Permafrost Strains

It is known that bacteria of various systematic groups are characterized by different intrinsic antibiotic resistance; moreover they may be cross-resistant to several classes of antimicrobial agents [Piddock, 2006]. Bacteria may also acquire resistance via the horizontal transfer of specific genes from other organisms [Davies, 1994; Tenover, 2006]. To study the mechanisms of antibiotic resistance in our collection of multidrug resistant permafrost bacteria we determined the localization of the resistance genes and their possible association with different mobile elements (plasmids and transposons).

We first analyzed whether the resistance determinants can have plasmid localization. Indeed, it was shown that most of the permafrost antibiotic resistant strains contained plasmids of different sizes (data not shown). For analysis of plasmid localization of the resistance genes, antibiotic resistant strains harbouring large plasmids were chosen (see Fig. 2). Plasmid DNA was extracted by an alkaline lysis method and analyzed by electrophoresis on 0.7 % (wt/vol) agarose gel in Tris-borate buffer, followed by staining with ethidium bromide and visualization under UV light [Sambrook et al., 1989]. The molecular sizes of the plasmids were evaluated by comparison with reference plasmids R388 (34 kb) [Datta & Hedges, 1972] and RP4 (60 kb) [Jacob & Grinter,1975].

Figure 2. Plasmids of bacterial strains isolated from permafrost. Horizontal agarose gel electrophoresis (run at 50V for 18h): 1=Tik1, 2=R388, 3=ED6-1, 4=RP4, 5=MR29-12, 6=ED94-71, 7=ED23-26, 8= ED23-35, 9=ED45-25

For detailed investigations we selected five strains of Pseudomonas, three strains of Acinetobacter and one strain of Psychrobacter psychrophilus, each containing a single large plasmid (about 25-55 kb) (see Fig. 2). The association of resistance genes with R plasmids was determined in conjugation and transformation experiments with laboratory recipient bacterial strains. For Acinetobacter and Psychrobacter strains, the Acinetobacter calcoaceticus strain BD413rif was used as a recipient; and for Pseudomonas strains, the Pseudomonas fluorescens strain P22-1-2 was used (Table 3). It should be noted that the latter strain was resistant to four different antibiotics (Cb, Cm, Sp, Tp). For this reason association of the corresponding resistance determinants with plasmids in Pseudomonas strains by conjugation experiments could not be evaluated with assurance.

We determined that in four strains, only mercury resistance was transferred to appropriate recipient; in three, both mercury and streptomycin resistance determinants were transferred, and in additional two plasmid localization was revealed for streptomycin and tetracycline resistance, and streptomycin and sulphathiazole resistance, respectively (Table 3).

Table 3. Plasmid localization of resistance determinants in permafrost strains

Resistance

Permafrost strains

Resistance pattern

determinants associated with R plasmids

Acinetobacter sp. ED23-35

Hg, Sm, Sp

Hg, Sm

Acinetobacter sp. ED45-25

Hg, Sm

Hg, Sm

Acinetobacter jonsonii M2-7

Ap, Hg, Tp

Hg

Psychrobacter psychrophilus MR29-12

Sm, Tc, Tp

Sm, Tc

Pseudomonas fluorescens ED23-26

Hg, Km, Cb, Cm, Sp, Tp

Hg

Pseudomonas sp. ED94-71

Hg, Km, Cb, Cm, Sp, Tp

Hg

Pseudomonas sp. (fluorescent) EDM6-1

Hg,Cb, Tp

Hg

Pseudomonas putida Tik1

Hg, Km, Cb, Cm, Sp, Tp

Hg, Sm

Pseudomonas sp. Tik3

Sm, Su, Cm, Sp, Tp

Sm, Su

Recipient strains:

Acinetobacter calcoaceticus BD413rif 1

[rif-r]

Pseudomonas fluorescens P22-1-22

Cb, Cm, Sp, Tp [str-r, rif-r]

Resistance determinants that were also present in Pseudomonas recipient strain, and therefore could not be analyzed by plasmid transfer, are shown in bold used in matings with strains of Acinetobacter and Psychrobacter psychrophilus; used in matings with strains of Pseudomonas; str-r - 30S-ribosome resistance; rif-r - mutations of RNA-polymerase gene rpoB

