Plasmidome, resistome, and virulence-associated gene characterization of <i>Acinetobacter johnsonii</i> in NASA cleanrooms and a clinical setting

Evidence suggests the persistence of non-spore-forming <i>Acinetobacter johnsonii</i> in high-stakes controlled and nutrient-limited environments. Here, we investigated the mechanisms underlying this adaptability through a comprehensive genomic analysis of 22 isolates of <i>A. johnsonii</i> from NASA's Payload Hazardous Servicing Facility (PHSF) and one carbapenem-resistant strain (E154408A) from patient colonization in Ireland. Core-genome phylogeny revealed clustering of PHSF-originating isolates in a monophyletic clade divergent from the main species lineage. Species-wide virulence-associated genes and metabolic reconstruction indicated the exclusive presence in PHSF-originating isolates of two complete efflux pumps and a conserved allantoin racemase, suggesting adaptability for multiple environmental stresses. The ubiquity of <i>bla</i>_OXA in genomes analyzed (<i>n</i> = 112) and the phenotypically validated multidrug-resistant profile of the E154408A strain highlight <i>A. johnsonii</i>'s potential as an antimicrobial resistance (AMR) reservoir. Plasmidome analysis suggested gain/loss events across the monophyletic population and potential AMR acquisition pathways. Genome-to-metagenome mapping identified genomic signatures of <i>A. johnsonii</i> in PHSF >10 years post-initial isolation.IMPORTANCE<i>Acinetobacter johnsonii</i> is increasingly recognized as an emerging human pathogen, with growing evidence of its ability to persist in controlled, high-stakes environments, posing risks as both a persistent environmental contaminant and an antimicrobial resistance (AMR) reservoir. Yet, gaps remain in our understanding of its AMR profile and the mechanisms that enable its enhanced environmental adaptability. This knowledge is necessary in contexts where biological cleanliness is a priority, such as clinical settings and spacecraft assembly facilities' cleanrooms, where contamination of hardware with terrestrial microorganisms is concerning. In this study, we aim to address some of the key knowledge gaps by providing genomic insights into a rare multidrug-resistant clinical isolate and 22 NASA cleanroom isolates that persisted for over a decade in extremely clean conditions. Our findings will help assess the contamination risk of <i>A. johnsonii</i> in high-stakes environments and ultimately strengthen our ability to manage this microbial contaminant across terrestrial and extraterrestrial settings.Cleanroom-derived <i>A. johnsonii</i> genomes show traits consistent with increased adaptability.Genomic signatures of <i>A. johnsonii</i> persisted in the cleanrooms for over 10 years.<i>bla</i>_OXA is ubiquitously found in all 112 <i>A. johnsonii</i> genomes analyzed.Isolate E154408A is the first reported patient colonization case by carbapenem-resistant <i>A. johnsonii</i> in Europe.