RU1819428C - PLASMID p741 DETERMINING MICROCINE SYNTHESIS, AND STRAIN OF BACTERIUM ESCHERICHIA COLI SHOWING ANTIBACTERIAL ACTIVITY - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: plasmid p74¹ (molecular mass is 30 mDa) is nonconjugative, its content in cells of strain E. coli C600 is 1-2 copies per a cell, it determines resistance for kanamycin (200 mcg/ml), synthesis of microcine and resistance to the latter. It does not correspond to wide-spread plasmid incompatibility. In cells of strains E. coli plasmid is sustained stably under nonselective conditions for at least 100 generations, it does not act on the growth rate and morphology of cells E. coli. At transformation of DNA of plasmid p74¹ in E. coli cells number of tranformants is 10² per 1 mcg DNA. Using plasmid p74¹ strain E. coli M17 BKM CR-322D is obtained which is harmless for newborn calves, it is adapted in intestine of newborn calves, prevents disease of calves with coli-bacteriosis, enhances viability of sick animals. EFFECT: increased effectiveness of plasmid indicated above. 3 cl, 2 tbl

Description

 The invention relates to medical and veterinary microbiology, relates to the production of a new plasmid and a new strain with antibacterial activity, and may find application for the prevention of intestinal diseases and the correction of the microflora of the digestive tract of farm animals and humans.

 The aim of the invention is to obtain a new plasmid that determines the synthesis of microcin with a wide spectrum of activity and obtain a new strain of Escherichla coli with a wide spectrum of action against pathogens of the digestive tract of farm animals and humans.

As a new plasmid, plasmid p74 ^{1 was} obtained. As a new strain, a strain of Escherichia coli VKM CR-322D was obtained.

To obtain a new plasmid p74 ¹ , the Tn5 transposon is introduced into the natural plasmid P1, which determines the synthesis of microcin, and clones of the bacterium Escherichia coli carrying the plasmid that are not transmitted by conjugation by cells of other bacteria are selected.

The new plasmid p74 ¹ has the following characteristics:
1. Has a meter 30 MDa (defined as the sum of the molecular weights of the fragments obtained by the action of restriction enzymes).

 2. Non-conjugative.

 3. In E. coli cells present in an amount of 1-2 copies.

 4. Determines: resistance to kanamycin (200 μg / ml); microcin synthesis; resistance to the produced microcin of cells carrying the plasmid.

 5. Not consistent with widespread plasmid incompatibility groups.

 6. In the cells of E. coli strains it is maintained stably under non-selective conditions for at least 100 generations (duration of the experiment).

 7. Does not affect the growth rate and morphology of E. coli cells.

8. During the transformation of E. coli C600 cells with deoxyribonucleic acid (DNA) of plasmid p74 ¹ , 10 ² transformants per 1 μg of DNA are formed.

9. The picture of the restriction of plasmid p74 ^{1 with} various restriction enzymes is given in table. 1.

To obtain a new strain of Escherichia coli VKM-332D, a strain of Escherichia coli M-17 was used, which was transformed with deoxyribocucleic acid of the new plasmid p74 ¹ and clones resistant to kanamycin and capable of synthesizing an antibiotic were selected.

 The new strain is characterized by the following features.

 Morphological signs. Gram-negative, non-spore-forming, movable sticks with rounded ends. Sizes 1.1-1.5x2.0-5.0 microns.

Cultural signs. The cells of the strain grow well on simple nutrient media meat-peptone agar (MPA), agarized Hottinger broth, minimal media with glucose, glycerin. When grown for 1 day at ³⁷ ° C in a liquid broth Hottinger or on meat broth (IIB) give uniform turbidity broth. When grown on agarized Hottinger broth or MPA, they form grayish-yellowish rounded colonies (sometimes with scalloped edges): on the Endo medium, red colonies with a metallic luster (1-2 days 37 ^° C) form.

Physiological and biochemical characteristics. Optional anaerobic. The temperature optimum during growth on the Hottinger broth or BCH 37 ^about C.

