Draft Genomes of Various Bacterial Phyla Isolated from the International Space Station

Abstract Whole-genome sequences were generated from 96 bacterial strains of 14 species isolated from the International Space Station surfaces during the Microbial Tracking-2 study. Continued characterization of this closed habitat's microbiome enables tracking the spread and evolution of secondary pathogens, which is vital for astronaut health. The International Space Station (ISS) is currently the only long-term human habitat in space. Microgravity disrupts human immune function (1), and close monitoring of the ISS microbiome for increased pathogenicity is therefore an ongoing critical task. Here, we report the draft genomes of 96 bacterial strains isolated from the ISS (Table 1). Eleven of the 14 species found are common members of the human microbiome, and most can act as opportunistic human pathogens. Four species from the phylum Actinobacteria were isolated, from the genera Pseudoclavibacter, Kocuria and Micrococcus, all of which are common in both environmental and human microbiomes (2-5). K. palustris and M. luteus are opportunistic pathogens (5, 6). M. luteus can survive in a dormant state in extreme oligotrophic conditions (7), and has both an increased growth rate and increased biomass yield in microgravity (8). Multiple coagulase-negative staphylococci (CoNS) were also found on ISS surfaces. CoNS are normal components of human skin flora (9) and are often resistant to antibiotics because of their ability to form biofilms (10); all CoNS in this report are documented as opportunistic pathogens (11-15). Three species from the phylum Proteobacteria were identified: Acinetobacter pittii, Pseudomonas fulva and Pseudomonas granadensis. A. pittii is a less-common nosocomial pathogen causing pneumonia and meningitis in intensive care patients (16), P.fulva is a commensal plant endophyte (17) which can also infect immunocompromised patients (18, 19), and P.granadensis is a recently discovered soil bacterium (20). Two other species in this report not associated with the human microbiome were Cytobacillus horneckiae, a gram-positive UV-resistant endospore-former isolated from a clean room at the Kennedy Space Center (21) and Methylobacterium organophilum, a facultative methylotroph (22). All strains reported here were collected aboard the ISS over the course of five flight missions between June 2017 and December 2018 (Table 1). Pre-moistened polyester wipes were used to collect surface samples from eight predetermined locations during each flight (see Table 1 for locations). After transport to Earth, wipes were agitated in sterile phosphate-buffered saline and the buffer concentrated with the InnovaPrep CP-150. Concentrates were plated on R2A (25°C, 7 days), PDA (25°C, 7 days), and blood agar (37°C, 2 days) using appropriate concentrations for microbial isolation. Genomic DNA from isolated colonies was extracted using the ZymoBIOMICS DNA MagBead kit according to the manufacturer’s instructions. Libraries for whole-genome shotgun sequencing were prepared using the Illumina Nextera DNA Flex Library Prep as in previous studies (23), and were sequenced using the NovaSeq 6000 S4 flow cell paired-end 2x150-bp platform. Sequencing reads were quality-filtered and trimmed, and adapter sequences removed, using fastQC v0.11.7 (24) and fastp v02.20.0 (25). Reads were assembled into scaffolds using SPAdes v3.11.1 (26). Quast v5.0.2 (27) was used to determine assembly quality, including number of contigs, genome size, and N50. Default settings were used for all assembly steps except for fastp which included 512 adapter screening. OrthoANIu (28) was used to confirm species identity for each strain against the species type strain sequence, with a minimum average nucleotide identity of 95% necessary for identification.