes adapt to the low oxygen potential of the gut and that they rapidly occupy all the intestinal niches, thus outcompeting any potential pathogens for available nutrients. The crosstalk between the gut microbiota and its metabolites also guarantee an intact gut barrier. These processes that protect against destabilization of gut microbes by potential entry of pathogenic bacteria are collectively called colonization resistance and are equally essential for liver health. In this review, we shall investigate how the mechanisms of colonization resistance influence the liver in health and disease and the microbial-liver crosstalk potential as therapeutic target areas.

Toward sustainable space exploration: a roadmap for harnessing the power of microorganisms.

Finding sustainable approaches to achieve independence from terrestrial resources is of pivotal importance for the future of space exploration. This is relevant not only to establish viable space exploration beyond low Earth-orbit, but also for ethical considerations associated with the generation of space waste and the preservation of extra-terrestrial environments. Here we propose and highlight a series of microbial biotechnologies uniquely suited to establish sustainable processes for in situ resource utilization and loop-closure. Microbial biotechnologies research and development for space sustainability will be translatable to Earth applications, tackling terrestrial environmental issues, thereby supporting the United Nations Sustainable Development Goals.

Microbial Tracking-2, a metagenomics analysis of bacteria and fungi onboard the International Space Station.

BACKGROUND: The International Space Station (ISS) is a unique and complex built environment with the ISS surface microbiome originating from crew and cargo or from life support recirculation in an almost entirely closed system. The Microbial Tracking 1 (MT-1) project was the first ISS environmental surface study to report on the metagenome profiles without using whole-genome amplification. The study surveyed the microbial communities from eight surfaces over a 14-month period. The Microbial Tracking 2 (MT-2) project aimed to continue the work of MT-1, sampling an additional four flights from the same locations, over another 14 months. METHODS: Eight surfaces across the ISS were sampled with sterile wipes and processed upon return to Earth. DNA extracted from the processed samples (and controls) were treated with propidium monoazide (PMA) to detect intact/viable cells or left untreated and to detect the total DNA population (free DNA/compromised cells/intact cells/viable cells). DNA extracted from PMA-treated and untreated samples were analyzed using shotgun metagenomics. Samples were cultured for bacteria and fungi to supplement the above results. RESULTS: Staphylococcus sp. and Malassezia sp. were the most represented bacterial and fungal species, respectively, on the ISS. Overall, the ISS surface microbiome was dominated by organisms associated with the human skin. Multi-dimensional scaling and differential abundance analysis showed significant temporal changes in the microbial population but no spatial differences. The ISS antimicrobial resistance gene profiles were however more stable over time, with no differences over the 5-year span of the MT-1 and MT-2 studies. Twenty-nine antimicrobial resistance genes were detected across all samples, with macrolide/lincosamide/streptogramin resistance being the most widespread. Metagenomic assembled genomes were reconstructed from the dataset, resulting in 82 MAGs. Functional assessment of the collective MAGs showed a propensity for amino acid utilization over carbohydrate metabolism. Co-occurrence analyses showed strong associations between bacterial and fungal genera. Culture analysis showed the microbial load to be on average 3.0 × 105 cfu/m2 CONCLUSIONS: Utilizing various metagenomics analyses and culture methods, we provided a comprehensive analysis of the ISS surface microbiome, showing microbial burden, bacterial and fungal species prevalence, changes in the microbiome, and resistome over time and space, as well as the functional capabilities and microbial interactions of this unique built microbiome. Data from this study may help to inform policies for future space missions to ensure an ISS surface microbiome that promotes astronaut health and spacecraft integrity. Video Abstract.

Understanding the Complexities and Changes of the Astronaut Microbiome for Successful Long-Duration Space Missions.