Resistance determinants that were also present in Pseudomonas recipient strain, and therefore could not be analyzed by plasmid transfer, are shown in bold used in matings with strains of Acinetobacter and Psychrobacter psychrophilus; used in matings with strains of Pseudomonas; str-r - 30S-ribosome resistance; rif-r - mutations of RNA-polymerase gene rpoB

In order to investigate the possible role of R-plasmids in resistance transfer between different bacteria we determined the host range of permafrost plasmids revealed. As recipients in conjugation experiments, laboratory strains P. fluorescens P22-1-2, A. calcoaceticus BD413rif and E. coli K-12 C600rif were used. It was revealed that none of the plasmid had the properties of broad host range plasmids capable to disseminate the resistance markers between unrelated bacteria (Table 4). At the same time, plasmids of some pseudomonads transferred into strains belonging to other Pseudomonas species. For instance, plasmids of P. putida Tik1 and P. sp. ED94-71 (non-fluorescent) could be transferred between various laboratory strains of P. putida and P. fluorescens (not shown).

It is of interest that among Pseudomonas and Acinetobacter plasmids studied we revealed several ones that harboured only mercury resistance genes. At the same time, we succeeded in detection of R plasmids harbouring both mercury-resistance and antibiotic-resistance determinants. In this connection it should be noted that in the history of clinical Pseudomonas plasmids, resistance to HgCl2 was the first marker recognized before 1969. Drug resistance plasmids of Pseudomonas studied thereafter conferred resistance to antibiotics of different families in addition to the mercury resistance [Kontomichalou, Papachristou & Angelatou, 1976]. Therefore, our results support the previous conclusions [Hughes & Datta, 1983;

Davies, 1994] that antibiotic resistance determinants could be "picked up" by bacterial plasmids as a consequence of introducing antibiotics in medicine and veterinary practice. The same phenomenon likely occurred in the environment but with a substantially lower rates.

Table 4. Determination of host-range of plasmids from permafrost bacterial strains

Donor strain carrying R-plasmid

Transfer ability of R-plasmids in crosses with recipient strains

Acinetobacter BD413rif

Pseudomonas P22-1-2

E.coli C600rif

Acinetobacter sp.ED23-35

+

-

-

Acinetobacter sp.ED45-25

+

-

-

P. psychrophilus MR29-12

+

-

-

P. fluorescens ED23-26

-

+

-

P. sp.(non-fluorescent) ED94-71

-

+

-

P. putida Tik1

-

+

-

+ transfer of R-plasmid is observed; - transfer is absent

+ transfer of R-plasmid is observed; - transfer is absent

We further analyzed whether the antibiotic resistance determinants with plasmid localization can be associated with transposons. We succeeded in isolation of two transposons containing antibiotic resistance genes from strains Psychrobacter psychrophilus MR29-12 and Pseudomonas sp. Tik3, respectively (see Tables 2 and 3). The first of these transposons carried determinants conferring resistance to streptomycin and tetracycline; the second - to streptomycin and sulphothiazole. [Petrova et al., 2008]. We further demonstrated that both transposons could translocate onto plasmids with broad-host-range spectrum and could be transferred into bacteria of different systematic groups including Escherichia coli and Pseudomonas spp. (data not shown). Further chracterization of R-plasmids and antibiotic resistance transposons identified in permafrost bacterial strains will be an important goal of future studies.

For a substantial fraction of the antibiotic resistant strains from our collection (in particular, various Pseudomonas and Acinetobacter strains), antibiotic resistance determinants could be transferred by neither conjugative transfer of plasmid DNA nor plasmid DNA transformation. One can suggest that in this case the resistance phenotype could result from chromosomally encoded intrinsic resistance [Gomez & Neyfakh, 2006]. To check this hypothesis, we analyzed whether the resistance determinants not associated with plasmids can be transferred between species by genomic DNA transformation.

Multidrug resistant Acinetobacter strains MR5-11 and VS15 which contained no plasmids were used as donor strains and model laboratory auxothrophic strain Acinetobacter calcoaceticus BD413ivl (Ivl-10) competent for transformation [Juni, 1972] was used as a recipient strain. For transformation of genomic DNA, a simple transformation assay procedure described for Acinetobacter spp was used [Juni, 1972]. Preparation of crude transforming DNA from donor strains was performed as described in [Sambrook et al., 1989]. As a marker phenotype to follow the transformation of antibiotic resistance, we used chloramphenicol resistance (CmR). To evaluate the efficiency of transformation, we compared the frequency of transformation of chloramphenicol resistance (CmS^CmR) with that of transformation from auxotrophy to prototrophy (Ivl-^ Ivl+).

