Novel spore-forming species exhibiting intrinsic resistance to third- and fourth-generation cephalosporins and description of Tigheibacillus jepli gen. nov., sp. nov.

A comprehensive microbial surveillance was conducted at NASA's Mars 2020 spacecraft assembly facility (SAF), where whole-genome sequencing (WGS) of 110 bacterial strains was performed. One isolate, designated 179-BFC-A-HST, exhibited less than 80% average nucleotide identity (ANI) to known species, suggesting a novel organism. This strain demonstrated high-level resistance [minimum inhibitory concentration (MIC) >256 mg/L] to third-generation cephalosporins, including ceftazidime, cefpodoxime, combination ceftazidime/avibactam, and the fourth-generation cephalosporin cefepime. The results of a comparative genomic analysis revealed that 179-BFC-A-HST is most closely related to Virgibacillus halophilus 5B73CT, sharing an ANI of 78.7% and a digital DNA-DNA hybridization (dDDH) value of 23.5%, while their 16S rRNA gene sequences shared 97.7% nucleotide identity. Based on these results and the recent recognition that the genus Virgibacillus is polyphyletic, strain 179-BFC-A-HST is proposed as a novel species of a novel genus, Tigheibacillus jepli gen. nov., sp. nov (type strain 179-BFC-A-HST = DSM 115946T = NRRL B-65666T), and its closest neighbor, V. halophilus, is proposed to be reassigned to this genus as Tigheibacillus halophilus comb. nov. (type strain 5B73CT = DSM 21623T = JCM 21758T = KCTC 13935T). It was also necessary to reclassify its second closest neighbor Virgibacillus soli, as a member of a novel genus Paracerasibacillus, reflecting its phylogenetic position relative to the genus Cerasibacillus, for which we propose Paracerasibacillus soli comb. nov. (type strain CC-YMP-6T = DSM 22952T = CCM 7714T). Within Amphibacillaceae (n = 64), P. soli exhibited 11 antibiotic resistance genes (ARG), while T. jepli encoded for 3, lacking any known ß-lactamases, suggesting resistance from variant penicillin-binding proteins, disrupting cephalosporin efficacy. P. soli was highly resistant to azithromycin (MIC >64 mg/L) yet susceptible to cephalosporins and penicillins. IMPORTANCE: The significance of this research extends to understanding microbial survival and adaptation in oligotrophic environments, such as those found in SAF. Whole-genome sequencing of several strains isolated from Mars 2020 mission assembly cleanroom facilities, including the discovery of the novel species Tigheibacillus jepli, highlights the resilience and antimicrobial resistance (AMR) in clinically relevant antibiotic classes of microbes in nutrient-scarce settings. The study also redefines the taxonomic classifications within the Amphibacillaceae family, aligning genetic identities with phylogenetic data. Investigating ARG and virulence factors (VF) across these strains illuminates the microbial capability for resistance under resource-limited conditions while emphasizing the role of human-associated VF in microbial survival, informing sterilization practices and microbial management in similar oligotrophic settings beyond spacecraft assembly cleanrooms such as pharmaceutical and medical industry cleanrooms.

Planetary Protection Knowledge Gap Closure Enabling Crewed Missions to Mars.

As focus for exploration of Mars transitions from current robotic explorers to development of crewed missions, it remains important to protect the integrity of scientific investigations at Mars, as well as protect the Earth's biosphere from any potential harmful effects from returned martian material. This is the discipline of planetary protection, and the Committee on Space Research (COSPAR) maintains the consensus international policy and guidelines on how this is implemented. Based on National Aeronautics and Space Administration (NASA) and European Space Agency (ESA) studies that began in 2001, COSPAR adopted principles and guidelines for human missions to Mars in 2008. At that point, it was clear that to move from those qualitative provisions, a great deal of work and interaction with spacecraft designers would be necessary to generate meaningful quantitative recommendations that could embody the intent of the Outer Space Treaty (Article IX) in the design of such missions. Beginning in 2016, COSPAR then sponsored a multiyear interdisciplinary meeting series to address planetary protection "knowledge gaps" (KGs) with the intent of adapting and extending the current robotic mission-focused Planetary Protection Policy to support the design and implementation of crewed and hybrid exploration missions. This article describes the outcome of the interdisciplinary COSPAR meeting series, to describe and address these KGs, as well as identify potential paths to gap closure. It includes the background scientific basis for each topic area and knowledge updates since the meeting series ended. In particular, credible solutions for KG closure are described for the three topic areas of (1) microbial monitoring of spacecraft and crew health; (2) natural transport (and survival) of terrestrial microbial contamination at Mars, and (3) the technology and operation of spacecraft systems for contamination control. The article includes a KG data table on these topic areas, which is intended to be a point of departure for making future progress in developing an end-to-end planetary protection requirements implementation solution for a crewed mission to Mars. Overall, the workshop series has provided evidence of the feasibility of planetary protection implementation for a crewed Mars mission, given (1) the establishment of needed zoning, emission, transport, and survival parameters for terrestrial biological contamination and (2) the creation of an accepted risk-based compliance approach for adoption by spacefaring actors including national space agencies and commercial/nongovernment organizations.

