Synergistic pathogenicity of Elizabethkingia miricola and Klebsiella pneumoniae in Black-Spotted Frogs and the Prophylactic Efficacy of a Novel Herbal Composite.

The black-spotted frog (Pelophylax nigromaculatus) is an ecologically and commercially important aquaculture species in China. However, intensive farming has escalated its vulnerability to diseases, particularly meningitis (torticollis-cataract syndrome). In August 2023, a severe disease outbreak characterized by neurological symptoms occurred on a frog farm in Huanggang (Hubei Province, China; 30.45° N, 114.08° E). In this study, Elizabethkingia miricola and Klebsiella pneumoniae were identified as co-pathogens from the kidney tissues of moribund frogs. Taxonomic confirmation and virulence characterization were performed through integrated physicochemical profiling, phylogenetic analysis, and whole-genome sequencing, complemented by artificial infection experiments. An innovative triple herbal formula comprising Rhus chinensis Mill., Terminalia chebula Retz., and Biancaea sappan L., was developed. Chemical characterization via high-performance liquid chromatography identified tannic acid as the predominant bioactive constituent. Ultrastructural analysis confirmed that the formula triggered irreversible cell envelope disruption and cytoplasmic leakage in both pathogens. In vivo feeding trials demonstrated superior prophylactic efficacy at a 15‰ dosage, reducing cumulative mortality from 42.62% (control) to 21.11% during a polybacterial challenge. This represents a 50.47% relative reduction in mortality, highlighting the formula's potent protective capacity. Our findings clarify the complex etiology of frog meningitis and provide a natural, high-efficiency strategy for sustainable disease management in aquaculture.