Aeromonas hydrophila is a potential zoonotic pathogen and primary fish pathogen. With overlapping characteristics, multiple isolates are often mislabelled and misclassified. Moreover, the potential pathogenic factors among the publicly available genomes in A. hydrophila strains of different origins have not yet been investigated. To identify the valid strains of A. hydrophila and their pathogenic factors, we performed a pan-genomic study. It revealed that there were 13 mislabelled strains and 49 valid strains that were further verified by Average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) and in silico multiple locus strain typing (MLST). Multiple numbers of phages were detected among the strains and among them Aeromonas phi 018 was frequently present. The diversity in type III secretion system (T3SS) and conservation of type II and type VI secretion systems (T2SS and T6SS, respectively) among all the strains are important to study for designing future strategies. The most prevalent antibiotic resistances were found to be beta-lactamase, polymyxin and colistin resistances. The comparative analyses of sequence type (ST) 251 and other ST groups revealed that there were higher numbers of virulence factors in ST-251 than in other STs group. Publicly available genomes have 13 mislabelled organisms, and there are only 49 valid A. hydrophila strains. This valid pan-genome identifies multiple prophages that can be further utilized. Different A. hydrophila strains harbour multiple virulence factors and antibiotic resistance genes. Identification of such factors is important for designing future treatment regimes.

NorV has been known to be an anaerobic nitric oxide reductase associated with nitric oxide (NO) detoxification. Recently, we showed that the norV gene of Aeromonas hydrophila was highly upregulated after co-culturing with Tetrahymena thermophila. Here, we demonstrated that the transcription and expression levels of norV were upregulated in a dose-dependent manner after exposure to NO under aerobic and anaerobic conditions. To investigate the roles of norV in resisting predatory protists and virulence of A. hydrophila, we constructed the norV gene-deletion mutant (ΔnorV). Compared to the wild type, the ΔnorV mutant showed no significant difference in growth at various NO concentrations under aerobic conditions but significantly stronger NO-mediated growth inhibition under anaerobic conditions. The deletion of norV exhibited markedly decreased cytotoxicity, hemolytic and protease activities under aerobic and anaerobic conditions. Also, the hemolysin co-regulated protein (Hcp) in the ΔnorV mutant showed increased secretion under aerobic conditions but decreased secretion under anaerobic conditions as compared to the wild-type. Moreover, the inactivation of norV led to reduced resistance to predation by T. thermophila, decreased survival within macrophages and highly attenuated virulence in zebrafish. Our data indicate a diverse role for norV in the expression of A. hydrophila virulence-associated traits that is not completely dependent on its function as a nitric oxide reductase. This study provides insights into an unexplored area of NorV, which will contribute to our understanding of bacterial pathogenesis and the development of new control strategies for A. hydrophila infection.

Pasteurella multocida (Pm) is a zoonotic pathogen that causes severe hemorrhagic pneumonia in pigs, characterized by vascular injury and systemic inflammatory responses. Our previous studies demonstrated that 18β-glycyrrhetinic acid (GA), a bioactive triterpenoid derived from liquorice, protected against Pm-induced vascular inflammatory injury through suppression of poly(ADP-ribose) polymerase 1 (PARP1)-mediated nuclear factor-kappa B (NF-κB) and high mobility group box 1 (HMGB1) signaling. However, whether GA modulates PARP1-mediated NF-κB p65 nuclear translocation, a critical step in NF-κB activation, remains unclear. This study evaluated the protective effects of GA and its underlying mechanism using a Pm-infected mouse model and immunofluorescence staining of porcine iliac artery endothelial cells. Our results demonstrated that GA treatment attenuated body weight loss, ameliorated hematological and biochemical parameters, and reduced vascular structural damage in Pm-infected mice. Mechanistically, Pm infection induced PARP1 upregulation, accompanied by phospho-p65 (p-p65) nuclear translocation, all of which were suppressed by GA. PARP1 overexpression drove p-p65 nuclear accumulation, which was reversed by GA, whereas PARP1 knockdown suppressed Pm-induced p-p65 nuclear translocation. The findings demonstrated that GA protected against Pm-induced vascular injury by inhibiting PARP1-mediated NF-κB p65 nuclear translocation, supporting its potential as a candidate therapeutic agent for Pm-associated vascular injury.