Study On 23S RRNA Point Mutation And Erm Gene In Macrolide-lincosamide-resistant Cutibacterium Acnes

Objectives:Acne vulgaris,commonly known as "pimples",is a common cosmetic skin disease that occurs more frequently in adolescents.The overproliferation of Cutibacterium acnes(C.acnes)is one of the key factors in the pathogenesis of acne,and anti-C.acnes therapy is an important means of acne treatment.The widespread use of macrolide-lincosamide(ML class)antibiotics in the treatment of acne and other infectious diseases has led to increasing resistance of C.acnes to ML class antibiotics,and there is a lack of information on the resistance of C.acnes to ML class antibiotics in our region in recent years.In this study,we monitored the resistance of C.acnes to ML class antibiotics isolated from acne lesions in our region,and detected the resistance genes of C.acnes to explore the resistance mechanism of C.acnes to ML class antibiotics in our region initially.Methods:Patients with acne vulgaris who met the inclusion criteria were collected from the First Affiliated Hospital of Kunming Medical University,and the contents of facial lesions were collected after obtaining informed consent,and C. acnes strains were identified by Optical Microscope and MALDI Biotyper after anaerobic culture.The standard strain ATCC 6919 was used as a control for in vitro ML class antibiotic susceptibility determination using a concentration gradient paper strip diffusion method.Some of the sensitive strains and all resistant strains were stored in 20% glycerol at-20℃,and 1ml of the frozen strain and the standard strain ATCC 6919 were used to extract their genomic DNA.Three pairs of primers were designed based on the sequence of transposon Tn5432 carrying resistance gene erm(X),and PCR amplified against resistance gene erm(X),transposon Tn5432 and IS1249 on both sides of transposon Tn5432,respectively.The corresponding fragments were confirmed by agarose gel electrophoresis to confirm the presence of the three fragments and to match the theoretical fragment length.Results:1.A total of 149 acne patients were included in this study,among which 139 cases of C.acnes(139 strains)were cultured,and the culture positivity rate was93.29%.2.Among 139 C.acnes strains,53 were resistant to ML class antibiotics(38.13%),and all were highly resistant(MIC value >256 μg/ml).3.23 S rRNA gene fragment point mutation detection:(1)In some resistant strains,the same resistance-associated point mutations as previously reported were detected:A→G mutation was present in 2 strains(3.77%)at locus 2058 of the 23 S rRNA gene fragment equivalent to E.coil;A→G mutation was present in 4 strains(7.55%)at locus 2059 of the 23 S rRNA gene fragment equivalent to E.coil;(2)In some drug-resistant strains,mutations different from those previously reported were detected,and an association with drug resistance could not be excluded:3 strains(3.75%)had the A→T mutation at the equivalent of the E.coil 23 S rRNA gene fragment 2058 locus;25 strains(31.25%)had the T→C mutation at locus 250;6 strains(7.50%)had the G→A mutation at locus 855.(3)In 18 sensitive strains and 1 standard strain,no point mutation of 23 S rRNA gene fragment was detected which was the same or different from previous reports.4.Detection of drug resistance genes erm(X),Tn5432 and IS1249:(1)The sequences of erm(X),Tn5432 and IS1249 detected were the same as those reported previously:All strains(resistant and sensitive strains)including the standard strain carried the erm(X)gene;(2)Among the 53 drug-resistant strains carrying the erm(X)gene,41(77.36%)had both transposon Tn5432 and IS1249 sequences.Conclusions:1.High detection rate of C.acnes in acne lesions in the region.2.The resistance rate of C.acnes to ML class antibiotics is high in this region.3.The molecular mechanism of resistance to ML class antibiotics in C.acnes in this region is more complex:(1)Point mutations consistent with known 23 S rRNA gene resistance sites were found,which showed high resistance to ML class antibiotics and were not detected in sensitive and standard strains,showing a clearer relationship between point mutations and resistance mechanisms;(2)All strains were found to carry the resistance gene erm(X),suggesting that the resistance mechanism may be related to various factors such as the copy number of erm(X)gene or the joint action of erm(X)gene and other resistance mechanisms;(3)The discovery of the drug resistance gene erm(X)carried by the non-transposon Tn5432 suggests that the erm(X)gene in this region may be carried by other transposons and/or plasmids.4.23 S rRNA point mutations different from those previously reported,which cannot be excluded from being associated with drug resistance,namely A2058 T,T250C,and G855 A.