The proposed study includes flight components to:

1) Characterize the frequency and genomic identity of antibiotic resistant organisms, including enterococci, on the ISS;

2) Assess the evolutionary selective pressure of the space environment (microgravity, space radiation) using EF as a model system;

3) Characterize the “natural” evolutionary history of EF on Earth and in space to reveal mechanisms of microbial adaption including natural selection.

The CS-05A: Genomic Enumeration of Antibiotic Resistance in Space (GEARS) payload is designed to fulfill specific aim 1, the characterization of the frequency and genomic identity of antibiotic resistant organisms on the ISS. The Co-Principal Investigators propose to carry out longitudinal sampling of ISS surfaces in a repeated measures design.

The CS-05B: Enterococcus Growth Advantage on ISS via Tn-seq (EnteroGAIT) payload is designed to fulfill specific aim 2: to assess the evolutionary selective pressure of the space environment. The flight experiment will utilize on-board long-duration microbial growth to measure the selective pressure of the space environment on a defined microbial population: Enterococcus faecalis mutants are created by transposon insertional mutagenesis; selection is measured by sequencing (Tn-Seq) and occurs on timescales far shorter than natural or experimental evolution.

The third study, Adaptation & Evolution of Resilient Enterococcus in Space (AERES) will generate complete whole genomes of Enterococcus isolates from the ISS and ground, combined with follow up characterization, to seek evidence of persistence and/or evolution in the space environment.

Enterococcus Growth Advantage on ISS via Tn-seq * (EnteroGAIT) Study: SVT work was carried out using E. faecalis OG1RF (BSL-1 strain). First, filter tests were carried out, resulting in selection of the media exchange filter of the two-chamber BioCell, under development by BioServe. The BioCells use an insert to distribute hydrostatic or hydrodynamic loads across the filter, and also to permit flexing of the outer window during loading/unloading of media or sampling of cells. The biocompatibility of the insert material was assessed using growth curves and coupons of material; this verified that either material can be used in contact with E. faecalis cells. Initial growth tests verified growth of E. faecalis in the hardware, in support of assessing validity of the nominal 8 hour post-media-addition sampling timepoint. Additional testing is planned to confirm this timepoint and to carry out longer-duration testing with the goal of retiring risk related to biofouling during long-duration culturing. Implementation Partner BioServe has already completed some long-duration culturing (e.g., nominal 8 hour growth period followed by 14 days of no new media, followed by new media and another growth and stasis period). As of early September 2023, it is anticipated that the current BioCell hardware is representative of flight and will soon be subjected to additional SVT and Experiment Verification Testing (EVT). [See future work, below.]

Adaptation & Evolution of Resilient Enterococcus in Space (AERES) Study: Oxford Nanopore Technologies (ONT) has recently developed a new kit (Kit 14) with higher accuracy. We tested this kit as part of GEARS SVT work. Based on these results, it seems likely we can simplify our AERES genome assembly work to perform nanopore only genome assemblies, instead of hybrid genome assemblies using Illumina and nanopore data.

Based on the current nominal requested flight schedule, a no cost extension request is underway.

The proposed study includes flight components to:

1) Characterize the frequency and genomic identity of antibiotic resistant organisms, including enterococci, on the ISS;

2) Assess the evolutionary selective pressure of the space environment (microgravity, space radiation) using EF as a model system;

3) Characterize the “natural” evolutionary history of EF on Earth and in space to reveal mechanisms of microbial adaption including natural selection.

The CS-05A: Genomic Enumeration of Antibiotic Resistance in Space (GEARS) payload is designed to fulfill specific aim 1, the characterization of the frequency and genomic identity of antibiotic resistant organisms on the ISS. The Co-Principal Investigators propose to carry out longitudinal sampling of ISS surfaces in a repeated measures design.

The CS-05B: Enterococcus Growth Advantage on ISS via Tn-seq (EnteroGAIT) payload is designed to fulfill specific aim 2: to assess the evolutionary selective pressure of the space environment. The flight experiment will utilize on-board long-duration microbial growth to measure the selective pressure of the space environment on a defined microbial population: Enterococcus faecalis mutants are created by transposon insertional mutagenesis; selection is measured by sequencing (Tn-Seq) and occurs on timescales far shorter than natural or experimental evolution.