Analysis of Microbiomes from Ultra-Low Biomass Surfaces Using Novel Surface Sampling and Nanopore Sequencing.

The rapid assessment of microbiomes from ultra-low biomass environments such as cleanrooms or hospital operating rooms has a number of applications for human health and spacecraft manufacturing. Current techniques often employ lengthy protocols using short-read DNA sequencing technology to analyze amplified DNA and have the disadvantage of a longer analysis time and lack of portability. Here, we demonstrate a rapid (~24 hours) on-site nanopore-based sequencing approach to characterize the microbiome of a NASA Class 100K cleanroom where spacecraft components are assembled. This approach employs a modified protocol of Oxford Nanopore's Rapid PCR Barcoding Kit in combination with the recently developed Squeegee-Aspirator for Large Sampling Area (SALSA) surface sampling device. Results for these ultra-low biomass samples revealed DNA amplification ~1 to 2 orders of magnitude above process control samples and were dominated primarily by Paracoccus and Acinetobacter species. Negative control samples were collected to provide critical data on background contamination, including Cutibacerium acnes, which most likely originated from the sampling reagents-associated microbiome (kitome). Overall, these results provide data on a novel approach for rapid low-biomass DNA profiling using the SALSA sampler combined with modified nanopore sequencing. These data highlight the critical need for employing multiple negative controls, along with using DNA-free reagents and techniques, to enable a proper assessment of ultra-low biomass samples.

Phylogenomics, phenotypic, and functional traits of five novel (Earth-derived) bacterial species isolated from the International Space Station and their prevalence in metagenomes.

With the advent of long-term human habitation in space and on the moon, understanding how the built environment microbiome of space habitats differs from Earth habitats, and how microbes survive, proliferate and spread in space conditions, is becoming more important. The microbial tracking mission series has been monitoring the microbiome of the International Space Station (ISS) for almost a decade. During this mission series, six unique strains of Gram-stain-positive bacteria, including two spore-forming and three non-spore-forming species, were isolated from the environmental surfaces of the ISS. The analysis of their 16S rRNA gene sequences revealed > 99% similarities with previously described bacterial species. To further explore their phylogenetic affiliation, whole genome sequencing was undertaken. For all strains, the gyrB gene exhibited < 93% similarity with closely related species, which proved effective in categorizing these ISS strains as novel species. Average nucleotide identity and digital DNA-DNA hybridization values, when compared to any known bacterial species, were < 94% and <50% respectively for all species described here. Traditional biochemical tests, fatty acid profiling, polar lipid, and cell wall composition analyses were performed to generate phenotypic characterization of these ISS strains. A study of the shotgun metagenomic reads from the ISS samples, from which the novel species were isolated, showed that only 0.1% of the total reads mapped to the novel species, supporting the idea that these novel species are rare in the ISS environments. In-depth annotation of the genomes unveiled a variety of genes linked to amino acid and derivative synthesis, carbohydrate metabolism, cofactors, vitamins, prosthetic groups, pigments, and protein metabolism. Further analysis of these ISS-isolated organisms revealed that, on average, they contain 46 genes associated with virulence, disease, and defense. The main predicted functions of these genes are: conferring resistance to antibiotics and toxic compounds, and enabling invasion and intracellular resistance. After conducting antiSMASH analysis, it was found that there are roughly 16 cluster types across the six strains, including ß-lactone and type III polyketide synthase (T3PKS) clusters. Based on these multi-faceted taxonomic methods, it was concluded that these six ISS strains represent five novel species, which we propose to name as follows: Arthrobacter burdickii IIF3SC-B10T (= NRRL B-65660T = DSM 115933T), Leifsonia virtsii F6_8S_P_1AT (= NRRL B-65661T = DSM 115931T), Leifsonia williamsii F6_8S_P_1BT (= NRRL B-65662T = DSM 115932T), Paenibacillus vandeheii F6_3S_P_1CT (= NRRL B-65663T = DSM 115940T), and Sporosarcina highlanderae F6_3S_P_2T (= NRRL B-65664T = DSM 115943T). Identifying and characterizing the genomes and phenotypes of novel microbes found in space habitats, like those explored in this study, is integral for expanding our genomic databases of space-relevant microbes. This approach offers the only reliable method to determine species composition, track microbial dispersion, and anticipate potential threats to human health from monitoring microbes on the surfaces and equipment within space habitats. By unraveling these microbial mysteries, we take a crucial step towards ensuring the safety and success of future space missions.

