# Nanochemical Cell-Surface Evaluation in Photothermal Spectroscopic Imaging of Antimicrobial Interactions in the Model System Bacillus subtilis and Vancomycin

## TL;DR

Researchers used a high-resolution imaging technique to study how vancomycin interacts with the cell walls of Bacillus subtilis bacteria.

## Contribution

The study introduces a novel method using photothermal spectroscopic imaging to visualize antibiotic interactions on bacterial surfaces with nanometer resolution.

## Key findings

- Chemical details of cell wall destruction in Bacillus subtilis after vancomycin treatment were observed with ∼5 nm resolution.
- Spectral shifts indicating hydrogen bond formation between vancomycin and peptidoglycan were detected in PiF-IR spectra.
- Vancomycin interaction signatures were localized in the septum region of B. subtilis with ∼10 nm resolution.

## Abstract

The power of photothermal spectroscopic imaging to visualize
antimicrobial
interactions on the surfaces of individual bacteria cells has been
demonstrated on the model system Bacillus subtilis and vanco­mycin using mid-infrared photoinduced force microscopy
(PiF-IR, also mid-IR PiFM). High-resolution PiF contrasts obtained
by merging subsequent PiF-IR scans at two different illumination frequencies
revealed chemical details of cell wall destruction after 30 and 60
min incubation with vanco­mycin with a spatial resolution of
∼5 nm. This approach compensates for local intensity variations
induced by near-field coupling of the illuminating electric field
with nanostructured surfaces, which appear in single-frequency contrasts
in photothermal imaging methods, as shown by Anindo et al. [J. Phys. Chem. C
2025, 129, 4517. DOI: 10.1021/acs.jpcc.4c08305]. Known spectral shifts associated with hydrogen bond formation
between vanco­mycin and the N-acyl-d-Ala4-d-Ala5 termini in the peptido­glycan cell
wall have been observed in chemometrics of PiF-IR spectra from treated
and untreated B. subtilis harvested after 30 min
from the same experiment. Spectral signatures of the vancomyin interaction
have been located in the piecrust of a progressing septum with ∼10
nm resolution using PiF contrasts of three selected bands of a PiF-IR
hyperspectral scan of an individual B. subtilis cell
harvested after 30 min incubation. Our results are complemented by
a discussion of imaging artifacts and the influence of parameter settings
supporting further development toward standardization in the application
of PiF-IR for visualizing the chemical interaction of antibiotics
on the surface of microbes with few nanometer resolution.