STEM Today by Abhishek Sinha

STEM TODAY February 2019, No. 41

CONTENTS Microbial Analysis of International Space Station (ISS) The major sources of microbiological risk factors for astronauts include food, drinking water, air, surfaces, payloads, research animals, crew members, and personnel in close contact with the astronauts.

Editorial Editor: Mr. Abhishek Kumar Sinha Editor / Technical Advisor: Mr. Martin Cabaniss

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Disclaimer ( Non-Commercial Research and Educational Use ) STEM Today is dedicated to STEM Education and Human Spaceflight. This newsletter is designed for Teachers and Students with interests in Human Spaceflight and learning about NASAâ&#x20AC;&#x2122;s Human Research Roadmap. The opinion expressed in this newsletter is the opinion based on fact or knowledge gathered from various research articles. The results or information included in this newsletter are from various research articles and appropriate credits are added. The citation of articles is included in Reference Section. The newsletter is not sold for a profit or included in another media or publication that is sold for a profit. Cover Page Apollo 12 Orbit Apollo 12 looks back at the Earth at the start of its 4 day journey to the moon. The shot includes the Lunar Module adapter panels shortly after jettison. Image # : AS12-50-7326 November 14, 1969 Image Credit: NASA

Back Cover View of Earth from Apollo 4 Earth as viewed from 10,000 miles. In 1969, the Apollo 4 (Spacecraft 017/Saturn 501) unmanned test flight made a great ellipse around Earth as a test of the translunar motors and of the high speed entry required of a human flight returning from the moon. A 70 mm camera was programmed to look out a window toward Earth, and take a series of photographs from "high apogee". Coastal Brazil, Atlantic Ocean, West Africa, Antarctica, looking west. This photograph was made when the Apollo 4 spacecraft, still attached to the S-IVB (third) stage, was orbiting Earth at an altitude of 9,544 miles. Image # : AS04-01-580 November 9, 1967 Image Credit: NASA

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Editorial Dear Reader

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All young people should be prepared to think deeply and to think well so that they have the chance to become the innovators, educators, researchers, and leaders who can solve the most pressing challenges facing our world, both today and tomorrow. But, right now, not enough of our youth have access to quality STEM learning opportunities and too few students see these disciplines as springboards for their careers. According to Marillyn Hewson, "Our children - the elementary, middle and high school students of today - make up a generation that will change our universe forever. This is the generation that will walk on Mars, explore deep space and unlock mysteries that we can’t yet imagine". "They won’t get there alone. It is our job to prepare, inspire and equip them to build the future." STEM Today will inspire and educate people about Spaceflight and effects of Spaceflight on Astronauts. Editor Mr. Abhishek Kumar Sinha

Editorial Dear Reader The Science, Technology, Engineering and Math (STEM) program is designed to inspire the next generation of innovators, explorers, inventors and pioneers to pursue STEM careers. According to former President Barack Obama, " Science is more than a school subject, or the periodic table, or the properties of waves. It is an approach to the world, a critical way to understand and explore and engage with the world, and then have the capacity to change that world..." STEM Today addresses the inadequate number of teachers skilled to educate in Human Spaceflight. It will prepare , inspire and educate teachers about Spaceflight. STEM Today will focus on NASA’S Human Research Road map. It will research on long duration spaceflight and put together latest research in Human Spaceflight in its monthly newsletter. Editor / Technical Advisor Mr. Martin Cabaniss

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MICROBIAL RESEARCH Microbial Analysis of International Space Station (ISS) The major sources of microbiological risk factors for astronauts include food, drinking water, air, surfaces, payloads, research animals, crew members, and personnel in close contact with the astronauts.

Multi-drug resistant Enterobacter bugandensis species isolated from the International Space Station

Enterobacter species are facultative anaerobic, Gram-stain-negative, and saprophytic microorganisms found in soil, sewage, and as a commensal enteric flora of the human gastrointestinal tract. They have been associated with nosocomial infection in humans, causing bacteremia, endocarditis, septic arthritis, osteomyelitis, skin and soft tissue infections, lower respiratory tract, urinary tract, and intra-abdominal infections. Some Enterobacter have also been reported plant pathogens. Antibiotic resistance and its clinical implications have been extensively studied in genus Enterobacter, especially Enterobacter cloacae, which is resistant to cephalosporins, ampicillin, amoxicillin, and cefoxitin. Five isolates belonging to the Enterobacter bugandensis group of bacteria from two different locations of the ISS were isolated.

