The environmental samples were collected with the polyester wipes from eight different locations in the International Space Station (ISS) during two consecutive sampling sessions (three months apart) within the ISS Microbial Observatory Experiment. DNA extracted from each of the samples was used to create amplicon libraries based on customized panel of 500 antimicrobial resistance genes followed by next-generation sequencing. This is the first study of that shows the reservoir of antimicrobial genes in the ISS. The International Space Station (ISS) as a closed built environment has its own environmental microbiome which is shaped by microgravity radiation and limited human presence. The microbial diversity associated with ISS environmental surfaces was investigated during this study. Polyester wipes and contact slides were used for sampling of eight various surface locations on the ISS at different time periods. The samples were retrieved and analyzed immediately upon the return to the Earth (via Soyuz TMA-14M or Dragon capsule from SpaceX). After surface sample collection contact slides containing nutrient media for the growth of bacteria and fungi were incubated at 25 xcb x9aC. The polyester wipes were processed to measure microbial burden (R2A Blood Agar and Potato Dextrose Agar) and recover cultivable bacteria as well as fungi. Subsequently viable microbial burden was assessed using Adenosine Triphosphate (ATP) assay and quantitative polymerase chain reaction (PCR) methods after propidium monoazide (PMA) treatment. The 16S-tag and metagenome analyses were used to elucidate viable microbial diversity. The cultivable bacterial population yield from the polyester wipes was very high (5 to 7-logs) when compared with the contact slides (102 to 103 CFU/m2). The PMA-qPCR analysis showed considerable variation of viable bacterial population (105 to 109 16S rDNA gene copies/m2) among locations sampled. Unlike contact slides polyester wipes cover much larger sample surface (~1 m2) and produce much more reliable results of the microbial diversity of the ISS covering both cultivable and non-cultivable species. The cultivable total and viable microbial diversity was determined utilizing state-of-the art molecular techniques. The implementation of the PMA assay before DNA extraction allowed distinguishing viable microorganisms which is crucial for determining their role to the crew health the ISS maintenance and the general knowledge of the closed environmentally controlled built systems.

As part of an ongoing effort to catalogue microbial communities inhabiting the International Space Station (ISS) crew-associated environmental samples were collected from the Japanese Kibo Russian and US research modules. Initial analysis based on 16S rRNA gene sequencing identified 11 Bacillus isolates (two from Kibo Japanese Experiment Module (JEM) four from US Segment Harmony Node 2 and five from Russian Segment Zvezda module sites) all belonging to the Bacillus anthracis-B. cereus-B. thuringiensis group. Isolates were further characterized by whole genome comparative analysis. Each isolate was sequenced assembled and aligned against all members of the B. cereus sensu lato group. Based on genome size estimates (5.2 - 5.3 Mbp) strain nucleotide identity (>99.99%) and maximum likelihood phylogenetic placement all isolates were found to exhibit a very high level of similarity. With respect to gene content all isolates were inspected for cry proteins common to B. thuringiensis as well as toxins specific to B. cereus. No significant hits were found to any known cry genes while full-length matches were found for multiple B. cereus toxin genes. Finally the isolates were closely screened against B. anthracis genomes and no B. anthracis signatures were identified. Notably all isolates contained the plcR ancestral C allele and lacked significant hits to pXO1 and pXO2 plasmids and toxins. The collective results from the analysis confirm that these strains belong to a previously uncharacterized Bacillus species hereafter referred to as Bacillus issensis. The type strain is ISSFR-003T (=NRRL B-65389T= DSMZ 101676T).

Microbes interact with humans in complex ways and understanding how they respond to the spaceflight environment is important to the success of future manned spaceflight missions. The BRIC-23 mission was designed to measure the response of Bacillus subtilis and Staphylococcus aureus to the spaceflight environment. This experiment aimed to produce high quality omics data from B. subtilis and S. aureus grown aboard the International Space Station (ISS) to allow comparison to matched ground controls. There were two primary objectives for this experiment: (1) Demonstrate all post-flight processes and operations required for successful completion of GeneLab Reference Missions conducted on ISS and (2) Generate high quality GeneLab Reference Mission omics data sets for two prokaryotic model organisms Bacillus subtilis and Staphylococcus aureus. Freezing Control Experiment: The BRIC hardware has significant thermal inertia thus the freezing rate of samples placed at -80 C is quite slow. This could affect RNA-sequencing proteomic and metabolic data sets. In an effort to understand how slow freezing could affect these data sets a control experiment was designed in which B. subtilis and S. aureus were grown in petri plates and either slow frozen to -80 C at a rate matching the BRIC-23 spaceflight samples or processed immediately to harvest RNA and protein. B.subtilis omics data is deposited in GLDS-138.