Genomic characterization and radiation tolerance of Naganishia kalamii sp. nov. and Cystobasidium onofrii sp. nov. from Mars 2020 mission assembly facilities.

During the construction and assembly of the Mars 2020 mission components at two different NASA cleanrooms, several fungal strains were isolated. Based on their colony morphology, two strains that showed yeast-like appearance were further characterized for their phylogenetic position. The species-level classification of these two novel strains, using traditional colony and cell morphology methods combined with the phylogenetic reconstructions using multi-locus sequence analysis (MLSA) based on several gene loci (ITS, LSU, SSU, RPB1, RPB2, CYTB and TEF1), and whole genome sequencing (WGS) was carried out. This polyphasic taxonomic approach supported the conclusion that the two basidiomycetous yeasts belong to hitherto undescribed species. The strain FJI-L2-BK-P3T, isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility, was placed in the Naganishia albida clade (Filobasidiales, Tremellomycetes), but is genetically and physiologically different from other members of the clade. Another yeast strain FKI-L6-BK-PAB1T, isolated from the Kennedy Space Center Payload Hazardous and Servicing Facility, was placed in the genus Cystobasidium (Cystobasidiales, Cystobasidiomycetes) and is distantly related to C. benthicum. Here we propose two novel species with the type strains, Naganishia kalamii sp. nov. (FJI-L2-BK-P3T = NRRL 64466 = DSM 115730) and Cystobasidium onofrii sp. nov. (FKI-L6-BK-PAB1T = NRRL 64426 = DSM 114625). The phylogenetic analyses revealed that single gene phylogenies (ITS or LSU) were not conclusive, and MLSA and WGS-based phylogenies were more advantageous for species discrimination in the two genera. The genomic analysis predicted proteins associated with dehydration and desiccation stress-response and the presence of genes that are directly related to osmotolerance and psychrotolerance in both novel yeasts described. Cells of these two newly-described yeasts were exposed to UV-C radiation and compared with N. onofrii, an extremophilic UV-C resistant cold-adapted Alpine yeast. Both novel species were UV resistant, emphasizing the need for collecting and characterizing extremotolerant microbes, including yeasts, to improve microbial reduction techniques used in NASA planetary protection programs.

Comparative genomic analysis of Cohnella hashimotonis sp. nov. isolated from the International Space Station.

A single strain from the family Paenibacillaceae was isolated from the wall behind the Waste Hygiene Compartment aboard the International Space Station (ISS) in April 2018, as part of the Microbial Tracking mission series. This strain was identified as a gram-positive, rod-shaped, oxidase-positive, catalase-negative motile bacterium in the genus Cohnella, designated as F6_2S_P_1T. The 16S sequence of the F6_2S_P_1T strain places it in a clade with C. rhizosphaerae and C. ginsengisoli, which were originally isolated from plant tissue or rhizosphere environments. The closest 16S and gyrB matches to strain F6_2S_P_1T are to C. rhizosphaerae with 98.84 and 93.99% sequence similarity, while a core single-copy gene phylogeny from all publicly available Cohnella genomes places it as more closely related to C. ginsengisoli. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values to any described Cohnella species are <89 and <22%, respectively. The major fatty acids for strain F6_2S_P_1T are anteiso-C15:0 (51.7%), iso-C16:0 (23.1%), and iso-C15:0 (10.5%), and it is able to metabolize a wide range of carbon compounds. Given the results of the ANI and dDDH analyses, this ISS strain is a novel species within the genus Cohnella for which we propose the name Cohnella hashimotonis, with the type strain F6_2S_P_1T (=NRRL B-65657T and DSMZ 115098T). Because no closely related Cohnella genomes were available, this study generated the whole-genome sequences (WGSs) of the type strains for C. rhizosphaerae and C. ginsengisoli. Phylogenetic and pangenomic analysis reveals that F6_2S_P_1T, C. rhizosphaerae, and C. ginsengisoli, along with two uncharacterized Cohnella strains, possess a shared set of 332 gene clusters which are not shared with any other WGS of Cohnella species, and form a distinct clade branching off from C. nanjingensis. Functional traits were predicted for the genomes of strain F6_2S_P_1T and other members of this clade.

Phylogenetic affiliations and genomic characterization of novel bacterial species and their abundance in the International Space Station.

