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(Image: Michael Kirkham)

Ancient microbial DNA on fossil teeth has opened a fresh window on our ancestors, revealing that civilisation has altered our mouth flora for the worse

ALMOST a millennium ago, a middle-aged man was buried in a graveyard in Dalheim, Germany. He had taken some hard knocks in his life – a fist fight had torn incisors from his jaw. And oral hygiene was clearly not a priority: his remaining teeth carried a thick coating of plaque, the gunk that modern people battle with toothbrush and dental floss. We should thank this unknown man for his grim chompers, however, for his dental plaque is now opening a surprising new window to the past. Inside it is a beautifully preserved record of the microbial community in his mouth when he was alive.

We have just begun to understand that the microscopic organisms that live inside and on us are not necessarily pests or freeloaders. Outnumbering our own cells by 10 to 1, they form ecosystems called microbiomes that play a vital role in keeping us healthy. Which microbes we harbour depend on our environment, diet and lifestyle. Since our ancestors co-evolved with an array of microbes and parasites that people in most developed countries no longer encounter, human microbiomes must have shifted over the millennia as we made the transitions from hunter-gatherers to farmers to urbanites. But bacterial remains rarely survive the ravages of decomposition and fossilisation, so direct study of ancient microbiomes seemed out of reach. The discovery that plaque holds the well-preserved remains of a rich microscopic community has been a revelation.

Looking at microbiomes of the past is more than mere voyeurism. In modern developed nations there has been a surge in inflammatory conditions such as asthma, allergies, diabetes and gum disease. A growing body of evidence suggests that this trend is the result of shifts in our internal ecosystems. “Life in urban environments, with antibiotics and advanced sanitation, represents a fundamental change in our relationship with microbes,” says anthropologist Cecil Lewis at the University of Oklahoma in Norman. “We’ve benefited from that change, but now we’re learning that we are also increasing our risk of inflammatory diseases.” Knowing how and why our microbiomes have changed could provide new ways to treating these conditions.

Unknown signatures

Just a decade ago, researchers believed they had identified all the dominant species of bacteria that live inside humans, by growing laboratory cultures of people’s microbiomes. That notion was proved drastically wrong when advanced DNA-sequencing techniques revealed the genetic signatures of a multitude of previously unknown bacterial species that do not grow under laboratory conditions. “Many of these are unnamed species, and we know nothing about them or what they do,” says Christina Warinner, an archaeological geneticist working in Lewis’s lab. At the same time, developments in the sequencing of ancient DNA have allowed us to reconstruct genomes from ever older and more degraded samples. This has made it possible to look at the microbiomes of long-dead people.

Lewis was among the first to try this. Intrigued by the idea that some of our health problems are caused by unbalanced modern microbiomes, he wanted to study ancient people and the communities of microorganisms they carried. He knew it wouldn’t be easy. DNA decays rapidly in the weeks after death and even extracting human genetic material from old bones can be an arduous, hit-and-miss proposition. Microbial DNA would be even harder to come by, as the soft tissue generally associated with microbiomes rarely survives for long after death. Nevertheless, DNA from ancient microbiomes has turned up in a handful of remains, including Ötzi the Iceman, a 5200-year-old frozen mummy found near a retreating glacier on Italy’s Alpine border with Austria. So, in 2007, Lewis began looking for more sources.

He searched several promising samples, including a 1600-year-old mummy from Chile and 3000-year-old fossilised faeces from Texas. In each case he drew a blank; the extracted DNA came from microbes that had contaminated the samples. when he managed to extract ancient microbial DNA from two samples of 1400-year-old human faeces found in a prehistoric cave dwelling at the edge of the Rio Zape in Durango, Mexico. This time, he found microbes reflecting ancient gut flora rather than contaminating bacteria from soil. The community of microorganisms was remarkably similar to that found in Ötzi’s remains. There were notable differences from the gut microbiome of modern city dwellers, however. In particular, people living in the cave at the Rio Zape harboured plenty of bacteria in the genus Treponema – a group that has disappeared from the innards of modern urbanites – and their gut communities were dominated by Prevotella, a group that today is most common in rural people living in remote undeveloped areas.