Aspergillus fumigatus is a saprophytic filamentous fungus that is ubiquitous outdoors (soil decaying vegetation) and indoors (hospitals simulated closed habitats etc.). A. fumigatus can adapt to various environmental conditions and form airborne conidia that are the inoculum for a variety of diseases (e.g. non- and invasive pulmonary infections allergic bronchopulmonary aspergillosis etc.) in immunocompromised hosts. In an on-going Microbial Observatory Experiments on the International Space Station (ISS) molecular phylogeny of several fungal isolates were characterized. Two strains ISSF 21 and IF1SW-F4 were isolated from the HEPA filter and the surface of the Cupola of the ISS respectively. Using primers targeting the internal transcribed spacers ITS1 and 2 both isolates were identified as A. fumigatus. The whole genome sequence analysis of ISSF 21 revealed increased number of single nucleotide polymorphisms (SNPs) when compared to the reference A. fumigatus 293. Knowing that A. fumigatus is an opportunistic pathogen and microgravity highly influences the antibiotic susceptibility and pathogenicity of microorganisms we examined pathogenicity of both ISS isolates using the zebrafish larval model. The space station isolates (ISSF-021 and IF1SW-F4) were more virulent than two clinical strains (Af293 and CEA10) whose pathogenicity was highly characterized. Here the whole genome sequences of ISSF-021 strain are being deposited.

As part of an ongoing effort to catalogue microbial communities inhabiting the International Space Station (ISS) crew-associated environmental samples were collected from the Japanese Kibo Russian and US research modules. Initial analysis based on 16S rRNA gene sequencing identified 11 Bacillus isolates (two from Kibo Japanese Experiment Module (JEM) four from US Segment Harmony Node 2 and five from Russian Segment Zvezda module sites) all belonging to the Bacillus anthracis-B. cereus-B. thuringiensis group. Isolates were further characterized by whole genome comparative analysis. Each isolate was sequenced assembled and aligned against all members of the B. cereus sensu lato group. Based on genome size estimates (5.2 - 5.3 Mbp) strain nucleotide identity (>99.99%) and maximum likelihood phylogenetic placement all isolates were found to exhibit a very high level of similarity. With respect to gene content all isolates were inspected for cry proteins common to B. thuringiensis as well as toxins specific to B. cereus. No significant hits were found to any known cry genes while full-length matches were found for multiple B. cereus toxin genes. Finally the isolates were closely screened against B. anthracis genomes and no B. anthracis signatures were identified. Notably all isolates contained the plcR ancestral C allele and lacked significant hits to pXO1 and pXO2 plasmids and toxins. The collective results from the analysis confirm that these strains belong to a previously uncharacterized Bacillus species hereafter referred to as Bacillus issensis. The type strain is ISSFR-003T (=NRRL B-65389T= DSMZ 101676T).

Microbes interact with humans in complex ways and understanding how they respond to the spaceflight environment is important to the success of future manned spaceflight missions. The BRIC-23 mission was designed to measure the response of Bacillus subtilis and Staphylococcus aureus to the spaceflight environment. This experiment aimed to produce high quality omics data from B. subtilis and S. aureus grown aboard the International Space Station (ISS) to allow comparison to matched ground controls. There were two primary objectives for this experiment: (1) Demonstrate all post-flight processes and operations required for successful completion of GeneLab Reference Missions conducted on ISS and (2) Generate high quality GeneLab Reference Mission omics data sets for two prokaryotic model organisms Bacillus subtilis and Staphylococcus aureus. Freezing Control Experiment: The BRIC hardware has significant thermal inertia thus the freezing rate of samples placed at -80 C is quite slow. This could affect RNA-sequencing proteomic and metabolic data sets. In an effort to understand how slow freezing could affect these data sets a control experiment was designed in which B. subtilis and S. aureus were grown in petri plates and either slow frozen to -80 C at a rate matching the BRIC-23 spaceflight samples or processed immediately to harvest RNA and protein.

As part of an ongoing effort to catalogue microbial communities inhabiting the International Space Station (ISS) crew-associated environmental samples were collected from the Japanese Kibo Russian and US research modules. Initial analysis based on 16S rRNA gene sequencing identified 11 Bacillus isolates (two from Kibo Japanese Experiment Module (JEM) four from US Segment Harmony Node 2 and five from Russian Segment Zvezda module sites) all belonging to the Bacillus anthracis-B. cereus-B. thuringiensis group. Isolates were further characterized by whole genome comparative analysis. Each isolate was sequenced assembled and aligned against all members of the B. cereus sensu lato group. Based on genome size estimates (5.2 - 5.3 Mbp) strain nucleotide identity (>99.99%) and maximum likelihood phylogenetic placement all isolates were found to exhibit a very high level of similarity. With respect to gene content all isolates were inspected for cry proteins common to B. thuringiensis as well as toxins specific to B. cereus. No significant hits were found to any known cry genes while full-length matches were found for multiple B. cereus toxin genes. Finally the isolates were closely screened against B. anthracis genomes and no B. anthracis signatures were identified. Notably all isolates contained the plcR ancestral C allele and lacked significant hits to pXO1 and pXO2 plasmids and toxins. The collective results from the analysis confirm that these strains belong to a previously uncharacterized Bacillus species hereafter referred to as Bacillus issensis. The type strain is ISSFR-003T (=NRRL B-65389T= DSMZ 101676T).

