NOTE: Extended to 9/30/2018 per NSSC information (Ed., 11/21/17)

NOTE: Element change to Human Health Countermeasures; previously Space Human Factors & Habitability (Ed., 1/18/17)

NOTE: Extended to 9/30/2017 per F. Hernandez/ARC (Ed., 3/11/16)

NOTE: Extended to 9/30/2016 per A. Chu/ARC (Ed., 8/5/14)

NOTE: Gap changes per IRP Rev E (Ed., 3/19/14)

Investigating the impact of stress and status of the immune system on the human microbiome, and potentially on human health, during a space mission is also applicable to equivalent stressful situations on Earth. Some of the conclusions of this project will also be useful in situations where a group of individuals are confined in a relatively small and closed space for a long period of time, such as a submarine crew.

1) Sample collection and processing.

During this period all samples have been already collected and processed.

Characterization of the overall spatial patterns of microbial community structure showed that the factor that most contributed to variation among all samples was sampling site, in agreement with previous studies showing that the skin and nose microbiomes are more similar to each other than to those from feces or mouth. In addition, samples collected from the same body site tended to cluster by crew member. Interestingly, ISS environmental samples overlapped with specimens from the two skin sites and the nose microbiota. Further analysis showed that there was no significant difference among bacterial species that were present/absent in ISS microbial communities and inflight forehead and forearm skin microbiomes.

2) Changes in beta diversity of the astronauts’ microbiota.

To determine the influence that the amount of time spent at ISS has in any observed changes in astronaut microbiomes, we compared microbial profiles of microbiome samples collected at every inflight and postflight time points to all preflight time points (as a baseline). This analysis showed that compositional changes of the nose, skin, and gastrointestinal (GI) microbiomes were rapid and became evident by FD7. These changes persisted for at least six months until the end of the mission at the ISS. Furthermore, beta diversity changes did not significantly increase with the time astronauts spent in space, although preflight-inflight dissimilarity distances of the two skin sites and the nose microbiota showed a very modest upward trend associated with time spent inflight. In addition, compositional shifts in the skin and nose microbiomes persisted for at least 60 days after the astronauts returned to Earth. However, the composition of the GI microbiota became similar to preflight samples within two months of the astronauts’ return from the ISS.

3) Alteration of the microbial composition of the crew microbiome associated with the space environment.

To investigate the influence of the ISS as a contained and human built environment on the crew members' microbiomes, differential abundance analysis was performed between samples collected before, during, and after the mission to the ISS. This analysis identified 15 gastrointestinal genera whose abundance significantly changed in space. Ten out of the 15 genera belonged to the phylum Firmicutes and most belonged to the order Clostridiales. Among these taxonomic groups, there was a more than five-fold inflight reduction in Akkermansia and Ruminococcus, and a ~3-fold drop in Pseudobutyrivibrio and Fusicatenibacter. Most of these compositional changes reverted to preflight levels after astronauts returned to Earth, with the exemption of four genera of the phylum Firmicutes. Examination of skin samples also revealed changes in the relative abundance of several bacterial groups corresponding to 43 and 31 genera in the forearm and forehead, respectively. Noteworthy, skin microbial communities whose abundance decreased in space were mostly Gram-negative Proteobacteria. These groups included bacteria from the genus Acinetobacter, Cloacibacterium, and Pseudomonas. In contrast, most of the skin bacteria that became more abundant inflight belonged to the phylum Firmicutes, Bacteroidetes, and Actinobacteria, including bacteria of the genus Streptococcus, Staphylococcus, and Corynebacterium. Postflight samples showed a similar trend as inflight samples, with lower Proteobacteria and higher Firmicutes, Bacteroidetes, and Actinobacteria compared to Preflight skin. In addition, we observed a similar but milder response of the nares microbiota to the space environment with a significant drop in three genera of Gram-negative Betaproteobacteria, all of which were also reduced in skin. Likewise, nose inflight samples showed increases in five Gram-positive genera that also became more abundant on the skin of astronauts inflight. Many of these changes dissipated after the astronauts returned to Earth.

4) Identification of microbial changes associated with Astronauts’ immune dysregulation in space.

