Stability of the individual gut microbiota profile in confined environment
The intestinal microbial communities of the six crewmembers of the MARS500 project (subject 5001 to 5006) were tracked over time during the 520 days of ground-based space simulation. Longitudinal faecal sampling also included collection of stools before entering the isolation facility, and a number of samples after the exit from the modules, up to 6 months later. A mean of 27 faecal samples per crewmember were collected. Each sample was characterized via next-generation sequencing of V3–V4 hypervariable region of the 16S rRNA gene, allowing for a total of 5,377,450 high-quality sequence reads (mean per subject, 33,820; range, 7759–91,366). Reads were clustered into 54,836 chimera- and singleton-filtered OTUs at 97% sequence similarity.
The gut microbiota dynamics were reconstructed across time and subjects, based on the variation of highly abundant OTUs, according to the normalization strategy described by David et al. [5]. As shown in Fig. 1, these trajectories revealed a strong individuality in the ecosystem structure and its dynamics during the prolonged isolated confinement in the MARS500 infrastructure, with a unique succession of individual microbial profiles. Even at phylum level, there were apparent microbial signatures that characterized each individual in his free-living conditions (i.e., before entering the isolation module) and accompanied him during and after the simulated interplanetary flight. The observed individual microbiota profiles at phylum level were consistent with the range of the phylum-level microbiota variation in the human population (Additional file 1: Figure S1). Specifically, Proteobacteria members were distinctive of subject 5002 (mean relative abundance across time series, 12.3%), Bacteroidetes were almost completely missing in subject 5004 (mean relative abundance across time series, 0.5%), and Verrucomicrobia were undetectable in the microbiota of subjects 5003 and 5006.
It should be pointed out that no detailed, individual-specific information on the micro- and macronutrient composition of the different variants of diet designed for the MARS500 astronauts throughout the whole project is available to the authors, making impossible any correlation with the microbial changes observed. However, based on available data, it is reasonable to assume that the diets were similar among astronauts across the mission and, thus, unlikely to make a major contribution to the inter-individual differences in the gut microbial dynamics.
Notwithstanding the strong individuality, the intestinal microbial communities of the six crewmembers shared 14 OTUs, all belonging to the Firmicutes phylum, and mainly to the Lachnospiraceae family (9 OTUs) (Additional file 2: Table S1; Additional file 3: Figure S2). All these OTUs were already present in the individual microbiota configurations prior to entry into the MARS500 module.
When exploring the variation of the beta (i.e., inter-astronaut) diversity of the gut microbiota over time, it is worthy to note that, unlike the weighted UniFrac distances that showed apparently random fluctuations, without a significant trend over time, the unweighted UniFrac values followed a downward trajectory, with a significant inverse association with the time spent in the MARS500 isolation facility (quantile median regression test: RC range, regression coefficients scaled to the full variation of UniFrac distances, −9362.98; RC sd, regression coefficients scaled to one standard deviation, 1900.01; P value generated by boot-strap analysis, 4E−5) (Additional file 4: Figure S3). In particular, immediately upon entry into the module, the median unweighted UniFrac distance was about 4% lower than the initial value (i.e., in free-living conditions) and reached a reduction of up to 9% after about 7 months spent in spacecraft.
Temporal dynamics of gut microbiota components
The highly abundant OTUs used to reconstruct the individual microbiota trajectories over the MARS500 study were further analyzed by evaluating the OTU propensity to variation over time, as the magnitude of change above or below the median relative abundance (Fig. 1 and Additional file 2: Table S1).
Interestingly, some behavioral patterns of OTUs were shared among subjects. Specifically, OTUs attributable to Roseburia faecis (OTU_ID 84029, family Lachnospiraceae), Faecalibacterium prausnitzii (OTU_ID 543524, family Ruminococcaeae), and when present, Akkermansia muciniphila (OTU_ID 35867, family Verrucomicrobiaceae) were identified as highly variable during the whole period of confinement, regardless of the initial microbial configuration. In particular, the F. prausnitzii OTU was especially affected in the second half of the MARS500 mission, showing for all subjects the lowest relative abundance values around about 1 year of confinement. On the other hand, where detected, the A. muciniphila OTU decreased dramatically in the gut microbiota of all crewmembers when returning to their own free-living conditions after the end of the mission. The OTUs associated with Bacteroides (including B. caccae, B. eggerthii, B. fragilis, B. ovatus, B. uniformis, and unclassified species) were instead among the most perturbed in the initial phase of the mission, typically reaching for each subject the highest relative abundance values within the first 30 days of confinement. Conversely, the OTUs classified as Dorea (when available, D. formicigenerans, OTU_ID 181619) and Coprococcus catus (OTU_ID 616283) were highly stable in the intestinal microbiota of all crewmembers.
