Investigating phenotypic changes in wound biofilms in response to antimicrobial treatment using Raman Spectroscopy.

Wound infections such as diabetic foot ulcers (DFUs) can be highly problematic in terms of clinical management. Such infections carry a significant socioeconomic burden, costing the NHS an estimated £8.3b in 2017/18. DFUs have significant mortality rates, with 5-year mortality rates comparable with cancer. However, worryingly this is not reflected in the level of funding for chronic wound compared to cancer research.

The main difficulty that clinicians face during the treatment of chronic wounds is the risk of infection at the wound site, arising from the accumulation of microbial aggregates in the wound bed. Infected wounds arise from the formation of complex biofilms that can be largely resistant to antibiotic therapy: most of which are polymicrobial in nature . This further complicates treatment regimes to alleviate specific pathogenic microorganisms within the mixed community. Given the increased complexity of the wound microbiome as well as the increasing threat of antimicrobial resistance, clinicians are faced with the challenging prospect of targeted topical therapy or systemic antibiotics. This can be extremely difficult as point-of-care technologies in wound care are limited with regards to microbial detection, with clinicians relying on basic culture or molecular methodologies that can take days for results. Thus there remains a requirement for rapid, sensitive, yet non-invasive methodologies for microbial identification at the bedside, to aid clinical therapy choices during wound management.

Raman is an information rich vibration technique that gives label-free biochemical information at the sub-cellular level, however high-resolution imaging at a single cell level is a relatively slow process making it challenging for live cell imaging. Stimulated Raman scattering (SRS) is a more sensitive and much faster technique which can provide high resolution information on larger cell populations compared with normal Raman. SRS provides detailed biochemical information with sub-cellular resolution including determination of protein, lipids and nucleic acid location. Due to its speed and ability to image larger areas in a label free manner, it has great potential for imaging bacterial biofilms giving both biochemical information as well as structural information on biofilm formation, as well as changes to biofilm in response to antimicrobial treatments. To date there is evidence that different bacterial species exhibit unique spectral profiles depending on their chemical structures at a cellular level. Existing research is largely limited to single cells, with few studies investigating biofilm kinetics using SRS.

The overall aims of the proposal are to test the feasibility of using SRS to monitor biofilm formation and assess unique Raman spectral profiles or signatures of different bacterial and fungal species related to wound infections.

1 Monitoring growth dynamics of mono-species wound biofilm models using SRS - comparing spectral profiles in different clinically-relevant microbial species (gram+, gram- bacterial and fungal organisms) during biofilm attachment, maturation and dispersal.
2 Investigating mono-species biofilm response to antibiotic therapy using a variety of microbiological, molecular, microscopic and spectroscopic techniques - SRS to be used to assess chemical signatures in the biofilms in response to bacterio- (or fungi-) static or -cidal antibiotics and other novel compounds.
3 Assessing changes in biofilm formation in a multi-species biofilm model using SRS. Visualizing phenotypic changes in biofilm chemical structures containing a consortia of different microbial species.
4 SRS to be used to profile real-world biofilms (those generated from clinical samples from wound infections which will be generated through an existing EPSRC grant EP/V005839/1). Characteristic changes in chemical structures identified by Raman Spectroscopy using simple biofilm models with known composition.