FTIR Spectroscopy of Bacterial Samples

Samples were harvested by centrifugation (8000× g, 20 min, RT) from 6 to 18 h in two-hour intervals (Supplementary Figure S1). The pellet was washed once with 1 mL PBS (5000× g, 2 min, RT) and resuspended in 100 μL of sterile deionised water. Subsequently, 30 μL each of the bacterial suspensions were spotted on a zinc selenite (ZnSe) optical plate, dried at 40 °C for 40 min, and measured with an HTS-XT microplate adapter coupled to a Tensor 27 FTIR spectrometer (Bruker Optics GmbH, Ettlingen, Germany). OPUS software (version 6.5; Bruker Optics GmbH) was used to process the measured FTIR spectra and perform chemometric analysis. Subtraction spectra were generated from second derivative, vector-normalised, average FTIR spectra by subtracting HA spectra from IN spectra for each time point. For a hierarchical cluster analysis (HCA), the spectral region that offers information about carbohydrate constituents (1200–800 cm⁻¹) was selected, which was shown to be highly discriminatory, based on the expression of CP and/or other cell surface glycans, such as wall teichoic acid [16 (link),56 (link)]. Bacterial strains were grown three times independently, each with three technical replicate measurements.