During space missions, astronauts are faced with a variety of challenges that are unique to spaceflight and that have been known to cause physiological changes in humans over a period of time. Several of these changes occur at the microbiome level, a complex ensemble of microbial communities residing in various anatomic sites of the human body, with a pivotal role in regulating the health and behavior of the host. The microbiome is essential for day-to-day physiological activities, and alterations in microbiome composition and function have been linked to various human diseases. For these reasons, understanding the impact of spaceflight and space conditions on the microbiome of astronauts is important to assess significant health risks that can emerge during long-term missions and to develop countermeasures. Here, we review various conditions that are caused by long-term space exploration and discuss the role of the microbiome in promoting or ameliorating these conditions, as well as space-related factors that impact microbiome composition. The topics explored pertain to microgravity, radiation, immunity, bone health, cognitive function, gender differences and pharmacomicrobiomics. Connections are made between the trifecta of spaceflight, the host and the microbiome, and the significance of these interactions for successful long-term space missions.

Clean room microbiome complexity impacts planetary protection bioburden.

BACKGROUND: The Spacecraft Assembly Facility (SAF) at the NASA's Jet Propulsion Laboratory is the primary cleanroom facility used in the construction of some of the planetary protection (PP)-sensitive missions developed by NASA, including the Mars 2020 Perseverance Rover that launched in July 2020. SAF floor samples (n=98) were collected, over a 6-month period in 2016 prior to the construction of the Mars rover subsystems, to better understand the temporal and spatial distribution of bacterial populations (total, viable, cultivable, and spore) in this unique cleanroom. RESULTS: Cleanroom samples were examined for total (living and dead) and viable (living only) microbial populations using molecular approaches and cultured isolates employing the traditional NASA standard spore assay (NSA), which predominantly isolated spores. The 130 NSA isolates were represented by 16 bacterial genera, of which 97% were identified as spore-formers via Sanger sequencing. The most spatially abundant isolate was Bacillus subtilis, and the most temporally abundant spore-former was Virgibacillus panthothenticus. The 16S rRNA gene-targeted amplicon sequencing detected 51 additional genera not found in the NSA method. The amplicon sequencing of the samples treated with propidium monoazide (PMA), which would differentiate between viable and dead organisms, revealed a total of 54 genera: 46 viable non-spore forming genera and 8 viable spore forming genera in these samples. The microbial diversity generated by the amplicon sequencing corresponded to ~86% non-spore-formers and ~14% spore-formers. The most common spatially distributed genera were Sphinigobium, Geobacillus, and Bacillus whereas temporally distributed common genera were Acinetobacter, Geobacilllus, and Bacillus. Single-cell genomics detected 6 genera in the sample analyzed, with the most prominent being Acinetobacter. CONCLUSION: This study clearly established that detecting spores via NSA does not provide a complete assessment for the cleanliness of spacecraft-associated environments since it failed to detect several PP-relevant genera that were only recovered via molecular methods. This highlights the importance of a methodological paradigm shift to appropriately monitor bioburden in cleanrooms for not only the aeronautical industry but also for pharmaceutical, medical industries, etc., and the need to employ molecular sequencing to complement traditional culture-based assays. Video abstract.

Characterizing the breast cancer lipidome and its interaction with the tissue microbiota.

Breast cancer is the most diagnosed cancer amongst women worldwide. We have previously shown that there is a breast microbiota which differs between women who have breast cancer and those who are disease-free. To better understand the local biochemical perturbations occurring with disease and the potential contribution of the breast microbiome, lipid profiling was performed on non-tumor breast tissue collected from 19 healthy women and 42 with breast cancer. Here we identified unique lipid signatures between the two groups with greater amounts of lysophosphatidylcholines and oxidized cholesteryl esters in the tissue from women with breast cancer and lower amounts of ceramides, diacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. By integrating these lipid signatures with the breast bacterial profiles, we observed that Gammaproteobacteria and those from the class Bacillus, were negatively correlated with ceramides, lipids with antiproliferative properties. In the healthy tissues, diacylglyerols were positively associated with Acinetobacter, Lactococcus, Corynebacterium, Prevotella and Streptococcus. These bacterial groups were found to possess the genetic potential to synthesize these lipids. The cause-effect relationships of these observations and their contribution to disease patho-mechanisms warrants further investigation for a disease afflicting millions of women around the world.