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Whole genome sequencing (WGS) and de novo assembly was performed on all five ISS E. bugandensis strains, creating MLST and genome variation profiles of the ISS strains. Furthermore, comparative genome alignment of the ISS. strains with all publicly available 1291 Enterobacter genomes revealed that genomes of these five ISS strains were highly similar to only three clinical E. bugandensis with very high genome similarities and formed a unique ecotype. They are (a) EB-247 strain, isolated from neonatal blood of a patient from Tanzania, (b) 153_ECLO strain, isolated from the urine of a neonatal patient strain admitted to the University of Washington Medical Center, Seattle, WA and (c) MBRL 1077 strain, a carbapenemase-producing strain isolated from the wound of a 72-yearold woman with a history of cutaneous scleroderma, medically complicated obesity, and venous insufficiency. In this study, comparative genomic analysis of five ISS strains and three clinical isolates were carried out to elucidate antimicrobial resistance (AMR) phenotypic properties, MDR gene profiles, and genes related to potential virulence and pathogenic potential of the ISS Enterobacter strains. The WGS data submitted to the National Center for Biotechnology Information (NCBI) GenBank and NASA GenLab databases were downloaded and characterized during this study. The complete genome sequences of all ISS strains were deposited in NCBI under Bioproject PRJNA319366 as well as at the NASA GeneLab system (GLDS-67). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence of isolated strains are: IF2SW-B1 (KY218809), IF2SW-B5 (KY218813), IF2SW-P2 (KY218815), IF2SW-P3 (KY218816), and IF3SWP2 (KY218819). Results Phenotypic characteristics The ISS strains showed aerobic, motile, rod shape, Gram stain negative characteristics; colonies were pale yellow in color, formed within 24-36 h at 35â&#x2014;ŚC on R2A, TSA, and blood agar. Growth was observed at 1-8% NaCl and in pH range 5-7. The Vitek and BioLog systems as well as MALDI-TOF profiles identified the ISS strains as E. ludwigii. The MALDI-TOF profile scores for the tested strains were 2.16(E. ludwigii) and 2.10 (E. asburiae). In general, no noticeable phenotypic differences were observed among the Enterobacter species tested including E. bugandensis EB-247T, whose genome is closer to ISS strains. As reported earlier, all these five ISS Enterobacter isolates were resistant to cefazolin, cefoxitin, oxacillin, penicillin and rifampin, while for ciprofloxacin and erythromycin, strains were either resistant or intermediate resistant. For gentamycin and tobramycin some strains were resistant, some intermediate resistant, and some susceptible. Molecular phylogeny The 16S rRNA gene sequencing of all five isolates placed them within the Enterobacter group and showed maximum similarity (99.6%) with E. bugandensis EB-247T , E. cancerogenus LMG 2693, E. ludwigii EN-119, and E.mori R18-2 (99 to 100%). Since 16S rRNA gene sequencing analysis is insufficient to differentiate Enterobacter species, polygenic and whole genome-based analyses were further attempted. All ISS strains were phylogenetically characterized by the gyrB locus (â&#x2C6;ź 1.9 kb) and showed that the ISS isolates form a close group with E. bugandensis EB-247T and 153_ECLO strains (> 99%) while MBRL 1077 isolate was exhibiting 97% similarity with high bootstrap value. 4

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MLST analysis The genomic contigs of the ISS isolates were searched for gene sequences of dnaA, fusA, gyrB. leuS, pyrG, rplB, and rpoB, which are standardized for the use of MLST analysis and reported for E. cloacae species. The good congruence between the single-gene reconstructions and the concatenate reinforced the stability of the genealogy were observed. The reconstruction was based on the RAxML algorithm and the resulting MLST tree (Fig. 1) shows that the ISS isolates are phlylogenetically related to E. bugandensis clinical strains (EB-247, strain 153_ECLO, and isolate MBRL 1077). SNP analysis Even though MLST analysis was clearly able to genomically resolve the ISS isolates to species level and distinguish them from other members of the genus Enterobacter, whole genome SNP analysis, SNP tree analysis excluding plasmid sequences, was carried out to validate these results. The snpTree does not ignore any nucleotide positions and is able to consider 100% of the chromosomal genome. All the available WGS of the Enterobacter genus reference genomes from GenBank were used for SNP analysis with snpTree. Of the 22 total nucleotide sequences; 58,121 positions were found in all analyzed genomes and 3832 positions in the dataset were used to confer the final tree (Fig. 2). The snpTree analyses confirmed and gave a strong validation to the MLST/gyrB data, confirming that all ISS isolates are E. bugandensis but strain MBRL 1077 grouped differently from the members of the E. bugandensis group. SNP identification within ISS strains was carried out using GATK HaplotypeCaller. Filtered SNP calls and indels (after removal of false positives) are given in the Additional file 1: Table S1. Post-filtration analyses showed that there were 9, 12, 15, 13, and 0 SNPs seen in IF2SWB1, IF2SWB5, IF2SWP2, IS2WP3 and IS3SWP2, re-

