Millennial-scale microbiome analysis reveals ancient antimicrobial resistance conserved despite modern selection pressures

Abstract Background Antimicrobial resistance presents a formidable challenge, yet its existence predates the introduction of antibiotics. Our study delves into the presence of antimicrobial resistance genes (ARGs) in ancient permafrost microbiomes, comparing them with contemporary soil and pristine environments. Majority of the samples are from regions around Beringia, encompassing parts of Russia and Alaska, with only one sample originating from the Tien Shan Mountain range in Kyrgyzstan. Results From over 2.3 tera basepairs of raw metagenomic data, retrieved from samples ranging in age from approximately 7,000 years to 1.1 million years, we assembled about 1.3 billion metagenomic contigs and explored the prevalence of ARGs within them. Our findings reveal a diverse array of ARGs in ancient microbiomes, akin to contemporary counterparts. On average, we identified 2 ARGs per rRNA gene in ancient samples. Actinomycetota, Bacillota, and several thermophiles were prominent carriers of ARGs in Chukochi and Kamchatkan samples. Conversely, ancient permafrost from the Tien Shan Mountain range exhibited no Thermophiles or Actinomycetota carrying ARGs. Both ancient and contemporary microbiomes showcased numerous divergent ARGs, majority of which have identity between 40 and 60% to genes in antibiotic resistance gene databases. To study the selection pressure on ARGs, we performed dN/dS analysis specifically on antibiotic inactivation-type ARGs, which exhibited purifying selection compared to contemporary genes. Conclusion Antibiotic resistance has existed throughout microbial evolution and will likely persist, as microbes have the capacity to develop and retain resistance genes through evolutionary processes. The classes of antimicrobial resistance genes profiled and the function of antibiotic-inactivating enzymes from ancient permafrost microbiomes do not seem to be very different from the genes found in the antibiotic era. Additionally, we retrieved 359 putative complete viruses from ancient microbiomes and none of them harboured any ARGs.