Characterization of Aeromonas hydrophila Wound Pathotypes by Comparative Genomic and Functional Analyses of Virulence Genes

Abstract

Aeromonas hydrophila has increasingly been implicated as a virulent and antibiotic-resistant etiologic agent in various human diseases. In a previously published case report, we described a subject with a polymicrobial wound infection that included a persistent and aggressive strain of A. hydrophila (E1), as well as a more antibiotic-resistant strain of A. hydrophila (E2). To better understand the differences between pathogenic and environmental strains of A. hydrophila, we conducted comparative genomic and functional analyses of virulence-associated genes of these two wound isolates (E1 and E2), the environmental type strain A. hydrophila ATCC 7966T, and four other isolates belonging to A. aquariorum, A. veronii, A. salmonicida, and A. caviae. Full-genome sequencing of strains E1 and E2 revealed extensive differences between the two and strain ATCC 7966T. The more persistent wound infection strain, E1, harbored coding sequences for a cytotoxic enterotoxin (Act), a type 3 secretion system (T3SS), flagella, hemolysins, and a homolog of exotoxin A found in Pseudomonas aeruginosa. Corresponding phenotypic analyses with A. hydrophila ATCC 7966T and SSU as reference strains demonstrated the functionality of these virulence genes, with strain E1 displaying enhanced swimming and swarming motility, lateral flagella on electron microscopy, the presence of T3SS effector AexU, and enhanced lethality in a mouse model of Aeromonas infection. By combining sequence-based analysis and functional assays, we characterized an A. hydrophila pathotype, exemplified by strain E1, that exhibited increased virulence in a mouse model of infection, likely because of encapsulation, enhanced motility, toxin secretion, and cellular toxicity.

Functional Genomic Characterization of Virulence Factors from Necrotizing Fasciitis-Causing Strains of Aeromonas hydrophila

“…For all draft genomes, genes at the end of a contig or interrupted by contig gaps were analyzed by using bidirectional BLASTN analysis against all other genomes. Genomic regions (GRs), defined as regions present in one (unique) or more genomes and missing from at least one other genome (dispensable), were identified as previously reported (42) for the genomes of A. hydrophila ATCC 7966…”

“…Luria-Bertani (LB) medium with 0.35% Difco Bacto agar (Difco Laboratories, Detroit, MI) was used to characterize swimming motility, while Difco nutrient broth with 0.5% Eiken agar (Eiken Chemical Co., Ltd., Tokyo, Japan) was used to measure the swarming motility of aeromonads (30,42,58). These methods provided better quantification of the motility data.…”

“…N-Acyl-homoserine lactone (AHL) production was detected by cross-streaking various Aeromonas strains against the biosensor Chromobacterium violaceum CV026 on LB agar plates (27,42). Violet pigment production from C. violaceum CV026 by AHLs produced by Aeromonas isolates was scored on the basis of color intensity after overnight incubation of the plates at 37°C.…”

“…Protease activity was measured in culture filtrates of Aeromonas isolates grown overnight (60). The hide powder azure substrate was used to measure protease activity (42). Protease activity was calculated per milliliter of culture filtrate per 10 8 CFU as determined by serial dilution of the samples and colony counting.…”

“…For the neutralization assay, culture filtrates of the strains studied were mixed with either preimmune (control) or hyperimmune rabbit serum (laboratory stock, 1:10 dilution) containing antibodies to Act (42,62) and incubated at 37°C for 1 h before use.…”