Identification of microbiota steady states and ecological succession in time
Temporal dynamics of the gut microbiota were further investigated for each crewmember, by identifying microbial steady states and characterizing their ecological succession over time (Fig. 2 and Additional file 5: Figure S4). Steady states were interpreted in the context of recent theories of microbiome ecology, predicting that ecosystems may exist under multiple states (to be intended as “stable” configurations/equilibria of the microbial community) [5]. For each subject, steady states were identified through the same method described by David et al. [5], i.e., using a cluster-defining height threshold in a Jensen-Shannon Distance (JSD)-based tree. A total of 5 different steady states were identified for individuals 5002 and 5004; 6 for 5001, 5003, and 5005; and 7 for 5006. As already described for the individual microbial profiles, the phylogenetic structures of steady states were largely dominated by Firmicutes but retained a strong individual fingerprint in terms of taxonomic composition of the microbial communities, with variable percentages of Bacteroidetes, Proteobacteria, Actinobacteria, and Verrucomicrobia members (Additional file 5: Figure S4). According to our data, the temporal succession of steady states followed a highly personalized non-linear trajectory, with different temporal persistence and times of recurrence for each of them. Except for 5006, whose microbiota was deeply unstable, with steady states continuously alternating with one another, the intestinal microbial communities of the other crewmembers remained stable for periods lasting on average 2 months, with steady states generally recurring one up to three times over the entire sampling period. Interestingly, only for subjects 5004 and 5006, we observed a return to the initial microbiota configuration, after exiting the module, while new steady states were consolidated for the other crewmembers.
Steady state-based temporal dynamics of the crewmembers differed from each other also for the microbial ecology of the establishment of a steady state compared to the previous one. Specifically, we used a SourceTracker/z-score combining approach to measure the relative contribution of microbial sources other than indigenous ones in defining the steady state structures. According to our data, within each time series, the transition from one steady state to the next generally retained a strong individual signature, indicative of a rearrangement of the autochthonous microbial asset (contribution of the individual microbial communities up to 96%). Nevertheless, in each steady state-based trajectory, we could identify a major change event (three for subject 5006) in the microbial configuration, associated with a higher relative contribution of allochthonous microbial sources than indigenous ones (median contribution of the individual microbial communities, 10%). These major events were variously distributed along the individual timelines but occurred on average after 340 days (range, 330–360 days) of isolated confinement for subjects 5002, 5004, and 5006.
According to a Random Forests analysis [12], the vast majority of steady state discriminatory OTUs belonged to the Lachnospiraceae and Ruminococcaceae families (Fig. 2). The main exceptions were represented by Bifidobacterium OTUs that discriminated two steady states in the first half of the MARS500 simulation, for subjects 5003 and 5006, and one steady state of subject 5004 in the second half of the study and after the return to real life. On the other hand, Proteobacteria OTUs, specifically belonging to Sutterella and Enterobacteriaceae, discriminated steady states between 2 and 7 months of confinement for subjects 5001 and 5002, respectively.
To further explore the patterns of microbiota variation across the individual steady states over the MARS500 study period, for each crewmember, we established co-abundance associations of the most abundant OTUs and then clustered correlated OTUs into three or four co-abundance groups (CAO; Fig. 2 and Additional file 5: Figure S4) [13]. The differences in the succession of individual CAO profiles, with intra- and inter-CAO rearrangements, confirmed the personalized alternating of multiple reversible configurations of the gut microbiota in a single time series, mainly characterized by different proportions of Ruminococcaceae and Lachnospiraceae SCFA producers.