Our study evaluated the hypothesis that yet-uncultured bacteria play a role in the pathogenesis of acne. This was made possible by applying a PCR-based strategy with a sensitivity and a discriminatory power surpassing those of previously used culture-based methods. The 16S rRNA-based method was recently developed and has been applied in several studies of complex bacterial communities associated with humans, including the microbiota of healthy skin (1
) The 16S rRNA gene is present in all known bacteria and contains a combination of conserved regions suited as primer sites and variable regions ideal for species assignment. The sensitivity of the method depends on primer specificity and the number of sequences determined. The coverage of the primers used in our study was found to be significant, with the primers matching more than 55,000 bacteria representing 1,061 genera in the RDP II database. Similar primers were used in previous studies and revealed that the microbiota of the superficial skin of healthy individuals has considerable diversity (10
As a validation of the method, the overall composition of the microbiota of superficial acne-affected skin that was demonstrated was very similar to that previously shown both for healthy human skin by a standard culture technique and for skin from the forearm and the forehead by the sequence-based technique used in this study (6
). The results of these studies combined show that the skin microbiota of both healthy and acne-affected individuals contains a ubiquitous and dominant core of bacterial species, i.e., S. epidermidis
and P. acnes
. As an exception, Grice et al. (14
) found Pseudomonas
species to be dominant in the more humid skin of the healthy human antecubital fossa, suggesting that significant regional differences may exist in the core bacterial species present on various skin surfaces. In addition, numerous species are present in minor proportions, and these proportions vary among individuals. The latter include bacteria that are recognized commensals of the oral cavity, which are presumably transiently present on the facial skin. In addition, our results and those reported by previous investigators (10
) clearly show that yet-uncultured phylotypes may be present but do not belong to the dominant core microbiota of the skin either in healthy individuals (10
) or in patients with acne (this study).
Sebaceous follicles, which are generally recognized to be the sites relevant to acne, must represent a uniquely harsh environment for bacteria, as evidenced by the surprising finding that only P. acnes
colonized the follicles of healthy individuals. Our coverage analysis demonstrates that sequencing of more clones would not have revealed additional diversity. No other exposed microenvironment of the human body is known to selectively allow colonization by a single bacterial species. It is conceivable that the more heterogeneous microbiota found in previous culture-based studies of healthy follicles (15
) was the result of differences in sampling methods. The cyanoacrylate method employed in the present study has the advantage, in addition to being noninvasive, that the upper part of the stratum corneum is captured in the applied cyanoacrylate, which minimizes the possibility of contamination with skin surface microorganisms when follicles are isolated by dissection. Microdissection of single follicles from biopsy punch specimens inevitably results in contamination with surface skin colonizers. Interestingly, Grice et al. (14
) detected the same composition of skin microbiota on the antecubital fossa by use of swap, scrape, and punch biopsy techniques, all of which are likely to include surface bacteria.
The bacterial microbiota of follicles from acne-affected subjects showed more, although still very limited, diversity (Table ). The microbiota was dominated by P. acnes and S. epidermidis, which were the only species consistently found. These results clearly exclude the possibility that yet-uncultured bacteria are associated with acne-affected skin follicles. Although the number of patients was limited, there was a complete absence of previously uncultured bacteria common to acne-affected follicles. It is unlikely that potential members of the microbiota present in numbers below the detection threshold of the method play a role in generating the intense inflammatory reaction typical of acne. We therefore conclude that yet-uncultured bacteria do not play an etiologic role in acne.
In contrast to previous culture-based studies (6
), our molecular analysis revealed differences in the microbiota of follicles from healthy and acne-affected subjects. The main difference was the presence of S. epidermidis
in acne-affected follicles (Table ). There is evidence from histological studies with fluorescently labeled species-specific antibodies that the majority of staphylococci, when they are present, colonize the dilated openings of follicles, an area that is unlikely to be included by our method as a result of the previously mentioned effect of cyanoacrylate (18
). Our detection of S. epidermidis
and occasional other bacterial species exclusively in acne-affected follicles may be explained by the facilitated access of the bacteria to dilated, inflamed follicles and by the previous histological demonstration that staphylococci infiltrate tissues surrounding inflamed follicles (18
). A possible alternative or additional explanation may be that the inflammatory reaction in the skin follicles of acne patients interferes with their apparently uniquely potent antimicrobial defense mechanisms.
The presence of S. epidermidis
exclusively in acne-affected follicles raises the question of the potential role of this species. Previous studies excluded staphylococci as agents that play a role in the pathogenesis of acne on the basis of their rapid development of resistance to therapeutic antibiotics (15
). It is possible that staphylococci are secondary colonizers, as inflamed and dilated follicles seem to be more conducive to colonization. However, on the basis of the findings of the present study, a reevaluation of the role of S. epidermidis
may be advisable. It is widely accepted that microbes interact and that such interactions may influence their production of biologically reactive substances. Further studies are required to elucidate the potential symbiotic effects of the two species.
In conclusion, by using a genetically based strategy with a sensitivity and a discriminatory power surpassing those of culture-based methods, we demonstrated that the follicles of healthy skin constitute a uniquely selective habitat for P. acnes, which was the only species detected. A microbiota that was more heterogeneous but that had limited complexity was demonstrated in acne-affected follicles. The results exclude the possibility that yet-uncultured bacteria are associated with acne.