Bioprospecting in Dental Tartar from Neanderthals for Novel Antibiotics and Revisiting the Discovery of Penicillin

Dense living communities of hundreds of bacterial species form biofilms on our teeth. Without careful brushing and flossing of this dental plaque, minerals seep in, hardening it into tartar. When proteins in saliva adhere tartar to tooth surfaces, a trip to the dentist is required to hack the stuff off.

Over time, the mineralized microbes of tooth tartar come to comprise a mouthful of tiny fossils, including snippets of degraded bacterial DNA. Because many antibiotic drugs come from or are based on modern bacteria, tooth tartar – aka dental calculus – from ancient people may hold genetic recipes for novel antibiotics from the past.

A team of researchers from the Leibniz Institute for Natural Product Research and Infection Biology, the Max Planck Institute for Evolutionary Anthropology, and Harvard University has reconstructed “paleogenomes” of previously unknown bacteria from the dental tartar of ancient and modern people. The work appears in Science.

Reconstructing Ancient Genomes

The technological challenge in isolating useful genes from the mishmash of time-tattered genomes in ancient tooth tartar is to collect large enough pieces of DNA to overlap to deduce the genome sequences of extinct bacteria. The hope is that some include genetic instructions for proteins that bacteria naturally manufacture that enable them to fight off other bacteria, but that we could use as promising new antibiotic drugs – or use the enzymes needed to manufacture them. That information lies in “biosynthetic gene clusters.”

But long DNA molecules fray as they’re copied as bacteria reproduce, from ancient times leaving pieces too small to match entries in DNA databases from modern species. But recent strides in computing have enabled overlapping tiny DNA fragments to piece together unknown genes and even genomes, from chunks exceeding 100,000 bases. Said co-lead author of the study Alexander Hübner of the three-year effort, “We had to completely rethink our approach. We can now start with billions of unknown ancient DNA fragments and systematically order them into long-lost bacterial genomes of the Ice Age.” Added first author Martin Klapper, “This is the first step towards accessing the hidden chemical diversity of earth’s past microbes, and it adds an exciting new time dimension to natural product discovery.”

The analysis yielded reconstructed “paleomes” from tartar, representing 459 bacterial species. The computational process is like cutting up a dictionary, a bible, and a textbook into multi-page pieces, mixing them, and then consulting the words at the beginnings and ends of the unbound pages to reconstruct the three books.

The researchers use the term “metagenome-assembled genomes,” or MAGs, to describe “a genome deduced and reconstructed from a sample containing many genomes (a metagenome).” They liken the strategy to “a billion-piece jigsaw puzzle.” The strategy can identify modern bacteria that won’t grow in a lab – or ancient ones.

Why seek clues to manufacturing drugs in ancient bacterial genes? Many of the natural products that we use as drugs are chemically complex and difficult to synthesize – but evolution has enabled microorganisms to figure it out. Bacterial genomes encode enzymes used to construct the useful molecules, so it is a little like we’re borrowing their weapons.

We’ve done well tapping the antibiotic potential of modern bacteria, but these single-celled organisms go back more than 3 million years.

Mouths Full of Fossils

The researchers collected tartar from 12 Neanderthals dating to 102,000–40,000 years ago; from 34 humans from 30,000 to 150 years ago; and from 18 modern humans.

An example that the team discussed in news releases prior to the publication of their report is a genome more than 90,000 years old related to bacteria of genus Chlorobium. That MAG harbored a biosynthetic gene cluster in tartar from seven Paleolithic humans and Neanderthals.

All seven reconstructed Chlorobium genomes yielded several unknown biosynthetic gene clusters that might include potential antibiotics. One in particular, fortunately renamed “paleofurans” from the initial 5-alkylfuran-3-carboxylic acid, joins the list of natural products that might yield novel antibiotic drugs, or suggest how to synthesize them.

Anan Ibrahim, co-lead author of the study explained. “The dental calculus of the 19,000-year-old Red Lady of El Mirón, Spain yielded a particularly well-preserved Chlorobium genome. Having discovered these enigmatic ancient genes, we wanted to take them to the lab to find out what they make.” The Red Lady was from a group of hunter-gatherers trapped in a cave.

Extracting information from her dental remains was possible because of the universality of the genetic code – in all organisms (and viruses), the same RNA triplets encode the same amino acids. So modern bacteria can produce the protein encoded in an extinct microbe’s DNA sequence.