To gain insights into the potential impact of changes to the microbiome during spaceflight on immune functioning, changes to cytokine abundance in plasma were compared to changes in the composition of the GI microbiome. This analysis identified strong evidence of association between changes in astronauts’ GI microbiome and changes in cytokine profiles. Most notably, the abundance of bacteria of the genus Fusicatenibacter was negatively correlated with the concentration of pro-inflammatory cytokines IL-8, IL-1b, IL4, and TNFa. In addition, changes in bacteria of the genus Dorea were also negatively correlated with changes in the level of several cytokines including IL-1b, IL-1ra VEGF, and MIP-1b, all of which were increased in space.

5) ISS environment and its interaction with the Astronauts Microbiome.

Given that the ISS microbiota resembled that of the astronauts’ skin (see above), we hypothesized temporal changes could be influenced by the arrival of new crew members to the ISS every three months. To test this hypothesis, we compared the microbial composition of the ISS samples from early and late time points to the skin microbiome of astronauts that traveled to the ISS at the beginning or end of the study. This analysis showed that while there were no differences in the species present/absent between skin and ISS samples at either early or late time points, early skin microbiome samples had a significantly different composition to late ISS microbiome samples and vice versa. Moreover, comparative analysis of taxonomic profiles showed that the skin and ISS samples collected by the time astronauts were leaving the ISS were more similar to each other than the ISS and skin samples collected at FD7, when crew members had just arrived at the ISS. Comparison of differences in microbial alpha diversity and richness revealed that the six ISS sites surveyed had similar richness and Shannon diversity values. Alpha diversity and richness were also similar across environmental and inflight skin samples. However, we found that alpha diversity and richness of ISS samples significantly fluctuated over time in a manner that correlated with changes in inflight alpha diversity and richness of the crew skin microbiota. In spite of the fluctuations in the composition of the ISS microbial communities over time, each of the assessed ISS surfaces had a small proportion of OTUs (< 4%) that were highly prevalent during the entire duration of our study, most of them from the phyla Firmicutes, Actinobacteria, and Proteobacteria, and therefore, may be long-term residents of the ISS environment.

6) Astronauts’ virus reactivation and hormone stress levels.

Reactivation and shedding of latent varicella zoster virus (VZV), Epstein bar virus (EBV), and Herpes simplex virus (HSV1) as well as diurnal salivary cortisol, alpha-amylase, and dehydroepiandrosterone (DHEA) were measured prospectively in 10 astronauts before, during, and after their mission at the ISS to assess astronauts’ stress. Two astronauts did not shed any virus in any of their samples collected during the study. VZV reactivation was detected in the saliva of four crew members. Except for one crew member that showed VZV reactivation by L-60, no VZV was detected in saliva before flight. However, VZV was significantly reactivated in space, reaching a peak by FD90. After 30 days of the return to Earth, three of the astronauts became negative for VZV except for one astronaut that remained positive for up to 180 days. EBV and HSV1 were detected in the saliva of three and five astronauts, respectively, but no association was found between detection in saliva and spaceflight. No significant changes in levels of salivary cortisol were detected during the entire mission in any of the crew members. The inflight salivary concentration of alpha-amylase, however, was higher than preflight values reaching a maximum by the end of the mission in the ISS. DHEA showed an opposite trend, becoming less abundant by FD90 at the ISS.

7) Changes in the metabolic capacity of the GI microbiome during space travel.

