Microbiome and Metagenome Analyses of a Closed Habitat during Human Occupation

Abstract

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.

Keywords: Analog habitat; closed habitat; extravehicular activity; functional metagenomics; metagenomics; microbial diversity; microbiome; spacecraft microbiome.

FIG 1

Culture-dependent and -independent analysis of Analog habitat surface samples. (A) Abundance of cultivable bacteria and fungi. Each dot in a column represents Analog habitat location sampled. No statistically significant differences in abundances were observed among flight missions and between bacteria and fungi (one-way ANOVA, P > 0.01). (B) The relative light unit (RLU) values for ATP counts for total ATP (small circles) and intracellular ATP (small squares). No statistically significant differences in abundances were observed among flight missions and between bacteria and fungi (one-way ANOVA, P > 0.01). (C) The qPCR-based microbial burden (total; non-PMA and viable; PMA) of various Analog habitat surface sample. The gene copies were measured by targeting 16S rRNA gene (bacteria) and ITS gene (fungi).

FIG 2

Multidimensional scaling plots of 16S rRNA gene amplicon sequencing data. NMDS ordination showing 99% confidence interval ellipses of non-PMA-treated (left panels) and PMA-treated (right panels) grouped based on surface material (A) and site categories (B). The various samples collected from across the Analog habitat are indicated by dots, and the closer the dots are to each other, the more similar their bacterial composition is in terms of types and number of bacteria.

FIG 3

Relative abundances of bacteria detected by 16S rRNA gene amplicon sequencing. The relative abundances of bacterial taxa identified in various samples across the Analog habitat were visualized by bar plots. Each bar represents a specific sample, and each colored box represents a particular taxon. The height of the colored box represents the relative abundance of that particular taxon within the sample. Taxa present in less than 1% abundance in a given sample are displayed in the “remaining fraction” at the top of the graph (gray box). The legend is read from bottom to top, with the bottom taxon in the legend corresponding to the bottom taxon on the graph. Non-PMA treated samples are displayed in panel A, and the PMA-treated samples are displayed in panel B.

FIG 4

Differential bacterial composition among various types of surfaces. Pie chart of the relative abundances of bacteria detected in the Analog habitat. The sequences obtained were summarized to the genus level. In total, 52 taxa were detected, but only the 10 most abundant taxa are displayed in the legends. The pie graphs are separated based on the site categories: LDP samples (left panels) and metal/glass (right panels) and treatment groups, no PMA (A) and PMA (B).

FIG 5

Metagenomic sequencing analysis of the Analog habitat. Cluster dendrogram of Euclidean distances was performed on non-PMA-treated (A, top) and PMA-treated samples (B, top). NMDS ordination showing the 95% confidence interval ellipse based on Unifrac distances in the matrix of all microbial species (bacteria and fungi) from both non-PMA-treated (middle left panel) and PMA-treated (middle right panel) samples. Similar treatment was performed for all bacterial species and NMDS ordination plots are depicted for non-PMA-treated (bottom left panel) and PMA-treated (bottom right panel) samples. The samples collected from various Analog habitat surfaces were indicated by various color circles.

FIG 6

Metagenomic sequencing analysis of bacteria of the Analog habitat. Heat map showing the relative abundance of each antimicrobial-associated gene (top) and virulence-associated genes (bottom) detected in each sample collected.

FIG 7

Locations and sampling tool kit feature for surface sampling. (A) Cantilever swab tool kit storage canister box (a) and swab head attached to the cantilever tool kit (b). (B) Two-dimensional (2D outline) of the Analog habitat and sampling locations (E1 to E16). (C) Photographs of Analog habitat sampling locations. The red circles indicated the areas where the samples were collected.