Screening, Mechanistic Investigation, and In Vivo Efficacy of a Compound Chinese Herbal Formulation Against Bovine-Derived Escherichia coli K99

Background:Escherichia coli (E. coli) K99 is one of the major pathogens responsible for neonatal calf diarrhea, characterized by widespread transmission and strong pathogenicity, leading to high mortality in calves and substantial economic losses in the cattle industry. Antibiotic-based therapies are increasingly challenged by drug residues and continuous emergence of antimicrobial resistance, which creates an urgent demand for safe and effective alternative antimicrobial agents. Chinese herbal medicines (CHMs), featuring low toxicity, abundant resources and synergistic effects of multiple bioactive constituents, have become promising candidates for new antibacterial agents. Objectives: This study aimed to screen Chinese herbal medicines for antibacterial activity against bovine-derived E. coli K99-2, develop a compound herbal formula F1 following the traditional “Jun-Chen-Zuo-Shi” compatibility principle, and systematically characterize its chemical composition, antibacterial mechanism, in-vivo safety and protective therapeutic efficacy. Materials and Methods: Seventy Chinese herbal medicines were subjected to in-vitro antibacterial screening using the Oxford cup agar diffusion assay. Candidate compound formulas were designed according to screening outcomes and “Jun-Chen-Zuo-Shi” theory. The MIC and MBC of the optimized formula F1 were determined via broth microdilution. LC-MS was performed to profile chemical constituents of F1. Growth curves, membrane potential, cell-wall permeability, intracellular protein and ATP levels, gel retardation assay and scanning electron microscopy were applied to investigate antibacterial mechanisms. A 28-day repeated oral gavage toxicity test in Kunming mice was conducted to evaluate biosafety. The LD₅₀ of E. coli K99-2 was determined by intraperitoneal challenge, and a mouse infection model was used to assess the in-vivo protective effect of F1. Results:Fructus Mume and Cornus officinalis displayed prominent antibacterial activity against K99-2. The optimized compound formula F1 produced an inhibition-zone diameter of 21 mm; both MIC and MBC were 62.5 mg/mL. Putatively annotated constituents of F1 included furan derivatives, amino acids, flavonoids, polyphenolic compounds and organic acids. F1 triggered bacterial cell-envelope damage, membrane depolarization, reduction of intracellular protein content and disturbance of ATP-associated energy metabolism, while it did not form stable complexes with bacterial genomic DNA. The 28-day oral toxicity assay revealed no obvious toxic manifestations within the tested dose range in mice. In the K99-2 infection-challenge experiment, oral administration of F1 raised the mouse survival rate from 40 % (untreated infected control) to 73 %. Discussion: Compound herbal formula F1 exerts multi-target antibacterial activity against E. coli K99-2 mainly by disrupting bacterial structural integrity and interfering with energy metabolism rather than directly binding to genomic DNA. F1 demonstrates favorable preliminary biosafety and can improve survival of K99-2-infected mice, indicating its potential as a veterinary herbal candidate for prevention and control of calf diarrhea caused by E. coli K99. Further work is required to identify key bioactive components, optimize dosage and verify efficacy in calf clinical settings. This research provides experimental basis for developing antimicrobial herbal veterinary preparations.