To investigate whether the observed changes in the microbial composition of the astronauts’ GI microbiome have any consequence on its metabolic capacity we carried out metagenomic sequence analysis of the GI microbiome of six crew members during their missions to the ISS. Comparative analysis of the enzyme profiles predicted for each astronaut metagenome showed that, within subjects, inflight enzyme profiles tend to be different from preflight or postflight samples. Further analysis at the individual enzyme level identified 10 enzymatic functions that were differentially abundant between pre and inflight metagenomes, while only EC3.7.1.3 changed and became less abundant in postflight samples compared with preflight samples. In addition, we identified five metabolic pathways that were either over or underrepresented in inflight samples of the GI microbiome. One of these pathways, which consistently increased in space, corresponded to the pathway for the biosynthesis of polysaccharides, or lipopolysaccharides (LPS), which is a typical component of the outer membrane of gram negative bacteria. Taxonomic analysis of the genes participating in the LPS pathway showed that the observed changes are mostly driven by changes in the abundance of bacteria from the genus Bacteroides. The direction of the changes in gene abundance in the other four pathways identified were less consistent than the LPS pathway, with different GI metagenomes responding differently to space travel. One of these pathways was the one responsible for the biosynthesis of the bacterial flagellum. GI bacteria encoding for this pathway were enriched in three crew members and reduced in the metagenomes of the other three astronauts analyzed. Further taxonomic analysis showed that for all astronauts changes in this pathway were mostly caused by increases or decreases of bacteria of the genus Eubacterium and Roseburia, two of the most abundant motile bacteria found in the GI tract of healthy individuals. In agreement, the abundance of genes involved in bacterial chemotaxis was found to significantly change during spaceflight in a fashion similar to the related bacterial flagellum biosynthesis pathway. Further taxonomic analysis showed that the changes were mainly driven by alterations in the relative abundance of bacteria from the genus Eubacterium and Roseburia. Also, we investigated how changes in the bacterial communities of the GI tract affected specific bacterial metabolic pathways that are relevant to human health. In particular, we assessed pathways involved in the biosynthesis of short chain fatty acids (SCFA) butyrate and propionate and of vitamins B1, B6, Biotin, and Folate. None of these pathways seemed to be significantly perturbed in space. However, two astronauts did show a reduction in space of some genes encoding for key enzymes relevant for the biosynthesis of vitamin B1 (EC2.5.1.3 and EC2.7.4.7).

ANNUAL REPORTING AUGUST 2017:

1 - Inform Consent Briefings, Recruitment of Astronauts, and Base Data Collection

We have recruited all 9 subjects requested for the study plus one backup volunteer and inform consents have been already signed by all of them. All preflight, inflight, and postflight samples required for the project have been already collected and are at different stages of the processing pipeline. All swab, stool, and water samples were delivered to J Craig Venter Institute (JCVI) for 16S and metagenomic sequencing and analysis. Saliva and Blood samples were sent to Dr. Ott’s laboratory for measurement of cytokines, virus reactivation, and cortisol levels in blood and saliva samples, respectively. Sample sets from each astronaut are being processed altogether to reduce variation due to methodological error.

2 Sequencing and analysis of 16S rRNA gene taxonomic profiles.

2.1. Sample processing.

During the current period we have finalized the sequencing and analysis of 16S taxonomic profiles for the entire set of swab and stool samples collected from Astronauts A to I, totaling 507 samples (88 forehead, forearm, nose, and tongue swab samples, 73 stool samples, and 82 ISS environmental swab samples).

2.2 Analysis of alpha diversity across all sampled sites.

It has been shown that changes in the microbial diversity of the gastrointestinal (GI) tract is associated with a number of human diseases. In addition, previous studies on culturable bacteria have shown that diversity of GI bacteria drops after a space mission. Therefore, we investigated how bacterial diversity of the five Astronauts’ microbiomes surveyed was affected by space-travel.

This analysis showed that in general the diversity of the GI microbiome significantly increased in space, contrary to what it was expected based on previous results on culturable bacteria. Our analysis also showed that gut bacterial diversity went back to its original preflight levels after the crew returned to Earth. These results underline the importance of collecting microbiome samples in space, given that differences in alpha diversity between pre and inflight stool samples were mostly erased once study subjects returned to Earth.

Alpha diversity from the two skin sites, forehead and forearm, significantly changed in space, although in this case the direction of the change was not as consistent as in stool samples. In general, the diversity of the forehead skin microbiota did not return to their preflight levels within 60 days after landing. Noteworthy, unlike the gut microbiome, alpha diversity of the nose microbiota tended to be lower in space and did not recover after flight. It is possible that the reduction in inflight alpha diversity is a consequence of the highly clean environment of the ISS.

The tongue microbiota is the only crew microbiome surveyed that did not show any overall significant changes during the entire mission. However, at the individual level, there were some exceptions.

2.3 Analysis of changes in the taxonomic profiles of the astronauts’ microbiome.

Next we investigated how the taxonomic profiles of the astronauts’ microbiome changed during a mission to the ISS. In all cases there was a significant change in the composition of the gut microbiota in space, but recovery of the gastrointestinal microbiome after flight was dissimilar among astronauts. A similar analysis on the nose microbiota showed that, in general, the composition of the nose microbiome did not significantly change in space, although a slight change was detected between postflight samples and either inflight and preflight samples.