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spectively. Further 6, 0, 4, 6, and 0 indels were seen in IF2SWB1, IF2SWB5, IF2SWP2, IS2WP3 and IS3SWP2, respectively (Additional file 1: Table S1). A maximum of 15 SNPs was observed among ISS isolates, probably being clonal in origin, with a very recent common ancestor. However, it should be noted that 4 strains were isolated from location #2 (space toilet) and one strain from the exercise platform (ARED).

ANI values and digital DNA-DNA hybridization The ANI values for the ISS strains were maximum against E. bugandensis EB-247, 153_ECLO, and MBRL 1077 strains (> 95%) as were those of MLST analyses, and the ANI values of rest of the Enterobacter genomes tested were < 91% (Table 1). The digital DNA-DNA hybridization (dDDH) results of the ISS strain showed high similarity with E. bugandensis EB-247 (89.2%), 153_ECLO (89.4%), and MBRL 1077 (64%) strains whereas dDDH value was < 44.6% to all the other available Enterobacter reference genomes (Table 1). Based on various molecular analyses attempted during this study all five ISS Enterobacter strains were phenotypically and genotypically identified as E. bugandensis. Functional characteristics A detailed genome analysis of all five ISS strains and 3 clinical isolates were carried out to understand its genetic makeup. A total of 4733 genes were classified as carbohydrate metabolism (635 genes), amino acid and derivatives (496 genes), protein metabolism (291 genes), cofactors, vitamins, prosthetic groups, pigments (275 genes), membrane transport (247 genes), and RNA metabolism (239 genes) (Fig. 3). To test antimicrobial resistance at genomic level, the ISS strains were further compared with nosocomial isolates (1291 genomes) having more than 95% ANI identity with the ISS strains, which taxonomically identified them as same species. Genomes of the clinical strains of E. bugandensis 247,153_ECLO, and MBRL-1077, whose ANI values were > 95%, were used for the genetic comparison to further broaden the picture. Features playing a broad role and implemented by the same domain such as Spectinomycin 9-O-adenylyltransferase and Streptomycin 3-O-adenylyltransferase (EC2.7.7.47) were only present in E. bugandensis 247 due to the probable lack of selective pressure that might have been encountered by the ISS isolates (Table 2). The pre-

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dicted arsenic resistance (arsenic resistance protein, ArsH) noticed in E. bugandensis 247 but not in other strains should be phenotypically tested to confirm the resistance properties conferred in strain E. bugandensis 247 and cross checked with the ISS strains for their inability to degrade arsenic. Trace metals detected in ISS potable water samples, but typically below potability requirements, included arsenic, barium, chromium, copper, iron, manganese, molybdenum, nickel, lead, selenium, and zinc. No mercury or cadmium was detected and the arsenic levels varied from nondetectable in water samples to a maximum of 3.8 Âľg/L.

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Global comparison of ISS genomes with other Enterobacter genomes A visualization program was reported to be invaluable in determining the genotypic differences between closely related prokaryotes. Visualizing a prokaryote genome as a circular image has become a powerful means of displaying informative comparisons of one genome to a number of others. Using BRIG, a global visual comparison of ISS isolates with other Enterobacter WGS from the GenBank Microbial Genomes Resource was carried out. The resulting output of the BRIG analysis, a visualization image, showed draft genome assembly information, read coverage, assembly breakpoints, and collapsed repeats. The mapping of unassembled sequencing reads of the ISS genomes against fully annotated E. cloacae central reference sequences is depicted in Fig. 4.

