In vivo transcriptomics analyzing Aeromonas dhakensis from fatal human cases to identify new virulence factors

Among the human-associated Aeromonas species, A. dhakensis is increasingly implicated in systemic infections and high rates of antimicrobial resistance. Two strains of A. dhakensis were recently isolated from separate clinical cases of fatal, monomicrobial bacteremia and we aimed to define the genes and pathways utilized by these isolates during infection. We performed comparative RNA sequencing of each isolate, alongside a third clinical A. dhakensis reference strain, grown in either a murine peritoneal cavity (in vivo), or in stagnant growth medium (in vitro) with in vitro expression as the baseline. Three genes upregulated in vivo with no previous association with A. dhakensis pathogenicity were selected for further evaluation–hdc, inhA, and asap1. Each were in-frame deleted and cis-complemented, and virulence attenuation was measured in vitro and in vivo. Here we show majority of previously characterized A. dhakensis virulence factor-encoding genes are upregulated in vivo including genes coding for iron acquisition, various secretion systems, exotoxin A, and the lateral flagella. Global metabolic shifts in A. dhakensis are also characterized, demonstrating adaptation to the nutrient-limiting host environment. Deletion of hdc and inhA genes significantly attenuates bacterial virulence both in vitro and in vivo, while asap1 gene deletion has no impact despite a previously established contribution to virulence in fish infection with A. salmonicida. Overall, we demonstrate the utility of this RNA-seq dataset in understanding the expression patterns of A. dhakensis during infection and in identifying novel virulence strategies and mechanisms employed by this pathogen to cause human disease(s). The bacterium Aeromonas dhakensis, originally a fish pathogen, can also cause serious disease in humans. Here, we compare the expression of bacterial genes during infection in mice to growth in laboratory medium. This allowed us to identify strategies the bacteria use to overcome host immunity and cause disease. Three of these genes activated during growth in mice were individually removed from Aeromonas dhakensis, and 2 were found important for causing infection, while the third was not. These findings improve our understanding of how A. dhakensis causes systemic infections and helps identify new bacterial targets that could be explored in future research to develop better therapies for difficult-to-treat infections. Neil et al. identify in vivo differentially expressed genes by comparative transcriptomics, and evaluate 3 by mutagenesis and phenotypic analysis. The results show widespread activation of known virulence pathways extensive metabolic reprogramming, and 2 upregulated genes are demonstrated as previously unrecognized contributors to virulence.