Lewis suspects that these bacteria are associated with the digestion of plant matter. Plant fossils found in the Rio Zape cave suggest the people there ate a diet rich in maize. Treponema are still found in rural people consuming a mostly vegetarian diet, and Prevotella remain common in the guts of modern people who eat diets rich in carbohydrate. Likewise, researchers have discovered that , which help them digest starchy foods such as potatoes and brown bread. So a move towards more refined foods in post-industrial societies is linked to changes in gut microbiomes – changes that can affect people’s health. Treponema and Prevotella bacteria may help protect against inflammatory diseases of the colon, which are common in the developed world.

Bulletproof plaque

Although these first analyses of prehistoric microbiomes are intriguing, Lewis needs more evidence to substantiate them. Well-preserved samples of ancient gut microbiomes are rare, but fortunately researchers have now discovered a near bulletproof source of ancient microbial DNA: calcified dental plaque. Plaque is a film made up of the microbes that thrive in the mouth. Before the days of efficient dentistry it calcified on teeth, forming a cement-like matrix called calculus. “Calculus is like a time capsule,” says Warinner. “It is mineralised while the person is alive, so it doesn’t decompose like faeces or soft tissues.” What’s more, calculus is common. It can be found on teeth from archaeological sites around the world – even on the teeth of skulls tens of thousands of years old.

Alan Cooper pioneered this approach, and his team at the (ACAD) at the University of Adelaide is now using dental calculus to probe the oral microbiome far back into human prehistory. In research published last year, they focused on 34 early European skeletons, ranging in age from 700 to 7500 years old, and compared their microbial DNA with the oral microbiomes of modern people living in Adelaide. The results show microbial communities shifting as lifestyles changed, with ecologies associated with disease becoming increasingly prevalent ().

The bacterium primarily associated with dental cavities, Streptococcus mutans, first appeared in the human mouth in the early Neolithic era, when our ancestors made the move from hunting and gathering to farming. At the same time, Porphyromonas gingivalis and other bacteria that cause gum disease became much more common (see graph). Oral microbiomes then remained surprisingly constant right up until the industrial revolution, when people started eating a diet rich in refined sugar. Today we carry dense populations of both Lactobacilli and Streptococci, bacteria that feed on sugars. The lactic acid they release as a waste product dissolves tooth enamel, causing cavities; it also lowers the pH inside the mouth, creating a hostile place for species adapted to more alkaline environments.

The fossil record shows that ancient hunter-gatherers had strong, intact teeth. These are found in remains of Poland’s last hunter-gatherers, who provided the oldest samples in the ACAD study, but also in more ancient hunter-gatherers including the Neanderthals, who died out about 28,000 years ago. Their bright smiles have traditionally been attributed to diets rich in meat and low in sugar, but the ancient DNA analysis suggests an alternative – our prehistoric ancestors may simply have lived in a time of better-balanced oral microbiomes. Cooper’s group is not alone in reaching this conclusion. Bioarchaeologist at the University of Warsaw, Poland, notes that . Populations living in the Near East who regularly ate sugary dates had an almost complete lack of decay, which Sołtysiak suggests was due to the absence of the major cavity-causing bacteria in their mouths. taken from the mouths of people around the world. These indicate that the species is relatively new, rapidly evolving, and specifically adapted to the human mouth. Estimates based on the genetic divergence from strains that infect other animals place the rise of human varieties of S. mutans at the dawn of agriculture, about 10,000 years ago.

