Experimental evaluation of biological risks of introduction of the gene-engineering-modified microorganism (GMM) B. subtilis VKPM V-7092 into the environment

      E.A. Stavskiy ¹, A.I. Lelyak ², N. M. Larina ³, O.N. Grishaeva ⁴, V.V. Serebrov ⁵, Yu. A. Gorbunov ¹,
      L. G. Duben’ ⁶, L. A. Timukhina ³, L. V. Mit’ko ³,
      R. S. Koval ³, V.P. Khodyrev ⁵

¹ State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Novosibirsk region;
² JSC SPC “Research Center”, Koltsovo, Novosibirsk region;
³ SVDI “Regional Veterinary Laboratory”, Accredited Testing Center, Affiliated Agency of Certification of Veterinary Preparations and Feeds FSA VSRCI, Novosibirsk;
⁴ JSC “Vector-Best”, Koltsovo, Novosibirsk region;
⁵ RI of Animal Systematic and Ecology SB RAS, RF, Novosibirsk;
⁶ Anti-plague Station, Medical and Sanitary Station 163 FSA “Medbioextrem”, Koltsovo, Novosibirsk region

Abstract

            Experimental evaluation of biological risks of introducing the genetically modified microorganism (GMM) B. subtilis VKPM B–7092, the active ingredient of the probiotic VETOM 1.1, into an open system has been performed. The survival of the above GMM in bovine gastroenteric tract, its influence on microbiocenosis, the species composition of microflora of the gastroenteric tract, the possibility of transfer of the DNA fragment cloned in B. subtilis bacterium and containing the gene of human leukocytic alpha-2 interferon to the representatives of intestinal microflora with administration of the probiotic VETOM 1.1, as well as its possible transfer to other microorganism species in the environment (soil) were studied. There were no negative effects of the GMM on the animal organism and the environment, including remote consequences.

Introduction

            Probiotics are widely used in medicine and veterinary for prophylaxis of dyspepsia, diarrhea, (dysbacteriosis) and gastroenteric diseases. They are often used to correct intestinal bacteriocenosis after antibiotic- or chemotherapy, as well as, to increase natural resistance and to stimulate growth in animals [9, 14, 16, 17, 23]. Probiotics are live active antagonistic microorganism cultures producing a favorable effect on intestinal bacteriocenosis. They are produced on the basis of one – three species of lactate bacteria and streptococci, Bifidobacteria, enterococci, nonpathogenic Escherichia, yeast and certain representatives of Bacillus genus. A large number of officially registered and approved probiotics is known [4, 9, 14, 16, 17, 23]. Recombinant probiotics with their broad prospects of medical and veterinary applications take a special place among probiotics [2, 5, 15, 22, 24]. However, the use of recombinant probiotics under the Law of Russian Federation “On the State Regulation in the Field of Gene Engineering” (N 86_3 of 05.07.96), as well as international regulations, require deep and substantial evidence of biological safety and demonstration of biological risks of using GMM (gene-engineering modified microorganisms) that are the active ingredient of this group of probiotics. This is especially urgent for open systems [6, 7, 28], where introduction of GMM into the environment is inevitable and predictable. 

            An evaluation of the possibility of transfer of the cloned DNA fragment to other microorganism species in the environment is required [8]. Available experience from analyzing the viabilities of selective producer strains and GMM in the natural environment suggests that the viabilities of these microorganisms in the environment are considerably inferior to those of microorganisms inhabiting natural ecological niches [1, 5, 10, 13, 19, 25]. Under conditions of natural habitat and artificial introduction, microorganisms including GMM can release chromosomal and plasmid DNA from lysed cells into the environment. Depending on external conditions, plasmids may preserve their transforming activity for a long time. This is one of the serious risk factors from the introduction, which potentially promotes uncontrolled dissemination of recombinant DNA with possible penetration into cells of new hosts [1, 6, 20, 25, 27]. Experimental data on risk evaluation and consequences of GMM introduction into the environment is urgent and practically important both from the points of view of obtaining and accumulating data on different aspects of biological safety using recombinant microorganisms and of mastering methods used to perform such studies.

            The goal of the present work was the evaluation of survival of a GMM (recombinant B. subtilis strain VKPM B–7092) in the bovine gastroenteric tract, determine its effect on microbiocenosis and species composition of the microflora of bovine gastroenteric tracts, examine the possibility of transfer of the DNA fragment cloned in B. subtilis bacterium, containing the gene of human leukocytic alpha-2 interferon, to the representatives of intestinal microflora when the probiotic VETOM 1.1 is given to animals, as well as its transfer to other microorganism species the environment (soil).

