Multiplex mismatch amplification mutation polymerase chain reaction assay for the rapid detection of antimicrobial-resistant determinants in Cutibacterium acnes.

The emergence of antimicrobial resistance in Cutibacterium acnes, a key factor that exacerbates acne vulgaris, has become a clinical concern. To prevent the progression of acne and the emergence of resistant strains, a rapid and easy method to assess antimicrobial resistance is needed. A multiplex polymerase chain reaction (PCR) assay was developed that could simultaneously detect multiple resistance factors associated with antimicrobial resistance in C. acnes. In total, 267 clinical C. acnes isolates were analysed based on their antimicrobial susceptibility and genetic characteristics. Mutation-specific primers targeting 23S rRNA, 16S rRNA, and GyrA were designed based on the principles of the mismatch amplification mutation assay-PCR (MAMA-PCR). A multiplex PCR incorporating the acquired resistance genes, erm(X), erm(50), and tet(W), was developed to evaluate the sensitivity and specificity of the method. The optimised MAMA-PCR protocol consisted of an initial denaturation step at 94°C for 3 min, followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 10 s, and extension at 72°C for 1 min 30 s, with a final extension step at 72°C for 5 min (total, 65 min). The newly-developed multiplex PCR assay for the comprehensive detection of major resistance determinants demonstrated 100% sensitivity and specificity, enabling rapid identification of resistance factors as compared to conventional methods. The multiplex PCR assay facilitated the rapid, accurate, and easily simultaneous detection of the main antimicrobial resistance determinants in C. acnes. This finding supports the use of antimicrobial agents for the treatment of acne.