To study the antibiotic resistance in ancient bacteria, five antibiotics including three aminoglycosides (gentamicin, kanamycin, streptomycin), tetracycline and chloramphenicol were chosen. For isolation of antibiotic resistance strains, bacteria from water suspensions of permafrost samples were plated on antibiotic supplemented solid media. Single colonies, formed on antibiotic containing plates were replated three times on the same media and incubated at 25°C. The concentrations of antibiotics in nutrient media were as follows (^g/ml): chloramphenicol (Cm), - 20; gentamicin (Gm), - 5-10; kanamycin (Km), - 25-50; streptomycin (Sm), - 50-100; tetracycline (Tc), - 10-20. At these concentrations, the fraction of bacteria, able to grow on selective nutrient media, to those unable to grow, was usually varied from between 1/10-2 and 1/10-3 CFU/g.
We succeeded in isolation of antibiotic resistant bacteria in all analyzed samples from both Arctic and Antarctic sediments. In general, the number of viable cells that were able to grow on media supplied with antibiotics was more than 103 CFU/g. It was noted that the number of antibiotic resistant bacteria did not decrease with the age of permafrost (data not shown).
In addition to direct selection of antibiotic resistant bacteria we isolated about 300 strains grown on nutrient media in the absence of antibiotics, and further studied their resistance to different antibiotics.
Study of cultural, morphological and biochemical properties of antibiotic resistant bacteria, as well as method of partial sequencing of 16S rRNA genes, allowed to identify the strains as representatives of genera Acinetobacter, Pseudomonas, Xanthomonas, Bacillus, Arthrobacter, Micrococcus, Flavobacterium, Psychrobacter, Stenotrophomonas, Sphingomonas, Brevundimonas, Paenibacillus. It was revealed, that the majority of the antibiotic resistant strains were Gram-positive bacteria. It is noteworthy that the bacterial diversity in our collection of antibiotic resistant permafrost strains was similar to that of strains isolated earlier from other cold Arctic and Antarctic environments [Vishnivetskaya et al., 2006, Gilichinsky et al., 2007]. In particular, antibiotic resistant strains included psychrophilicbacteria.
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