Materials and methods

            B. subtilis VKPM B–7092 strain is a recombinant strain which cells contain a plasmid with the structural gene of the mature form of human leukocytic alpha-2 interferon. The above plasmid provides for the synthesis and secretion of biologically active interferon by B. subtilis VKPM B–7092 cells [18, 25]. Bacteria of B. subtilis VKPM B–7092 strain are aerobes. When grown on LA medium, they form chamomile-like colonies. The colonies are whitish. The bacteria are Gram positive baculiform. The cells of a one-day agar culture are (2 – 4)·(0.5 – 0.8) mm large. They form spores, but do not form capsules. Bacteria of this strain can reproduce at 15 – 50°C, optimal growth is observed at the temperature of 36 – 37°C. They hydrolyze starch and reduce nitrates. They split glucose, sucrose, mannose, maltose and lactose. The strain is canamycin-resistant; besides alpha-2 interferon, it synthesizes an antibiotic of broad spectrum of action inhibiting the growth of fungi, staphylococci, streptococci and Pseudomonas aeruginosa  [18].

            The probiotic VETOM 1.1 manufactured by the JSC Scientific and Production Company “Research Center” (Koltsovo, Novosibirsk region) was used in the experiments.

            A solid medium with canamycin was used to isolate B. subtilis VKPM B–7092 from bovine feces [18], and generally accepted methods and standard bacterial media were used for intestinal bacteria, etc. [11, 18, 21].

            Animals that were healthy by veterinary indices were used in the work. The animals were given the probiotic VETOM 1.1 two times a day at the rate of 75 mg of the preparation (not less than 7.5·104 of bacterial spores) per 1.0 kg of bodyweight for 9 days. The survival of the recombinant strain was estimated by the period of isolation from gastroenteric tract (till complete elimination) via seeding the contents of feces of experimental animals on nutrient bacterial media. Fecal samples were selected before giving the preparation, daily for the whole period of giving the preparation and up the moment of complete excretion of B. subtilis VKPM B–7092 from the animal gastroenteric tract. Besides, the probiotic effect on the microbiocenosis structure in the animal, the gastroenteric tract was evaluated by changes in the numbers of dominating microbial populations making part of the microbiocenosis determined by microbiological analyses of the above samples and those collected 30 days after the preparation was first given [11, 21].

            The study of the possibility of transfer of a DNA fragment cloned in B. subtilis bacterium to the representatives of intestinal microflora was performed using the method of two-round PCR with specific primers. Specific primers were developed and synthesized for the detection of specific DNA fragments with the program Oligo-6 (for artificial marker of the gene of leukocytic human interferon with the lengths of fragments of 452 bp and 328 bp, respectively; for the marker of canamycin resistance gene – 834 bp and 314 bp, respectively). The specificity of primers was confirmed using the program BLAST (/gi:396443/, / gi: 31415690/, /gi: 29568850/, /gi: 3953638/) of the National Institute of Health, USA (http://www.ncbi.nlm.nih.gov/blast/). PCR conditions for these primers were selected and perfected. As it was known that the sought genetic fragments were in plasmid structures that had been used to transform the B. subtilis VKPM B–7092 strain, plasmid DNAs were isolated from bacterial cultures [26] obtained from animal feces and were analyzed in PCR. Standard reagents, solutions and equipment were used to perform the two-round PCR.

            Amplification was carried out in 30 ml of a mixture containing 10X PCR-buffer (10 mM of Tris-HCl, pH 8.3, 50 mM of KCl ),  2 mM of MgCl , 0.2 mM of each dNTP, 20 pM of each primer, 2.5 U of Taq- DNA-polymerase and 5 ml of the tested sample. Thirty amplification cycles were performed for all the used primers in the following mode:

The first round of PCR
95°_ – 3 min   – 1 cycle.
94°_ – 30 sec
58°_ – 30 sec
72°_ – 30 sec – 30 cycles.
72°_ – 3 min – 1 cycle.

            After the first round, amounts of 1.0 µl of amplificate were transferred to test tubes with the reactive mixture for the second round of PCR.

The second round of PCR
95°_  - 3 min – 1 cycle.
94°_ – 30 sec
68°_ – 30 sec
72°_ – 30 sec – 30 cycles.
72°_ – 3 min – 1 cycle.