 It breaks down glucose, lactose, sucrose, maltose, rhamnose, arabinose, mannitol, inositol with the formation of acid and gas, and very poorly utilizes sorbitol. Does not recycle sodium citrate and sodium malonate. Urease activity is absent. Hydrogen sulfide is not formed. Synthesizes β-galactosidase. Indole forms. Does not synthesize phenylalanine deaminase. There is no gelatinase activity, milk ferments. Lysine decarboxylase and ornithine decarboxylase activities are detected. Arginine dihydrolase is absent.

 The strain belongs to the serological type 02: K1 (L): H6. There are no adhesive antigens K88, K99, F41, Att25, 987P.

 The strain is resistant to kanamycin (100-200 μg / ml).

 The strain is stored in freeze-dried state for at least one year. Cells grown on jambs with Hottinger agar broth during the day are washed off with a lyophilization solution of the following composition: 1 ml of physiological solution + 1 ml of a mixture (10 ml of cattle blood serum without preservative + 1 ml of sucrose solution 30 g / 30 ml).

PRI me R 1. Obtaining plasmids p74 ¹ .

 To obtain a new mutant plasmid incapable of conjugative transfer, E. coli C 600 (pl, RSF 1010) was used. Plasmid pl, determine the synthesis of broad-spectrum microcin, plasmid RSF 1010 resistance to streptomycin (100 μg / ml) and sulfonamides.

 a) transfer of plasmids p1 and RSF 1010 by conjugation into E. coli MKD5208 cells.

 Donor strain of E. coli C 600 (p1, RSF 1010).

Recipient strain of E. coli MKD 5208F ^- , prototroph, sup ^o , val-r, sts-s. Transfer of plasmid pl into E. coli MKD 5208 cells was judged by mobilization of pl non-conjugative plasmid RSF 1010, which determines streptomycin resistance (100 μg / ml). The cells of the donor and recipient strains were grown in LD broth to a titer of 1 x 10 ⁸ cells / ml, 5 ml of each strain were taken and mixed in a 100 ml flask for conjugation. Bacterial cells grown for 18 hours at ³⁷ ° C without rocking. Then the cells were centrifuged, washed 2 times with physiological saline, resuspended in physiological saline to the original volume and plated on a plate with agarized minimal medium of the following composition (per 1 liter of distilled water), g: NH ₄ Cl 1; KH ₂ PO ₄ 1.5; Na ₂ HPO ₄ 3.5; MgSO ₄ 0.1; glucose 24, pH 7.0-7.5, to which streptomycin was added (100 μg / ml). Only E. coli MKD 5208 cells carrying plasmid RSF 1010 could grow on this medium. As a rule, these cells also contained plasmid pl, which was determined by the formation of microcin (it was detected by the presence of melting zones of E. coli cell growth around the injections of the test strain) as well as plasmid DNA analysis. As a result of this work, the strain E. coli MKD 5208 (p, RSF 1010) was obtained.

 b) the introduction of transposon Tn 5 in the natural plasmid pl.

The overnight culture of E. coli strain MKD 5208 (RSF 1010, pl) was diluted with LD medium to an optical density of 0.1 (λ560 nm), incubated with aeration to a density of 0.8, precipitated by centrifugation (4500 rpm) for 10 min, suspended in former volume of LD medium with MgCl ₂ (0.01 M). To 0.1 ml of cells was added 0.1 ml of a suspension of phage λ: Tn 5 (. Phage titer 5.0 × 10 ⁸ units / ml) were incubated with aeration for 20 minutes at ³⁰ ° C, was added 0.2 ml of medium LB + MgCl _2, incubated for an additional 20 minutes at 30 ^C. The cells were plated on LB agar with kanamycin (200 mg / ml) and triturated with a spatula and were grown 1 day.

 The grown colonies were washed off with physiological saline and plasmid DNA was isolated (as described below, section c).

