Enzymatic pretreatment gives a very precise image of skin bacteria

Healthy skin has a bacterial shield to protect itself from germs: the microbiome. This complex assembly of microorganisms was previously considered difficult to decipher. A team of researchers has now successfully used the enzyme benzonase to identify living bacteria in skin swabs through sequencing. Their method opens up new possibilities for diagnosis and treatment in dermatology.

In many skin diseases such as atopic dermatitis and acne, the bacterial layer protecting the skin is damaged. “Our aim is to learn the role played in these diseases by the different types of skin bacteria”, explains Dr Martin Köberle, head of the dermato-infectology laboratory of the Klinikum Rechts der Isar of the Technical University of Munich (TUM ).

Past efforts by dermatologists to investigate the detailed makeup of the microbiome have encountered obstacles. The reason: In conventional cultures grown on agar, not all bacteria grow and multiply as well. As a result, some slow growing species can be completely ignored. The downside of newer genetic analysis methods is that large amounts of DNA sequences from skin cells and fragments of dead bacteria are captured. This reduces the informative value of the results.

Separating the genetic wheat from the chaff

Dr Köberle and biologist Dr Yacine Amar, both members of Prof. Biedermann’s team at the TUM Clinic and Polyclinic of Dermatology and Allergology, have developed a method to remove DNA from non-target species by cooperation with an international and interdisciplinary team. . They used a special feature of the enzyme benzonase. It destroys the nucleotide chains that carry hereditary information in all living things by breaking them down into short fragments. Only living bacteria whose DNA is protected by an outer cell wall escape destruction by the enzyme.

Benzonase has been used for some time, for example to purify proteins: enzymes break down all fragments of foreign DNA and RNA. These can then be removed in a centrifuge, leaving the proteins behind. The selection of skin bacteria works on the same principle: the genetic material of skin cells or dead bacteria is broken down by the enzyme and can then be separated from the sample. The remaining bacteria can be mechanically destroyed, allowing the study of their DNA.

“Our experiments have shown that with this method, we can indeed completely eliminate non-target DNA and select the skin microbiome”, explains project manager Yacine Amar. In the laboratory, he first studied artificial samples containing a mixture of human cells and dead and living bacteria created according to a strict protocol and pretreated with benzonase. “The process then used – known as 16S sequencing – gave a very precise picture of the composition of intact bacteria,” explains the researcher. The analysis of real skin swabs was equally successful: no residual DNA from dead bacteria was found in the samples.

Dr Köberle is convinced that this approach will also play a key role in future research: “Enzymatic selection of live skin bacteria can help us find microbial biomarkers for certain skin diseases and also identify bacteria that have a positive influence. on the course of the disease. Maybe they will be used in treatments one day. The new method of microbiome analysis is already being used in numerous cohort studies of skin diseases at the TUM Clinic and the Polyclinic of Dermatology.

Source:

Technical University of Munich (TUM)

Journal reference:

Amar, Y., et al. (2021) Pre-digestion of unprotected DNA with benzonase improves the representation of live skin bacteria and efficiently depletes host DNA. Microbiome. doi.org/10.1186/s40168-021-01067-0.

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