Background: With the advent of long-term human habitation in space and on the moon, understanding how the built environment microbiome of space habitats differs from Earth habits, and how microbes survive, proliferate and spread in space conditions, is coming more and more important. The Microbial Tracking mission series has been monitoring the microbiome of the International Space Station (ISS) for almost a decade. During this mission series, six unique strains of Gram-positive bacteria, including two spore-forming and three non-spore-forming species, were isolated from the environmental surfaces of the International Space Station (ISS). Results: The analysis of their 16S rRNA gene sequences revealed <99% similarities with previously described bacterial species. To further explore their phylogenetic affiliation, whole genome sequencing (WGS) was undertaken. For all strains, the gyrB gene exhibited <93% similarity with closely related species, which proved effective in categorizing these ISS strains as novel species. Average ucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values, when compared to any known bacterial species, were less than <94% and 50% respectively for all species described here. Traditional biochemical tests, fatty acid profiling, polar lipid, and cell wall composition analyses were performed to generate phenotypic characterization of these ISS strains. A study of the shotgun metagenomic reads from the ISS samples, from which the novel species were isolated, showed that only 0.1% of the total reads mapped to the novel species, supporting the idea that these novel species are rare in the ISS environments. In-depth annotation of the genomes unveiled a variety of genes linked to amino acid and derivative synthesis, carbohydrate metabolism, cofactors, vitamins, prosthetic groups, pigments, and protein metabolism. Further analysis of these ISS-isolated organisms revealed that, on average, they contain 46 genes associated with virulence, disease, and defense. The main predicted functions of these genes are: conferring resistance to antibiotics and toxic compounds, and enabling invasion and intracellular resistance. After conducting antiSMASH analysis, it was found that there are roughly 16 cluster types across the six strains, including ß-lactone and type III polyketide synthase (T3PKS) clusters. Conclusions: Based on these multi-faceted taxonomic methods, it was concluded that these six ISS strains represent five novel species, which we propose to name as follows: Arthrobacter burdickii IIF3SC-B10T (=NRRL B-65660T), Leifsonia virtsii, F6_8S_P_1AT (=NRRL B-65661T), Leifsonia williamsii, F6_8S_P_1BT (=NRRL B- 65662T and DSMZ 115932T), Paenibacillus vandeheii, F6_3S_P_1CT(=NRRL B-65663T and DSMZ 115940T), and Sporosarcina highlanderae F6_3S_P_2 T(=NRRL B-65664T and DSMZ 115943T). Identifying and characterizing the genomes and phenotypes of novel microbes found in space habitats, like those explored in this study, is integral for expanding our genomic databases of space-relevant microbes. This approach offers the only reliable method to determine species composition, track microbial dispersion, and anticipate potential threats to human health from monitoring microbes on the surfaces and equipment within space habitats. By unraveling these microbial mysteries, we take a crucial step towards ensuring the safety and success of future space missions.

Dynamics of Hydrogen Bond Breaking Induced by Outer-Valence Intermolecular Coulombic Decay.

The photoexcitation of weakly bound complexes can lead to several decay pathways, depending on the nature of the potential energy surfaces. Upon excitation of a chromophore in a weakly bound complex, ionization of its neighbor upon energy transfer can occur due to a unique relaxation process known as intermolecular Coulombic decay (ICD), a phenomenon of renewed focus owing to its relevance in biological systems. Herein, we report the evidence for outer-valence ICD induced by multiphoton excitation by near-ultraviolet radiation of 4.4 eV photons, hitherto unknown in molecular systems. In the binary complexes of 2,6-difluorophenylacetylene with aliphatic amines, a resonant two-photon excitation localized on the 2,6-difluorophenylacetylene chromophore results in the formation of an amine cation following an outer-valence ICD process. The unique trends in experimentally observed translational energy distribution profiles of the amine cations following hydrogen bond dissociation, analyzed with the help of electronic structure and ab initio molecular dynamics calculations, revealed the presence of a delicate interplay of roaming dynamics, methyl-rotor dynamics, and binding energy.

Characterization of metagenome-assembled genomes from the International Space Station.

BACKGROUND: Several investigations on the microbial diversity and functional properties of the International Space Station (ISS) environment were carried out to understand the influence of spaceflight conditions on the microbial population. However, metagenome-assembled genomes (MAGs) of ISS samples are yet to be generated and subjected to various genomic analyses, including phylogenetic affiliation, predicted functional pathways, antimicrobial resistance, and virulence characteristics. RESULTS: In total, 46 MAGs were assembled from 21 ISS environmental metagenomes, in which metaSPAdes yielded 20 MAGs and metaWRAP generated 26 MAGs. Among 46 MAGs retrieved, 18 bacterial species were identified, including one novel genus/species combination (Kalamiella piersonii) and one novel bacterial species (Methylobacterium ajmalii). In addition, four bins exhibited fungal genomes; this is the first-time fungal genomes were assembled from ISS metagenomes. Phylogenetic analyses of five bacterial species showed ISS-specific evolution. The genes pertaining to cell membranes, such as transmembrane transport, cell wall organization, and regulation of cell shape, were enriched. Variations in the antimicrobial-resistant (AMR) and virulence genes of the selected 20 MAGs were characterized to predict the ecology and evolution of biosafety level (BSL) 2 microorganisms in space. Since microbial virulence increases in microgravity, AMR gene sequences of MAGs were compared with genomes of respective ISS isolates and corresponding type strains. Among these 20 MAGs characterized, AMR genes were more prevalent in the Enterobacter bugandensis MAG, which has been predominantly isolated from clinical samples. MAGs were further used to analyze if genes involved in AMR and biofilm formation of viable microbes in ISS have variation due to generational evolution in microgravity and radiation pressure. CONCLUSIONS: Comparative analyses of MAGs and whole-genome sequences of related ISS isolates and their type strains were characterized to understand the variation related to the microbial evolution under microgravity. The Pantoea/Kalamiella strains have the maximum single-nucleotide polymorphisms found within the ISS strains examined. This may suggest that Pantoea/Kalamiella strains are much more subjective to microgravity changes. The reconstructed genomes will enable researchers to study the evolution of genomes under microgravity and low-dose irradiation compared to the evolution of microbes here on Earth. Video Abstract.