 Plasmid DNA was transformed (an example of section d) cells of the strain E. coli MKD 5208. Clones were selected for resistance to kanamycin (200 μg / ml) and sensitivity to streptomycin (100 μg / ml). As a result, E. coli MKD 5208 clones containing plasmids pl Tn 5 and not containing plasmids RSF 1010 were selected.

 c) isolation of plasmid DNA.

Cells from an overnight culture of E. coli were precipitated by centrifugation, suspended in 1 ml of solution A (50 mm glucose, 10 mm EDTA, pH 8.0 25 mm Tris-HCl, pH 8.0, lysozyme 2 mg / ml), 2 ml was added The solution (1 SDS, 0,2 n. NaOH), gently stirred, was added 1.5 ml of 3M acetate Na (pH 4.8), incubated 1 h at 0 ^C. The mixture was centrifuged 20 min at 16000 rev / min, isopropyl alcohol (0.6 of the initial mixture volume) was added to the supernatant. The precipitate was washed with 70% ethanol, dried and dissolved in 100 μl of TE buffer (10 mM Tris-Hcl, pH 8.0; 1 mM EDTA, pH 8.0). Samples were analyzed on a 0.7% agarose gel in TBE buffer (50 mM Trisborate, 4 mM EDTA, pH 8.0).

 d) transformation of E. coli MKD 5208 plasmid DNA cells.

Plasmid DNA isolated from kanamycin-resistant (200 μg / ml) E. coli MKD 5208 cells (pl, RSF 1010) was transformed with E. coli MKD 5208 cells after Tn 5 administration. The overnight culture of E. coli MKD 5208 strain was bred into 100 times with LB medium and were grown up to the middle of the logarithmic growth phase. Cells in a volume of 400 ml were precipitated by centrifugation, washed with 0.14 M NaCl, suspended in 2 ml of 50 mM CaCl ₂ , and incubated in an ice bath for 20 minutes. To 50 μl of competent cells, 10 μl of total plasmid DNA (at a concentration of 0.1 mg / ml) was added and incubated for 40 min in an ice bath. After heat shock (42 ^{° C,} 3 min), the cells were diluted into 0.5 ml of LB medium and incubated with aeration for 1-1.5 h at 37 ^C. The cells Sowing was performed on LB agar medium with kanamycin (200 ug / ml) and incubated for 1 day at 37 ^about C.

d) selection of a clone containing a new non-conjugative plasmid p74 ¹ .

In order to select a mutant non-conjugative plasmid, conjugated crosses of donor clones of E. coli MKD 5208 (pl Tn5) were carried out with the recipient strain E. coli C 600 str-r, as described in example a). After 18-20 hours of growth of the mixed culture, cells without washing with saline were plated on plates with Hottinger agar broth, to which kanamycin (200 μg / ml) and streptomycin (100 μg / ml) were added. If donor clones contained a non-conjugative plasmid, then no colonies grew on plates with this medium. Thus, a clone of E. coli 5208 (pl Tn 5) carrying a non-conjugative plasmid named p74 ^{1 was} selected.

PRI me R 2. Obtaining a new strain of Escherichia coli VKM CR-322D. Plasmid DNA (as described above) was isolated from the cells of the selected E. coli MKD 5208 clone (p74 ¹ ) and transformed using Escherichia coli M-17 cells (transformation method above). Clones resistant to kanamycin (100 μg / ml) and capable of synthesizing microcin were selected. Cells were scattered twice to separate colonies on a medium with kanamycin and tested for prototrophy, the ability to synthesize microcin, and the presence of plasmid p74 ¹ . The strain exhibiting these symptoms was named Escherichia coli BKM CR-322D.

 The properties of the new strain are illustrated by the following examples.

