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Background
The antimicrobial resistance (AMR) phenotypic properties, multiple drug resistance (MDR) gene profiles, and genes related to potential virulence and pathogenic properties of five Enterobacter bugandensis strains isolated from the International Space Station (ISS) were identified and compared with genomes of three clinical strains. Whole genome sequences of ISS strains were characterized using the hybrid de novo assembly of Nanopore and Illumina reads. In addition to traditional microbial taxonomic approaches, multilocus sequence typing (MLST) analysis was performed to classify the phylogenetic lineage. The agar diffusion disc assay was performed to test antibiotic susceptibility. The draft genomes after assembly and scaffolding were annotated with the Rapid Annotations using Subsystems Technology and RNAmmer servers for downstream analysis.

Results
Molecular phylogeny and whole genome analysis of the ISS strains with all publicly available Enterobacter genomes revealed that the ISS strains were E. bugandensis and similar to the type strain EB-247^T and two clinical isolates (153_ECLO and MBRL 1077). Comparative genomic analyses of all eight E. bungandensis strains showed that a total of 4733 genes were associated with carbohydrate metabolism (635 genes), amino acids and derivatives (496 genes), protein metabolism (291 genes), cofactors, vitamins, prosthetic groups, pigments (275 genes), membrane transport (247 genes), and RNA metabolism (239 genes). In addition, 112 genes identified in the ISS strains were involved in virulence, disease, and defense. Genes associated with resistance to antibiotics and toxic compounds, including the MDR tripartite system, were also identified in the ISS strains. A multiple antibiotic resistance (MAR) locus or MAR operon encoding MarA, MarB, MarC, and MarR, which regulates more than 60 genes, including upregulation of drug efflux systems that have been reported in Escherichia coli K12, was also observed in the ISS strains.

Conclusion
Given the MDR results for these ISS Enterobacter genomes and increased chance of pathogenicity (PathogenFinder algorithm with > 79% probability), these species pose important health considerations for future missions. Thorough genomic characterization of the strains isolated from ISS can help to understand the pathogenic potential, and inform future missions, but analyzing them in in vivo systems is required to discern the influence of microgravity on their pathogenicity.