Co-dissemination of anti-phage defence systems and multidrug resistance is associated with the seventh-pandemic Vibrio cholerae defensome

Anti-phage defence systems in Vibrio cholerae are recognized as cargo on mobile genetic elements carrying antimicrobial resistance (AMR) and virulence genes, but a population-scale view of how the V. cholerae defensome partitions between the pandemic O1 clade and the environmental non-O1 reservoir has been missing. We analysed 1,740 RefSeq V. cholerae genomes (773 O1, 967 non-O1) sampled from 1934 to 2022 across 60 countries. Cataloguing defence systems with DefenseFinder, we found that the non-O1 reservoir harboured 283 subtypes and 55 families absent from any O1. This asymmetry persisted after collapsing each core-genome lineage to a single representative, although it partly reflected the greater diversity of non-O1. O1 favoured abortive infection systems (36.8% vs 14.1% in non-O1; P <0.001). Defence and prophage burden were inversely related within O1 (R=−0.15) but weakly positive in non-O1, and a five-decade reconstruction revealed mirror trajectories of rising defence diversity and declining prophage burden in O1, coincident with SXT/R391 integrative conjugative element-borne AMR. A bipartite network identified strong defence–AMR associations (Phi up to 0.80), nominating AbiE, AbiJ, AvcID, Menshen, Retron-I_A and Lamassu-Fam as candidate co-acquired cargo; though much of this is clonal, the strongest associations falling to 0.51 to 0.68 after de-duplication. Uniform Manifold Approximation and Projection clustering resolved seven defensome lineages, and a Random Forest classifier trained on defence profile predicted multidrug resistance status with cross-validated area under the receiver operating characteristic curve (AUC) of 0.96, generalizing across O1 (0.92), non-O1 (0.93), decades (0.88) and regions (AUC=0.92); a five-system signature (AbiE, BREX-I, CBASS-II, AvcID and AbiJ) achieved AUC=0.91. Together, these data show that the pandemic defensome has expanded in step with the AMR-bearing mobilome rather than with prophage burden, with defence and resistance genes co-inherited on seventh pandemic El Tor elements. Their association reflects shared lineage history rather than a causal or mechanistic link, and the defensome serves as a descriptive genomic correlate of multidrug resistance.