The third study, Adaptation & Evolution of Resilient Enterococcus in Space (AERES) will generate complete whole genomes of Enterococcus isolates from the ISS and ground, combined with follow up characterization, to seek evidence of persistence and/or evolution in the space environment.

The Science Requirements Document (SRD) for CS05A was completed and signed in December 2021. The Principal Investigator's (PI’s) lab was completed in April-May 2022, and the lab was moved in preparation for Science Verification Test (SVT) and Experiment Verification Test (EVT) studies. Co-PI Wallace’s lab continued optimization of key aspects in the sample collection and preparation process. SVT studies were initiated, including an antibiotic stability and shelf-life assessment assay. Recently, the compliance review package for GEARS was developed in concert with the Mission Scientist (MS), and nominal flight candidates were identified.

Enterococcus Growth Advantage on ISS via Tn-seq (EnteroGAIT) progress:

The subaward to Massachusetts Eye and Ear (Co-PI Gilmore) was initiated (initially delayed to preserve funds), the SRD was signed/approved, and BioServe was selected as the payload developer (PD) for the EnteroGAIT/CS05B hardware. A kickoff meeting was held in late July 2022. Mass estimates for the payload were made by the PD. Total crew time was estimated. The compliance review package is currently in development in cooperation with the MS. Nominal flight candidates have been identified. Initial SVT testing is underway and will include selection of the membrane material and pore size required to retain cells during media exchange, biocompatibility and growth tests, protocol verification, media stability, and optimization of sample storage, prior to a full EVT.

Adaptation and Evolution of Resilient Enterococcus in Space (AERES) progress:

This sub-project was delayed until after the PI moved to his permanent lab (May 2022). Initiation is now underway and will include nanopore sequencing of isolates from multiple sources in combination with analysis of whole genomes now available via the National Center for Biotechnology Information (NCBI). Near-term evaluation of new higher-accuracy sequencing kits (ONT Kit 14) will be performed to evaluate the potential for nanopore-only genome assembly.

We submitted an abstract to the American Society for Gravitational and Space Research (ASGSR) Annual Meeting, scheduled for November, 2022, to share progress on SVT work for GEARS.

The proposed study includes flight components to:

1) Characterize the frequency and genomic identity of antibiotic resistant organisms, including enterococci, on the ISS;

2) Assess the evolutionary selective pressure of the space environment (microgravity, space radiation) using EF as a model system;

3) Characterize the “natural” evolutionary history of EF on Earth and in space to reveal mechanisms of microbial adaption including natural selection.

The CS-05A: Genomic Enumeration of Antibiotic Resistance in Space (GEARS) payload is designed to fulfill specific aim 1, the characterization of the frequency and genomic identity of antibiotic resistant organisms on the ISS. The Co-Principal Investigators propose to carry out longitudinal sampling of ISS surfaces in a repeated measures design.

The CS-05B: Enterococcus Growth Advantage on ISS via Tn-seq (EnteroGAIT) payload is designed to fulfill specific aim 2: to assess the evolutionary selective pressure of the space environment. The flight experiment will utilize on-board long-duration microbial growth to measure the selective pressure of the space environment on a defined microbial population: Enterococcus faecalis mutants are created by transposon insertional mutagenesis; selection is measured by sequencing (Tn-Seq) and occurs on timescales far shorter than natural or experimental evolution.

The third study, Adaptation & Evolution of Resilient Enterococcus in Space (AERES) will generate complete whole genomes of Enterococcus isolates from the ISS and ground, combined with follow up characterization, to seek evidence of persistence and/or evolution in the space environment.

A formal project kickoff was conducted on March 1, 2021, involving a NASA-PI interchange meeting with a seminar given by PI Carr. Carr is a new faculty member at the Georgia Institute of Technology (Georgia Tech) and so initial project activities involved lab set up for microbiology, including incubator in biosafety cabinet (for later BSL-2 Enterococcus cultures), smoke testing, certification, PPE (personal protective equipment) provisioning. Jordan McKaig, a graduate student in the Georgia Tech School of Earth and Atmospheric Sciences, joined the project. We also re-established our nanopore sequencing pipeline in the new lab (hardware, software, reagents, personnel, training). Nanopore sequencing is used in GEARS and Adaptation & Evolution of Resilient Enterococcus in Space (AERES) to obtain complete whole genome information from colonies and isolates, respectively.

