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Abstract
Introduction
The goal of the present work was the evaluation of survival of a GMM
(recombinant B. subtilis strain VKPM B7092) 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).
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 B7092
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 7.5104 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 B7092 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].
After the first round, amounts of 1.0 l of amplificate were transferred to test
tubes with the reactive mixture for the second round of PCR.
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 B7092 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.
Table 1
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
Before After
30 days after
Composition of microflora giving giving
preparation
of bovine gastroenteric tract VETOM VETOM
was first given
1.1 1.1
COW (Note: ND = Not
Determined)
Pathogenic enterobacteria ND ND ND
6 5
E.coli with normal 5_10 3_10 9_104
enzymatic activity (62.8%) (100%)
6
E.coli with low activity 3_10 ND ND
(lactosonegative) (37.2%)
Hemolytic E.coli ND ND ND
5
Protei ND 3_10 ND
Other conventionally ND ND ND
pathogenic enterobacteria
Plasmonegative staphylococci 3_105 1.2_105 ND
6 4
Hemolytic streptococci 1_10 10 104
Lactobacteria 104 104 104
Fungi ND ND ND
7 7
Bifidobacteria 10 10 108
Clostridia ND ND ND
CALF
Pathogenic enterobacteria ND ND ND
5 5
E.coli with normal 1_10 4_10 1_105
enzymatic activity (3,4%)
E.coli with low activity 29_105 ND ND
(lactosonegative) (96.6%)
Hemolytic E.coli ND ND ND
7
Protei ND 3_10 ND
Other conventionally ND ND ND
pathogenic enterobacteria
Plasmonegative staphylococci 2_105 1.3_104 ND
6 4
Hemolytic streptococci 1_10 10 104
Lactobacteria 104 104 104
Fungi ND ND ND
7 5
Bifidobacteria 10 10 108
Clostridia ND ND ND
BULL
Pathogenic enterobacteria ND ND ND
4 4
E.coli with normal 10 7_10 2_105
enzymatic
(3%) (100%) (100%)
activity
E.coli with low activity 5_107 ND ND
(lactosonegative) (97%)
Hemolytic E.coli ND ND ND
Protei 12_107 8_105 ND
Other conventionally ND ND ND
pathogenic enterobacteria
Plasmonegative staphylococci 8_105 ND ND
4 4
Hemolytic streptococci 1_10 10 104
Lactobacteria 105 104 105
Fungi ND ND ND
Bifidobacteria 108 106 108
Clostridia ND ND ND
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 -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 marker
Determination of marker
ANIMAL Time of of canamyncin-resistance
of _-2 interferon gene
cultures gene
isolation, days Cultures Cultures
Cultures Cultures
after the obtained on obtained on
obtained on obtained on
preparation medium medium
medium with medium with
was first given without without
canamycin canamycin
canamycin canamycin
BULL 0 - - - -
- - - -
- - + +
- - + +
12 - - + +
- - + -
- - - -
23 - - + -
- +
- -
COW 0 - - + +
- - + +
- - + +
- - + -
12 - - + -
- - + -
- - - -
- -
CALF 0 - - + +
- - + +
- - -
12 - - + -
- -
- -
23 - - + +
- - + +
- -
- -
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.
Table 4
Dull, grayish-white,
round, small colonies
2 - +
with round edges,
convex. Very small
Gram-negative rods.
Flat, large with rough
edges. Gram-positive
3 rods with and without - +
spores with central and
terminal arrangement.
Large, grayish-white,
4 dull colonies. Thin and - +
long Gram-positive
rods.
5 Small with smooth - +
edges, convex, shining,
grayish-white colonies.
Individual Gram-
positive cocci.
Small, grayish-yellow,
convex, round colonies.
6 - +
Individual Gram-
positive cocci.
Small, convex, grayish-
white, transparent,
7 - +
shining, round colonies.
Gram-negative rods.
Large, whitish
camomile-like colonies.
VKPM _-7092
Gram-positive + +
(control)
sporiferous rods. Do not
form capsules.
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.
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.
References
ASA thanks Dr. Stavskiy and his Vector group for sharing this information
with the ASA family of professionals.