Simulated Microgravity Promotes Horizontal Gene Transfer of Antimicrobial Resistance Genes between Bacterial Genera in the Absence of Antibiotic Selective Pressure.

Bacteria are able to adapt and survive in harsh and changing environments through many mechanisms, with one of them being horizontal gene transfer (HGT). This process is one of the leading culprits in the spread of antimicrobial resistance (AMR) within bacterial communities and could pose a significant health threat to astronauts if they fell ill, especially on long-duration space missions. In order to better understand the degree of HGT activity that could occur in space, biosafety level-2, donor and recipient bacteria were co-cultured under simulated microgravity (SMG) on Earth with concomitant 1G controls. Two AMR genes, blaOXA-500 and ISAba1, from the donor Acinetobacter pittii, were tracked in four recipient strains of Staphylococcus aureus (which did not harbor those genes) using polymerase chain reaction. All four S. aureus strains that were co-cultured with A. pittii under SMG had a significantly higher number of isolates that were now blaOXA-500- and ISAba1-positive compared to growth at 1G. The acquisition of these genes by the recipient induced a phenotypic change, as these isolates were now resistant to oxacillin, which they were previously susceptible to. This is a novel study, presenting, for the first time, increased HGT activity under SMG and the potential impact of the space environment in promoting increased gene dissemination within bacterial communities.

Draft Genome Sequences of Fungi Isolated from the International Space Station during the Microbial Tracking-2 Experiment.

As part of the Microbial Tracking-2 study, 94 fungal strains were isolated from surfaces on the International Space Station, and whole-genome sequences were assembled. Characterization of these draft genomes will allow evaluation of microgravity adaption, risks to human health and spacecraft functioning, and biotechnological applications of fungi.

Investigation of Spaceflight Induced Changes to Astronaut Microbiomes.

The International Space Station (ISS) is a uniquely enclosed environment that has been continuously occupied for the last two decades. Throughout its operation, protecting the health of the astronauts on-board has been a high priority. The human microbiome plays a significant role in maintaining human health, and disruptions in the microbiome have been linked to various diseases. To evaluate the effects of spaceflight on the human microbiome, body swabs and saliva samples were collected from four ISS astronauts on consecutive expeditions. Astronaut samples were analyzed using shotgun metagenomic sequencing and microarrays to characterize the microbial biodiversity before, during, and after the astronauts' time onboard the ISS. Samples were evaluated at an individual and population level to identify changes in microbial diversity and abundance. No significant changes in the number or relative abundance of taxa were observed between collection time points when samples from all four astronauts were analyzed together. When the astronauts' saliva samples were analyzed individually, the saliva samples of some astronauts showed significant changes in the relative abundance of taxa during and after spaceflight. The relative abundance of Prevotella in saliva samples increased during two astronauts' time onboard the ISS while the relative abundance of other commensal taxa such as Neisseria, Rothia, and Haemophilus decreased. The abundance of some antimicrobial resistance genes within the saliva samples also showed significant changes. Most notably, elfamycin resistance gene significantly increased in all four astronauts post-flight and a CfxA6 beta-lactam marker significantly increased during spaceflight but returned to normal levels post-flight. The combination of both shotgun metagenomic sequencing and microarrays showed the benefit of both technologies in monitoring microbes on board the ISS. There were some changes in each astronaut's microbiome during spaceflight, but these changes were not universal for all four astronauts. Two antimicrobial resistance gene markers did show a significant change in abundance in the saliva samples of all four astronauts across their collection times. These results provide insight for future ISS microbial monitoring studies and targets for antimicrobial resistance screenings.

Draft Genome Sequences of Various Bacterial Phyla Isolated from the International Space Station.