A comparative phenotypic and genotypic analyses of ISS isolates identified as E. bugandensis were carried out. Additional genomic analyses revealed a close genetic relatedness between ISS isolates and nosocomial earth isolates. MLST and whole genome SNP tree placed ISS and nosocomial isolates to a separate clade when phylogenetically aligned with other member of genus Enterobacter. A detailed functional and antimicrobial resistance analysis reveals that the ISS isolates have a 79% probability of being a human pathogen and share similar antimicrobial resistance pattern with E. bugandensis EB-247, MBRL-1077 and 153_ECLO strains, making them relevant for future missions and crew health considerations.

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A total of 112 identified genes of the ISS strains were involved in virulence, disease, and defense. Genes associated with resistance to antibiotics and toxic compounds, including the multidrug resistance tripartite system (also known as 3-protein systems) as shown in a polychlorinated biphenyl-degrader, Burkholderia xenovorans LB400, was noticed in the ISS strain. This protein forms the basic structure and plays a crucial role in, the functioning of an efflux pump rendering a microbe drug resistant. A multiple antibiotic resistance (MAR) locus or MAR operon was observed in ISS strains, which codes for protein MarA, MarB, MarC, and MarR, and regulate more than 60 genes, including upregulation of drug efflux systems that have been reported in Escherichia coli K12. Aminoglycoside adenylyltransferases, whose role is spectinomycin 9-O-adenylyltransferases, which confers microbial resistance to the aminoglycosides in Salmonella enterica, was also seen in ISS strains. Similarly, resistance to fluoroquinolones due to a mutation in gyrA gene in S. enterica, and fosfomycin resistance due to the presence of FosA protein-coding gene, which catalyzes the addition of glutathione to C1 of the oxirane in Serratia marcescens, were observed in ISS strains. Multiple copies of multi-drug resistance (MDR) genes highly homologous to S. marcescens, a pathogen, were identified in the ISS Enterobacter genomes, which gives an indication that these strains may be a potential human pathogen. When tested with PathogenFinder algorithm, strain IF2SW-P2T had > 77% probability to be a human pathogen. When compared with E. cloacae ATCC 13047, which is a well-described human pathogen, all five ISS strains showed a > 79% probability score. Astronauts have been taking beta-lactam based medical drugs for approximately two decades, and Î˛-lactamase (superfamily I [metal dependent hydrolases] and E.C.3.5.2.6) was present in all strains under study, while penicillin-binding proteins (PPB4B) were only present in MBRL-1077. Fluoroquinolone resistance due to gyrase and topoisomerase mutation was present in all the strains. Metal-dependent hydrolases, cation efflux system protein CusA, cobalt-zinc-cadmium resistance protein, cobalt-zinc-cadmium resistance protein CzcA, DNAbinding heavy metal response regulator, Co/Zn/Cd efflux system membrane fusion protein, zinc transporter ZitB were found in both ISS isolate and nosocomial organism understudy. These genes principally help in detoxification of periplasm by exporting toxic metal cation outside the cell. Determinants of the metal resistance are usually located on the plasmid and readily acquired from the environment and also complement antibiotic resistance. The plasmid encoded putative transcriptional regulators containing the CopG/Arc/MetJ DNA-binding domain and a metal-binding domain were present in the ISS strains (Additional file 2: TableS2). Further studies are required for phenotypic characterization to confirm this trait. Presence of active beta lactamase gene, efflux pump, and RND (resistance, nodulation and cell division protein family) protein family renders broad-spectrum resistance to ISS isolates from drugs and natural inhibitors.

Detection of antimicrobial resistance genes associated with the International Space Station environmental surfaces

