Introduction:
Acne vulgaris, more commonly known as acne, is a disease that mostly everyone deals with at some point in their life, most likely in their adolescent years, and can lead to self-esteem issues and negative impacts on mental health (Lee, et. al., 2019). Of course, there are some people who maybe get the occasional pimple, but generally do not have acne-prone skin and to them, people might say they are blessed with good genetics. But is genetics really the outstanding cause of whether you have acne or not? Microbiologists would suggest it is the microbes inhabiting our skin, which are microscopic sized organisms. The skin microbiome, or collection of microbes living on and in your skin, is diverse and can contribute to a number of skin conditions, acne being one of them. There is one specific microbe known to inhabit the skin quite pervasively and progress acne: Propionibacterium acnes. This microbe is able to survive without oxygen within a layer beneath your skin and deep within the hair follicles of the skin.
A study done by Jacob Hall and colleagues in 2018 offers a comparative analysis of the three methods best suited for sampling the skin to determine the presence of microbes. The use of pore strips is one method used in this study, but it is stated that previous studies have emphasized concern they do not reach deep enough to obtain an accurate analysis (Fitz-Gibbon, et al., 2013). Another method used was swabbing the skin surface with a cotton swab, which only obtains information from the surface of the skin and not internal layers, where we know P. acnes to be most abundant. The third method used was cyanoacrylate glue which was able to obtain 50% deeper into the hair follicle than the pore strips were able to. The two methods of data analysis used in this study were 16S rRNA sequencing and whole-genome sequencing (WGS). 16S rRNA sequencing offers microbial identification by amplifying a highly conserved region within all bacteria, while WGS takes the entire DNA sequence and matches the parts of DNA to sequences that are available on public databases to determine which taxa are present.
Central Question
Of the three skin sampling methods identified, which is the optimal method for sampling and sequencing the skin microbiome? Additionally, which method of data analysis provides the more sensitive and precise representation of the skin microbiome?
Methods:
The researchers split 27 volunteers into two groups, each with 10-12 individuals without acne and 3 individuals with mild acne. The sampling methods differed between each group. Cohort A was sampled with a pore strip on the left cheek and glue on the right cheek while Group B was sampled by a surface swab on the right cheek and glue on the left cheek (Figure 1). Comparing the glue and pore strip methods used on cohort A individuals, the researchers found the glue method collected complete follicles, while the pore strip method was able to collect only incomplete follicles. This determined the glue method could obtain a greater depth than the pore strip. The microbial composition of these two samples were analyzed via 16S rRNA sequencing and WGS. Next, the skin microbiomes of the individuals that were subject to the glue method and surface swab method were compared using both 16S rRNA sequencing and WGS.
Figure 1. A visualization of skin sampling method used on each cohort and subsequent analysis. Additionally, a representation of the sampling depth for each sampling method used (Hall, et. al., 2018).
Results
Pore Strips vs Glue
Using both WGS and 16S rRNA sequencing approaches, the researchers used the DNA extracted from the samples to observe what types of microbes were present when samples were collected via pore strips and glue. No differences were found in alpha diversity and beta diversity of the microbes present, regardless of the sequencing method. This finding was supported by identifying the same types of microbes in the skin samples that were collected via pore strips and glue. They also found P. acnes to be the most abundant species detected, as expected. These results concluded that, in general, WGS and 16S rRNA sequencing gave a similar representation of the microorganisms present within the hair follicle. However, the WGS analysis gave a more precise representation of the microbes present at the taxonomic level than 16S rRNA sequencing did.
Figure 2. A heat map displaying the 20 most abundant microorganisms collected via pore strips and glue methods for both a) 16S and b) WGS (Hall, et. al., 2018).
Glue vs Swabs
The DNA collected from the surface swab method and the glue method underwent the same data analysis methods as pore strips and glue methods. From the WGS data, the researchers found the two sampling methods had little differences in alpha and beta diversity, but noticed an increasing trend in the alpha diversity of the surface microbiome. This meant that, although both sampling types determined the same highly abundant microbes, they also both found different types of microbes, including viruses and fungi. The surface swab method found a larger variety of microbes while the glue method found only a few types of microbes. Among the types of microbes found on the skin were various bacteria and viruses. Viruses were found both on the surface environment of the skin and within the hair follicle.
