Quantitation of Major Human Cutaneous Bacterial and Fungal Populations

Abstract

Because the human skin microbiota may play roles in the causation or modification of skin diseases, we sought to provide initial quantitative analysis from different cutaneous locations. We developed quantitative PCRs to enumerate the total bacterial and fungal populations, as well as the most common bacterial and fungal genera present in six locales, in eight healthy subjects. We used a set of primers and TaqMan MGB probes based on the bacterial 16S rRNA and fungal internally transcribed spacer region, as well as bacterial genus-specific probes for Propionibacterium, Corynebacterium, Streptococcus, and Staphylococcus and a fungal genus-specific probe for Malassezia. The extent of human DNA contamination of the specimen was determined by quantitating the human housekeeping GAPDH gene. The highest level of 16S rRNA copies of bacteria was present in the axilla (4.44 ؎ 0.18 log 10 copies/l [mean ؎ standard error of the mean]), with normalization based on GAPDH levels, but the other five locations were similar to one another (range, 2.48 to 2.89 log 10 copies/l). There was strong symmetry between the left and right sides. The four bacterial genera accounted for 31% to 59% of total bacteria, with the highest percent composition in the axilla and the lowest in the forearm. Streptococcus was the most common genus present on the forehead and behind the ear. Corynebacterium spp. were predominant in the axilla. Fungal levels were 1 to 2 log 10 lower than for bacteria, with Malassezia spp. accounting for the majority of fungal gene copies. These results provide the first quantitation of the site and host specificities of major bacterial and fungal populations in human skin and present simple methods for their assessment in studies of disease.Human skin harbors a diverse group of microorganisms that form complex communities and occupy specific niches and microenvironments (5,9,12,14,15). While most organisms colonizing the human body may be beneficial for health (4, 9, 18), some relationships with the host can change from commensal to pathogenic for reasons that are poorly understood (1). The global composition and presence of specific organisms also is relevant to the biological effects of the skin microbiota. The human cutaneous surface includes regions with diverse pH, temperature, moisture, and sebum content (14, 17), and skin structures such as hair follicles, sebaceous, eccrine, and apocrine glands comprise subhabitats that may be associated with their own unique microbiota (20).Until recently, our knowledge of the bacterial biota in human skin has been based mostly on cultivation studies, which are insufficient because many organisms cannot be cultured (6). The direct PCR amplification and sequencing of bacterial genes encoding the small subunit rRNA (16S rRNA) or variable region fragments thereof has been a powerful method to analyze the enormous variation in the human microbiome (8,24,30), showing important differences among a number of cutaneous sites (5, 9, 12, 15). However, analysis of ribosomal genes (16S rRNA ...

Distinct cutaneous bacterial assemblages in a sampling of South American Amerindians and US residents

“…The similarities between the samples from the two US locations to one another in relation to the Amerindian samples (Supplemental Figure 1), despite some differences in study methods, are consistent with prior US studies of the cutaneous microbiota that show conservation of the major taxa (Gao et al, 2007(Gao et al, , 2010Grice et al, 2009;Costello et al, 2009). In contrast, the microbiota from the Amerindians was substantially different from the United States, and was divided into two major clusters that were essentially not represented in the US samples.…”

“…Sample collection and DNA extraction For Platanillal and New York residents, the volar aspect of the forearms were sampled as described in prior publications (Gao et al, 2007(Gao et al, , 2010Grice et al, 2009;Staudinger et al, 2011) by applying a cotton pledget soaked in ST solution (0.15 M NaCl with 0.1% Tween 20). After swabbing for B30 s, the head of each swab was aseptically cut from the handle, centrifuged for 10 min at 5900 g, and then removed.…”

“…Investigators now are using molecular, DNA-based methodologies to assess the microbial populations present in and on human skin (Fredricks, 2001;Paulino et al, 2006Paulino et al, , 2008Gao et al, 2007Gao et al, , 2008Gao et al, , 2010Fierer et al, 2008Fierer et al, , 2010Grice et al, 2008Grice et al, , 2009Costello et al, 2009;Grice and Segre, 2011). However, these studies were all performed in humans who live in developed countries, as does the large-scale National Institute of Health (NIH)-initiated Human Microbiome Project (Peterson et al, 2009), that shares this focus on humans living lifestyles associated with postmodern socioeconomic development.…”

“…We sought to describe the composition and uniformity of the sampled Amerindian cutaneous microbiome in a community of Amerindian subjects, to identify correlates with particular clustering patterns, and to determine whether Amerindian and US populations could be distinguished. For these studies, we sampled a single cutaneous site, the volar mid-forearm, as this site is easily identified and sampled, permits examining laterality of populations, is washed less than hands, and is subject to an extensive sequence database that is currently growing (Paulino et al, 2006;Gao et al, 2007Gao et al, , 2008Gao et al, , 2010Paulino et al, 2008;Costello et al, 2009;Staudinger et al, 2011).…”

Traumatic Wound Microbiome Workshop

“…Blaser discussed some methodological innovations and concerns. One innovation was the use of eukaryotic DNA quantification to provide a standard allowing the relative quantification of bacterial DNA between samples [8]. Using skin swabs, the variability of the eukaryotic to bacterial DNA concentrations is approximately two orders of magnitude; this may correlate with actual bacterial cell density on the skin surface.…”