Virulence and Antibiotic Resistance of Pathogenic Aeromonas caviae from Diseased Macrobrachium rosenbergii

Abstract

In recent years, viral and bacterial diseases have posed serious challenges to the sustainable development of Macrobrachium rosenbergii (giant freshwater prawn) aquaculture, resulting in considerable economic losses across China. Among the bacterial pathogens, Aeromonas caviae has emerged as a notable opportunistic agent capable of causing large-scale mortality in various aquatic species. In this study, a highly virulent strain of A. caviae (designated GMRS4) was isolated from diseased M. rosenbergii exhibiting mass mortality in Yangzhou, Jiangsu Province. The isolate, a Gram-negative bacillus, was identified as the causative agent based on morphological, molecular, and histopathological analyses. Pathogenicity was confirmed through experimental infection, with the strain displaying marked virulence, evidenced by an LD₅₀ of 1.91 × 10⁶ CFU/mL at 96 h. Whole-genome sequencing of GMRS4 revealed 4078 coding sequences, including a suite of virulence-associated genes encoding extracellular enzymes (DNase, hemolysin, caseinase, and lecithinase) and toxins (serine protease, elastase, and flagellin). Antimicrobial susceptibility testing indicated resistance to several antibiotics, particularly those in the penicillin and sulfonamide classes, while maintaining sensitivity to quinolones. Genomic analysis further revealed multiple antibiotic resistance genes and virulence genes, offering insights into the pathogenic mechanisms and resistance profile of the strain. This study underscores the threat posed by A. caviae to freshwater prawn aquaculture and provides a genetic basis for developing targeted disease management strategies.

Keywords: Aeromonas caviae; Macrobrachium rosenbergii; complete genome; pathogenesis.

Figure 1

Electron micrograph of GMRS4, scale bar = 0.5 μm. The bacterium appears rod-shaped with blunt, rounded ends and a single polar flagellum. Cell dimension range: length, 1.4–1.8 μm; width, 0.7–1.1 μm.

Figure 2

Histological changes in the Hepatopancreas (A,B), gills (C,D), and intestines (E,F) of M. rosenbergii infected with isolate GMRS4. GS represents disrupted glandular structure; IS represents enlarged intratubular spaces; IC represents inflammatory cells; FS represents reduced fold structures; ML represents ruptured muscle layer.

Figure 3

Phylogenetic tree of Aeromonas species based on 16S rRNA sequences. Bootstrap values are shown beside the clades. The names of bacteria are indicated beside the accession numbers.

Figure 4

Survival rates of M. rosenbergii challenged by GMRS4 at concentrations of 2.1 × 10⁸, 2.1 × 10⁷, 2.1 × 10⁶, 2.1 × 10⁵, and 2.1 × 10⁴ CFU/mL. PBS was set as the control group. Each group included three parallel replicates.

Figure 5

Virulence genes of A. caviae GMRS4 via PCR amplification. M, marker; 1, ahp; 2, ahyB; 3, flgM; 4, hly; 5, alt; 6, aer.

Figure 6

Extracellular enzyme test results of A. caviae GMRS4. (a) DNase activity; (b) hemolysin activity; (c) caseinase activity; (d) lecithinase activity; (e) amylase activity.

Figure 7

Circular genome map (A) and plasmid map (B) of A. caviae GMRS4. There are coding genes, COG databases, genome size, GC content, and the distribution of GC-skew value from outside to inside.

Figure 8

Clusters of Orthologous Gene (COG) functional annotation of the whole genome of A. caviae GMRS4. A total of 3951 genes has a COG classification among the 24 categories. The x-axis indicates the COG categories, and the y-axis indicates the number of genes.

Figure 9

Gene Oncology (GO) functional annotation of the whole genome of A. caviae GMRS4. All annotated genes were grouped into 3 functional subcategories: biological process, cellular component, and molecular function.

Figure 10

Gene distribution based on Kyoto Encyclopedia of Genes and Genomes (KEGG) classification of A. caviae GMRS4. All annotated genes were grouped into 5 functional subcategories: cellular process, environmental information processing, genetic information processing, metabolism, and organismal systems.