Dogs were domesticated from wolves around 15,000 years ago and have lived with us through the shifts to agriculture and urbanisation. However, the inside of a dog’s mouth is alkaline, and so unfavourable for the microbes adapted to the acidic, sugar-stoked environment of modern human mouths. Our carbohydrate-rich diet has transformed our oral ecosystems. “In humans, Streptococci are the most common genera in the mouth. We have about 20 species of Streptococci,” says microbiologist Floyd Dewhirst at the Forsyth Institute in Cambridge, Massachusetts. He has recently analysed and shown it to be dramatically different from ours, containing just two or three Streptococci species.

“Our carbohydrate-rich diet has transformed our oral ecosystems for the worse”

Cavities are a nuisance, but an unbalanced oral microbiome may be responsible for far greater problems. Periodontitis is a disease of the gums that plagues many modern people – about 13 per cent of US residents will suffer from it during their lifetime, for example. An inflammation that occurs when the immune system reacts to some of the bacteria in plaque, it is connected to other health problems, including type 2 diabetes. if oral bacteria make their way into the bloodstream and form plaques that block major arteries. Researchers have identified a trio of pathogenic microbes involved in periodontal disease, known as the “red complex” bacteria. Warinner realised she could analyse ancient DNA to find out more about the emergence of the disease. And this is where the pugilistic fellow unearthed in Germany comes in.

In research published in February, Warinner and her colleagues examined the calculus of four people buried almost a millennium ago in the graveyard of Saint Petri church in Dalheim (). They chose individuals who had died in middle age, since younger people had less dental plaque and older ones had lost most of their teeth. The team also focused on people whose remains showed signs of periodontal disease.

DNA analysis of the calculus left no doubt that red-complex bacteria were around in medieval times. The calculus also contained proteins released by neutrophils – immune cells that mobilise to destroy invading bacteria – suggesting that the individuals had mounted the same immune response as is seen in periodontitis in modern people. “The course of the disease in medieval times was similar to what we see today,” says Warinner.

Periodontitis may have even deeper roots. In the ACAD study, red-complex bacteria showed up in the mouths of some of Europe’s earliest farmers, who lived as much as 7000 years ago. “It used to be thought that periodontitis is caused by infection,” says Warinner. “Now we realise that the pathogens are always there, but the microbial community shifts from health to disease.” And, like cavities, the rot seems to have set in when our ancestors began farming, shifting to a diet rich in carbohydrates, which fuelled varieties of microbes more likely to turn pathogenic.

“Like cavities, gum disease seems to have set in when people began farming”

“Extracting so much information out of ancient dental calculus is a huge accomplishment,” says Dewhirst. “Archaeologists used to prepare skulls by cleaning off the dental plaque and throwing it away. Nobody realised it contained such an incredible biological record.”

Still, many questions remain. A key one is whether modern microbiomes are less diverse than their apparently healthier predecessors. The suspicion that they might be is fed by recent studies showing that inflammatory bowel disease and obesity are associated with low microbial diversity in the gut. In their 2013 work on early European skeletons, the ACAD team concluded that oral ecosystems have lost biodiversity as humanity moved through its major dietary and cultural transitions. But the researchers now acknowledge this conclusion may have been premature. “We made some assumptions that could have led us to misconstrue that argument of diversity declining through time,” says team member Laura Weyrich.

There is also the question of how modern antibiotics affect our microbiomes. Warinner found that the bacteria in medieval mouths had the means to resist antibiotics centuries before we started using them. This is not surprising, given that antibiotics are simply pharmaceutical versions of chemicals used by bacteria to communicate and perform other functions. But it does make clear that our microbiomes can be rich reservoirs for drug resistance, and underscores the need to carefully manage antibiotic use.

It is early days for the analysis of ancient calculus, but already the research leaves little doubt that the modern oral microbiome is out of whack. A deeper knowledge of the inner ecosystems of our ancestors may reveal ways to better balance our microbiomes and reduce our susceptibility to a range of diseases. To that end, the ACAD team is currently delving even further back into human prehistory, working with teeth up to 50,000 years old, to chart the changes over time. “We need to understand ancient microbiomes because modern ones are so important in health and disease,” says Weyrich.

This article appeared in print under the headline “Pearly time capsule”