Unraveling the virulence and host immune responses to Enterococcus gallinarum during a mass mortality event in bighead carp (Hypophthalmichthys nobilis)

The pathogenicity of Enterococcus gallinarum in cultured fish is poorly characterized and emerging opportunistic bacteria pose an escalating threat to sustainable freshwater aquaculture. This study provides the first comprehensive phenotypic, histopathological and targeted candidate-gene expression analysis of E. gallinarum as an emerging multidrug-resistant (MDR) pathogen of bighead carp ( Hypophthalmichthys nobilis ) associated with a mass mortality event (~50% cumulative loss) in a sewage-fed system of the East Kolkata Wetlands, India. The disease was characterized by deep ulcerative lesions and cutaneous hemorrhage indicative of systemic infection. The clinical relevance of the dominant isolate is confirmed by robust identification using biochemical assays, 16S rRNA gene sequencing and phylogenetic analysis. Functional assays showed significant virulence, including biofilm formation greater than that of the Aeromonas hydrophila positive control, high swimming motility, and strong β-hemolysis. The in vivo pathogenic potential of the isolate was supported by 100% mortality following experimental challenge and recovery of E. gallinarum from internal tissues of challenged fish. The resistance to seven of 26 antimicrobials, including frontline drugs such as tetracyclines, macrolides, ansamycins, phenicols and carbapenems, and the high multiple antibiotic resistance (MAR) index of 0.34, suggest a significant therapeutic constraint and that antibiotics have been under constant selection pressure in the source environment. Histopathological examination showed widespread multi-organ injury, including hepatocellular necrosis, renal tubular necrosis, myofibrillar fragmentation, and splenic lymphoid depletion with melanomacrophage aggregates. Transcriptional profiling of brain, liver, kidney and muscle revealed a high but uncoordinated host response: NF-κB was uniformly up-regulated, MHC II was consistently down-regulated, TLR4 and chemokine receptor transcripts were down-regulated in all tissues except muscle, the most responsive tissue, and Hsp70, C3, IgM and NOD1 were regulated in a tissue-specific manner, suggesting that E. gallinarum not only uses host tissues but also interferes with pathogen recognition and antigen presentation. Taken together, these results outline the multifactorial nature of E. gallinarum virulence and MDR, highlighting its importance as an emerging pathogen in sewage-fed aquaculture. Future studies should focus on genomic dissection of its resistance and virulence determinants, longitudinal surveillance to understand transmission dynamics and the spread of AMR across the wetland aquaculture interface, and experimental testing of alternative mitigation strategies such as probiotics, phage therapy, and immune-modulating interventions to decrease the need for antibiotic use and support antimicrobial stewardship in sewage-fed aquaculture systems.