Developing novel technologies to model the effects of therapies on microbial biofilms

Adams, Jennifer 2023. Developing novel technologies to model the effects of therapies on microbial biofilms. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

The development of antimicrobial resistance against multiple antibiotic and antifungal classes in bacterial and fungal pathogens respectively is of global clinical concern. With “last resort” antibiotics and antifungal treatments having associated toxicity issues and with a drying-up of new antimicrobials in the development pipeline, (especially for Gram-negative organisms), the development of novel therapies is urgently needed. OligoG CF-5/20 is a low molecular weight alginate oligosaccharide that has been shown to have antimicrobial and anti-biofilm properties against a range of multi-drug resistant pathogens. OligoG CF-5/20 was tested against a range of Candida spp., in addition to being used as a combination therapy with the antifungal nystatin. Minimum inhibitory concentration (MIC) assays and growth curves showed that OligoG CF-5/20 synergistically enhanced the effect of nystatin against planktonic cells. Furthermore, confocal laser scanning microscopy (CLSM) and COMSTAT analysis showed that it was also effective at preventing biofilm formation, as well as disrupting established biofilms. This study demonstrated that OligoG CF-5/20 can act as a stand-alone treatment against Candida spp., as well as showing enhanced effectiveness when used as a combination therapy with the antifungal nystatin (P <0.0001). To investigate the contribution of calcium (Ca2+)-binding in its antimicrobial activity, G-block (OligoG CF-5/20) and an M-block alginate oligosaccharide (OligoM) with comparable average size (DPn 19) and contrasting Ca2+ binding properties (that of OligoG being greater), were compared. The tailored alginate OligoM was shown to be comparable to OligoG CF-5/20 at reducing planktonic and biofilm growth of P. aeruginosa; also demonstrating similar reductions in virulence iii factor production of pyocyanin (P <0.05), elastase (P <0.0001), and protease (P <0.0001) when tested at 6%. Compared to OligoM, OligoG CF-5/20, exhibited a significantly stronger inhibitory effect on quorum sensing (QS) signaling, and displayed increased potentiation of the antibiotic azithromycin in MIC and biofilm assays. These results with OligoM highlight the fact that the antimicrobial effects of alginate oligosaccharides were not purely dependent upon Ca2+ binding. Colistin is often referred to as the antibiotic of last resort for the treatment of e.g., carbapenem-resistant Enterobacteriaceae. The discovery of the plasmid-encoded colistin resistance, mcr, and its rapid dissemination into the environment in recent years heralds the risk of a post-antibiotic era. The resistance profiles associated with the mcr-1 and mcr-3 genes were apparent in MIC and minimum biofilm eradication concentration (MBEC) assays, and the associated fitness costs of mcr plasmid carriage were highlighted in the growth curves with mcr-1 strains showing significantly lower growth rates than those carrying mcr-3. A bead biofilm model was adapted to monitor the stability of mcr-1 and mcr-3 genes over time, using isogenic E. coli J53 hosts. After serial passage (every 2 days for a month), the mcr-1 gene was lost by day 23, while the mcr-3 gene was stably maintained for up to 31 days. CLSM and COMSTAT analysis demonstrated the antibiofilm effect of OligoG CF-5/20 against mcr carrying E. coli (P <0.05), whilst also showing changes in extracellular polymeric substance (EPS) such as a reduction of proteins (P <0.05) and the release of eDNA. These results suggest that OligoG CF-5/20 has considerable potential as an antimicrobial agent, either as a stand-alone treatment or in combination with existing therapies, such as colistin or nystatin, to treat multidrug resistant pathogens that are responsible for hard-to-treat chronic infections.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Dentistry
Subjects:	Q Science > QR Microbiology R Medicine > RM Therapeutics. Pharmacology
Funders:	Algipharma AS, KESS2
Date of First Compliant Deposit:	6 March 2024
Last Modified:	07 Mar 2024 12:09
URI:	https://orca.cardiff.ac.uk/id/eprint/166905

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