Genome analysis of predominant aeromonas species isolated from diseased freshwater fishes and their virulence in striped snakehead fish (Channa Striata)

Abstract

Aeromonas is an important bacterial pathogen in freshwater fishes. This study aimed to investigate the distribution and diversity of Aeromonas spp. including their antimicrobial resistance patterns. 86 Aeromonas isolates were collected from diseased fish in 13 fish farms around Thailand. Biochemical tests, MALDI-TOF MS, PCR assays, and the gyrB gene sequence analysis were used to identify all isolates. MALDI-TOF MS confirmed 100% (86 isolates) at the genus level and 88.4% (76 isolates) at the species level. Six Aeromonas species were confirmed based on sequences of gyrB gene, including A. veronii (72.1%), A. jandaei (11.6%), A. schubertii (9.3%), A. diversa (3.5%), A. hydrophila (2.3%), and A. caviae (1.2%). Antimicrobial susceptibility test for all Aeromonas isolates exhibited resistance against amoxicillin (99%), ampicillin (98%), oxolinic acid (81.4%), oxytetracycline (77%), trimethoprim-sulfamethoxazole (24%), and enrofloxacin (21%). Our findings indicated that A. veronii is the predominant species in cultured freshwater fishes in Thailand. The genetic diversity expressed by the gyrB gene exhibited A. veronii isolates from different fish species in the same cluster. It indicates that A. veronii, which has genetic closeness, can infect other fish species. Out of 62 A. veronii isolates, only 33 isolates showed more than one band, and the others were untypable when typing using ERIC-PCR. However, 33 ERIC profiles were observed, and the isolates were categorized into four clusters with 95% similarity. Experimental challenge evaluation using striped snakehead fish revealed that all A. veronii strains isolated from different fishes are susceptible (mortality ≥50%), and put A. veronii strains NBCF28 (walking catfish) is the most virulent strain with LD50 1.2 x105 CFU/ml. The genomic comparisons analysis among five A. veronii strains of distinct origins in Thailand revealed the main factors contributing to A. veronii virulence, including fimbriae, flagella, toxins, type II, type III, and type VI secretion systems, CRISPR-Cas system, and the number of prophages. These results confirm the genetic information of A. veronii strains that recently caused high mortality in cultured freshwater fishes.