Antibiofilm activity against common pathogenic bacteria in Thailand and liquid chromatography-quadrupole time-of-flight mass spectrometry chemical profiling of Ha-Rak formula and its components: an in vitro study.

BACKGROUND

Bacterial biofilms reduce antibiotic penetration, create tolerant bacterial subpopulations, and contribute to difficult-to-treat infections and antimicrobial resistance (AMR). The Ha-Rak Formula (HRF), a traditional Thai polyherbal remedy used for febrile illnesses, has not been systematically evaluated for antibiofilm activity.

OBJECTIVE

This study assessed the in vitro antibiofilm effects of HRF and its five constituent herbs against clinically relevant bacterial pathogens in Thailand and characterized their chemical profiles using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS).

METHODS

Water and 80% ethanol extracts were prepared from authenticated roots and commercially sourced root-stem materials. Minimum inhibitory concentrations (MICs) against six pathogenic bacteria were determined by broth microdilution at final concentrations of 64 - 16,384 µg/mL. Biofilm biomass was quantified by crystal violet staining at final extract concentrations of 400 and 1000 µg/mL, and untargeted LC-QTOF-MS with multivariate analysis was used for chemical profiling.

RESULTS

All extracts had MIC values > 16,384 µg/mL against planktonic cells, except the ethanol extract of root-derived HRF, which inhibited Klebsiella pneumoniae ATCC 13883 only at the highest tested concentration (MIC = 16,384 µg/mL). In contrast, root-derived extracts reduced biofilm biomass across multiple species at sub-MIC concentrations. Ethanol extracts displayed distinct chemical profiles enriched in putative carbohydrates and conjugates.

CONCLUSIONS

These findings indicate that HRF has stronger activity against biofilm formation than against planktonic bacterial growth under the conditions tested. Root-only and root-stem formulations both exhibited antibiofilm activity under the conditions tested, while water and ethanol extracts showed distinct chemical profiles. Further standardization, safety assessment, and mechanistic studies are required before clinical translation.