Exploring caffeine as a disruptor of membrane integrity and genomic stability in Staphylococcus aureus: functional...

To explore the mechanistic underpinnings of caffeine as a potent antibacterial against Staphylococcus aureus ATCC 25923 via in vitro functional assays, whole-genome sequencing, and in silico docking studies. In vitro studies established that caffeine's minimum inhibitory concentration (MIC) against S. aureus ATCC 25923 is 0.01544 mmol/mL. Functional assays along with Scanning Electron Microscopy confirmed that caffeine at 0.030089 mmol/mL (2MIC) released nucleotide constituents (nucleotide leakage assay) and effluxed potassium ions (potassium efflux assay) thereby, further validating caffeine's role as a membrane-active antimicrobial agent. Whole genome sequencing of control versus caffeine treated samples revealed a significant drop in read mapping percentage from 99.96 to 23.68% and GC content from 30.69 to 6.93%. This massive reduction in the treated sample was a consequence of single nucleotide polymorphisms (SNPs, 50,303), along with insertions and deletions (InDels, 62). Several of these caffeine-induced mutations were found to be harbouring the coding regions of genes involved in processes such as cell membrane organization, bacterial virulence, and DNA repair processes. Thus, implying a caffeine-mediated genomic rearrangement and instability. In silico docking studies revealed a strong binding affinity of caffeine to key cell wall proteins ltaA (-6.9 kcal/mol) and ltaS (-6.5 kcal/mol) respectively. The dynamic simulation studies revealed caffeine's interaction with receptor ltaS remained stable, with low deviations and minimal fluctuations. Although caffeine has been widely investigated for its antibacterial properties, its specific mechanisms of action, notably its effects on the cell membrane and genomic integrity in S. aureus ATCC 25923, are little understood. This study thus offers a comprehensive functional genomic analysis of caffeine as an antibacterial against S. aureus.