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Microbial monitoring of air and surfaces have shown that many different microbes exist in this closed, harsh environment, some of which are opportunistic pathogens of human origin. The presence of such microorganisms within this environment poses a serious risk to crew health, as opportunistic pathogens have shown increased virulence while grown in space and prolonged spaceflight weakens an astronautâ&#x20AC;&#x2122;s immune system, rendering him/her more susceptible to infection. Some human associated opportunistic pathogens that have been isolated from the ISS include Staphylococcus aureus, S. haemolyticus, S. hominis, Acinetobacter pitti, Pantoea conspicua and members of the family Enterobacteriaceae, while others such as Propionibacterium and Corynebacterium have been detected with culture-independent, molecular based methods. This study however, does not discuss virulence- a key factor that determines whether or not a microbe could be harmful to humans. Thus, the AMR gene carrying bacteria reported in this study do not necessarily pose a threat to astronauts on the ISS. Antibiotics are the primary treatment strategy to combat infections, but this approach may not be as easy to implement on the ISS or on future deep space missions. Studies have shown that some organisms become less susceptible to antibiotic action when exposed to spaceflight. For example, the minimum inhibitory concentration (MIC) of colistin and kanamycin for E. coli, isolated from an astronaut aboard Salyut 7, increased from 4 Âľg/ml to >16 Âľg/ml, compared to the ground control, while that of S. aureus increased two fold for oxacillin, chloramphenicol, and erythromycin. Increased antibiotic resistance has also been noted on Discovery shuttle and Mir space station flight missions. The mode of action for this resistance is unclear, but it could be due to increased mutations in bacterial genes. A study comparing S. epidermidis grown on the ISS vs Earth, for 122 hours, showed a 24-fold higher frequency in mutations in the rpoB gene (beta subunit of RNA polymerase) when grown on the ISS, which rendered the isolates resistant to rifampicin. In a study comparing anti-microbial resistance in bacteria isolated from the ISS and the Antarctic Research Station Concordia, Schiwon et al. found that 75.8% of the ISS isolates were resistant to one or more antibiotics, whereas only 43.6% of the Antarctic isolates were resistant. From the ISS, 86.2% of the isolates had relaxase and/or transfer genes encoded on plasmids which can promote the dissemination of AMR genes from one microorganism to another. Importantly, the same genera and even most of the same species were examined between the two locations. The two major differences between these two confined, hostile and extreme environments are microgravity and radiation, which may contribute to the higher prevalence of antibiotic resistant microorganisms on the ISS. However, unlike the Schiwon et al. study that measured AMR profiles from isolated strains, this study has examined AMR gene profiles directly from DNA, without culturing. It is well established that only a small proportion of microorganisms in any given environment can be cultured with standard laboratory techniques and the ISS is no different. A recent microbial tracking study performed by the lab showed that less than 30% of intact, potentially viable microorganisms (i.e. samples treated with propidium monoazide (PMA) which prevent amplification of dead cells in molecular analysis) could be cultured, leaving 70% of organisms and their resistomes (i.e. the collection of AMR genes within a genome) unaccounted for. In a recent study, propidium iodide ions were shown to move across intact cell membranes for Dinoroseobacter shibae and Bacillus subtilis using BacLight fuorescence staining and hypothesized that high membrane potential facilitated breakthrough of the double-charged propidium iodide and marked viable cells as dead. In contrast, using polymerase chain reaction (PCR) we have shown that PMA was able to intercalate DNA in compromised cells which eventually blocked the amplification. In this study, authors performed a thorough analysis of anti-microbial resistance genes within the ISS environment, using traditional and state-of-the art molecular techniques. Samples from eight ISS locations were obtained from three separate sampling events. The bacteria isolated from the samples were tested for resistance to several antibiotics. Concurrently, DNA from PMA treated samples was subjected to a target amplification of 518 antimicrobial genes, designed to match the content of the Antimicrobial Resistance Determinant Microarray (ARDM). Samples were treated with PMA before DNA extraction so that our focus would be on intact, potentially viable cells and not dead ones as live cells pose a greater risk to astronaut health. In addition, metagenome data generated from the DNA of the same PMA treated samples were mined for 12

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the presence of AMR and compared. This is the first report that presents the identification of the most common AMR genes on the ISS without culturing and correlates the results with phenotypic susceptibility and WGS data of cultured isolates. The study of the ISS resistome is crucial for the development of better surveillance and mitigation procedures on future long-duration spaceflight.

Results Twenty-four samples were collected during three sampling events, over a span of a year, from eight locations onboard the ISS (Table 1). Samples were both (i) cultured, to obtain isolates for phenotypic antibiotic susceptibility testing and WGS and (ii) subjected to DNA extraction, after propidium monoazide (PMA) treatment, to allow for Ion AmpliSeqT M and metagenomics analyses. PMA is a dye that intercalates with nucleic acid from dead cells and free DNA, allowing one to assess only intact bacteria within a sample. A summary of the analysis work flow is presented in Fig. 1.

