Distribution of the type VI secretion systems and the plasmid encoding pirAB genes in Vibrio parahaemolyticus and Vibrio campbellii

Acute Hepatopancreatic Necrosis Disease (AHPND) is one of the most devastating bacterial diseases affecting shrimp aquaculture, causing severe and sustained economic losses worldwide. AHPND is caused by the binary toxin PirAB, encoded on a ~ 70-kb plasmid. However, potential contributions of other bacterial factors to AHPND pathogenesis remain poorly understood. The Type VI Secretion System (T6SS), a conserved bacterial nanomachine involved in interbacterial competition and host virulence, has previously been reported to occur exclusively in AHPND-causing strains of Vibrio parahaemolyticus . We investigated this proposed association using newly sequenced V. parahaemolyticus PH1339 and PH1273 and V. campbellii PH1401 and PH1409, which were isolated from Philippine shrimp farms with documented mortality outbreaks, together with 807 publicly available genomes. Whole-genome analysis revealed multiple T6SS gene clusters - T6SS1, T6SS2, and T6SS3 among the newly sequenced strains. T6SS1 was present in the AHPND-causing strains PH1339 and PH1401 and in the non-AHPND strain PH1409. T6SS2 was identified in all four strains, whereas T6SS3 was detected exclusively in V. campbellii . Genome analysis showed that all pirAB ⁺ strains encoded T6SS1 and T6SS2, while pirAB ⁺ V. campbellii strains also encoded T6SS3. However, phylogeny-aware co-occurrence analysis detected no significant associations between pirAB and T6SS gene clusters, suggesting that their observed co-occurrence reflects lineage dependence rather than a direct functional association. These findings clarify the distribution of T6SS gene clusters among AHPND-associated Vibrio lineages and provides a genomic framework for further investigation of their potential roles in AHPND pathogenesis.