Whole-genome sequences were generated from 96 bacterial strains of 14 species that were isolated from International Space Station surfaces during the Microbial Tracking 2 study. Continued characterization of this closed habitat's microbiome enables tracking of the spread and evolution of secondary pathogens, which is vital for astronaut health.

End-to-End Protocol for the Detection of SARS-CoV-2 from Built Environments.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is a respiratory virus primarily transmitted person to person through inhalation of droplets or aerosols, laden with viral particles. However, as recent studies have shown, virions can remain infectious for up to 72 h on surfaces, which can lead to transmission through contact. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end (E2E) study showed that the effective combination for monitoring SARS-CoV-2 on surfaces includes using an Isohelix swab collection tool, DNA/RNA Shield as a preservative, an automated system for RNA extraction, and reverse transcriptase quantitative PCR (RT-qPCR) as the detection assay. Using this E2E approach, this study showed that, in some cases, noninfectious viral fragments of SARS-CoV-2 persisted on surfaces for as long as 8 days even after bleach treatment. Additionally, debris associated with specific built environment surfaces appeared to inhibit and negatively impact the recovery of RNA; Amerstat demonstrated the highest inhibition (>90%) when challenged with an inactivated viral control. Overall, it was determined that this E2E protocol required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from test surfaces. Despite our findings of viral fragment longevity on surfaces, when this method was employed to evaluate 368 samples collected from various built environmental surfaces, all samples tested negative, indicating that the surfaces were either void of virus or below the detection limit of the assay.IMPORTANCE The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus responsible for coronavirus disease 2019 [COVID-19]) pandemic has led to a global slowdown with far-reaching financial and social impacts. The SARS-CoV-2 respiratory virus is primarily transmitted from person to person through inhalation of infected droplets or aerosols. However, some studies have shown that virions can remain infectious on surfaces for days and can lead to human infection from contact with infected surfaces. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end study showed that the effective combination for monitoring SARS-CoV-2 on surfaces required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from surfaces. This comprehensive study can provide valuable information regarding surface monitoring of various materials as well as the capacity to retain viral RNA and allow for effective disinfection.

Validation of the International Space Station Smart Sample Concentrator for Microbial Monitoring of Low Biomass Water Samples.

Microbial monitoring on the International Space Station (ISS) is essential to keep astronauts healthy. Current practice involves culture-based methods, but future directives by the National Aeronautics and Space Administration (NASA) will require the use of molecular-based approaches, such as quantitative PCR (qPCR). However, in order to successfully and reliably detect the allowable limit of 5 × 104 colony forming units (CFUs) of bacteria per liter in potable water on the ISS with qPCR, water concentration must first be performed. This report presents the data from a validation study of a NASA-sponsored small business research initiative to develop a microgravity-compatible, automated water concentrator to be used on the ISS, which has been named the ISS Smart Sample Concentrator (iSSC). Efficiency and reproducibility of the iSSC were compared with a ground-based automated water concentrator and the standard Millipore manual filtration. Using 104 CFU/L of Sphingomonas paucimobilis, Ralstonia pickettii, and Cupriavidus basilensis and a mixed microbial community, we have shown, through culture and qPCR, that the iSSC is comparable, if not better, at recovering and concentrating bacteria from large volumes of water, with good reproducibility.

Validating an Automated Nucleic Acid Extraction Device for Omics in Space Using Whole Cell Microbial Reference Standards.

NASA has made great strides in the past five years to develop a suite of instruments for the International Space Station in order to perform molecular biology in space. However, a key piece of equipment that has been lacking is an instrument that can extract nucleic acids from an array of complex human and environmental samples. The Omics in Space team has developed the µTitan (simulated micro(µ) gravity tested instrument for automated nucleic acid) system capable of automated, streamlined, nucleic acid extraction that is adapted for use under microgravity. The µTitan system was validated using a whole cell microbial reference (WCMR) standard comprised of a suspension of nine bacterial strains, titrated to concentrations that would challenge the performance of the instrument, as well as to determine the detection limits for isolating DNA. Quantitative assessment of system performance was measured by comparing instrument input challenge dose vs recovery by Qubit spectrofluorometry, qPCR, Bioanalyzer, and Next Generation Sequencing. Overall, results indicate that the µTitan system performs equal to or greater than a similar commercially available, earth-based, automated nucleic acid extraction device. The µTitan system was also tested in Yellowstone National Park (YNP) with the WCMR, to mimic a remote setting, with limited resources. The performance of the device at YNP was comparable to that in a laboratory setting. Such a portable, field-deployable, nucleic extraction system will be valuable for environmental microbiology, as well as in health care diagnostics.

