Prevalence, Virulence Gene Distribution and Alarming the Multidrug Resistance of Aeromonas hydrophila Associated with Disease Outbreaks in Freshwater Aquaculture

Abstract

The study aims to evaluate the infection prevalence, virulence gene distribution and antimicrobial resistance of Aeromonas hydrophila associated in diseased outbreaks of cultured freshwater fish in Northern Vietnam. The confirmed A. hydrophila were screened for the presence of the five pitutative-virulence genes including aerolysin (aerA), hemolysin (hlyA), cytotonic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), and heat-stable enterotoxin (ast), and examined the susceptibility to 16 antibiotics. A total of 236 A. hydrophila isolates were recovered and confirmed from 506 diseased fish by phenotypic tests, PCR assays, and gyrB, rpoB sequenced analyses, corresponding to the infection prevalence at 46.4%. A total of 88.9% of A. hydrophila isolates harbored at least one of the tested virulence genes. The genes aerA and act were most frequently found (80.5% and 80.1%, respectively) while the ast gene was absent in all isolates. The resistance to oxacillin, amoxicillin and vancomycin exhibited the highest frequencies (>70%), followed by erythromycin, oxytetracycline, florfenicol, and sulfamethoxazole/trimethoprim (9.3-47.2%). The multiple antibiotic resistance (MAR) index ranged between 0.13-0.88 with 74.7% of the isolates having MAR values higher than 0.2. The results present a warning for aquaculture farmers and managers in preventing the spread of A. hydrophila and minimizing antibiotic resistance of this pathogen in fish farming systems.

Keywords: Aeromonas hydrophila; Vietnam; antimicrobial resistances; freshwater fish; infection prevalence; virulence genes.

Figure 1

Clinical symptoms and gross lesions of diseased fish infected with A. hydrophila. The clinical signs of heavy hemorrhage around the fish’s mouth, operculum, and fin bases, and fin erosion of tilapia (A1), grass carp (B1) and channel catfish (C1); and gross lesions of enlarged gall bladder and liver, enlarged and darkened spleen, hemorrhage and empty stomach and intestines in infected tilapia (A2), grass carp (B2), channel catfish (C2).

Figure 2

Amplified products of representative A. hydrophila isolates from tilapia (n = 5), carp (n = 4) and channel catfish (n = 4) of Aeromonas genus (953 bp)—upper ranges and A. hydrophila species (625 bp)—lower ranges: M: DNA ladder; lane 1–13 representative isolates from diseased fish; lane 14-negative control; lane 15-positive control (A. hydrophila ATCC 7966).

Figure 3

Phenotypic analysis based on gyrB sequences of the representative isolates recovered from the three fish hosts with those of other of Aeromonas species retrieved from Genbank using the neighbor-joining method. Bootstraps of 2000 replicates were performed.

Figure 4

Phenotypic analysis based on rpoB sequences of the representative isolates recovered from the three fish hosts with those of other of Aeromonas species retrieved from Genbank using the neighbor-joining method. Bootstraps of 2000 replicates were performed.

Figure 5

The detection frequencies of the A. hydrophila isolates from cultured tilapia, carp, and channel catfish carrying 0–4 virulence genes out of the 5 tested genes in the study.

Figure 6

PCR amplification of virulence genes carried in representative A. hydrophila isolates. M: DNA ladder; lane 1–7 representative amplified products of act gene (232 bp) (A); aerA gene (431 bp) (B); hlyA gene (597 bp) (C); and alt gene (442 bp) (D).

Figure 7

The distributions of multiple antibiotic resistances (MAR) of A. hydrophila isolates from cultured tilapia, carp, and channel catfish. None of the isolates was resistant to only 0 or 1 agent. The values 2/16 to 14/16 mean that the isolates showed resistance to 2 to 14 out of 16 antimicrobial agents with the corresponding MAR values.

Figure 8

A map showing the provincial sites (indicated by red color) from which diseased fish were collected in the study.