Phenotypic susceptibility tests of cultured organisms

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Fifty-seven Biosafety Level-2 (BSL-2) microorganisms, isolated from the ISS, were tested for their resistance against the following 9 antibiotics using the disc diffusion method: cefazolin, cefoxitin, ciprofloxacin, erythromycin, gentamycin, oxacillin, penicillin, rifampin and tobramycin. Table S1 shows the measured zone of inhibition along with the percentage of resistant, intermediate resistant and susceptible strains to each antibiotic. Out of the total 57 strains tested, 92% were resistant to penicillin while only 14% were resistant to gentamicin and ciprofloxacin. For the remaining 6 antibiotics the resistance percentages were as follows: oxacillin (68%), rifampin (66%), erythromycin (64%), cefoxitin (49%), cefazolin (29%) and tobramycin (19%).

Enterobacter bugandensis, a novel species isolated from the ISS, was resistant to the most antibiotics: all 6 E. bugandensis isolates tested were resistant against cefazolin, cefoxitin, erythromycin, oxacillin, penicillin and rifampin, while for ciprofloxacin and gentamycin, strains were either resistant or intermediate resistant. For tobramycin some strains were resistant, some intermediate resistant and some susceptible. S. haemolyticus on the other hand, was not resistant to any of the antibiotics tested. Due to the serious health concerns of methicillin resistant S. aureus (MRSA), which are resistant to a variety of antibiotics, all 12 S. aureus isolates obtained on the ISS were further tested with the Vitek system, which included a more comprehensive panel of antibiotics. Antibiotics that were tested with both the disc diffusion assay and Vitek showed the same results (with the exception of three isolates which were susceptible with Vitek but resistant with the disc assay) and the additional antibiotics tested with Vitek showed all strains to be susceptible, except four that were resistant to clindamycin (Table S1). In addition, assessment of methicillin resistance (i.e. MRSA) was performed using the HardyCHROM MRSA biplates and all 12 S. aureus strains tested were shown to not be MRSA.

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Examining bacterial genomes for antibiotic resistance genes WGS was performed on 15 of the 57 BSL-2 organisms that were isolated from the ISS. In addition, six S. aureus mutant colonies that were present within the clear zone of inhibition after exposure to rifampin, also had their genomes sequenced. The presence or absence of AMR genes in each isolate is displayed in Fig. 2A. It is evident that S. aureus genomes show a distinct AMR gene profile compared to other staphylococcal species and other genera. Furthermore, amongst S. aureus isolates, the two that were resistant to erythromycin (IF4SW_P1_Sau and IF7SW_P3_Sau), as determined by the disc diffusion assay and Vitek testing, display a distinct AMR gene profile compared to all the others, indicating a possible genetic basis for resistance. Mutations in the rpoB gene have been implicated in rifampin resistance , therefore the rpoB gene sequence from the spontaneous rifampin resistant mutants and the sensitive parent strains were compared. With the exception of a base pair change from C to T at position 144, there were no other differences between parent and mutant strains. Seven antibiotic resistance gene clusters were analyzed in the novel species E. bugandensis, isolated from the waste and hygiene compartment on the ISS. These AMR gene clusters were associated with known pathogens and were not found in Enterobacter species isolated on Earth (Fig. 2B). This suggests that horizontal gene transfer (HGT) might have occurred but more research is needed to determine if HGT happened on the ISS. Anti-microbial resistance gene profiles on the ISS using Ion AmpliSeqT M To survey the AMR gene content of a large collection of samples and independent of culture restrictions, DNA isolated from the 24 ISS samples was analyzed using Ion AmpliSeqT M . Ion AmpliSeqT M technology consists of a pool of primer pairs, with each pair designed to amplify a specific gene. In this study, this panel consisted of primer pairs designed against 518 known antibiotic resistance genes from a variety of bacteria. Of the 518 genes that were tested for, a total of 123 were detected, but only those that were over 100 reads from the Ion Torrent sequencer were kept for further analysis. This was a threshold setting to remove potential "false positives" which can arise from non-specific primer binding, mutations during amplification and Ion Torrent sequencing error. A comprehensive summary of the 63 detected genes (those with read counts over 100), their annotation numbers in NCBI (GI), the microorganisms they are related to, their function and the resistance they confer are found in Table S2. Figure 3 shows the relative proportion of each detected AMR gene within a sample, with each colored bar representing a different AMR gene. The different colors and color patterns for each location suggest differences in spatial distribution of AMR genes at each location. This variability was confirmed with biplots, which show the distribution of the samples in relation to each other, and the contribution of each variable (i.e. gene) to the overall composition of that sample (Fig. S1). In addition to spatial differences, the dendrogram in Fig. 4A, shows temporal differences as well, with a clear separation of Flight 3 (F3) samples from Flight 1 (F1) and Flight 2 (F2). While there was no overall separation between F1 and F2, most paired samples (i.e. F1_8 vs F2_8) do cluster apart between the two sampling events (i.e. locations 1, 2, 4, 6, 7, 8). The separation between F1/F2 and F3 may be attributed to the low relative abundances of AMR genes present on the ISS during F3. As the heatmap in Fig. 4B shows, F3 samples had a higher number of genes shaded yellow, an indicator of abundances lower than the geometric mean abundance of all the 24 samples combined. At the functional level, the genes for beta-lactam and trimethoprim resistance were abundant and widespread across the ISS (Fig. 5A). However, when a metagenomics approach was used to examine the AMR gene profiles, genes for beta-lactam resistance, while found in every sample, were not as abundant compared to other func15

