Objective: To prepare in-house susceptibility test discs containing HuanglianJiedu Tang (HLJDT) and evaluate their in vitro antibacterial activity against Staphylococcus aureus (S. aureus). Methods: 1) The content of berberine in HLJDT was quantified using high-performance liquid chromatography (HPLC) to ensure quality control. 2) The antibacterial activity of HLJDT susceptibility discs was assessed against S. aureus at varying concentrations, disc thicknesses, and culture times to evaluate their stability, uniformity, and efficacy. 3) The inhibitory effects of the discs were tested on 64 clinical S. aureus isolates, and the correlation between inhibition zone diameter and minimum inhibitory concentration (MIC), determined via broth microdilution, was analyzed. Results: 1) The berberine concentration in HLJDT, as determined by HPLC, was 2358.0 μg/mL. 2) In the disc diffusion assay, high (45.0 mg) and medium (22.5 mg) concentration HLJDT discs produced significant inhibition zones against S. aureus strains USA300 and ATCC29213, while the low concentration (11.25 mg) discs showed negligible activity. Discs with a thickness of 0.7 mm exhibited significantly better antibacterial effects against MRSA compared to those with a thickness of 1.0 mm (P < 0.05). 3) The inhibition zones for methicillin-resistant S. aureus (MRSA) were significantly larger than those for methicillin-susceptible S. aureus (MSSA) at both high and medium concentrations (15.33 ± 2.37 mm and 11.82 ± 2.48 mm vs. 13.50 ± 1.68 mm and 9.87 ± 1.60 mm, respectively; P < 0.01). Furthermore, the inhibition zone diameter for MRSA using high-concentration discs was strongly negatively correlated with MIC values (r = ？0.501, P < 0.01), and moderate negative correlations were observed at medium and low concentrations (r = ？0.327 and r = ？0.353, respectively; P < 0.05). Conclusion: The HLJDT susceptibility test discs demonstrated reliable quality, good uniformity, and stable antibacterial performance. They showed notable antibacterial activity against S. aureus, particularly MRSA, with a strong correlation between inhibition zone diameter and MIC. These findings provide a robust methodological foundation for further research and potential clinical applications of HLJDT in the treatment of S. aureus infections.
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