18ß-Glycyrrhetinic Acid Induces Metabolic Changes and Reduces Staphylococcus aureus Bacterial Cell-to-Cell Interactions.

The rise in bacterial resistance to common antibiotics has raised an increased need for alternative treatment strategies. The natural antibacterial product, 18ß-glycyrrhetinic acid (GRA) has shown efficacy against community-associated methicillin-resistant Staphylococcus aureus (MRSA), although its interactions against planktonic and biofilm modes of growth remain poorly understood. This investigation utilized biochemical and metabolic approaches to further elucidate the effects of GRA on MRSA. Prolonged exposure of planktonic MRSA cell cultures to GRA resulted in increased production of staphyloxanthin, a pigment known to exhibit antioxidant and membrane-stabilizing functions. Then, 1D 1H NMR analyses of intracellular metabolite extracts from MRSA treated with GRA revealed significant changes in intracellular polar metabolite profiles, including increased levels of succinate and citrate, and significant reductions in several amino acids, including branch chain amino acids. These changes reflect the MRSA response to GRA exposure, including potentially altering its membrane composition, which consumes branched chain amino acids and leads to significant energy expenditure. Although GRA itself had no significant effect of biofilm viability, it seems to be an effective biofilm disruptor. This may be related to interference with cell-cell aggregation, as treatment of planktonic MRSA cultures with GRA leads to a significant reduction in micro-aggregation. The dispersive nature of GRA on MRSA biofilms may prove valuable for treatment of such infections and could be used to increase susceptibility to complementary antibiotic therapeutics.

Exposure of Methicillin-Resistant Staphylococcus aureus to Low Levels of the Antibacterial THAM-3ΦG Generates a Small Colony Drug-Resistant Phenotype.

This study investigated resistance against trishexylaminomelamine trisphenylguanide (THAM-3ΦG), a novel antibacterial compound with selective microbicidal activity against Staphylococcus aureus. Resistance development was examined by culturing methicillin resistant S. aureus (MRSA) with sub-lethal doses of THAM-3ΦG. This quickly resulted in the formation of normal (WT) and small colonies (SC) of S. aureus exhibiting minimal inhibitory concentrations (MICs) 2× and 4× greater than the original MIC. Continuous cell passaging with increasing concentrations of THAM-3ΦG resulted in an exclusively SC phenotype with MIC >64 mg/L. Nuclear magnetic resonance (NMR)-based metabolomics and multivariate statistical analysis revealed three distinct metabolic profiles for THAM-3ΦG treated WT, untreated WT, and SC (both treated and untreated). The metabolome patterns of the SC sample groups match those reported for other small colony variants (SCV) of S. aureus. Supplementation of the SCV with menadione resulted in almost complete recovery of growth rate. This auxotrophism was corroborated by NMR analysis revealing the absence of menaquinone production in the SCV. In conclusion, MRSA rapidly acquires resistance to THAM-3ΦG through selection of a slow-growing menaquinone auxotroph. This study highlights the importance of evaluating and monitoring resistance to novel antibacterials during development.