Aspergillus fumigatus is a saprophytic filamentous fungus that is ubiquitous outdoors (soil decaying vegetation) and indoors (hospitals simulated closed habitats etc.). A. fumigatus can adapt to various environmental conditions and form airborne conidia that are the inoculum for a variety of diseases (e.g. non- and invasive pulmonary infections allergic bronchopulmonary aspergillosis etc.) in immunocompromised hosts. In an on-going Microbial Observatory Experiments on the International Space Station (ISS) molecular phylogeny of several fungal isolates were characterized. Two strains ISSF 21 and IF1SW-F4 were isolated from the HEPA filter and the surface of the Cupola of the ISS respectively. Using primers targeting the internal transcribed spacers ITS1 and 2 both isolates were identified as A. fumigatus. The whole genome sequence analysis of ISSF 21 revealed increased number of single nucleotide polymorphisms (SNPs) when compared to the reference A. fumigatus 293. Knowing that A. fumigatus is an opportunistic pathogen and microgravity highly influences the antibiotic susceptibility and pathogenicity of microorganisms we examined pathogenicity of both ISS isolates using the zebrafish larval model. The space station isolates (ISSF-021 and IF1SW-F4) were more virulent than two clinical strains (Af293 and CEA10) whose pathogenicity was highly characterized. Here the whole genome sequences of ISSF-021 strain are being deposited.

The environmental samples were collected with the polyester wipes from eight different locations in the International Space Station (ISS) during two consecutive sampling sessions (three months apart) within the ISS Microbial Observatory Experiment. DNA extracted from each of the samples was used to create amplicon libraries based on customized panel of 500 antimicrobial resistance genes followed by next-generation sequencing. This is the first study of that shows the reservoir of antimicrobial genes in the ISS. The International Space Station (ISS) as a closed built environment has its own environmental microbiome which is shaped by microgravity radiation and limited human presence. The microbial diversity associated with ISS environmental surfaces was investigated during this study. Polyester wipes and contact slides were used for sampling of eight various surface locations on the ISS at different time periods. The samples were retrieved and analyzed immediately upon the return to the Earth (via Soyuz TMA-14M or Dragon capsule from SpaceX). After surface sample collection contact slides containing nutrient media for the growth of bacteria and fungi were incubated at 25 xcb x9aC. The polyester wipes were processed to measure microbial burden (R2A Blood Agar and Potato Dextrose Agar) and recover cultivable bacteria as well as fungi. Subsequently viable microbial burden was assessed using Adenosine Triphosphate (ATP) assay and quantitative polymerase chain reaction (PCR) methods after propidium monoazide (PMA) treatment. The 16S-tag and metagenome analyses were used to elucidate viable microbial diversity. The cultivable bacterial population yield from the polyester wipes was very high (5 to 7-logs) when compared with the contact slides (102 to 103 CFU/m2). The PMA-qPCR analysis showed considerable variation of viable bacterial population (105 to 109 16S rDNA gene copies/m2) among locations sampled. Unlike contact slides polyester wipes cover much larger sample surface (~1 m2) and produce much more reliable results of the microbial diversity of the ISS covering both cultivable and non-cultivable species. The cultivable total and viable microbial diversity was determined utilizing state-of-the art molecular techniques. The implementation of the PMA assay before DNA extraction allowed distinguishing viable microorganisms which is crucial for determining their role to the crew health the ISS maintenance and the general knowledge of the closed environmentally controlled built systems.

Microbes interact with humans in complex ways and understanding how they respond to the spaceflight environment is important to the success of future manned spaceflight missions. The BRIC-23 mission was designed to measure the response of Bacillus subtilis and Staphylococcus aureus to the spaceflight environment. This experiment aimed to produce high quality omics data from B. subtilis and S. aureus grown aboard the International Space Station (ISS) to allow comparison to matched ground controls. There were two primary objectives for this experiment: (1) Demonstrate all post-flight processes and operations required for successful completion of GeneLab Reference Missions conducted on ISS and (2) Generate high quality GeneLab Reference Mission omics data sets for two prokaryotic model organisms Bacillus subtilis and Staphylococcus aureus. Freezing Control Experiment: The BRIC hardware has significant thermal inertia thus the freezing rate of samples placed at -80 C is quite slow. This could affect RNA-sequencing proteomic and metabolic data sets. In an effort to understand how slow freezing could affect these data sets a control experiment was designed in which B. subtilis and S. aureus were grown in petri plates and either slow frozen to -80 C at a rate matching the BRIC-23 spaceflight samples or processed immediately to harvest RNA and protein.