Culturing Ancient Bacteria Carrying Resistance Genes from Permafrost and Comparative Genomics with Modern Isolates

Long considered to be a consequence of human antibiotics use by deduction, antibiotic resistance mechanisms appear to be in fact a much older phenomenon as antibiotic resistance genes have previously been detected from millions of year-old permafrost samples. As these specimens guarantee the viability of archaic bacteria, we herein propose to apply the culturomics approach to recover the bacterial content of a Siberian permafrost sample dated, using the in situ-produced cosmogenic nuclide chlorine36 (³⁶Cl), at 2.7 million years to study the dynamics of bacterial evolution in an evolutionary perspective. As a result, we cultured and sequenced the genomes of 28 ancient bacterial species including one new species. To perform genome comparison between permafrost strains and modern isolates we selected 7 of these species (i.e., Achromobacter insolitus, Bacillus idriensis, Brevundimonas aurantiaca, Janibacter melonis, Kocuria rhizophila, Microbacterium hydrocarbonoxydans and Paracoccus yeei). We observed a high level of variability in genomic content with a percentage of shared genes in the core genomes ranging from 21.23% to 55.59%. In addition, the Single Nucleotide Polymorphism (SNP) comparison between permafrost and modern strains for the same species did not allow a dating of ancient strains based on genomic content. There were no significant differences in antibiotic resistance profiles between modern and ancient isolates of each species. Acquired resistance to antibiotics was phenotypically detected in all gram-negative bacterial species recovered from permafrost, with a significant number of genes coding for antibiotic resistance detected. Taken together, these findings confirm previously obtained data that antibiotic resistance predates humanity as most of antimicrobial agents are natural weapons used in inter-microbial conflicts within the biosphere.