            The detection of the amplified DNA after PCR was performed with gel electrophoresis method in 2% agarose gel. The obtained results were considered satisfactory when there was a clearly seen DNA band of the calculated length in the gel. Samples were considered positive when they had a DNA band in the gel corresponding to the DNA fragment of the control sample by length as well as to an appropriate band in the marker of the fragments lengths.

            Soil samples were collected at agricultural enterprises that had used the probiotic VETOM 1.1 for treatment and prophylaxis of bovine diseases, in particular, at the JSC “Kirzinskoye”, Ordynsk district, Novosibirsk region, where the preparation had been used for eight years. Control soils were from APC “Rogalevskoye”, Ordynsk district, Novosibirsk region, that had not used the VETOM 1.1. Soil samples were collected from plots where manure from the farms of the JSC “Kirzinskoye” (samples # 1, 2 and 3, respectively) had been added eight, five and one year ago. Samples from the plot of APC “Rogalevskoye” (sample 4) where manure from the farms had been added six years ago served as samples for comparison. Bacterial cultures from soil samples were isolated on liquid and solid bacterial media supplemented with canamycin according to generally accepted technique [11, 25, 18, 21].

            Initial identification of the GMM strain among bacterial cultures isolated from soil samples was performed by its cultural and morphological characteristics [18]. Exact identification of the GMM B. subtilis VKPM B–7092 strain was carried out with the method of two-round PCR using specific primers. Cultures of microorganisms isolated from soil were grown on a solid medium supplemented with canamycin. Plasmid DNAs were isolated from the cell culture obtained at the end of the exponential – the beginning of the stationary phase of development with a modified Birnboim - Doly method of alkaline lysis [26]. The obtained DNA was analyzed with the two-round PCR method. Aqueous extracts were obtained from soil samples # 1-3 to reveal the possible presence of the plasmid DNA that entered the environment from lysed bacterial cells of the GMM and was preserved in soil during the analyzed periods. Plasmid DNA was isolated from aqueous extracts and analyzed with the two-round PCR method.

            Standard statistical methods were employed for processing and analysis of obtained results [12].

Results and discussion.

            The results of evaluating the survival of recombinant B. subtilis VKPM B–7092 strain in bovine gastroenteric tract when the probiotic VETOM 1.1 was given to animals, its effect on microbiocenosis and species composition of microflora in gastroenteric tract of these animals are presented in Tables 1 and 2. The number of bacterial cultures isolated from the contents of feces of each of experimental animals, as well as the results of evaluating the possible of transfer of the GMM plasmid genes into these bacterial cultures, are presented in Tables 3 and 4.

Table 1

            The dynamics of isolation of B. subtilis VKPM V-7092 strain from bovine gastroenteric tract when the probiotic VETOM 1.1 is given to animals.

            The data of Table 1 show that the excretion of B. subtilis from gastroenteric tracts of all experimental animals ceases 7 days after the cycle of administration of the probiotic VETOM 1.1 is completed. The obtained results are close to the data of determining the terms of elimination of bacteria of B. subtilis 2335 pBMB105 strain, the active ingredient of another recombinant probiotic Subalin, from gastroenteric tracts of some animal species [1, 2, 3, 5, 15, 22].

Table 2

            The study of the effect of B. subtilis VKPM V-7092 on the microflora of bovine gastroenteric tract when the probiotic VETOM 1.1 is given to animals

            Microbial maps presented in Table 2 showed that after the 9-day cycle of introducing of B. subtilis VKPM V-7092 cells into gastroenteric tract, the number and the ratio in the dominating taxonomic groups of intestinal microflora remained practically unchanged as compared to the initial indices. However, 30 days after the probiotic VETOM 1.1 was first given the data demonstrating the improvement of qualitative and quantitative compositions of the microflora of bovine gastroenteric tract were obtained.

             Table 3 presents the results of determining the markers of the genes of human leukocytic α-2 interferon and canamycin-resistance in 48 isolated bacterial cultures from feces contents of a bull, a cow and a calf after giving them the preparation VETOM 1.1 and full elimination of B. subtilis VKPM V-7092 cells from gastroenteric tracts of these animals. 

Table 3

            Determination of the markers of the gene of human leukocytic α-2 interferon and canamycin-resistance gene in bacterial cultures isolated from the contents of feces of experimental animals with the two-round PCR method

            Note: “ + ” – presence of the sought markers;
              “ - ” – absence of the sought markers.

