Type IV ‐Dominated Plasmid‐Encoded CRISPR ‐Cas Systems Are Enriched in Multireplicon and Multi‐Stressor Resistance Plasmids Across the Global Plasmidome

Plasmids are central vectors of antimicrobial resistance (AMR). However, the features that distinguish the most epidemiologically successful resistance plasmids remain incompletely understood. CRISPR-Cas systems are classically regarded as barriers to horizontal gene transfer, which makes their occurrence within resistance plasmids yet unexplored at large scale. Here we analyzed 72,555 curated plasmid sequences from the Plasmid database (PLSDB) to test whether plasmid-encoded CRISPR-Cas systems are non-randomly associated with plasmid architecture and multi-stressor resistance across a large scale plasmidome. Using a three-tier classification of CRISPR-Cas system integrity based on array quality and Cas gene presence, we found that intact systems, defined by the co-occurrence of a high-confidence array and cas genes within the same replicon, were rare (1.03%) but strongly and specifically enriched in multireplicon plasmids (OR = 12.36) and in plasmids resistant to multiple stressor classes (MSR; OR = 6.25). These associations were robust to clonal redundancy: after dereplicating near-identical sequences over 100 iterations, both remained significant in every iteration, whereas the association with antimicrobial multidrug resistance (MDR) collapsed, identifying it as an artifact of clonal expansion. Intact systems were concentrated in high-risk IncF and IncH backbones and were more prevalent in plasmids from environmental sources than in those from clinical sources. Type IV-A was the predominant subtype (38.8% of intact systems), consistent with its near-exclusive association with mobile genetic elements and its proposed role in inter-plasmid competition rather than host defense. Together, these findings suggest that plasmid-encoded CRISPR-Cas systems, whether retaining defense-associated functions or acting as intra-MGE competition elements, are non-randomly retained in structurally complex, multi-stressor plasmid backbones that are particularly effective at accumulating and disseminating antimicrobial resistance.