Multi-faceted metagenomic analysis of spacecraft associated surfaces reveal planetary protection relevant microbial composition.

The National Aeronautics and Space Administration (NASA) has been monitoring the microbial burden of spacecraft since the 1970's Viking missions. Originally culture-based and then focused 16S sequencing techniques were used, but we have now applied whole metagenomic sequencing to a variety of cleanroom samples at the Jet Propulsion Lab (JPL), including the Spacecraft Assembly Facility (SAF) with the goals of taxonomic identification and for functional assignment. Our samples included facility pre-filters, cleanroom vacuum debris, and surface wipes. The taxonomic composition was carried out by three different analysis tools to contrast marker, k-mer, and true alignment approaches. Hierarchical clustering analysis of the data separated vacuum particles from other SAF DNA samples. Vacuum particle samples were the most diverse while DNA samples from the ISO (International Standards Organization) compliant facilities and the SAF were the least diverse; all three were dominated by Proteobacteria. Wipe samples had higher diversity and were predominated by Actinobacteria, including human commensals Cutibacterium acnes and Corynebacterium spp. Taxa identified by the three methods were not identical, supporting the use of multiple methods for metagenome characterization. Likewise, functional annotation was performed using multiple methods. Vacuum particles and SAF samples contained strong signals of the tricarboxylic acid cycle and of amino acid biosynthesis, suggesting that many of the identified microorganisms have the ability to grow in nutrient-limited environments. In total, 18 samples generated high quality metagenome assembled genomes (MAG), which were dominated by Moraxella osloensis or Malassezia restricta. One M. osloensis MAG was assembled into a single circular scaffold and gene annotated. This study includes a rigorous quantitative determination of microbial loads and a qualitative dissection of microbial composition. Assembly of multiple specimens led to greater confidence for the identification of particular species and their predicted functional roles.

Multidrug-resistant Acinetobacter pittii is adapting to and exhibiting potential succession aboard the International Space Station.

BACKGROUND: Monitoring the adaptation of microorganisms to the extreme environment of the International Space Station (ISS) is crucial to understanding microbial evolution and infection prevention. Acinetobacter pittii is an opportunistic nosocomial pathogen, primarily impacting immunocompromised patients, that was recently isolated from two missions aboard the ISS. RESULTS: Here, we report how ISS-associated A. pittii (n = 20 genomes) has formed its own genetically and functionally discrete clade distinct from most Earth-bound isolates (n = 291 genomes). The antimicrobial susceptibility testing of ISS strains and two related clinical isolates demonstrated that ISS strains acquired more resistance, specifically with regard to expanded-spectrum cephalosporins, despite no prediction of increased resistance based on genomic analysis of resistance genes. By investigating 402 longitudinal environmental and host-associated ISS metagenomes, we observed that viable A. pittii is increasing in relative abundance and therefore potentially exhibiting succession, being identified in >2X more metagenomic samples in back-to-back missions. ISS strains additionally contain functions that enable them to survive in harsh environments, including the transcriptional regulator LexA. Via a genome-wide association study, we identified a high level of mutational burden in methionine sulfoxide reductase genes relative to the most closely related Earth strains. CONCLUSIONS: Overall, these results indicated a step forward in understanding how microorganisms might evolve and alter their antibiotic resistance phenotype in extreme, resource-limited, human-built environments. Video Abstract.

Draft Genome Sequences of Fungi Isolated from Mars 2020 Spacecraft Assembly Facilities.

During the Mars 2020 mission, several fungal strains were isolated from surfaces where spacecraft components were assembled. Draft genome sequencing and characterization will help identify the genes responsible for radiation resistance, supporting the development of countermeasures to prevent fungal contamination of extraterrestrial environments.

Machine learning algorithm to characterize antimicrobial resistance associated with the International Space Station surface microbiome.