Mucosal or systemic microbiota exposures shape the B cell repertoire.

Colonization by the microbiota causes a marked stimulation of B cells and induction of immunoglobulin, but mammals colonized with many taxa have highly complex and individualized immunoglobulin repertoires1,2. Here we use a simplified model of defined transient exposures to different microbial taxa in germ-free mice3 to deconstruct how the microbiota shapes the B cell pool and its functional responsiveness. We followed the development of the immunoglobulin repertoire in B cell populations, as well as single cells by deep sequencing. Microbial exposures at the intestinal mucosa generated oligoclonal responses that differed from those of germ-free mice, and from the diverse repertoire that was generated after intravenous systemic exposure to microbiota. The IgA repertoire-predominantly to cell-surface antigens-did not expand after dose escalation, whereas increased systemic exposure broadened the IgG repertoire to both microbial cytoplasmic and cell-surface antigens. These microbial exposures induced characteristic immunoglobulin heavy-chain repertoires in B cells, mainly at memory and plasma cell stages. Whereas sequential systemic exposure to different microbial taxa diversified the IgG repertoire and facilitated alternative specific responses, sequential mucosal exposure produced limited overlapping repertoires and the attrition of initial IgA binding specificities. This shows a contrast between a flexible response to systemic exposure with the need to avoid fatal sepsis, and a restricted response to mucosal exposure that reflects the generic nature of host-microbial mutualism in the mucosa.

Microbiome and Metagenome Analyses of a Closed Habitat during Human Occupation.

Microbial contamination during long-term confinements of space exploration presents potential risks for both crew members and spacecraft life support systems. A novel swab kit was used to sample various surfaces from a submerged, closed, analog habitat to characterize the microbial populations. Samples were collected from various locations across the habitat which were constructed from various surface materials (linoleum, dry wall, particle board, glass, and metal), and microbial populations were examined by culture, quantitative PCR (qPCR), microbiome 16S rRNA gene sequencing, and shotgun metagenomics. Propidium monoazide (PMA)-treated samples identified the viable/intact microbial population of the habitat. The cultivable microbial population ranged from below the detection limit to 106 CFU/sample, and their identity was characterized using Sanger sequencing. Both 16S rRNA amplicon and shotgun sequencing were used to characterize the microbial dynamics, community profiles, and functional attributes (metabolism, virulence, and antimicrobial resistance). The 16S rRNA amplicon sequencing revealed abundance of viable (after PMA treatment) Actinobacteria (Brevibacterium, Nesternkonia, Mycobacterium, Pseudonocardia, and Corynebacterium), Firmicutes (Virgibacillus, Staphylococcus, and Oceanobacillus), and Proteobacteria (especially Acinetobacter) on linoleum, dry wall, and particle board (LDP) surfaces, while members of Firmicutes (Leuconostocaceae) and Proteobacteria (Enterobacteriaceae) were high on the glass/metal surfaces. Nonmetric multidimensional scaling determined from both 16S rRNA and metagenomic analyses revealed differential microbial species on LDP surfaces and glass/metal surfaces. The shotgun metagenomic sequencing of samples after PMA treatment showed bacterial predominance of viable Brevibacterium (53.6%), Brachybacterium (7.8%), Pseudonocardia (9.9%), Mycobacterium (3.7%), and Staphylococcus (2.1%), while fungal analyses revealed Aspergillus and Penicillium dominance.IMPORTANCE This study provides the first assessment of monitoring cultivable and viable microorganisms on surfaces within a submerged, closed, analog habitat. The results of the analyses presented herein suggest that the surface material plays a role in microbial community structure, as the microbial populations differed between LDP and metal/glass surfaces. The metal/glass surfaces had less-complex community, lower bioburden, and more closely resembled the controls. These results indicated that material choice is crucial when building closed habitats, even if they are simply analogs. Finally, while a few species were associated with previously cultivated isolates from the International Space Station and MIR spacecraft, the majority of the microbial ecology of the submerged analog habitat differs greatly from that of previously studied analog habitats.

