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

Abstract

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.

Metaplasmidome-encoded functions of Siberian low-centered polygonal tundra soils

“…Finally, it is also possible that these genes are naturally present within Siberian bacteria as a response to naturally produced antibiotic-like compounds. Antibiotic-resistant genes have now been reported in various soil samples from Arctic and Antarctic regions, including millions-year-old permafrost, i.e., these genes originate from the pre-antibiotic era in medicine [24,62,71,72]. Detection of antibiotic resistance genes in bacterial species recovered from permafrost indicates that antibiotic resistance predates humanity and these are natural resistance mechanisms to antimicrobial agents produced and used by microorganisms as weapons in intermicrobial competition within the biosphere [72].…”

Genome Analysis of Acinetobacter lwoffii Strains Isolated from Permafrost Soils Aged from 15 Thousand to 1.8 Million Years Revealed Their Close Relationships with Present-Day Environmental and Clinical Isolates

“…In this regard, the search for antibiotic resistance genes in microorganisms that have not previously come into contact with the human population is of great interest [ 12 ]. Many studies on permafrost have reported the presence of genes coding for resistance to several classes of antibiotics [ 13 , 14 , 15 , 16 , 17 , 18 ] and heavy metals [ 19 , 20 ] similar to ones observed in present day clinical and environmental strains. The Psychrobacter maritimus strain, resistant to tetracycline and streptomycin, was isolated from 15,000–35,000 years old permafrost sediments sampled from the coast of the East Siberian Sea.…”

“…Perron et al used functional metagenomics to retrieve antibiotic resistance genes from bacteria isolated from Canadian high Arctic permafrost and identified genes conferring clinical levels of resistance against aminoglycoside, β-lactam and tetracycline antibiotics [ 17 ]. Twenty eight bacterial species were cultured form a Siberian permafrost sample dated at 2.7 million years and resistance to antibiotics was phenotypically detected in all 10 Gram-negative species [ 18 ]. No significant differences in antibiotic resistance profiles between modern and ancient isolates of each species were found [ 18 ].…”

“…Twenty eight bacterial species were cultured form a Siberian permafrost sample dated at 2.7 million years and resistance to antibiotics was phenotypically detected in all 10 Gram-negative species [ 18 ]. No significant differences in antibiotic resistance profiles between modern and ancient isolates of each species were found [ 18 ]. Direct evidence for the presence of antibiotic resistance genes in the permafrost microbiome has been obtained as a result of metagenomic analyses of 30,000-year-old permafrost sediments in Alaska [ 15 ].…”