Multi-drug resistant bacteria are currently undermining our health care system worldwide. While novel antimicrobial drugs, such as antimicrobial peptides, are urgently needed, identification of new modes of action is money and time consuming, and in addition current approaches are not available in a high throughput manner. Here we explore how small angle X-ray scattering (SAXS) as high throughput method can contribute to classify the mode of action for novel antimicrobials and therefore supports fast decision making in drug development. Using data bases for natural occurring antimicrobial peptides or predicting novel artificial peptides, many candidates can be discovered that will kill a selected target bacterium. However, in order to narrow down the selection it is important to know if these peptides follow all the same mode of action. In addition, the mode of action should be different from conventional antibiotics, in consequence peptide candidates can be developed further into drugs against multi-drug resistant bacteria. Here we used one short antimicrobial peptide with unknown mode of action and compared the ultrastructural changes of Escherichia coli cells after treatment with the peptide to cells treated with classic antibiotics. The key finding is that SAXS as a structure sensitive tool provides a rapid feedback on drug induced ultrastructural alterations in whole E. coli cells. We could demonstrate that ultrastructural changes depend on the used antibiotics and their specific mode of action. This is demonstrated using several well characterized antimicrobial compounds and the analysis of resulting SAXS curves by principal component analysis. To understand the result of the PCA analysis, the data is correlated with TEM images. In contrast to real space imaging techniques, SAXS allows to obtain nanoscale information averaged over approximately one million cells. The measurement takes only seconds, while conventional tests to identify a mode of action require days or weeks per single substance. The antimicrobial peptide showed a different mode of action as all tested antibiotics including polymyxin B and is therefore a good candidate for further drug development. We envision SAXS to become a useful tool within the high-throughput screening pipeline of modern drug discovery. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

A new Rococo 2 X-ray fluorescence detector was implemented into the cryogenic sample environment at the Hard X-ray Micro/Nano-Probe beamline P06 at PETRA III, DESY, Hamburg, Germany. It features a high solid angle of up to 1.10 steradian and high count rates of 1 Mcounts s−1 per sensor.

A combination of small- and ultra-small-angle X-ray scattering enabled the resolution of important intracellular constituents in Escherichia coli (ribosomes, DNA and proteins). The impact of treatment with three antibiotic agents was monitored.

Coherent X-ray ptychography is a tool for highly dose efficient lensless nano-imaging of biological samples. We have used partially coherent soft X-ray synchrotron radiation to obtain a quantitative image of a laterally extended, dried, and unstained fibroblast cell by ptychography. We used data with and without a beam stop that allowed us to measure coherent diffraction with a high-dynamic range of 1.7·10. As a quantitative result, we obtained the refractive index values for two regions of the cell with respect to a reference area. Due to the photon energy in the water window we obtained an extremely high contrast of 53% at 71 nm half-period resolution. The dose applied in our experiment was 9.5·10 Gy and is well below the radiation damage threshold. The concept for dynamic range improvement for low dynamic range detectors with a beam stop opens the path for high resolution nano-imaging of a variety of samples including cryo-preserved, hydrated and unstained biological cells.

Soft X-ray SAXS and ASAXS reveal nanostructural properties and temperature induced morphological changes in catalyst materials. The stabilizing effect of cerium oxide deposits on the gold catalyst and the morphological properties of the cerium oxide were determined.