Succession and persistence of microbial communities and antimicrobial resistance genes associated with International Space Station environmental surfaces

Abstract

Background: The International Space Station (ISS) is an ideal test bed for studying the effects of microbial persistence and succession on a closed system during long space flight. Culture-based analyses, targeted gene-based amplicon sequencing (bacteriome, mycobiome, and resistome), and shotgun metagenomics approaches have previously been performed on ISS environmental sample sets using whole genome amplification (WGA). However, this is the first study reporting on the metagenomes sampled from ISS environmental surfaces without the use of WGA. Metagenome sequences generated from eight defined ISS environmental locations in three consecutive flights were analyzed to assess the succession and persistence of microbial communities, their antimicrobial resistance (AMR) profiles, and virulence properties. Metagenomic sequences were produced from the samples treated with propidium monoazide (PMA) to measure intact microorganisms.

Results: The intact microbial communities detected in Flight 1 and Flight 2 samples were significantly more similar to each other than to Flight 3 samples. Among 318 microbial species detected, 46 species constituting 18 genera were common in all flight samples. Risk group or biosafety level 2 microorganisms that persisted among all three flights were Acinetobacter baumannii, Haemophilus influenzae, Klebsiella pneumoniae, Salmonella enterica, Shigella sonnei, Staphylococcus aureus, Yersinia frederiksenii, and Aspergillus lentulus. Even though Rhodotorula and Pantoea dominated the ISS microbiome, Pantoea exhibited succession and persistence. K. pneumoniae persisted in one location (US Node 1) of all three flights and might have spread to six out of the eight locations sampled on Flight 3. The AMR signatures associated with β-lactam, cationic antimicrobial peptide, and vancomycin were detected. Prominent virulence factors were cobalt-zinc-cadmium resistance and multidrug-resistance efflux pumps.

Conclusions: There was an increase in AMR and virulence gene factors detected over the period sampled, and metagenome sequences of human pathogens persisted over time. Comparative analysis of the microbial compositions of ISS with Earth analogs revealed that the ISS environmental surfaces were different in microbial composition. Metagenomics coupled with PMA treatment would help future space missions to estimate problematic risk group microbial pathogens. Cataloging AMR/virulence characteristics, succession, accumulation, and persistence of microorganisms would facilitate the development of suitable countermeasures to reduce their presence in the closed built environment.

Keywords: Built environment; Functional metagenomics; International Space Station; Metagenome; Propidium monoazide.

Fig. 1

a Species-level abundance analysis based on normalized reads. b Venn diagram representing the common species between all three flights. c Species-based NMDS analysis representing various sampling sites in 2D ordinate as per the microbiome composition

Fig. 2

Species-level ordinate analysis of all three flight samples of ISS. Species-level diversity was determined using normalized reads. a Diversity analysis using the Chao1 estimator, b Shannon-Weaver index, and c Simpson’s reciprocal index. d Principal component analysis

Fig. 3

Abundance of metagenomics reads retrieved in PMA-treated samples that showed the presence of cultured bacteria (blue) and fungi (green). Three microbial species observed in culture analyses of PMA-treated samples were not observed in the metagenomic data set

Fig. 4

Relative abundance of metagenomics reads associated with a metabolism and b virulence

Fig. 5

Distribution of antibiotic resistance across samples as seen in metagenomics analysis. Read counts of AMR genes grouped together based on the class of antibiotic they are resistant to, as detected by metagenomics. White boxes indicate genes that were absent in a particular sample. Red indicates the highest read count and pink, the lowest read count. Columns represent samples and rows represent antibiotic resistance features

Fig. 6

a Abundance profile of biosafety level 2 bacterial and fungal organisms in the ISS microbiome based on metagenomics sequences of the PMA-treated samples. b Venn diagram representing the common BSL-2 species between all three flights. c Species-based NMDS analysis representing various sampling sites in 2D ordinate as per the microbiome composition

Fig. 7

Dominant and persistent BSL-2 microbial species of ISS environmental surfaces on a Flight 1, b Flight 2, and c Flight 3

Fig. 8

a PCoA plot depicting dissimilarity between the ISS samples and associated microbiome. b Venn plot visualization of common species found in Flight 1, Flight 2, Flight 3, ISS dust, and ISS-HEPA