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Study of Antibiotic Resistance Spectrum of Permafrost Strains

About half of the antibiotic resistant strains obtained after initial selection, were chosen for further work. Among them we identified 53 strains of Gram-positive and 30 strains of Gram-negative bacteria. For each of these strains we determined the spectrum of resistance to all antibiotics used previously (Km, Gm, Sm, Tc, Cm). This analysis revealed not only strains resistant to a single antibiotic, but also strains resistant to two and three different antibiotics. The frequency of strains with double antibiotic resistance exceeded 20% both in Grampositive and Gram-negative bacteria, while the strains with triple resistance were found only among Gram-negative bacteria with 20% frequency [Mindlin et. al., 2008].

Twenty five strains of Gram-negative bacteria and four strains of Gram-positive bacteria, most of them resistant to at least two antibiotics, were chosen for detailed analysis of resistance determinants (Table 2). Phylogenetic affiliation of all isolates using 16S rRNA gene sequencing allowed to identify ten strains belonging to genus Pseudomonas, eight Acinetobacter, two Stenotrophomonas, two Sphingomonas strains, and four strains belonging to Gram-positive genus Paenibacillus. The remaining three strains were found to belong to Xanthomonas, Psychrobacter and Brevundimonas genera, respectively (Table 2).

For each of these strains we determined the spectrum of resistance to wide range of antibiotics (beta-lactams, aminoglycosides, tetracyclines, sulphonamide drugs, trimethoprim and quinolones), including those used during the first step of selection. Besides, we determined the mercury resistance of these strains. Susceptibility of the resistant strains to different antimicrobial agents was determined by the agar diffusion and the agar dilution methods using the Mueller-Hinton agar [Hirai et al., 1986; Andrews, 2007]. This analysis revealed that the most permafrost strains studied were resistant to antibiotics of different classes (Table 2). For instance, strain MR29-12 of Psychrobacter psychrophilus was simultaneously resistant to streptomycin, tetracycline and trimethoprim; strain MR5-8 of Acinetobacter sp. was resistant to five antibiotics (Ap, Cm, Gm, Km, Tb); and strain ED23-35 of Acinetobacter sp. was simultaneously resistant to HgCl2 and streptomycin. It is interesting to note that, independently of the isolation procedure (the presence or absence of particular antibiotics during isolation), many strains were simultaneously resistant to two or more structurally unrelated antibiotics. As an example, strain Tiki of Pseudomonasputida that was isolated in the absence of antimicrobial agents was simultaneously resistant to HgCl2, carbenicillin, chloramphenicol, streptomycin and trimethoprim, which belong to different classes of antibacterial agents (Table 2).

Antibiotic designations and concentrations used (|g/ml): ampicillin (Ap), -200; chloramphenicol (Cm), -20; gentamicin (Gm), -5; HgCl2 (Hg), -5; carbenicillin (Cb), -400; kanamycin (Km), -25 (50-200); nalidixic acid (Nal), -20; neomycin (Nm), -25; streptomycin (Sm), -50(100); spectinomycin (Sp), -100; sulphathiazole (Su), -25; tobramycin (Tb), -40; tetracycline (Tc), -15; trimethoprim (Tp), -25. Resistance to Cb always acompany Ap-resistance; Parentheses indicate low-level resistance A-Arctic, B-Antarctic; Age in years before present (YBP). K=10 years; M=10 years; Sphingomonas were originally named Pseudomonas paucimobilis but were later reclassified into distinct species within a new genus Sphingomonas [Yabuuchi et al., 1990]; A new Pseudomonas species, Pseudomonas libanensis sp. nov. is part of the Pseudomonas fluorescens intrageneric cluster [Dabboussi et al., 1999]; Bacteria belonging to Brevundimonas were previously classified in the genus Pseudomonas but later were included into a deeply branching cluster in the stem of gram-negative bacteria [Ochi, 1995].

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