Contrary to the nose microbiota, both skin microbiomes surveyed showed a strong compositional change in space that did not recover after the crew returned to Earth. This result suggests that the skin microbiota is more sensitive to environmental changes compared to the other three microbiomes studied. One possible explanation is that the skin surface is more exposed to external conditions than the nose, tongue, and gut, making the skin microbiota more susceptible to changes in the environmental conditions.

Lastly, the composition of the tongue microbiome showed a slight change in space, but most of those changes reverted to their original preflight composition by R+60.

2.4 Study of the ISS environmental microbiota.

To study the stability of the ISS microbiome over time, we collected samples from six different sites on the ISS over a period of about three years. This analysis showed that the diversity of the ISS environmental microbiota is relatively stable over time, with some sites being more variable than others. This difference in microbial stability might be caused by the fact that some sites in the ISS are cleaned more frequently than others or are accessed by different crew members.

Study of the microbial composition of the ISS environment also showed that the microbes from the air filter inlet sampled in this study were different from those microorganisms inhabiting the rest of the ISS sites surveyed. This analysis also revealed that the composition of the ISS environmental microbiota significantly change over time and that those changes are likely to be driven by the skin microbiota of the crew.

3 Changes in cytokine concentration in plasma.

During this period we have also finalized with the cytokine analysis of plasma samples. This analysis revealed a number of cytokines whose concentration changed in space, most of them associated with the inflammatory response. These results are in line with previous studies on the same topic.

4. Future directions.

We are currently working on the integration of metagenomic-based microbiome functional data with cytokine profiles, astronauts’ stress information, and additional metadata collected for this project. Given the importance of the human GI microbiome on human health we are also generating 16S taxonomic profiles from the backup crew member to increase the significance of the results obtained so far.

NOTE: Element change to Human Health Countermeasures; previously Space Human Factors & Habitability (Ed., 1/18/17)

NOTE: Extended to 9/30/2017 per F. Hernandez/ARC (Ed., 3/11/16)

NOTE: Extended to 9/30/2016 per A. Chu/ARC (Ed., 8/5/14)

NOTE: Gap changes per IRP Rev E (Ed., 3/19/14)

Investigating the impact of stress and status of the immune system on the human microbiome, and potentially on human health, during a space mission is also applicable to equivalent stressful situations on Earth. Some of the conclusions of this project will also be useful in situations where a group of individuals are confined in a relatively small and closed space for a long period of time, such as a submarine crew.

We have recruited all 9 subjects requested for the study, named A to I, plus one backup volunteer, and inform consents have been already signed by all of them. Almost all pre-flight, in-flight and post-flight sample sets have been already collected and are at different degrees of processing, except for one set of blood and saliva samples from astronaut F. Swab, stool, and water samples were delivered to J Craig Venter Institute (JCVI) for 16S and metagenomic sequencing and analysis. Saliva and Blood samples were sent to Johnson Space Center (JSC) for measurement of cytokines (immune response), and virus reactivation and cortisol levels (to evaluate astronauts’ stress levels) in blood and saliva samples, respectively. Sample sets from each astronaut are being processed altogether to reduce variation due to methodological error.

2- Sequencing and preliminary analysis of 16S rRNA gene taxonomic profiles.

A variable region from the bacterial 16S rRNA gene was amplified by total DNA extracted from 399 participant swab samples (75 forehead, 75 forearm, 75 nares, 75 tongue, and 75 negative controls), 63 fecal samples, and 63 International Space Station (ISS) environmental samples (60 from different surfaces and 3 from the water tank) and sequenced in batches of 250 samples per Illumina MiSeq run. The last batch of 140 samples, received on June 22 2016, is currently being amplified by PCR for sequencing.

Of the 399 samples sequenced, 156 were processed through an in-house 16S sequence analysis pipeline for a preliminary analysis of the 16S data. These preliminary results, mostly derived from astronauts C and H, indicated that the gastrointestinal (GI) tract microbiota of astronaut C changed during space flight, showing an increase in alpha diversity, mostly explained by a change in the relative abundance of bacterial species and not due to a gain or lost of species. This result was further confirmed by Unifrac analysis. The alteration in relative abundance seems to revert to its original pre-flight state 30 days after the return from space. Similar trends were observed for the skin, nose, and tongue microbiomes of astronauts C and H, although for some sites the microbiomes did not revert to their original composition, once the astronaut returned from space.