Figure 3. A heat map displaying the 20 most abundant microorganisms collected via glue method and surface swab method for both a) 16S and b) WGS (Hall, et. al., 2018).
P. acnes presence
Because of the link that previous studies had already established between acne and the bacterium, P. acnes, the researchers shifted their focus to the presence of this specific bacterium between each sampling method (pore strip, glue, and cotton swab) (Alexeyev, et. al. 2012). They found that when the skin sampling methods were compared that none of the methods had a concern of missing the presence of P. acnes. This meant that regardless of sampling method, P. acnes is present in all layers of the skin, including the surface layer.
Data analysis comparison
When comparing the two analysis methods, 16S rRNA sequencing or WGS, the researchers suggest WGS for studies that aim to accurately identify a variety of microbe types within samples. Because the 16S region is specific to bacteria, it would not have detected the fungi and viruses present in the skin. The researchers speculated that WGS also may become more cost-effective in the near future as advancements continue, which was true as WGS has become more affordable since this study was published in 2018.
Further Thoughts and Questions
The results of this study may have implications for further acne research and provide a resource for those considering skin sampling and data analysis methods. It provides a comparison of the best suited sampling method for capturing a representation of the microorganisms present on and in the hair follicles of the face. However, it was mentioned in the study that limitations include the small sampling size and the predominance of individuals without acne. In future studies, I would like to see this replicated with the majority of the sampling size displaying mild or severe acne to gain a better understanding of the role microorganisms, specifically P. acnes, have in the progression of Acne vulgaris. How will this affect the results? Would we still see P. acnes present in all environments of the skin? Furthermore, it is extremely interesting that the researchers found not just bacteria present on and within the skin, but fungi and viruses as well. A deeper analysis of how these types of microbes are connected to Acne vulgaris would be beneficial to those studying acne as well as those affected by it.
Overall, the emphasis of this study was that when studying the microorganisms on and within the skin, it is best to consider which skin sampling method and which data analysis method to use. Their study suggests the cyanoacrylate glue sampling method and data analysis of the entire DNA sequence of each microbe found within each sample provide the most accurate analysis.
Further Reading
For more information on P. acnes:
This is a great article that provides details on the type of bacteria P. acnes is and the other implications for human health it may have.
P. Propionibacterium acnes: infection beyond the skin
For more information on the epithelial layers and hair follicles:
This article provides additional information on how our hair follicles may have a major impact on skin health. It is helpful to understand the unique features of hair follicles in order to understand their effect on skin health.
Hair follicles and their role in skin health
For more information on 16S rRNA sequencing:
This is a great introductory article to 16S rRNA amplicon sequencing and its importance in the world of microbial research. It provides useful information on the fundamental principles of 16S rRNA sequencing and the molecular biology to support it.
Introduce to 16S rRNA and 16S rRNA Sequencing
For more information on whole genome sequencing (WGS) in comparison to 16S rRNA sequencing:
This article is great for understanding the fundamental differences between 16S rRNA sequencing and whole genome sequencing. It highlights the advantages of WGS in comparison to 16S rRNA sequencing.
Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing.
For more information on the microorganisms and their impact on skin:
This article clearly illustrates the link between the skin microbiome and the progression of Acne vulgaris. It is a great article to reference when trying to understand the factors that potentially cause acne.
Potential Role of the Microbiome in Acne: A Comprehensive Review
References:
Jacob B. Hall, et. al. Isolation and Identification of the Follicular Microbiome: Implications for Acne Research, Journal of Investigative Dermatology, Volume 138, Issue 9, 2018, Pages 2033-2040, ISSN 0022-202X, doi:10.1016/j.jid.2018.02.038
Sorel Fitz-Gibbon, et. al. Propionibacterium acnes Strain Populations in the Human Skin Microbiome Associated with Acne, Journal of Investigative Dermatology, Volume 133, Issue 9,2013,Pages 2152-2160,ISSN 0022-202X, https://doi.org/10.1038/jid.2013.21.
Lee YB, Byun EJ, Kim HS. Potential Role of the Microbiome in Acne: A Comprehensive Review. J Clin Med. 2019 Jul 7;8(7):987. PMID: 31284694; PMCID: PMC6678709. doi: 10.3390/jcm8070987