Ancient DNA from İnönü Cave reveals 5,000-year-old antibiotic resistance and early medical use of coal derivatives
Antibiotic resistance history
Extracted from soil samples
Early gastrointestinal treatments
When archaeologists began excavating İnönü Cave in Zonguldak, Turkey, they expected to find the usual artifacts of ancient human life: tools, pottery, and animal bones. Instead, they uncovered a medical mystery preserved in soil—one that would push back the recorded history of antibiotic resistance by thousands of years and reveal surprisingly sophisticated ancient medical treatments using an unexpected substance: coal.
Their findings reveal that antibiotic resistance isn't merely a modern consequence of medical overprescribing, but an ancient phenomenon with deep environmental roots 1 5 .
In a groundbreaking study bridging archaeology and microbiology, researchers have extracted genetic secrets from soil samples dating back to the Chalcolithic Age (approximately 4300 BCE). Even more remarkably, they discovered evidence that inhabitants of the cave 5,000 years ago used coal derivatives to treat gastrointestinal illnesses—a finding that rewrites both medical history and our understanding of ancient human ingenuity 3 7 .
Antibiotic resistance genes identified dating back 6,300 years, challenging modern assumptions about their origins.
Evidence of early Bronze Age inhabitants using coal derivatives for gastrointestinal treatments.
Soil acts as a remarkable biological archive because it accumulates evidence of human activities over time. As the researchers note, "Human activity affects soil properties and is accompanied by the introduction of specific organic matter into the soil. After removing the anthropogenic influence, soil microorganisms lose organic matter due to mineralization and transformation. However, the former anthropogenic influence on soils can be preserved in the soil microbiota and their activities" 1 8 .
Soil samples were carefully collected from four distinct cultural layers in İnönü Cave, spanning from the Chalcolithic Age to the Early Iron Age 1 5 . This stratification allowed researchers to track changes in microbial communities and resistance genes across different historical periods.
Using the DNeasy PowerSoil Pro Kit, researchers isolated genetic material from the soil samples, taking special precautions to prevent contamination with modern DNA 1 .
The team employed next-generation sequencing (NGS) methods, specifically targeting the 16S rRNA gene for identifying bacterial communities and conducting metagenomic analyses to detect antibiotic resistance genes 1 5 .
The sequenced DNA was compared against known genetic databases to identify both microbial species and antibiotic resistance genes. The presence of specific resistance genes was confirmed through multiple rounds of analysis 1 .
The results were startling. The researchers identified antibiotic resistance genes in three different historical periods within the cave:
Simultaneously, analysis of the Early Bronze Age layer (approximately 3000 BCE) revealed that inhabitants suffered from gastrointestinal illnesses and apparently used coal derivatives for treatment—an astonishing finding that predates the documented discovery of coal in the region by thousands of years 3 7 .
"This tells us that resistance genes are not actually the problem of our century, but have a history dating back 5,000 years" 3 .
The genetic analysis revealed a diverse community of microorganisms that called İnönü Cave home across millennia.
| Bacterial Group | Ecological Role & Significance |
|---|---|
| Acidobacteriota | Common in soil, involved in organic matter decomposition |
| Actinobacteriota | Known for producing natural antibiotics; important in drug discovery |
| Bacteroidota | Play role in carbon cycling and organic matter decomposition |
| Chloroflexi | Photosynthetic capabilities; diverse metabolic functions |
| Cyanobacteria | Oxygen-producing photosynthetic bacteria |
| Firmicutes | Includes many human gut bacteria; some can form protective spores |
| Myxococcota | Predatory bacteria that hunt other microbes |
| Proteobacteria | Large, diverse phylum including many pathogens |
This microbial diversity underscores how soil serves as a reservoir for bacteria with diverse capabilities, including natural antibiotic production and resistance mechanisms.
The discovery of specific antibiotic resistance genes across different historical periods provides compelling evidence for the ancient origins of this phenomenon.
| Historical Period | Approximate Date | Resistance Gene Identified | Function & Significance |
|---|---|---|---|
| Chalcolithic Age | 4300 BCE | tetA | Confers resistance to tetracycline antibiotics |
| Early Bronze Age | 3000 BCE | class 1 integron intl1 | Associated with mobile genetic elements that can capture and spread resistance genes |
| Late Bronze Age | 1500 BCE | OXA58 | Oxacillinase gene providing resistance to beta-lactam antibiotics |
As one researcher noted, "This bize aslında direnç genlerinin çağımızın problemi olmadığı, bundan 5 bin yıl öncesine dayanan geçmişinin olduğu bilgisini verdi" ("This tells us that resistance genes are not actually the problem of our century, but have a history dating back 5,000 years") 3 .
Conducting such sophisticated ancient DNA research requires specialized materials and reagents.
| Reagent/Kit | Function in Research |
|---|---|
| DNeasy PowerSoil Pro Kit | Specialized DNA extraction optimized for soil samples which often contain substances that inhibit PCR |
| SmartChip Real-Time PCR System | High-throughput platform for simultaneously quantifying numerous target genes |
| 16S rRNA Primers | Target conserved bacterial genes to identify and classify microorganisms |
| Qubit Fluorometer with dsDNA HS Assay | Precisely measures DNA concentration in small or dilute samples |
| Custom ARG Primer Sets | Designed to detect specific antibiotic resistance genes in environmental samples |
Recent research has revealed another worrying dimension to environmental antibiotic resistance. A 2025 study published in Nature Ecology & Evolution demonstrated that climate warming is making soil bacteria more likely to carry antibiotic resistance genes 9 . As temperatures rise, soil bacteria become more adaptable, more active, and better equipped to survive medicines meant to kill them.
"Most people don't realize that many infections come from bacteria that start in the natural environment. As soil bacteria become more resistant, the chance of untreatable infections rises" 9 .
The study predicts that soil antibiotic resistance gene levels could rise by up to 23% by 2100 if high greenhouse gas emissions continue.
The İnönü Cave project exemplifies an emerging trend in scientific research: the integration of disparate disciplines to answer complex questions about our past and present.
Follow the evolution of specific pathogens over millennial timescales to understand how they've adapted and changed.
Learn how historical human activities shaped modern microbial ecosystems and resistance patterns.
Create better predictive models for antibiotic resistance spread based on historical patterns.
Uncover ancient therapeutic compounds lost to history that might inform modern medicine.
The soil of İnönü Cave has yielded insights far beyond what its ancient inhabitants could have imagined.
What we often consider modern problems frequently have deep historical roots.
Human interactions with microbes—and our attempts to control them—have been shaping microbial evolution for millennia.
The discovery of 5,000-year-old antibiotic resistance genes and evidence of early medical innovation with coal derivatives teaches us two profound lessons, as Doç. Dr. Şükran Öztürk reflected: "Antibiyotik direnci sadece kullanıma bağlı değil, günümüzden 6300 yıl öncesine ait bir dönemden itibaren aslında var olmuş" ("Antibiotic resistance is not only related to use, but has actually existed since a period 6300 years ago") 7 .
The interdisciplinary approach pioneered in studies like the İnönü Cave project—merging archaeology, microbiology, and environmental science—offers our best hope for untangling the complex relationship between human activities and the microbial world that surrounds, inhabits, and profoundly influences us.