Screening and Characterization of a Probiotic Fish-Derived Enterococcus faecium and Its Co-Fermentation with...

Under intensive aquaculture practices, the frequent occurrence of diseases exacerbates the risk of antibiotic overuse. Therefore, screening safe and efficacious probiotics originating from the host fish themselves represents one of the key strategies to address this issue. In this study, lactic acid bacteria were isolated from the intestinal contents of healthy crucian carp and identified by morphological, physiological, and biochemical characterization, as well as 16SrDNA sequencing. Their probiotic potential was comprehensively evaluated by antimicrobial assays, in vitro tolerance assays, and auto-aggregation assays, among others. Furthermore, their biosafety was assessed via antimicrobial susceptibility testing, hemolysis tests, and PCR detection of virulence and transferable antibiotic resistance genes. Based on the immunoenhancing effects of Acanthopanax senticosus extract identified in previous studies, the selected strain was co-fermented with this extract. Subsequently, the effects of the composite preparation on growth, immunity, and disease resistance of crucian carp were evaluated through a 28-day feeding trial and a challenge test with Aeromonas hydrophila. From the 130 isolated strains, Strain No.19 was finally selected and identified as Enterococcus faecium. It exhibited inhibition zone diameters of ≥10 mm against all four tested pathogenic bacteria, and showed a survival rate of 76.9% under 0.3% bile salt stress. The strain also demonstrated good resistance to acid, alkali, trypsin, and heat treatment at 60 °C. Its auto-aggregation rate and hydrophobicity rate were 48.90% and 56.71%, respectively, and it possessed strong biofilm-forming ability. Antimicrobial susceptibility testing revealed that it was sensitive to 16 tested antibiotics, with resistance only to cefazolin. Moreover, PCR detection of virulence and transferable antibiotic resistance genes yielded negative results, and no hemolytic activity was observed. The animal trial demonstrated that the group supplemented with 6% bacterial preparation and 0.5% A. senticosus extract achieved the highest weight gain rate (66.58%), while the group with 4% bacterial preparation (Group C) exhibited the optimal immunomodulatory function, as evidenced by significantly increased activities of serum catalase (CAT), superoxide dismutase (SOD), lysozyme (LZM), and complement C3 and C4, along with upregulated expression of TNF-α and IL-1β in the kidney, liver, and intestine. Following the challenge, this group also showed the highest survival rate, with a relative percent survival (RPS) of 35.29%. In summary, E. faecium strain 19 derived from crucian carp exhibited excellent probiotic properties and a favourable safety profile. Its combination with A. senticosus extract significantly promoted growth, enhanced immune responses, and improved disease resistance in crucian carp. Although the present study was conducted under laboratory conditions, these findings have confirmed its practical value as a green feed additive for aquaculture. Nevertheless, further validation is still required regarding its specific application effects under actual farming conditions.