Aeromonas hydrophila ST251 and Aeromonas dhakensis are major emerging pathogens of striped catfish in Vietnam

Abstract

Introduction: Aeromonads are ubiquitous in aquatic environments and several species are opportunistic pathogens of fish. Disease losses caused by motile Aeromonas species, particularly Aeromonas hydrophila, can be challenging in intensive aquaculture, such as at striped catfish (Pangasianodon hypophthalmus) farms in Vietnam. Outbreaks require antibiotic treatments, but their application is undesirable due to risks posed by resistance. Vaccines are an attractive prophylactic and they must protect against the prevalent strains responsible for ongoing outbreaks.

Methods: This present study aimed to characterize A. hydrophila strains associated with mortalities in striped catfish culture in the Mekong Delta by a polyphasic genotyping approach, with a view to developing more effective vaccines.

Results: During 2013-2019, 345 presumptive Aeromonas spp. isolates were collected at farms in eight provinces. Repetitive element sequence-based PCR, multi-locus sequence typing and whole-genome sequencing revealed most of the suspected 202 A. hydrophila isolates to belong to ST656 (n = 151), which corresponds to the closely-related species Aeromonas dhakensis, with a lesser proportion belonging to ST251 (n = 51), a hypervirulent lineage (vAh) of A. hydrophila already causing concern in global aquaculture. The A. dhakensis ST656 and vAh ST251 isolates from outbreaks possessed unique gene sets compared to published A. dhakensis and vAh ST251 genomes, including antibiotic-resistance genes. The sharing of resistance determinants to sulphonamides (sul1) and trimethoprim (dfrA1) suggests similar selection pressures acting on A. dhakensis ST656 and vAh ST251 lineages. The earliest isolate (a vAh ST251 from 2013) lacked most resistance genes, suggesting relatively recent acquisition and selection, and this underscores the need to reduce antibiotics use where possible to prolong their effectiveness. A novel PCR assay was designed and validated to distinguish A. dhakensis and vAh ST251 strains.

Discussion: This present study highlights for the first time A. dhakensis, a zoonotic species that can cause fatal human infection, to be an emerging pathogen in aquaculture in Vietnam, with widespread distribution in recent outbreaks of motile Aeromonas septicaemia in striped catfish. It also confirms vAh ST251 to have been present in the Mekong Delta since at least 2013. Appropriate isolates of A. dhakensis and vAh should be included in vaccines to prevent outbreaks and reduce the threat posed by antibiotic resistance.

Keywords: Pangasianodon hypophthalmus; antibiotic resistance; antimicrobial resistance (AMR); aquaculture; comparative genomics; hypervirulent Aeromonas hydrophila (vAh); motile Aeromonas septicaemia; pangasius.

Figure 1

Multilocus phylogenetic analysis by sequence comparison of concatenated housekeeping genes at 10 loci for 87 representative field isolates of Aeromonas from striped catfish, five reference isolates, and 12 type strains. rep-PCR type, sequence type (ST), and alleles derived from ngsMLST analysis of 10 genes including 16S rRNA are color coded. *Gene sequences (when available) were retrieved from the genome assembly at NCBI.

Figure 2

Phylogenetic tree of 14 field isolates, one reference isolate, and 116 published A. hydrophila and A. dhakensis genomes. Four Aeromonas spp. genomes are included in the analysis as an outlier group. Tree is based on the pan-genome analysis. Average nucleotide identity (ANI) values were calculated compared to the three labeled type strains (A. dhakensis CECT 7289^T assembly GCF_000819705.1; A. dhakensis CIP 107500^T assembly GCF_000820305.1; and A. hydrophila subsp. hydrophila ATCC 7966^T assembly GCF_000014805.1). The origin (Ori) of each isolate and the presence/absence of 17 antibiotic resistance genes in the pan-genome analysis is provided. *The quinoline resistance gene was found on a plasmid and plasmid sequences were available only for the genomes sequenced in this present study. Full list of the abbreviations and detailed gene names is provided in Supplementary Table 5.

Figure 3

eBurst network based on multilocus sequence typing (MLST) patterns of publicly available A. hydrophila and A. dhakensis genomes. Circles denote a distinct MLST: small and unlabelled if undefined and unique; small and labeled if undefined but identified multiple times (“u”); and larger and labeled if present in the PubMLST database. Links show single allele variants. Sequence types (STs) containing field isolates are outlined in black and these correspond to A. hydrophila vAh ST251 and A. dhakensis ST656.

Figure 4

Distribution of protein-encoding genes in the pan-genome of A. hydrophila and A. dhakensis. Tree is based on pan-genome analysis, calculated by PIRATE using the accessory genes. Gray background indicates isolates sequenced de novo in this present study, with field isolates labeled “outbreak”. Shared protein-encoding genes constitute the core genome (95% presence; 2,517 genes). A. dhakensis has species-specific genes (698 genes) whilst the outbreak strains (ST656, from this present study) share a cluster of clone-specific genes (additional 769 genes). Similarly, A. hydrophila possesses 439 unique genes (95% presence), whilst the vAh ST251 strains exhibit a specific gene cluster distinct from all other specimens (additional 425 genes). A 4-way Venn diagram of the separate pan-genomes for A. dhakensis, A. dhakensis ST656, A. hydrophila and A. hydrophila vAh ST251 is available in Supplementary Figure 4.