INVESTIGATION OF BACTERIAL CELL PERTURBATION USING FT-IR SPECTROPHOTOMETER

Chemical compounds with diverse mechanisms of action are constantly been described as growth inhibitors intended to control bacterial infections, and it is necessary to search and/or develop new strategies to evaluate their effects on cell perturbation. A high-sensitivity physicochemical technique that has gained importance in several biological analyzes is the Fourier transform infrared (FT-IR) spectroscopy, which enables the identification and differentiation of microorganisms, the characterization of extracellular substances produced by them, and it can also be used to study the formation and development of microbial biofilms. Among the advantages of the FT-IR spectrophotometer technique we can cite: it is a safe technique, fairly easy to be applied and analyzed. Moreover, the FT-IR spectra can be considered molecular fingerprints useful for the characterization of the chemical composition of complex biological mixtures. Here, we show the use of FT-IR spectrophotometer to study the effects of some antimicrobial agents in bacterial cells. Bacillus subtilis cells were exposed to three antibiotics (kanamycin, nisin and vancomycin), to which the mechanisms of action have been described. Subsequently, untreated and treated B. subtilis were analyzed by FT-IR and the spectra generated were compared in order to detect variations that could be related to the exposure to the chemical compounds. Our results show that it is possible to observe perturbations in the bacterial membrane and cell wall by analyzing spectral data in the range of 2000 to 800 cm-1. FT-IR data allowed us to tentatively identify the major molecular complexes that were affected by the disappearance or deformation of peaks in the spectrum. In addition, we confirmed those internal cellular structures such as enzymes or the genetic material cannot be analyzed using such a technique; consequently antibiotics such as kanamycin (action on the translation) do not induce changes in their FT-IR spectra. However, for those compounds that recognizably target structures on the bacterial surface (nisin that produces pores in the cytoplasmic membrane, and vancomycin that acts on the cell wall precursors) we detected changes in the FT-IR spectra that are consistent with their modes of action. Finally, the technique proved feasible for the evaluation of cell disruption within its limitations and it can be used as a complementary methodology for cell analysis.