Characterisation and disruption of extracellular polymeric substances in clinically relevant biofilms

Albarrak, Abdullah 2025. Characterisation and disruption of extracellular polymeric substances in clinically relevant biofilms. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Antimicrobial resistance and biofilms contribute to treatment failure and pose a global burden on healthcare systems. Biofilms are microbial communities attached to surfaces with the cells encased within self-produced extracellular polymeric substance (EPS) matrix. The EPS is involved in the biofilm’s structural stability, nutrient retention, and protection against external stresses including the presence of antimicrobial agents. Persistence of biofilm infections and their increased tolerance have made them a major focus of current microbiological and clinical research. In this PhD, the overarching research aim was to assess the role of EPS in promoting tolerance of in vitro biofilms to antimicrobials. Initial studies sought to determine suitable in vitro conditions to grow biofilms using selected bacterial species as well as Candida albicans. Assessment of biofilm quantity was done by culture and confocal laser scanning microscopy (CLSM) with live/dead staining. It was determined that polycarbonate was a suitable substrate to support biofilm growth in full strength medium under continuous flow in a CDC biofilm reactor. There were minor exceptions depending on the combinations of microorganisms used. Having established biofilm growth parameters, CLSM coupled with appropriate fluorescent probes was used to detect and quantify specific EPS components of clinically relevant biofilm-forming microorganisms including S. sanguinis, S. gordonii, S. salivarius, C. albicans and N. gonorrhoeae, providing details on their spatial distribution. The most frequently detected EPS components were carbohydrates, followed by proteins. Extracellular DNA was not detected in in vitro biofilms. It was however found that N. gonorrhoea biofilms produced by mutants lacking Lig E (and considered unable to generate eDNA) were less pathogenic in a reconstituted vaginal epithelium model. Typically, in unsupplemented media, biofilms had limited EPS and when present it was closely associated with cells. However, research found that when Streptococcus mutans was present in biofilms, both biofilm and EPS production increased when the culture medium was supplemented with 1% (w/v) sucrose and dextran (1 μM). Using CSLM, Raman microscopy and carbohydrate quantification it was shown that dextranase (0.25%) degraded the EPS of S. mutans biofilms. For antimicrobial testing, biofilms exhibited higher tolerance to chlorhexidine (CHX) compared to planktonic growth, with EPS rich biofilms being less susceptible compared to biofilms with limited EPS. Furthermore, EPS degradation during biofilm formation using 0.25% dextranase, reduced tolerance of 1% sucrose and 1 μM dextran supplemented biofilm to levels comparable to biofilm with limited EPS. Biofilms treated with dextranase post formation for 1 h, followed by CHX exposure, resulted in increased numbers of dead cells compared to biofilms with intact EPS. In conclusion, results of this research highlighted the role of EPS in antimicrobial tolerance and showed the potential of enzymatic degradation in rendering biofilms more susceptible to CHX. This suggests that EPS targeting may not only inhibit the establishment of complex biofilms but may facilitate their eradication when coupled with conventional antimicrobial treatments.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Dentistry
Subjects:	Q Science > QR Microbiology
Date of First Compliant Deposit:	26 January 2026
Last Modified:	28 Jan 2026 11:52
URI:	https://orca.cardiff.ac.uk/id/eprint/184192

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