            The data of Table 3 demonstrate that 48 studied bacterial cultures do not contain the gene of human leukocytic _-2 interferon in the cell genomes. In a portion of the analyzed bacterial cultures isolated from the contents of feces of experimental animals the marker of canamycin-resistance gene was detected. However, in the control initial samples of bacterial cultures isolated from the contents of feces of the above animals before the cycle of VETOM 1.1 administration, the presence of the marker of canamycin-resistance gene was revealed even in a greater number of cultures (in 2, 4 and 2 cultures, respectively) than after the cycle had been completed. Most likely, the obtained results and the available literature data on rather frequent occurrence of canamycin-resistance both in soil populations and in the microflora of animal gastroenteric tract [1, 3, 25] are indicative of the circulation of canamycin resistance genes in native populations.

            Nineteen canamycin-resistant bacterial cultures were isolated from soil samples as a result of the study of remote consequences of using the probiotic VETOM 1.1 in the areas of potential getting of GMM into the environment. Table 4 presents some of their cultural and morphological properties, the results of PCR analysis of DNA samples obtained from the above cultures as well as aqueous extracts from soil samples # 1-3.

Table 4

            Cultural and morphological properties of bacterial strains isolated from soil samples, results of PCR analysis DNA samples obtained from the above cultures as well as aqueous extracts from soil samples # 1- 3 

Note: * - a mixture of cultures of two strains obtained by taking of a colony of one strain grown on a colony of another strain or two confluent colonies using a bacterial loop.

            The data of Table 4 demonstrate that 7 of 19 species of canamycin-resistant bacterial cultures were isolated from soil samples from the plot of APC “Rogalevskoye”. This farm had never used the probiotic VETOM 1.1 for prophylaxis and treatment of animals. Four different bacterial cultures were isolated from the soils of the JSC “Kirzinskoye” where the probiotic had been used for one - eight years. The study of 19 isolated bacterial cultures by the main cultural and morphological properties [18] did not reveal the presence of B. subtilis culture VKPM V-7092 among them. The presence of the marker of canamycin-resistance gene was revealed in the genomes of the cells of 19 isolated bacterial cultures. Most likely, the obtained results and the available literature data are indicative of the circulation of canamycin-resistance genes in native microbial populations of genes and frequent occurrence of canamycin resistance in soil populations [1, 3, 20, 22]. The studies bacterial cultures do not contain the marker of the gene of human leukocytic α-2 interferon. The cultural and morphological data of Table 4 and the results of two-round PCR demonstrated the absence of B. subtilis bacteria VKPM V-7092 among the 19 studied bacterial cultures. According to PCR data, the analyzed aqueous extracts of soil samples from the JSC “Kirzinskoye”, did not contain the recombinant plasmid DNA of the above GMM, which could be preserved in soil during the whole observation period (from one to eight years).

Conclusions

1. Bacteria of the GMM B. subtilis strain VKPM V-7092 do not colonize bovine gastroenteric tract when the probiotic VETOM 1.1 is given to the above animals and are fully excreted from the intestines of experimental animals four-seven days after the probiotic administration had been completed.

2. The introduction of the probiotic VETOM 1.1 does not produce a negative effect on intestinal microbiocenosis of experimental animals and the number and ratio in the dominating taxonomic groups of normoflora. It was shown that introducing of the probiotic promoted the normalization of the microflora of bovine gastroenteric tract.

3. The absence of transfer of plasmid genes of GMM B. subtilis VKPM V-7092 strain to bacteria of intestinal microflora at its introduction into the animal organism was shown in experiments.

4. Experimental evaluation of remote consequences of risks of introducing the GMM B. subtilis VKPM V-7092 strain into the environment at observation periods from 1 to 8 years showed that bacteria of the given GMM were not detected in soil samples of the agricultural enterprise that had used the probiotic VETOM 1.1 for treatment and prophylaxis of cattle diseases, with methods of initial and exact identification. The introduction of B. subtilis VKPM _-7092 strain into the environment did not result in its unlimited growth and spread in soil.

5. The absence of transfer of plasmid genes (the genes of human leukocytic α-2 interferon) from the GMM B. subtilis VKPM V-7092 strain to other microorganism species spread in the areas of the GMM getting into the environment (soil) was shown in experiments. The presence of recombinant plasmid DNA from lysed cells of the above GMM was not revealed in soil samples.

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    • ASA thanks Dr. Stavskiy and his “Vector” group for sharing this information with the ASA family of professionals.