Escherichia coli Antibiotic Uptake Analysis

Escherichia coli JM109 cells (overnight culture in liquid nutrient medium Luria–Bertani (pH 7.2), 10⁸ CFU) were washed twice with 0.01 M sterile PBS from the culture medium by centrifuging (Eppendorf centrifuge 5415C, 10 min, 12,000× g). Cell suspensions (10⁷ CFU/mL) were incubated with antibiotic samples; then, after 1-2-12-24 h, the cell’s samples were suspended and aliquots of 0.5 mL were taken. The cells are precipitated by centrifugation and separated from the supernatant, washed twice, and resuspended in 50 µL PBS to register the IR spectra. The supernatant is separated to determine the amounts of unabsorbed substances. ATR-FTIR spectra of cells samples suspension were recorded using a Bruker Tensor 27 spectrometer equipped with a liquid nitrogen-cooled MCT (mercury cadmium telluride) detector. Samples were placed in a thermostatic cell BioATR-II with a ZnSe ATR element (Bruker, Bremen, Germany). The FTIR spectrometer was purged with a constant flow of dry air (Jun-Air, Michigan, USA). FTIR spectra were acquired from 900 to 3000 cm⁻¹ with 1 cm⁻¹ spectral resolution. For each spectrum, 50–70 scans were accumulated at a 20 kHz scanning speed and averaged. Spectral data were processed using the Bruker software system Opus 8.2.28 (Bruker, Bremen, Germany), which includes linear blank subtraction, baseline correction, differentiation (second order, 9 smoothing points), min-max normalization, and atmosphere compensation. When necessary, 11-point Savitsky–Golay smoothing was used to remove noise. Peaks were identified by the standard Bruker picking-peak procedure. The concentration of Rif inside the cells was calculated from the material balance considering the unabsorbed Rif by UV-vis spectroscopy.