Persistent multispecies dissemination of <i>armA</i> -carrying IncR plasmids among clinical and environmental bacterial populations in a Spanish veterinary hospital

Aminoglycoside resistance mediated by 16S rRNA methyltransferases poses a growing threat in both human and veterinary medicine. Here, we conducted a retrospective genomic study of Enterobacterales isolates (<i>n</i> = 789) collected from clinical samples at a veterinary teaching hospital in Spain between 2011 and 2020. We identified four high-level aminoglycoside-resistant ST171 <i>Enterobacter hormaechei</i> subsp. <i>xiangfangensis</i> isolates carrying the <i>armA</i> gene, all from horses. Using Illumina and Nanopore sequencing, we determined that <i>armA</i> was located on a 70 kb IncR plasmid, also carrying other resistance genes such as <i>msr(E</i>), <i>mph(E</i>), <i>bla</i>_DHA-1, and <i>qnrB4</i>, embedded within a Tn<i>1548</i>-like element. Genomic comparisons indicated that the IncR plasmid was linked to a 2008-2010 ST11 <i>Klebsiella pneumoniae</i> outbreak in companion animals at the same hospital, with >95% sequence plasmid identity. Although the IncR plasmid lacked conjugative genes, it was mobilizable <i>in vitro</i> via co-resident conjugative plasmids. To probe for an environmental reservoir, we sampled three horse stalls in 2022 and recovered 19 <i>armA</i>-positive isolates (<i>E. hormaechei</i>, <i>Klebsiella pneumoniae,</i> and <i>Mixta calida</i>) whose IncR plasmids were nearly identical to that found in clinical clones. Broader analysis of 1,330 IncR plasmids from the genomic plasmid database PLSDB revealed that most were mobilizable, frequently co-integrated with other replicons, and carried diverse resistance genes, though <i>armA</i> was uncommon. These findings demonstrate that non-conjugative IncR plasmids can persist in the environment and be horizontally disseminated to clinical isolates over a long period of time, underscoring the need for routine, plasmid-focused genomic surveillance in veterinary healthcare settings within a One Health framework.IMPORTANCEThe spread of antimicrobial resistance threatens both human and animal health. In veterinary hospitals, bacteria can share resistance genes not only through direct transmission but also via mobile plasmids that persist in the environment. In this study, we uncovered a decade-long persistence of a non-conjugative IncR plasmid carrying the <i>armA</i> gene, which confers high-level aminoglycoside resistance, in a Spanish veterinary teaching hospital. This plasmid was found in clinical isolates of <i>Enterobacter hormaechei</i>, <i>Klebsiella pneumoniae,</i> and <i>Mixta calida</i> from horses and from the hospital environment. Our findings show that even plasmids lacking self-transfer capability can be maintained and disseminated across bacterial species over many years. These results highlight the need for routine genomic surveillance of plasmids in veterinary healthcare settings to prevent long-term environmental reservoirs from fueling recurrent outbreaks.