Nested PCR-denaturing gradient gel electrophoresis analysis of human skin microbial diversity with age

Abstract

To determine whether the composition and structure of skin microbiota differ with age, cutaneous bacteria were isolated from the axillary fossa of 37 healthy human adults in two age groups (old people and young adults). Bacterial genomic DNA was extracted and characterized by nested PCR-denaturing gradient gel electrophoresis (PCR-DGGE) with primers specifically targeting V3 region of the 16S rRNA gene. The excised gel bands were sequenced to identify bacterial categories. The total bacteria, Staphylococcus spp., Staphylococcus epidermidis and Corynebacterium spp. were further enumerated by quantitative PCR. There were no significant differences in the species diversity profiles between age groups. The similarity index was lower across age groups than that it was intra-group. This indicates that the composition of skin flora is more similar to others of the same age than across age groups. While Staphylococcus spp. and Corynebacterium spp. were the dominant bacteria in both groups, sequencing and quantitative PCR revealed that skin bacterial composition differed by age. The copy number of total bacteria and Corynebacterium spp. were significantly lower in younger subjects, whereas there were no statistical differences in the quantity of Staphylococcus spp. and Staphylococcus epidermidis. These results suggest that the skin flora undergo both quantitative and qualitative changes related to aging.

Keywords: 16S rRNA gene; Bacterial diversity; Nested PCR-DGGE; Quantitative PCR; Skin microbiota.