BACKGROUND: Antimicrobial resistance (AMR) has a detrimental impact on human health on Earth and it is equally concerning in other environments such as space habitat due to microgravity, radiation and confinement, especially for long-distance space travel. The International Space Station (ISS) is ideal for investigating microbial diversity and virulence associated with spaceflight. The shotgun metagenomics data of the ISS generated during the Microbial Tracking-1 (MT-1) project and resulting metagenome-assembled genomes (MAGs) across three flights in eight different locations during 12 months were used in this study. The objective of this study was to identify the AMR genes associated with whole genomes of 226 cultivable strains, 21 shotgun metagenome sequences, and 24 MAGs retrieved from the ISS environmental samples that were treated with propidium monoazide (PMA; viable microbes). RESULTS: We have analyzed the data using a deep learning model, allowing us to go beyond traditional cut-offs based only on high DNA sequence similarity and extending the catalog of AMR genes. Our results in PMA treated samples revealed AMR dominance in the last flight for Kalamiella piersonii, a bacteria related to urinary tract infection in humans. The analysis of 226 pure strains isolated from the MT-1 project revealed hundreds of antibiotic resistance genes from many isolates, including two top-ranking species that corresponded to strains of Enterobacter bugandensis and Bacillus cereus. Computational predictions were experimentally validated by antibiotic resistance profiles in these two species, showing a high degree of concordance. Specifically, disc assay data confirmed the high resistance of these two pathogens to various beta-lactam antibiotics. CONCLUSION: Overall, our computational predictions and validation analyses demonstrate the advantages of machine learning to uncover concealed AMR determinants in metagenomics datasets, expanding the understanding of the ISS environmental microbiomes and their pathogenic potential in humans. Video Abstract.

Complete Genome Sequence of Klebsiella quasipneumoniae subsp. similipneumoniae Strain IF3SW-P1, Isolated from the International Space Station.

The 5.2-Mb circular genome of Klebsiella quasipneumoniae subsp. similipneumoniae strain IF3SW-P1, isolated from the International Space Station, was sequenced using Oxford Nanopore Technologies. The genome lacks a megaplasmid typical of hypervirulent and multidrug-resistant Klebsiella strains but does contain a chromosomally encoded OqxAB efflux pump associated with carbapenem resistance.

Genomic Characterization of the Titan-like Cell Producing Naganishia tulchinskyi, the First Novel Eukaryote Isolated from the International Space Station.

Multiple strains of a novel yeast belonging to genus Naganishia were isolated from environmental surfaces aboard the International Space Station (ISS). These strains exhibited a phenotype similar to Titan cell (~10 µm diameter) morphology when grown under a combination of simulated microgravity and 5% CO2 conditions. Confocal, scanning, and transmission electron microscopy revealed distinct morphological differences between the microgravity-grown cells and the standard Earth gravity-grown cells, including larger cells and thicker cell walls, altered intracellular morphology, modifications to extracellular fimbriae, budding, and the shedding of bud scars. Phylogenetic analyses via multi-locus sequence typing indicated that these ISS strains represented a single species in the genus Naganishia and were clustered with Naganishia diffluens. The name Naganishia tulchinskyi is proposed to accommodate these strains, with IF6SW-B1T as the holotype. The gene ontologies were assigned to the cell morphogenesis, microtubule-based response, and response to UV light, suggesting a variety of phenotypes that are well suited to respond to microgravity and radiation. Genomic analyses also indicated that the extracellular region, outer membrane, and cell wall were among the highest cellular component results, thus implying a set of genes associated with Titan-like cell plasticity. Finally, the highest molecular function matches included cytoskeletal motor activity, microtubule motor activity, and nuclear export signal receptor activity.

Genomic Characterization of Potential Plant Growth-Promoting Features of Sphingomonas Strains Isolated from the International Space Station.