Interestingly, we found at least one case of bacteria, genus Acidocella, that were abundant in both in-flight and post-flight skin samples from astronauts H, E, and C but absent or at very low abundance in pre-flight samples. This genus is not commonly found in human skin but was found in significant quantities in the ISS environmental samples. This strongly suggests that the exposure of astronauts to the ISS environment may lead to a long-term acquisition of environmental bacteria that are not usually part of the human microbiome, and that the acquired bacteria may persist for long periods of time in the microbiome after the astronauts return to Earth.

Comparisons between the astronauts’ and the ISS microbiota also revealed an association between the skin and the ISS microbial communities. The only exception was the bacteria collected from the ISS Intermodular Ventilation Inlet (IMV) that resembled the astronauts’ nose microbiome.

Because the clustering of 16S sequencing data into Operational Taxonomic Units (OTUs) is affected by the initial collection of sequencing reads, it is not recommended to compare taxonomic profiles generated from two different batches of sequences that were processed independently from each other through the 16S sequence analysis pipeline. Therefore, we will proceed with a second (and final) round of taxonomic profiling, which will include all the microbiome samples collected for the project, once the last batch of samples is sequenced at JCVI (expected by the end of August 2016).

3- Metagenomic sequencing of astronauts’ microbiome isolates.

Although the sequencing and posterior comparative analysis of 16S-based taxonomic profiles allow the characterization of compositional changes of the crew microbiome, this type of analysis does not provide in-depth information about how alterations in commensal bacteria may affect their functional interaction with the human host. To fill this gap, we are carrying out a metagenomic sequencing approach on microbial samples collected from the GI (gastrointestinal) tract, nose, and tongue of up to five astronauts, totaling 123 microbiome samples. Currently all the samples have been sequenced with Illumina NextSeq technology using 150 bp paired-end reads at a sequencing depth of approximately 1 Gbp of sequencing data per sample (~164 Gbp total).

Metagenomic sequencing reads derived from each astronaut were assembled into contigs with SPAdes, followed by bacterial gene identification. Gene sequences were then processed through a JCVI functional annotation pipeline to predict gene functions and metabolic pathways likely to be encoded by each of the astronauts’ assembled metagenomes.

Because sequencing reads from nose microbiomes are underrepresented in the pool of microbial reads generated from each astronaut, the resulting metagenome assemblies contained a low amount of contigs representing nose microbiomes. To tackle this issue, we performed a nares-specific assembly by pooling and assembling all the nose sequencing reads generated from the five astronauts.

Predicted gene functions were used to identify biological features, known as genome properties, that were likely to be encoded by each of the assembled metagenomes. Genome properties include functional features of interest such as virulence factors, metabolic pathways, and biofilm formation, encoded by microbial genes, whose relative abundance may change during a space mission. Therefore, this type of analysis may shed light into how changes in the human microbiota associated with space travel may affect crew health. To investigate how genome properties encoded by each metagenome vary throughout a mission, microbial metagenomic sequencing reads from each sample were mapped onto their corresponding assemblies with the program BWA and mapping information was then used to quantify how the relative abundance of individual microbial genes changed during the mission. Mapping of all reads is already completed and we are currently in the process of performing comparative analysis of the microbial gene frequencies to identify those genome properties that are affected by space travel.

4- Changes in cytokine concentration in plasma

Changes in the level of different cytokines in plasma samples collected at five time points during the mission (L-180, FD10, R-1, R+0, and R+180) are being measured in order to assess the immune response of participating astronauts and to investigate its potential association with taxonomic or functional changes in the crew microbiomes. Thirty-nine out of the 45 plasma samples to be collected in this study have been already processed and cytokine measurements completed. Four out of the five remaining samples (all from astronaut F) have been collected and will be processed once the last sample from astronaut F is delivered to the JSC to avoid batch effects that may arise from technical sources of variation.

Comparative analysis of cytokine profiles from the eight completed set of samples using Principal Component Analysis (PCA) revealed that the cytokine profiles from samples collected after 180 days in space (FD180 or R-1) are significantly different from those of pre-flight samples (Time point L-180).