The results of experiments (Table 5) showed that frequency of homologous transformation of BD413ivl to prototrophy with DNA isolated from prototrophic BD413 strain (2.3 x 10-5) exceeds that of heterologous transformation with DNA isolated from permafrost Acinetobacter strains (1.6x10-6 and 7.0x10-6, Table 5). This agrees with published data on the efficiencies of homologous and heterologous transformation for Acinetobacter species [Juni, 1972]. We also observed transformation of chloramphenicol resistance (Cm-r) from the resistant permafrost strains to the sensitive BD413 ivl strain. The frequency of Cm-r transformation (2.8-9.0x10-7) was comparable to the frequency of the Ivl-^ Ivl+ transformation. Remarkably, no transformation of chloramphenicol resistance was observed when control chloramphenicol sensitive strain was used as DNA source (Table 5).

Table 5. Transformation of Acinetobacter calcoaceticus BD413ivl with genomic DNA

Source of DNA

Frequency of transformations (per 1p.L" DNA)

Ivl+

Cm-r

VS15 (Ivl+,Cm-r)

1.6x 1Q-6

9.Q x 1Q-7

MR5-11 (Ivl+,Cm-r)

7.Qx 1Q-6

2.8 x1Q-7

BD413 (Ivl+,Cm-s)

2.3 x 1Q-5

< 2.Q x1Q-8

Frequency of transformation was determined as a ratio of the number of transformants to the total number of recipient strain colonies

Frequency of transformation was determined as a ratio of the number of transformants to the total number of recipient strain colonies

Taking into account that a single chromosomal gene can afford intrinsic resistance to different antibiotics [Poole, 2005; Piddock, 2006], we compared the resistance pattern of the BD413 Cm-r transformants to the resistance pattern of original antibiotic resistant Acinetobacter strains. In particular, we analyzed resistance of the transformants to streptomycin, spectinomycin, trimethoprim and nonspecific DNA intercalating agent ethidium bromide. To assess the level of antibiotic resistance, we used a twofold serial dilution technique [Hirai et al., 1986]. The acquisition of resistance was defined as at least fourfold increase in the minimal inhibition concentration (MIC). Analysis of antibiotic susceptibility patterns showed that Cm-r transformants also displayed higher levels of resistance to streptomycin, spectinomycin and trimethoprim. Moreover, they were characterized by increased resistance to ethidium bromide (Table 6). Thus, the antibiotic resistance pattern of wild type Acinetobacter strains VS15 and MR5-8 could be transferred to the laboratory strain of the same genus by transformation of their chromosomal DNA, suggesting that the multidrug resistance phenotype can be explained by chromosomally encoded intrinsic resistance. Indeed, such intrinsic resistant has been previously described for some species of Acinetobacter [Magnet et al., 2001; Gomez & Neyfakh, 2006].

Table 6. Transfer of antibiotic resistance pattern between permafrost and laboratory strains of Acinetobacter

Table 6. Transfer of antibiotic resistance pattern between permafrost and laboratory strains of Acinetobacter

Strain

MIC (|g/ml)

Cm

Sm

Sp

Tp

EthBr

Permafrost resistant strains

MR5-11

64-128

>256

256

32

128

VS15

>32

>256

>256

32

64

Laboratory sensitive strain

BD413ivl

8

4-8

8-16

8

16-32

Transformants

BD413ivl/VS15

64

>128

128

32

128

BD413ivl/MR5-11

64

>128

128

32

128

MICs were determined in three independent experiments performed by the agar dilution method using the Mueller-Hinton medium; Cm-chloramphenicol; Sm-streptomycin; Sp-spectinomycin; Tp-trimethoprim; EthBr - ethidium bromide.

MICs were determined in three independent experiments performed by the agar dilution method using the Mueller-Hinton medium; Cm-chloramphenicol; Sm-streptomycin; Sp-spectinomycin; Tp-trimethoprim; EthBr - ethidium bromide.

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