In an ongoing microbial tracking investigation of the International Space Station (ISS), several Sphingomonas strains were isolated. Based on the 16S rRNA gene sequence, phylogenetic analysis identified the ISS strains as Sphingomonas sanguinis (n = 2) and one strain isolated from the Kennedy Space Center cleanroom (used to assemble various Mars mission spacecraft components) as Sphingomonas paucimobilis. Metagenomic sequence analyses of different ISS locations identified 23 Sphingomonas species. An abundance of shotgun metagenomic reads were detected for S. sanguinis in the location from where the ISS strains were isolated. A complete metagenome-assembled genome was generated from the shotgun reads metagenome, and its comparison with the whole-genome sequences (WGS) of the ISS S. sanguinis isolates revealed that they were highly similar. In addition to the phylogeny, the WGS of these Sphingomonas strains were compared with the WGS of the type strains to elucidate genes that can potentially aid in plant growth promotion. Furthermore, the WGS comparison of these strains with the well-characterized Sphingomonas sp. LK11, an arid desert strain, identified several genes responsible for the production of phytohormones and for stress tolerance. Production of one of the phytohormones, indole-3-acetic acid, was further confirmed in the ISS strains using liquid chromatography-mass spectrometry. Pathways associated with phosphate uptake, metabolism, and solubilization in soil were conserved across all the S. sanguinis and S. paucimobilis strains tested. Furthermore, genes thought to promote plant resistance to abiotic stress, including heat/cold shock response, heavy metal resistance, and oxidative and osmotic stress resistance, appear to be present in these space-related S. sanguinis and S. paucimobilis strains. Characterizing these biotechnologically important microorganisms found on the ISS and harnessing their key features will aid in the development of self-sustainable long-term space missions in the future. IMPORTANCESphingomonas is ubiquitous in nature, including the anthropogenically contaminated extreme environments. Members of the Sphingomonas genus have been identified as potential candidates for space biomining beyond earth. This study describes the isolation and identification of Sphingomonas members from the ISS, which are capable of producing the phytohormone indole-3-acetic acid. Microbial production of phytohormones will help future in situ studies, grow plants beyond low earth orbit, and establish self-sustainable life support systems. Beyond phytohormone production, stable genomic elements of abiotic stress resistance, heavy metal resistance, and oxidative and osmotic stress resistance were identified, rendering the ISS Sphingomonas isolate a strong candidate for biotechnology-related applications.

Genomic Characterization of Parengyodontium torokii sp. nov., a Biofilm-Forming Fungus Isolated from Mars 2020 Assembly Facility.

A fungal strain (FJII-L10-SW-P1) was isolated from the Mars 2020 spacecraft assembly facility and exhibited biofilm formation on spacecraft-qualified Teflon surfaces. The reconstruction of a six-loci gene tree (ITS, LSU, SSU, RPB1 and RPB2, and TEF1) using multi-locus sequence typing (MLST) analyses of the strain FJII-L10-SW-P1 supported a close relationship to other known Parengyodontium album subclade 3 isolates while being phylogenetically distinct from subclade 1 strains. The zig-zag rachides morphology of the conidiogenous cells and spindle-shaped conidia were the distinct morphological characteristics of the P. album subclade 3 strains. The MLST data and morphological analysis supported the conclusion that the P. album subclade 3 strains could be classified as a new species of the genus Parengyodontium and placed in the family Cordycipitaceae. The name Parengyodontium torokii sp. nov. is proposed to accommodate the strain, with FJII-L10-SW-P1 as the holotype. The genome of the FJII-L10-SW-P1 strain was sequenced, annotated, and the secondary metabolite clusters were identified. Genes predicted to be responsible for biofilm formation and adhesion to surfaces were identified. Homology-based assignment of gene ontologies to the predicted proteome of P. torokii revealed the presence of gene clusters responsible for synthesizing several metabolic compounds, including a cytochalasin that was also verified using traditional metabolomic analysis.

Description and Genome Characterization of Three Novel Fungal Strains Isolated from Mars 2020 Mission-Associated Spacecraft Assembly Facility Surfaces-Recommendations for Two New Genera and One Species.

National Aeronautics and Space Administration's (NASA) spacecraft assembly facilities are monitored for the presence of any bacteria or fungi that might conceivably survive a transfer to an extraterrestrial environment. Fungi present a broad and diverse range of phenotypic and functional traits to adapt to extreme conditions, hence the detection of fungi and subsequent eradication of them are needed to prevent forward contamination for future NASA missions. During the construction and assembly for the Mars 2020 mission, three fungal strains with unique morphological and phylogenetic properties were isolated from spacecraft assembly facilities. The reconstruction of phylogenetic trees based on several gene loci (ITS, LSU, SSU, RPB, TUB, TEF1) using multi-locus sequence typing (MLST) and whole genome sequencing (WGS) analyses supported the hypothesis that these were novel species. Here we report the genus or species-level classification of these three novel strains via a polyphasic approach using phylogenetic analysis, colony and cell morphology, and comparative analysis of WGS. The strain FJI-L9-BK-P1 isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) exhibited a putative phylogenetic relationship with the strain Aaosphaeria arxii CBS175.79 but showed distinct morphology and microscopic features. Another JPL-SAF strain, FJII-L3-CM-DR1, was phylogenetically distinct from members of the family Trichomeriaceae and exhibited morphologically different features from the genera Lithohypha and Strelitziana. The strain FKI-L1-BK-DR1 isolated from the Kennedy Space Center facility was identified as a member of Dothideomycetes incertae sedis and is closely related to the family Kirschsteiniotheliaceae according to a phylogenetic analysis. The polyphasic taxonomic approach supported the recommendation for establishing two novel genera and one novel species. The names Aaosphaeria pasadenensis (FJI-L9-BK-P1 = NRRL 64424 = DSM 114621), Pasadenomyces melaninifex (FJII-L3-CM-DR1 = NRRL 64433 = DSM 114623), and Floridaphiala radiotolerans (FKI-L1-BK-DR1 = NRRL 64434 = DSM 114624) are proposed as type species. Furthermore, resistance to ultraviolet-C and presence of specific biosynthetic gene cluster(s) coding for metabolically active compounds are unique to these strains.