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tional groups and genes for trimethoprim resistance was not detected (Fig. 5B). The metagenomics approach also showed that genes conferring resistance to metals (i.e. zinc, copper etc)and those involved in multi-drug efflux pumps were the most abundant across the ISS, both of which were not detected with Ion AmpliSeqT M .

Fluoroquinolone resistance was also highly represented across the ISS, while Ion AmpliSeqT M only detected this gene family in only 3 samples. Ion AmpliSeqT M detected 23 antibiotic groups and metagenomics 13, with 6 shared between the two methods: aminoglycosides, beta-lactams, quinols (i.e. fluoroquinolones), fosfomycin, methicillin and streptomycin. At the gene level AmpliSeqT M and metagenomics both detected tetR, macB, fosA, and femB. As the two methods use a different set of reference genes and the raw data is processed differently, the fact that there are differences is not surprising, but it does highlight the usefulness of using a variety of

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analytical approaches.

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Multiple AMR genes can be transferred on a single plasmid, giving rise to multi-drug resistance in the recipient. Authors were therefore interested in assessing whether any genes were associated with each other and thus possibly found together. A correlation analysis showed statistically significant negative and positive correlations between the genetic determinants of AMR (see Table 2 for Spearman rho and P values). Authors have provided a comprehensive summary of the AMR genes present across the ISS over a span of a year using phenotypic assays, WGS, Ion AmpliSeqT M and metagenomics. The WGS of BSL-2 isolates showed

the collective presence of 51 genes conferring resistance to 18 antibiotics.

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In most cases, the genotype of the sequenced strains could predict the phenotype, such as the presence of ermA and blaZ in S. aureus, which protects against erythromycin and penicillin respectively and the presence of oqxA and oqxB in K. quasipneumoniae, which protects against ciprofloxacin, all of which were resistant to their respective antibiotics in the disc diffusion assay.

In other cases, the AMR profiles were not as straightforward to interpret. For example, one S. aureus isolate, (IF4SW.P1_Sau) lacking the blaZ gene was still resistant to penicillin. In another case, the presence of norA in multiple S. aureus isolates did not protect against ciprofloxacin, even though transfer of this gene to susceptible strains has been proven to be enough to confer resistance. The lack of consensus between genotype and phenotype in this case could be due to inadequate gene expression, which has been shown in numerous studies to be a barrier against resistance. Moreover, this study was focused on the detection of genomic sequences of known AMR genes so any mutations and variations in gene expression of AMR genes were not analyzed in this study. E. bugandensis was the most antibiotic resistant species tested with our phenotypic assay, with all six isolates being resistant to all antibiotics tested (with the exception of two that were susceptible to tobramycin). Enterobacter species, a member of the coliform group of bacteria, have been reported to be pathogenic and cause opportunistic infections in immunocompromised individuals and are commonly associated with urinary and respiratory tract infections. Of note, approximately 6% of the isolates cultured from the ISS were identified as Enterobacter. Carbapenems, a class of beta-lactams, are the most effective treatment against Enterobacter infections as they remain stable against extended spectrum beta lactamases, unlike penicillin and oxacillin. Ertapanem is the only carbapenem included in the medical kit onboard the ISS, however this may need to be re-evaluated, as numerous clinical isolates of Enterobacter species have shown resistance to ertapanem, though not other carbapenems, such as imipenem and meropenem. There is the possibility that the ISS Enterobacter isolates, like the clinical isolates, are resistant to ertapanem, and potentially other carbapenems, as the IMP-2 gene, a metallo beta-lactamase that is able to inhibit the action of this class of antibiotic, was detected in our samples. Further evaluation of this resistance profile and any other multi-drug resistant microorganisms, onboard the ISS, should be made a priority, in order to assess risk of infection and to ensure that proper strategies are in place to combat such infections. Ion AmpliSeqT M revealed a collective of 65AMR genes onboard the ISS, representing resistance to 23 different antibiotics. Nine of those detected genes (qacB, blaZ, bla- OKP, oqxA, oqxB, fos, tet38, norA and bla1) were 19