Further analysis of changes in the plasma concentration of individual cytokines at different time points during the mission, compared with the initial pre-flight concentration at L-180, revealed similar findings as those described previously in another study, carried out by Crucian et al. in 2014, for ISS astronauts during spaceflight. In agreement with that study, we detected significant in-flight increases in the C-C motif chemokine ligand 2 (CCL2, p=0.01) and the anti-inflammatory interleukin 1-ra (IL-1ra, p=0.003) as well as trends toward in-flight increases for two inflammatory cytokines, tumor necrosis factor alpha (TNFa, p=0.03) and interleukin 8 (IL-8, p=0.03). All these cytokines participate in the inflammatory response. Unexpectedly, our analysis also identified a significant in-flight increase in interleukin 2 (IL-2, p=0.007) that was not seen before in the study by Crucian et al. Interleukin 2 participates in adaptive immunity, primarily by promoting the differentiation of T cells. Also, some changes associated with spaceflight reported previously (Tpo, VEGF) were not observed in this study. This discordance between the two investigations may be due to differences in sample handling and processing.

5- Detection of viral DNA and cortisol levels in saliva samples

Processing of saliva samples is still ongoing. As reported in 2015, measurement of cortisol levels and virus reactivation in saliva samples are already done for four astronauts, C, E, G, and H, and we are currently processing four additional complete sets of saliva samples from astronauts A, B, D, and I. As with the cytokine samples, it is recommended to do these measurements once all the samples from a particular astronaut are collected and available for processing to reduce methodological errors and batch effects. Processing of samples from astronaut F will start once the last set of saliva samples are collected for time point R+180.

NOTE: Extended to 9/30/2017 per F. Hernandez/ARC (Ed., 3/11/16)

NOTE: Extended to 9/30/2016 per A. Chu/ARC (Ed., 8/5/14)

NOTE: Gap changes per IRP Rev E (Ed., 3/19/14)

Investigating the impact of stress and status of the immune system on the human microbiome, and potentially on human health, during a space mission is also applicable to equivalent stressful situations on Earth. Some of the conclusions of this project will also be useful in situations where a group of individuals are confined in a relatively small and closed space for a long period of time, such as a submarine crew.

Collection of pre-flight, in-flight, and post-flight samples started on February 2013 and approximately two thirds of the projected samples are already collected and delivered to JCVI (J Craig Venter Institute) or the Johnson Space Center (JSC) for sample processing and analysis.

2. Preliminary analysis of 16S rRNA gene taxonomic profiles.

2.1 Global comparative analysis of phylogenetic profiles across sites.

To investigate the composition of the microbial communities present in the samples recollected by the astronauts we amplified and sequenced the V4 variable region from the bacterial 16S rRNA gene by PCR from total DNA extracted from 156 participant swab samples (37 forehead, 36 forearm, 41 nares, and 42 tongue), 31 fecal samples, and 30 ISS environmental samples (27 from different surfaces and 3 from the water tank). Then PCR amplicons were sequenced in an Illumina MiSeq machine and sequencing data was used to identify the bacterial species that were present in each of the samples. All samples analyzed in this report corresponded to baseline pre-flight (L-240, L-150, L-90, and L-60), in-flight (FD7, FD90, and R-14), and post-flight (R+1, R+30, and R+60) time points.

Preliminary analysis of the 16S sequencing data from the 187 human samples described above demonstrated that our data were consistent with previous human microbiome studies and findings from the Human Microbiome Project. Indeed, our analysis showed that the main source of compositional variation across microbial communities was the isolation site. Our results are also in agreement with previous studies showing that the skin and nose microbiomes were more similar to each other than to those from feces or tongue [1,2]. In addition, for samples derived from the same body site, those collected from the same participant were more similar to each other than those coming from different subjects, except for forearm samples (stool p < 0.001, forehead p-value = 0.027, nose p-value < 0.001, tongue p-value < 0.001).

Similarly, microbial composition analysis of 30 environmental microbial samples collected at different time points from the ISS also clustered by sampling site based on their beta diversity values (p < 0.001). Beta diversity is a measurement of the proportion of species shared by two samples. Interestingly, the microbial composition of environmental samples from the Intermodular Ventilation (IMV) Inlet of the ISS significantly overlapped with the nose microbiome while samples collected from the ISS ARED (Advanced Resistive Exercise Device) Handle Bar, Cupola Nadir Window Shade Knob, Crew Quarters Stationary Light Knob, and Handheld Microphone handles/grips showed a distribution that overlapped with the two skin samples, forehead, and forearm (p < 0.001, pairwise error rate for multiple testing = 0.005 [ Bonferroni ] ).

