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

Abstract

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 (n = 789) collected from clinical samples at a veterinary teaching hospital in Spain between 2011 and 2020. We identified four high-level aminoglycoside-resistant ST171 Enterobacter hormaechei subsp. xiangfangensis isolates carrying the armA gene, all from horses. Using Illumina and Nanopore sequencing, we determined that armA was located on a 70 kb IncR plasmid, also carrying other resistance genes such as msr(E), mph(E), blaDHA-1, and qnrB4, embedded within a Tn1548-like element. Genomic comparisons indicated that the IncR plasmid was linked to a 2008–2010 ST11 Klebsiella pneumoniae outbreak in companion animals at the same hospital, with >95% sequence plasmid identity. Although the IncR plasmid lacked conjugative genes, it was mobilizable in vitro via co-resident conjugative plasmids. To probe for an environmental reservoir, we sampled three horse stalls in 2022 and recovered 19 armA-positive isolates (E. hormaechei, Klebsiella pneumoniae, and Mixta calida) 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 armA 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.