Development of DNA-based tools to study cutibacteria in skin health and disease

Abstract

Cutibacteria are a group of organisms known for their role in skin health and disease, although they are now increasingly recognised and studied in the context of conditions beyond the skin. In particular, the bacterium Cutibacterium acnes has had a long association with the inflammatory skin condition acne vulgaris which is ranked as the eighth most prevalent disease worldwide. The major aims of the work described here were to 1) develop new, and improve existing, DNA-based tools for metagenomic and culture-based identification and analysis of cutibacteria populations associated with skin disease, specifically severe recalcitrant acne (SRA) associated with isotretinoin resistance in the case of this thesis, and 2) examine a number of non-antibiotic-based approaches as novel treatment strategies for acne.

In the first part of this thesis, we developed a new diagnostic system for cutibacteria, known as CUTI-ID (CUTIbacterium Identification), which was centred on an Amplified Ribosomal DNA Restriction Analysis (ARDRA) platform. CUTI-ID displays high specificity and sensitivity for all current members of the genus, including the more recently added spp. C. namnetense and C. modestum, and will provide researchers with a robust and flexible, yet simple and low-cost, method to identify these bacteria when isolated from clinical samples. In addition, a novel ARDRA approach, called ARES (Antimicrobial REsistance SNP) analysis, was also developed for the identification of SNPs in the 16S and 23S rRNA genes of C. acnes that are the major drivers of high-level resistance to
tetracycline (16S rRNA; 1058G>C) and macrolides/ lincosamines (23S rRNA; 2058A>G, 2059A>G) antibiotics commonly used in primary practice for the treatment of moderate-to-severe acne. ARES will be a replacement for targeted nucleotide sequencing and also provide a low cost and facile approach for molecular screening of isolates for these SNPs. We also describe a flexible high resolution bilocus sequence typing scheme for C. acnes, known as CUTIS-SEQ (CUTIbacterium acnes BilocuS SEQuence typing), which displays good discriminately power for strains belonging to all subspp. of the bacterium, but which should also be applicable for metagenomic analysis of intraspecies communities of the bacteria within clinical samples. This scheme is a significant improvement on a previously described single locus typing method for C. acnes which has lately become the default method for high-resolution intraspecies analysis of the organism.

Date of Award	Jun 2022
Original language	English
Sponsors	British Skin Foundation
Supervisor	Andrew McDowell (Supervisor)