Research on the application of a high-throughput fully automated bacterial growth curve monitor for screening natural antibacterial compounds.

backgroundThe emergence of multidrug-resistant bacteria poses a severe challenge to infection treatment, creating an urgent need for novel antimicrobial agents. Monomers derived from natural products exhibit multi-mechanism antibacterial properties with low resistance development potential. However, evaluation of these monomers is frequently hampered by color interference in traditional turbidimetric methods. This study developed a high-throughput fully automated bacterial growth curve monitor (HTFA-BGM) to provide a new tool for screening and assessing antimicrobial monomers.

methodsThe HTFA-BGM was designed and developed based on the principle of scattering nephelometry. Its performance in determining the minimum inhibitory concentration (MIC) was compared with the microdilution and the tube dilution methods. After establishing the instrument's MIC determination criterion, it was used to evaluate and screen the antibacterial effects of Traditional Chinese Medicine (TCM) monomers.

resultsThe HTFA-BGM could screen MICs of 40 monomers against one strain or one monomer against 40 strains simultaneously, unaffected by compound color. When a threshold of ≤ 10% for the ratio of sample turbidity change to bacterial suspension turbidity change was used as the MIC criterion, there was no significant difference between the HTFA-BGM method and the microdilution or tube dilution methods. The HTFA-BGM was used to evaluate 15 monomers (including polyphenolic, quinonoid, and other types of compounds), and the MIC of rhein against 90 strains of Methicillin-Resistant Staphylococcus aureus (MRSA) was 8–64 μg/mL. The synergistic rates of rhein combined with penicillin, cefoxitin, cefazolin or oxacillin against MRSA in vitro were 30% (27/90), 63% (57/90), 57% (51/90), and 58% (52/90), respectively, whereas the additive rates were 70% (63/90), 37% (33/90), 43% (39/90), and 42% (38/90), respectively. The MIC of oleanolic acid against 13 isolates of vancomycin-resistant Enterococcus (VRE) was 16–32 μg/mL. When combined with vancomycin, vancomycin MIC decreased from 256–1024 to 1 μg/mL.

conclusionThe HTFA-BGM enables automatic, real-time, and dynamic monitoring of bacterial growth curves. Compared with the microdilution method, which determines the MIC on the basis of endpoint turbidity, this instrument not only has improved efficiency but also yields more objective and accurate results. This method specifically resolves the interference caused by the intrinsic color of colored TCM monomers such as rhein during their determination tests. Therefore, it is better suited for evaluating and screening antibacterial TCM monomers. The HTFA-BGM demonstrates excellent practical applicability and warrants broad promotion and application.