2.2 Analysis of alpha diversity in human samples.

To further investigate the effect of space travel on the human microbiome we evaluated the stability of the inverse Simpson alpha diversity index, that is a quantitative measure of the species richness and evenness in an ecosystem. This index increases with an increase in the number of species or when species are more evenly distributed in a particular ecological niche.

Although our sample collections are still incomplete, alpha diversity measurements of almost-complete datasets showed some preliminary trends that are worth mentioning. For example, the gut microbiome from astronaut C showed a markedly increase in in-flight alpha diversity. A similar increase in alpha diversity was observed for forearm in-flight samples from astronauts C and H. Astronauts B and E, however, showed an opposite trend, although more time points need to be incorporated in order to have a more complete picture about the fluctuations in forearm alpha diversity.

2.3. Analysis of beta diversity in human samples.

While alpha diversity measures the species diversity within each sample, beta diversity estimates the difference in species diversity between ecological niches and gives an idea of how much diversity is being shared between them. For this study we used the Yue & Clayton measure of dissimilarity index [3], that takes into account the proportion of shared and unshared species between two microbial communities.

Beta diversity analysis of stools samples from astronaut C revealed that in-flight samples were more similar to each other than to pre-flight samples (p < 0.001) or post-flight samples (p = 0.006). A similar analysis on forearm and forehead samples from astronauts C and H indicated that in-flight samples had a lower beta diversity index compared to pre-flight samples (p = 0.007 forearm; p = 0.047 forehead) but no significant differences were found between in- and post-flight samples. On the other hand, nose and tongue samples from the same two astronauts did not show any statistical difference in beta diversity among pre-, in-, and post-flight samples.

2.4 Analysis of the microbial composition of environmental samples from the ISS.

The analysis of 16S taxonomic profiles from 27 ISS surface samples and 3 samples from the water tank showed that the microbial composition of the five ISS sites surveyed resemble that of the two astronauts skin sites, forearm and forehead. Interestingly, some of the samples from the Intermodular Ventilation (IMV) Inlet and the Smoke Detector are enriched in some of the most abundant genera in the nose microbiome of the astronauts. On the other hand the water tank sample presented a less diverse microbial community compared to ISS surfaces.

3. Discussion.

In this report we presented preliminary results from the first 156 human microbiome samples and 30 ISS environmental samples received at JCVI and collected before, during, and after a mission to the ISS. Our analysis showed that the taxonomic data generated from sequencing PCR amplicons spanning the v4 region of the 16S rRNA gene from astronauts’ samples were consistent with previous human microbiome studies and the HMP project. Indeed, each of the five human habitats surveyed, tongue, gut, forehead, forearm, and nose, harbored a distinctive microbiota, with forehead and forearm being the most similar and gut and tongue (dorsal) the most different. Also, in agreement with other human microbiome studies, samples collected from the same subject usually clustered together and taxonomic profiles from each sample were enriched in microbial genera typically found in the environments surveyed.

Even though most of the sequenced datasets lack several data points, it was still possible to identify some trends in those sample collections that were almost complete, mostly from astronauts C and H. Our analysis of taxonomic profiles showed that the composition of the microbial communities from subjects C and H changed during their stay in the ISS and in some cases those communities seemed to get back to their original composition 30 days after return.

In addition, analysis of alpha diversity seemed to indicate that the response of the human microbiome to the space environment is site-specific and in some cases subject-specific. Also, our study of beta diversity on subjects C and H showed that in-flight samples were more similar to each other than to pre-flight samples, supporting the hypothesis that long stays in the ISS affect the composition of the human microbiome.

Lastly, taxonomic profiling of 30 environmental samples indicated that the microbiota that inhabits different ecological niches in the ISS is most likely of human origin and mainly from human skin.

4. References

1. Structure, function and diversity of the healthy human microbiome. Nature 486, 207-14 (2012).

2. Costello, E.K. et al. Bacterial community variation in human body habitats across space and time. Science 326, 1694-7 (2009).