Discovery of the First Antibiotic Drugs

Metagenomics is a modern biotechnology that discovers potentially useful genes among collections of diverse genomes sampled from a natural environment, such as soil or seawater – or an old tooth.

Soil is a particularly rich resource for finding compounds with medicinal properties. The second, third, and fourth antibiotics discovered came from soil bacteria – streptomycin, chloramphenicol, and tetracycline. Finding the first, of course, began with Alexander Fleming’s observation that a fleck of fungus (Penicillium notatum) killed Staphylococcus bacteria growing on a petri dish. Many references call the organism a bread mold, for its green fuzzy appearance on baked goods, which isn’t correct, taxonomically speaking. But whatever the organisms are called, ancient peoples, like the Mayans and Ukrainians, used the fungi to dress wounds.

In 1945, Alexander Fleming, Boris Chain, and Howard Florey were awarded the Nobel prize for developing penicillin.

The story began in 1921 at St. Mary’s Hospital in London, where Fleming discovered that a substance from when he blew his nose killed certain types of bacteria. His mucus musings led to his identifying lysozyme, an enzyme that kills bacteria by shattering their cell walls. Mutations that enable bacteria to evade lysozyme would eventually explain some antibiotic resistance.

In the late 1920s, Florey, a pathologist from Australia doing research at the University of Sheffield, was working on lysozyme too. He was intrigued by finding that gut bacteria, E. coli, stopped other types of bacteria from growing, a phenomenon called antibiosis. It involved lysozyme.

Then in 1928, Alexander Fleming returned from holiday and noticed the soon-to-be famous fungus clearing out staph bacteria on a petri dish. He tried to isolate the responsible compound but gave up on the complex chemistry of the natural product.

“His basic insight into penicillin was thus at once both decisive and defective, a potential breakthrough thwarted by a failure of vision,” wrote John Galbraith Simmons in Doctors & Discoveries: Lives That Created Today’s Medicine. Fleming’s finding was published in 1929 in The British Journal of Experimental Pathology. Here’s info from his notebooks.

The other thread of the story picks up in 1935, when chemist Boris Ernst Chain joined Florey, at Oxford, working on how lysozyme dismantles bacterial cell walls. When they probed the medical literature for reports of natural substances that induce the effect, they came upon Fleming’s penicillin paper. Florey and Chain purified and formulated the fungal extract into an injectable form and treated mice infected with strep.

Results in the sick mice were striking. In 1941 came the first test on a human – a man who’d lost an eye to a strep infection. He nearly died, but recovered after getting the new miracle drug.

With the value of penicillin quickly realized and the world at war, efforts ramped up to get it to the troops: clinical trials in 1942, large-scale production in 1943, widespread use within a few years. Early versions were injected – I remember getting shots in the tush as a kid – but soon were reinvented as pink powdery pills and liquids.

But evidence of resistance to penicillin soon arose. By 1950, 40 percent of staph infections were resistant and by 1960, 80 percent. In response, by 1962, more than 20 classes of antibiotics had been developed.

Fleming might not have been the world’s best synthetic organic chemist at the time, but he was prophetic, noting that “microbes are educated to resist penicillin.” A decade earlier, Barbara McClintock had described the mechanism of resistance through her discovery of transposable elements in corn – “jumping genes” – that would explain how stretches of DNA can move from one genome to another, even across species. Then natural selection would take over, favoring an adaptive trait.

Spots on corn revealed jumping genes; in bacteria, it was the transfer of antibiotic resistance. About 45 percent of a human genome consists of these transposable elements, most of them silenced into inactivity.

Barbara McClintock received the Nobel Prize in 1983. I got my PhD in 1980 and was trained by maize geneticists so knew McClintock’s story well. I recall that when biotech companies began springing up a few years later, many news reports credited discovery of jumping genes to molecular biologists working with microorganisms. But it was a woman and her spotted corn plants that revealed the transposons that fuel antibiotic resistance.

Coda

The metagenomic approach to discovering new antibiotics is a giant leap forward, systematizing the ages-old practice of researchers bringing soil samples home from trips. Technology can now sort through the DNA, like identifying the components of a complex vegetable soup. Extending that capability to microbial remnants on teeth from Neanderthals and ancient ancestors may open up an entire new source of antibiotic drugs.