PRI me R 3. The study of the antagonistic properties of a new strain. To evaluate the antagonistic action prick strain cells were plated onto plates of the agar medium and grown 2 days at 37 ^C. The grown colonies were killed in pairs of chloroform, after which the surface of the medium filled in the second layer 3 ml of 0.7% strength agar medium of the same composition, mixed with 0.1 ml of indicator culture of E. coli B, grown to an exponential growth phase (1-3 x 10 ⁸ cells / ml) on a rocking chair in a liquid medium of the same composition. Under these conditions, transparent zones of inhibition of the growth of the indicator strain up to 400 mm in diameter formed around the colony of the strain. The optimal activity of the strain on the minimum salt medium (g / l of distilled water): NH ₄ Cl 1 g; KH ₂ PO ₄ 1.5 g; Na ₂ HPO ₄ 3.5 g; MgSO ₄ 0.1 g; agar (Difco or Ferrac) 1.5% glucose 0.2% yeast extract (Difco) 0.3% pH 7.0-7.5.

 The strain isolated a substance with an antibacterial effect, diffusing through cellophane and sensitive to proteases.

 The strain showed antagonistic activity against E. coli (acted on all E. coli strains of various origins studied in this respect, including pathogens of colibacteriosis of farm animals), as well as Shigella, Salmonella, Proteus, Enterobacter, Serratia, Citrobacter, Klebsiella, Pseudmonas, Bacillus megaterium, Bacillus pumilus.

 In the table. Figure 2 shows the results of one of the experiments on the action of cells of a new strain on various bacteria.

PRI me R 4. The study of virulence and survival of a new strain in the body of laboratory animals and calves. The strain did not cause the death of mice weighing 16-20 g when they were injected subcutaneously with 10 ⁹ cells, and also when administered intraperitoneally with 5 x 10 ⁸ cells. Oral administration of the strain to 7-day old sucker rats (5 x 10 ⁸ cells), as well as to the anal administration of 3-day old sucker mice (2-5 x 10 ⁸ cells), showed its harmlessness. At autopsy, no inflammatory or dystrophic changes in the intestine were detected microscopically and macroscopically.

 It was shown that the strain does not produce thermolabile and thermostable enterotoxins.

 A good survival rate of the strain in the intestines of sucker rats has been established; cells were isolated from the intestines of rat pups for 30 days (observation period).

 The strain is harmless to calves when administered orally and does not cause pathological changes in them.

 PRI me R 5. Obtaining the drug from cells of Escherichia coli VKM CR-322D. To obtain from the cells a new strain of live bacterial preparation used to prevent colibacteriosis in calves, the culture was grown in one of two generally accepted ways.

Cells were grown in cups or arrays on the surface of agar (1.5% agar) Hottinger broth for 20 hours at 37 ^C, after which the cells were washed with physiological saline. From 1 cm ^{2 the} surface of the nutrient medium receive 0.8-1.0 x 10 ⁹ bacterial cells.

The culture is grown in a fermenter together in a liquid nutrient medium, Hottinger broth or broth diluted 4 times with a minimum salt medium of the following composition (per 1 liter of distilled water): NH ₄ Cl 0.5 g, KH ₂ PO ₄ 0.75 g, Na ₂ HPO ₄ 1.75 g; MgSO ₄ 0.05; glucose 0.3% pH 7.0-7.5. 7-10 x 10 ⁹ cells were obtained with 1 ml of medium.

The drug was used as a suspension of cells in physiological saline. It can also be used as a lyophilized culture, diluted before use with boiled water or saline. For lyophilization, the cells were suspended in a sugar gelatin medium (1% gelatin and 10% sucrose in distilled water). The suspension of cells in saline or boiled water kept refrigerated at 4-12 ^{° C} and was suitable for use for not less than 10 days; during this time period, the cell concentration decreased by no more than 2.5 times.

PRI me R 6. The use of the drug from the cells of a new strain for the prevention and treatment of colibacillosis in calves. Newborn calves were given per os 15 ml of the drug (a suspension of cells in saline or boiled water, in total from 2 x 10 ¹⁰ to 1 x 10 ¹¹ cells) 30 minutes before feeding 2 times a day, starting from the first colostrum. In order to prevent colibacteriosis, the living culture preparation was given for 4 days, with the aim of treatment for 6 days.