3. Parks, D.H. & Beiko, R.G. Measures of phylogenetic differentiation provide robust and complementary insights into microbial communities. ISME J 7, 173-83 (2013).

NOTE: Extended to 9/30/2016 per A. Chu/ARC (Ed., 8/5/14)

NOTE: Gap changes per IRP Rev E (Ed., 3/19/14)

Investigating the impact of stress and status of the immune system on the human microbiome, and potentially on human health, during a space mission is also applicable to equivalent stressful situations on Earth. Some of the conclusions of this project will also be useful in situations where a group of individuals are confined in a relatively small and closed space for a long period of time, such as a submarine crew.

All nine subjects required for the study have been already recruited. Collection of pre-flight, in-flight, and post-flight samples started in February 2013. Three batches of swab and fecal samples have been delivered to JCVI and are currently being processed. Collected saliva and blood samples have been sent to JSC for measurement of cytokines and stress levels.

2. Comparative analysis of phylogenetic profiles

The V1-V3 variable region of the bacterial 16S rRNA gene was amplified by PCR from total DNA extracted from 28 swab samples (7 forehead, 7 forearm, 7 nares, and 7 tongue) and 11 fecal samples derived from six different subjects and sequenced using Illumina technology. All samples correspond to baseline pre-flight time points (L-240, L-150, L-90, and L-60).

To characterize spatial and temporal changes in the taxonomic composition of the samples, 16S rRNA sequences were run through the JCVI 16S pipeline to identify operational taxonomic units present in each of the pre-flight samples analyzed. The results show that microbial communities isolated from the same body-site presented similar taxonomic profiles that were consistent across subjects. As it has been previously described for the Human Microbiome Project, the baseline composition of microbial communities varied from site to site, with skin and nose microbiomes being more similar to each other than to those from feces or mouth. A more in-depth analysis of the most frequent genera present in each of the five body habitats sampled also demonstrated that taxonomic profiles were consistent with those from other 16S-based studies.

To evaluate the stability of microbial communities assessed in our study we performed a spatial and temporal comparison of alpha and beta diversity. Our analysis did not reveal any significant change in alpha diversity (Shannon diversity index) between time points L-90 and L-60 for any of the five habitats tested (p-value > 0.05). Similarly, we did not find any significant shift in beta diversity (Sørensen similarity index) between time points L-90 and L-60 compared to the baseline community L-150. However, comparisons of intra- versus inter-site beta diversity confirmed that the human microbiota from the five sampled sites was significantly different (p-value < 0.01), with the exception of nares and forehead (p-value = 0.3). These results supported our previous observations that taxonomic profiles are similar among samples from the same body-site but not between different habitats. Finally, we did not find any significant difference between intra-vs-interpersonal variability of beta diversity.

In conclusion, our results from baseline samples did not show any significant temporal change in alpha or beta diversity. Similarly, we did not observe any significant difference between beta diversity values within and between subjects, although it is known that interpersonal microbiome variability tends to be higher than samples isolated from the same individual. This apparent microbiome stability at the baseline is most likely to be the result of the small number of samples analyzed and it is expected that with the incorporation of additional samples we will be able to reach more conclusive results. Currently, we are processing two additional set of samples that include in-flight and post-flight samples as well as environmental samples from the ISS. The incorporation of these samples will provide the first indication of whether and to what extent space travel alters the human microbiome.

NOTE: Gap changes per IRP Rev E (Ed., 3/19/14)

Investigating the impact of stress and status of the immune system on the human microbiome, and potentially on human health, during a space mission is also applicable to equivalent stressful situations on Earth. Some of the conclusions of this project will also be useful in situations where a group of individuals are confined in a relatively small and closed space for a long period of time, such as a submarine crew.

Collected swab and fecal samples were submitted to the J. Craig Venter Institute for further processing and analysis. Those samples are currently being processed for DNA extraction.

Blood and saliva samples were submitted to Dr. Ott's laboratory at the JSC and will be processed once all samples have been collected.

Investigating the impact of stress and status of the immune system on the human microbiome, and potentially on human health, during a space mission is also applicable to equivalent stressful situations on Earth. Some of the conclusions of this project will also be useful in situations where a group of individuals are confined in a relatively small and closed space for a long period of time, such as a submarine crew.