also identified in the WGS analysis of our selected cultured isolates. One of the detected genes was mecA, a biomarker for methicillin resistant strains of S. aureus, commonly referred to as MRSA. MRSA are strains of Staphylococcus that have become resistant to methicillin and other beta-lactams (i.e. oxacillin, penicillin) used to treat ordinary Staphylococcus infections can cause serious complications (skin infections, sepsis and pneumoniae) in immune-compromised individuals. Even though mecA was detected in numerous samples across the ISS via AmpliSeqTM, none of the 12 S. aureus isolates tested with Vitek were MRSA positive nor was mecA detected in their genomes. While mecA is commonly associated with S. aureus, it has also been detected in other Staphylococcus species which may have been the source of the detected mecA during this study. Despite the fact that none of the S. aureus isolates were MRSA at the time of collection, there is the possibility that they acquire mecA from other staphylococci. Indeed, the results of a hospital in the Netherlands, showed that mecA gene transfer occurred in a patient between mecA+ S. epidermidis and a mecA− strain of S. aureus.

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This is not surprising as there is mounting evidence in favor of the "resistance gene reservoir" where commensal organisms in the body act as reservoirs for AMR genes that can be transferred to resident or transient bacteria, including pathogens. Further studies assessing the proportion and types of mobile genetic elements within the ISS metagenome could shed some light on how readily AMR (and possibly virulence genes) may be acquired and transferred by bacteria on the ISS and if space conditions promote the transfer of AMR genes between different species and even different genera more readily then when these organisms are grown together on Earth. A positive correlation was observed between oliquadox (oqxA and oqxB) and fosfomycin (fosA) resistance on the ISS, which conforms with ground studies showing the co-transfer of oqxA/oqxB and fosA genes from single plasmids. A positive correlation was also observed between mupirocin and lincosamide/streptograminA resistance genes on the ISS. While studies have documented the co-transfer of β-lactam, quinolone, aminoglycoside, amphenicol and fosfomycin resistance genes to transconjugants, this is the first study, to author’s knowledge, that has observed an association between muprirocin and lincosamide/streptogtanimA resistance genes. It has been documented that metal exposure and the resulting resistance towards those metals leads to coselection of unrelated antibiotic resistance, most likely a consequence of metal resistance genes and AMR genes present on the same plasmid and/or transposon. Indeed, bacterioplankton isolated from metal contaminated Ash basins were significantly more tolerant to a variety of antibiotics, which were not found in these basins, compared to bacterioplankton collected from basins void of metals. Similar phenomena have been reported from metal contaminated freshwater and soil samples. The metagenomics data showed that the highest resistance genes across all time points and all locations were in fact metal resistance genes. It is unknown whether the material of the ISS surfaces that these organisms were collected from promotes metal resistance dissemination amongst the microbiota but if that is the case, this metal resistance may play a role in the spread and abundance of AMR genes on the ISS. The sample processing took place in a 10 K class cleanroom at JPL immediately upon return to Earth. Each wipe was aseptically taken out from the zip lock bag and transferred to a 500-mL bottle containing 200 mL of sterile PBS. The bottle with the wipe was shaken for two minutes followed by concentration with a Concentrating Pipette (Innova Prep, Drexel, MO) using 0.45 µm Hollow Fiber Polysulfone tips and PBS elution fluid. The environmental control and each sample were concentrated to 5 mL. The data presented in this manuscript are available in the NCBI Sequence Read Archive under accession number: SRP082440. The raw metagenomics sequences can be downloaded from the following GeneLab website.

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