DNA analysis reveals diet and microbiome composition of prehistoric people

26 Jan 2024 --- A recent DNA analysis of archeological finds at Huseby Klev, Sweden, pulled from a masticated chewing gum consistency of a teenage girl, observed that the diet of prehistoric people included eukaryotic species such as red fox, hazelnut, red deer and apple. But more interestingly it provided enough information for further studies into the bacteria and microbiota of the ancient microbiome.

The study is a collaboration between Stockholm University and the Swedish Museum of Natural History where the researchers present the metagenomic analysis of three pieces of chewed pitch that were dated to 9,890–9,540 before present. The prehistoric chewed pitch has been a useful source of ancient DNA from humans and their microbiomes.

“We are curious — what did our ancestors eat? And also because it helps us understand what big variety of foods we can use,” Nils Anders Götherström, professor of molecular genetics, in the department of archaeology and classical studies at Stockholm University, tells Nutrition Insight.

“This is mostly because we are interested in our history and prehistory. If we want to develop future diets, then it may be interesting to have an idea of how diverse our diets have been over the millenia, but modern knowledge is better for developing diets than prehistoric foods.”

The metagenomic profile exposes a Mesolithic oral microbiome that includes opportunistic oral pathogens. “This provides a snapshot of the life of a small group of hunter-gatherers on the Scandinavian west coast. I think it is amazing, there are other well established methods to work out what nutrition and diet relates to the Stone Age, but here we know that these teenagers were eating deer, trout and hazelnuts 9,700 years ago on the west coast of Scandinavia, while at least one of them had severe problems with his teeth.”

Dysbiotic microbiome datasets created
The chewed material from Huseby Klev has already generated a study on the human genetic data from three individuals and the DNA in the material that was not of human origin has also been analyzed and published. Metagenomics on ancient DNA is an expanding area, but there have only been a few studies on this type of chewed material.

The researchers compared the data with healthy and dysbiotic microbiome datasets and identified an abundance of periodontitis-associated microbes. In addition, trained machine learning models predicted dysbiosis with 70–80% probability. The findings were published in Scientific Reports. 

“I think it shows us that humans are fairly omnivorous (as in eating very different things). These specific groups of people were living by the coast with access to animals, fish and plants. And that is what we find,” says Anders Götherström.

“We find other bacteria as well that are pathological, involved in caries and heart diseases. But not enough to say that she was actually suffering from these diseases, the small amounts we find could be present without causing pathologies. The only thing we know is that she did have a gum infection.”

Picture the scene
Approximately 9,700 years ago, a group of people were camping on the west coast of Scandinavia, north of what is Göteborg today. They were fishing, hunting and collecting food resources. A few teenagers were chewing resin to produce glue, after having eaten trout, deer and hazelnuts. 

Cut to the present day where the site on the island Orust was excavated 30 years prior. Chewed resin was found together with the remains of stone tools. The stone material also indicated a Mesolithic chronology.

“There is a richness of DNA sequences in the chewed mastic from Huseby Klev, and in it we find both the bacteria that we know are related to periodontitis, and DNA from plants and animals that they had chewed before,” says Dr. Emrah Kırdök, from the department of biotechnology at Mersin University, Türkiye, who coordinated the metagenomic work on the Mesolithic chewing gum. 

The masticates (pitch pieces) were another promising source of ancient microbiome DNA investigated as they contained material from the oral cavity. Previous studies investigated the oral microbiome and dietary components from 5,700 years old chewed pitch from Denmark and successfully proved its potential use in ancient DNA research.DNA analysis of prehistoric chewing gum found on a teenage girl revealed information about the composition of the oral microbiome (Credit: Bengt Nordquist).

The bacterial species present in ancient chewing gum is useful to explore the oral microbiome, which is in concordance with recent studies where some 15% of the salivary microbiome seems to get encapsulated in modern chewing gums. 

Exploring microbes
The  scientists explored the presence of differentially abundant microbes that are specific to dysbiosis conditions using modern periodontitis, caries and healthy salivary datasets. The analysis showed 27 differentially abundant bacteria that could be used as markers for periodontitis and caries conditions. In relation to dental caries, they found an abundance of Streptococcus sobrinus and Parascardovia denticolens compared to a healthy oral microbiome profile.

“We had to apply several computational heavy analytical tools to single out the different species and organisms. All the tools we needed were not ready to be applied to ancient DNA but much of our time was spent on adjusting them so that we could apply them,” says Andrés Aravena, from the department of molecular biology and genetics at Istanbul University, Türkiye.

Non-metric multidimensional scaling on Bray-Curtis distances grouped chewed pitch materials close to human oral microbiome and ancient dental calculus samples. In addition, SourceTracker analysis showed a contribution of around 50% of modern oral microbiome and ancient dental calculus to ancient pitch samples.

Genomic sequencing
To begin they compiled a database from full length genomic sequences of archaea, bacteria, viruses and fungi and used malt — an alignment-based metagenomic classification method — to confirm the presence of known oral commensal bacteria. 

They performed a de novo assembly of the DNA reads and created draft ancient bacterial genomes. According to the researchers the analysis proves that the metagenomic content in ancient pitch pieces is closer to the oral microbiome profile and contains commensal oral bacterial species.

A general problem with ancient microbial paleopathology is that there can be bacterial presence without physical symptoms. Typically, the presence of pathogenic species correlates with dysbiotic states but not necessarily in a causal way, therefore, the results suggest dysbiosis but do not show disease symptoms.

By Inga de Jong