 The live culture preparation was tested on newborn calves belonging to various state farms, which differed in the conditions of keeping and feeding the animals, as well as in the nature of the course of the disease and the observed departure of animals.

 Household N 1. Diarrhea was observed in the farm of newborn calves. The drug was given for 4 days. In the first 4 days of 10 experimental calves, there were no clinical signs of the disease; on days 5-7, some calves had mild diarrhea, which disappeared after 2 days. All 10 calves in the control group developed diarrhea and were treated with anticolibacteriosis serum.

 Farm N 2. The farm was observed colibacillosis of newborn calves. The drug was given for 4 days. Mild diarrhea was observed in 12 calves, which stopped after 2-3 days. All 12 control calves suffered from severe diarrhea, of which 2 calves (16.7%) fell, the rest recovered after prolonged treatment.

 Farm No. 3. At the livestock complex, newborn calves observed colibacteriosis. The drug was given for 4 days. The drug was tested on 15 calves. Calves fell ill on the third day, diarrhea lasted 24-32 hours in a mild form. The use of decoctions and tea was enough to stop the disease. Of the 15 control calves, all suffered from diarrhea, with 3 in very severe form. For treatment, antibiotics and sulfa drugs were used, as well as colibacteriosis serum, Kadykov serum and glucose.

 Household N 4. Inpatiently dysfunctional for colibacillosis. The drug was given for 6 days. The drug was tested on 16 calves. Of these, 7 (43.7%) animals fell ill, 1 (6.25%) calf fell. Of the 16 control calves, 12 (75%) calves developed severe diarrhea and 4 (25%) calves fell. All calves in the control group underwent prolonged and intensive treatment.

 Farm N 5. The farm was observed diarrhea in newborn calves. The drug was given for 4 days. Daily samples of feces were taken, which were sown on the differential diagnostic medium Endo with kanamycin, determining the presence of cells of a new strain in feces. The drug was tested on 7 calves. It was established that the cells of the new strain remained viable in the intestine 6-8 days after the last dose of the drug. Digestion returned to 6-7 days.

The test results presented in 5 farms show that the preparation prepared from the living culture of the new strain:
harmless to newborn calves;
takes root in the intestines of newborn calves;
prevents calf disease with colibacillosis,
the disease proceeds in a mild form (mild diarrhea, stops after 2-3 days), which does not require the use of antibiotics, sulfanilamides and other expensive drugs; diet, decoctions and tea are sufficient to treat diarrhea;
increases the survival of sick animals.

 The above examples show that the new strain can be successfully used for the prevention and treatment of colibacteriosis of newborn calves.

Claims (2)

1. Plasmid p74 ¹ , which determines the synthesis of microcin, mol.m. ZOMDa containing DNA of plasmid pl of strain Enterobacter sp. a phage λ DNA fragment containing the transposon Tn 5 in the region of the gene tra, 6 fragments with sizes 25.0; 9.0; 6.8; 2.5; 2.2; 2.2 formed by digestion with restriction enzyme Eco RI, 5 fragments with a size of 24.0; 23.0; 3.3; 2.5; 2.0 formed by restriction enzyme Sal Gl 8 fragment with a size of 15.0; 14.0; 7.5; 4.0; 3.5; 3.3; 3.0; 1.8, formed by cleavage with the restriction enzyme Hindl III, 7 fragments with a size of 20.0; 14.0; 4,5; 3.8; 3.5; 2.5; 2.0, formed by restriction enzyme digestion with Pvu II, genes for the synthesis of microcin, resistance to microcin, resistance to kanamycin (100 μg / ml), is non-conjugative, in the cell of E. coli strains under non-selective conditions for at least 100 generations is stable, in cells E. coli is present in an amount of 1 2 copies per cell, does not correspond to the widespread incompatibility groups of plasmids, does not affect the growth rate and morphology of E. coli cells; upon transformation of E. coli cells With 600 DNA plasmid p74 ¹ forms 10 ² transformants per 1 mcg of DNA.  2. The bacterial strain Escherichia coli VKM CR-322D with antibacterial activity.

Images