The influence of spaceflight on the astronaut salivary microbiome and the search for a microbiome biomarker for viral reactivation.

BACKGROUND: Spaceflight impacts astronauts in many ways but little is known on how spaceflight affects the salivary microbiome and the consequences of these changes on astronaut health, such as viral reactivation. In order to understand this, the salivary microbiome was analyzed with 16S rRNA gene amplicon sequencing, and saliva viral titers were analyzed with quantitative polymerase chain reaction (qPCR) with primers specific for Epstein-Barr virus (EBV), herpes simplex virus (HSV), and varicella zoster virus (VZV) from 10 astronauts pre-flight, in-flight, and post-flight. RESULTS: Streptococcus was the most abundant organism in the saliva, making up 8% of the total organisms detected, and their diversity decreased during spaceflight. Other organisms that had statistically significant changes were Proteobacteria and Fusobacteria which increased during flight and Actinobacteria which decreased during flight. At the genus level, Catonella, Megasphera, and Actinobacillus were absent in more than half of saliva samples collected pre-flight but were then detected during flight. In those subjects that already had these genera pre-flight, their relative abundances increased during flight. Correlation analyses between the microbiome and viral titers revealed a positive correlation with Gracilibacteria, Absconditabacteria, and Abiotrophia and a negative correlation between Oribacterium, Veillonella, and Haemophilus. There was also a significant positive correlation between microbiome richness and EBV viral titers. CONCLUSIONS: This is the first study to look at how the salivary microbiome changes as a result of spaceflight and the search for bacterial biomarkers for viral reactivation. Further studies examining the role of specific organisms that were shown to be correlative and predictive in viral reactivation, a serious problem in astronauts during spaceflight, could lead to mitigation strategies to help prevent disease during both short and long duration space missions. Video abstract.

Crewmember microbiome may influence microbial composition of ISS habitable surfaces.

The International Space Station (ISS) is a complex built environment physically isolated from Earth. Assessing the interplay between the microbial community of the ISS and its crew is important for preventing biomedical and structural complications for long term human spaceflight missions. In this study, we describe one crewmember's microbial profile from body swabs of mouth, nose, ear, skin and saliva that were collected at eight different time points pre-, during and post-flight. Additionally, environmental surface samples from eight different habitable locations in the ISS were collected from two flights. Environmental samples from one flight were collected by the crewmember and samples from the next flight were collected after the crewmember departed. The microbial composition in both environment and crewmember samples was measured using shotgun metagenomic sequencing and processed using the Livermore Metagenomics Analysis Toolkit. Ordination of sample to sample distances showed that of the eight crew body sites analyzed, skin, nostril, and ear samples are more similar in microbial composition to the ISS surfaces than mouth and saliva samples; and that the microbial composition of the crewmember's skin samples are more closely related to the ISS surface samples collected by the crewmember on the same flight than ISS surface samples collected by other crewmembers on different flights. In these collections, species alpha diversity in saliva samples appears to decrease during flight and rebound after returning to Earth. This is the first study to compare the ISS microbiome to a crewmember's microbiome via shotgun metagenomic sequencing. We observed that the microbiome of the surfaces inside the ISS resemble those of the crew's skin. These data support future crew and ISS microbial surveillance efforts and the design of preventive measures to maintain crew habitat onboard spacecraft destined for long term space travel.