In support of the GEARS project, initial testing of the protocol to be used for on-orbit culturing and sequencing was evaluated in the lab of GEARS Co-PI Sarah Wallace (NASA Johnson Space Center). This initial data was analyzed and used to assemble two microbial genomes, simulating one potential outcome of the GEARS on-orbit culturing and sequencing experiments.

We submitted an abstract to the American Society for Gravitational and Space Research (ASGSR) Annual Meeting, scheduled for November 3-6, 2021, to share details about these investigations including our progress to date.

Note that the supporting work behind this publication was completed during Postdoc Noelle Bryan’s Space Biology Fellowship and provides important background for the project: Bryan NC, Lebreton F, Gilmore M, Ruvkun G, Zuber MT, Carr CE. Genomic and Functional Characterization of Enterococcus faecalis Isolates Recovered From the International Space Station and Their Potential for Pathogenicity. Front Microbiol. 2021 Jan 11;11:515319. https://doi.org/10.3389/fmicb.2020.515319 ; PMID: 33505359

Specific Aims: Here we propose a three-year Principal Investigator (PI) team research program, including flight components, to: 1) Characterize the frequency and genomic identity of antibiotic resistant organisms, including enterococci, on the ISS; 2) Assess the evolutionary selective pressure of the space environment (microgravity, space radiation) using EF as a model system; and 3) Characterize the natural evolutionary history of EF on Earth and in space to reveal mechanisms of microbial adaptation including natural selection, and refine crew health risk assessment and countermeasures for future space missions. Each aim is addressed by a PI-led investigation.

Genomic Enumeration of Antibiotic Resistance in Space (GEARS): Led by Co-PIs Burton and Wallace (NASA), this flight experiment will leverage existing ISS facilities to enable detection, species-level identification (via nanopore sequencing), and isolate selection (for Earth return and characterization), complementing ongoing routine microbial monitoring by focusing on the clinically-relevant subset of antibiotic resistant microbes.

Enterococcus Growth Advantage on ISS via Tn-seq (EnteroGAIT): Led by Co-PI Gilmore (Massachusetts Eye And Ear Infirmary, MEEI), this flight experiment will utilize the Bioculture System, currently undergoing validation on the ISS, to measure the selective pressure of the space environment on a defined microbial population: a library of EF mutants, created by transposon insertional mutagenesis, will undergo several rounds of growth and starvation on ISS with a synchronous ground control; selection is measured for each gene knockout by sequencing (Tn-Seq) and occurs on timescales far shorter than natural or experimental evolution.

Adaptation and Evolution of Resilient Enterococcus in Space (AERES): Led by PI Carr (Georgia Tech), this ground-based data and experiment-driven study will use an extensive collection of existing (>500) and newly generated (~100) whole genomes of Earth and space-derived enterococci isolates to characterize horizontal gene transfer, mutation, and implications for community-acquired infection in space and on Earth. Genetic techniques will be used to establish causality.

Significance and Relevance: The proposed work will improve our fundamental understanding of microbial adaptation to the space environment and crew health risks. Our work directly addresses the solicitation research foci A.3.1.a "a. Conduct long-term, multigenerational studies of microbes using the ISS or other appropriate flight platforms as a Microbial Observatory to study and understand the population and community dynamics of the microbes that inhabit that unique environment" (primary) and A.3.1.b "b. Determine the influence of spaceflight on defined microbial populations and communities. Space Biology studies will determine the effects of spaceflight on dynamics of microbes in mono or mixed cultures with respect to cell processes (including virulence and antibiotic resistance, evolution, biofilm formation, and community development)" (secondary) and the 2011 Decadal Survey recommendation to use the ISS as a Microbial Observatory (P1). Furthermore, our work addresses multiple Human Research Roadmap (HRR) gaps (MICRO-1 to MICRO-5 and Med01).