Comparative Genomics of Novel Agrobacterium G3 Strains Isolated From the International Space Station and Description of Agrobacterium tomkonis sp. nov.

Strains of Agrobacterium genomospecies 3 (i.e., genomovar G3 of the Agrobacterium tumefaciens species complex) have been previously isolated from diverse environments, including in association with plant roots, with algae, as part of a lignocellulose degrading community, from a hospital environment, as a human opportunistic pathogen, or as reported in this study, from a surface within the International Space Station. Polyphasic taxonomic methods revealed the relationship of Agrobacterium G3 strains to other Agrobacterium spp., which supports the description of a novel species. The G3 strains tested (n = 9) were phenotypically distinguishable among the strains from other genomospecies of the genus Agrobacterium. Phylogenetic analyses of the 16S rRNA gene, gyrB gene, multi-locus sequence analysis, and 1,089-gene core-genome gene concatenate concur that tested G3 strains belong to the Agrobacterium genus and they form a clade distinct from other validly described Agrobacterium species. The distinctiveness of this clade was confirmed by average nucleotide identity (ANI) and in silico digital DNA-DNA hybridization (dDDH) comparisons between the G3 tested strains and all known Agrobacterium species type strains, since obtained values were considerably below the 95% (ANI) and 70% (dDDH) thresholds used for the species delineation. According to the core-genome phylogeny and ANI comparisons, the closest relatives of G3 strains were Agrobacterium sp. strains UGM030330-04 and K599, members of a novel genomospecies we propose to call genomovar G21. Using this polyphasic approach, we characterized the phenotypic and genotypic synapomorphies of Agrobacterium G3, showing it is a bona fide bacterial species, well separated from previously named Agrobacterium species or other recognized genomic species. We thus propose the name Agrobacterium tomkonis for this species previously referred to as Agrobacterium genomospecies 3. The type strain of A. tomkonis is IIF1SW-B1T (= LMG 32164 = NRRL B-65602). Comparative genomic analysis show A. tomkonis strains have species-specific genes associated with secretion of secondary metabolites, including an exopolysaccharide and putative adhesins and resistance to copper. A. tomkonis specific gene functions notably relate to surface adhesion and could be involved to colonize nutrient-poor and harsh habitats. The A. tomkonis strains from the ISS showed presence of a 40-kbp plasmid and several other potential mobile genetic elements detected that could also be part of conjugative elements or integrated prophages.

Performance of Multiple Metagenomics Pipelines in Understanding Microbial Diversity of a Low-Biomass Spacecraft Assembly Facility.

NASA planetary protection (PP) requires an assessment of the biological contamination of the potential microbial burden on spacecraft destined to explore planetary bodies that may harbor signs of life, like Mars and Europa. To help meet these goals, the performance of multiple metagenomic pipelines were compared and assessed for their ability to detect microbial diversity of a low-biomass clean room environment used to build spacecraft destined to these planetary bodies. Four vendors were chosen to implement their own metagenomic analysis pipeline on the shotgun sequences retrieved from environmental surfaces in the relevant environments at NASA's Jet Propulsion Laboratory. None of the vendors showed the same microbial profile patterns when analyzing same raw dataset since each vendor used different pipelines, which begs the question of the validity of a single pipeline to be recommended for future NASA missions. All four vendors detected species of interest, including spore-forming and extremotolerant bacteria, that have the potential to hitch-hike on spacecraft and contaminate the planetary bodies explored. Some vendors demonstrated through functional analysis of the metagenomes that the molecular mechanisms for spore-formation and extremotolerance were represented in the data. However, relative abundances of these microorganisms varied drastically between vendor analyses, questioning the ability of these pipelines to quantify the number of PP-relevant microorganisms on a spacecraft surface. Metagenomics offers tantalizing access to the genetic and functional potential of a microbial community that may offer NASA a viable method for microbial burden assays for planetary protection purposes. However, future development of technologies such as streamlining the processing of shotgun metagenome sequence data, long read sequencing, and all-inclusive larger curated and annotated microbial genome databases will be required to validate and translate relative abundances into an actionable assessment of PP-related microbes of interest. Additionally, the future development of machine learning and artificial intelligence techniques could help enhance the quality of these metagenomic analyses by providing more accurate identification of the genetic and functional potential of a microbial community.