Work package 1

The aim of this work package is to develop a robust and interchangeable protocol for sample preparation in order to determine the dynamics of antibacterial agent penetration into bacteria in biofilms.

To aid the metrology developments in WP2, WP3 and WP4, a simple non‑biological system will be prepared to enable a metrological inter‑comparison. This will consist of samples with a uniform layer of exopolysaccharide with known thickness and with different concentrations of antimicrobial.

Measurements on the full biological systems will incorporate microorganisms such as non-pathogenic, laboratory adapted Gram negative Escherichia coli or pathogens: Pseudomonas aeruginosa (Gram negative), Staphylococcus aureus (Gram positive) and Mycobacterium bovis bacille Calmette‑Guerin (BCG) as a model of the acid fast positive Mycobacterium tuberculosis. Mycobacteria are included as their unique cell wall contributes directly to antimicrobial resistance. All the bacteria chosen are tractable to genetic modifications, thus enabling, for example, the inactivation of efflux pumps. This will provide a well‑controlled system to be studied.

Work package 2

The overall aim of this work package is to develop metrology for label‑free chemical imaging of microorganisms and biofilms, and for the localisation of microbial metabolites and antimicrobial agents in bacteria and biofilms. These will be accomplished by (1) a revolutionary new instrument 3D OrbiSIMS which is capable of chemical imaging with 100 times better sensitivity (with laser post‑ionisation developed in this project), a high‑spatial resolution (100 nm), mass resolution of >100 000, and the ability to sample from sub‑micron areas simultaneously and (2) nanoscale 2D imaging, at <50 nm resolution using s‑SNOM. The latter will be reinforced by the development of numerical modelling and algorithms to support high resolution spectroscopies and to probe microscopic measurements in complex biological environments.

This work package will develop advanced sample preparation methods for drugs in bacteria and biofilms. This will include cryo‑preparation methods to allow water into the vacuum of high‑performance instruments without ultrastructural reorganisation of biological systems. In addition, nanofabrication and nano‑manipulation of sample surfaces will be explored to enhance sensitivity and to enable the specific localisation and arrangement of clusters of bacteria for high‑resolution analysis.

Work package 3

The overall aim of this work package is to develop new metrological methodologies based on vibrational spectroscopy and innovative optical microscopy techniques in order to follow and study the penetration and efficiency of drugs inside bacterial cells over time. Optical and vibrational spectroscopies (e.g. RAMAN, TERS) are important for measuring the dynamic interaction between bacterial cells and biocides as they can provide short time data collection, non‑destructive analysis and they can be used as imaging techniques to locate lipids, nucleic acids and drug molecules in bacterial cells, even with a potential nanometre resolution.

Work package 4

The aim of this work package is to establish traceable measurements for the characterisation and quantification of the uptake of biocides in a biofilm and beyond that its concentration depth profile in the biofilm. Whilst these methods typically rely on a unique chemical element they are essential to calibrate the label‑free methods such as SIMS and Raman which are of more general applicability in the pharmaceutical and medical device industry. The aim is to establish novel approaches to quantitatively measure the biofilm and its biocide uptake in its natural liquid environment.

To get access to the quantification of the biocide concentration depth‑profile of biofilms two complementary approaches with Near‑Ambient Pressure X‑ray Photoelectron Spectroscopy (NAP‑XPS) and Grazing‑Incidence XRF (GIXRF) will be used. To get access to the biofilm in its liquid environment for (GI‑)XRF analysis a novel liquid cell will be adapted for use with biofilms. Complementary analysis will be performed using the same liquid cell and infra‑red spectroscopy.

Work package 5

Creating impact

Work package 6

Management and coordination