An optimized physical-chemical method for analyzing cell perturbations using FTIR-ATR spectrophotometry

The present work proposes an optimization of a physical-chemical technique for assessing the perturbation of bacterial cells caused by antimicrobial compounds. We adapted an analysis method using FTIR-ATR spectrophotometry to evaluate any damage or molecular changes in the structure of bacterial cells. Three antibiotics (ciprofloxacin, vancomycin, and nisin), with their mechanisms of action already known, were used and analyzed using the proposed methodology. Bacillus subtilis was the microorganism used for testing. To validate the proposed technique, fluorescence microscopy with fluorescent marker dyes and the B . subtilis spo0J-gfp mutant was used. The results showed that perturbations in the bacterial membrane and cell wall can be observed in the range of 2000 to 800 cm -1 . This allows for the evaluation of which major molecules were affected by the disappearance or deformation of peaks in the FTIR-ATR spectrum. It has also been confirmed that other structures, such as DNA, cell division, or specific intracellular structures like enzymes or proteins, cannot be analyzed. The use of FTIR-ATR has also proved inadequate in confirming whether a cell is alive or dead after exposure to antibiotics that affect cell division, such as ciprofloxacin. Nevertheless, the technique proved to be viable for evaluating cell disruption, despite its limitations concerning cell surface structures. The technique proved to serve as an efficacy complementary approach to identify the mechanisms of action of antimicrobial compounds. • An optimization of a biophysical technique for identifying cellular perturbations; • Proposal for a more cost-effective method to analyze the mechanisms of action of antibiotics; • FTIR-ATR fingerprinting is highly effective for visualizing the surface structures of bacterial cells; • FTIR-ATR is not effective or valid for analyzing changes in cellular DNA.