Strain of micromycete trichoderma hamatum, having antibacterial activity against anthrax causative agent bacillus anthracis

FIELD: biotechnology.

SUBSTANCE: strain is deposited in the State collection of pathogenic microorganisms "SCIM-Obolensky" under the number F-11. The culture fluid centrate obtained as a result of culturing of said the strain on the liquid nutrient medium has high activity against anthrax causative agent. The zone of inhibition of growth of Bacillus anthracis is 20-25 mm and 35-45 mm when the culture fluid centrate of the said strain is used in an amount of 10 mcl and 100 mcl, respectively.

EFFECT: strain of micromycete Trichoderma hamatum has antibacterial activity against anthrax causative agent Bacillus anthracis.

4 tbl, 3 ex

 

The invention relates to the field of medical Microbiology and biotechnology.

The fight against anthrax in natural foci of its distribution is the most important task for the prevention of infection by a bacterial infection of humans and animals. One of the challenges in carrying out anti-epidemic measures against anthrax is the introduction in practice of new Disinfectology drugs and technologies of their application [On measures to improve interventions for the prevention of anthrax in the Russian Federation. The resolution of the Chief state sanitary doctor of the Russian Federation G. G. Onishchenko No. 41 of June 27, 2008 ].

To date the primary means of decontamination of contaminated anthrax microbe soil remain chemical disinfectants [Sokolova N. F. Modern means and methods for disinfection in anthrax // Materials of VIII all-Russian Congress of epidemiologists, microbiologists and Parasitologists: Sat. articles. -M.: Resinex. - 2002. - P. 56-57]. However, chemicals have a number of disadvantages environmental and sanitary nature [Fedorova L. S. Disinfectant properties of characterwhich connections and tools on their basis (literature review) /L. S. Fedorova, I. M. Zvereva, A. S. Belov, N. With. Lunch� / / Zh. Disinfection Case. - 2011. - No. 4. - S. 11-19].

It is known that in the list of disinfectants used for decontamination of the soil, are completely absent biological drugs [Disinfectants]. M: Trading company BINGO GRAND. - 2007. - 336]. However it is known that the soil is rich microbial antagonists that performs a significant role in the elimination of bacterial pathogens [Cherkassky, B. L., " Principles and prospects of rehabilitation of soil foci of anthrax// Materials of the International. Symposium. disinfection and sterilization. - M.: Medicine, 1972. - S. 54-56].

It is known that in respect of the bacterium Bacillus anthracis most potential antagonistic effects have culture Strcptomyces roseus, Bacillus subtilis, Escherichia coli [Semenov S. A., Galiullin A. K. Microbial antagonists for biological rehabilitation of farm animals // scientific notes of KHAWM. - Kazan. - 2010. - T. 204. - S. 246 through 251].

It is also known that among the microbes, inhibit the development of anthrax, the most studied actinomycetes [Kalyuzhnaya, L. D. a Study of the antagonistic action of actinomycetes on the bacilli of anthrax / Kalyuzhnaya, L. D., A. M. Bryansk, S. A. Korotich, V. P. Kosachenko // J. Antibiotics. -1975. - No. 7. - Pp. 617-623]. Micromycetes as antagonists of anthrax is not known.

The object of the invention is the obtaining of a new strain of micromic�that Trichoderma hamatum (Bonordcn) Bainicr with pronounced antibacterial properties against the anthrax Bacillus anthracis.

The problem is solved in that the proposed strain of micromycetes Trichoderma hamatum FX-1714/2 (Bonorden) Bainier with pronounced antibacterial properties against the anthrax Bacillus anthracis.

The strain of the fungus Trichoderma hamatum VL-1714/2 selected from the surface of the spider (Acarina), was found on the Bank of the Samara river in the village of Andreevka, Novomoskovsk district, Dnipropetrovsk region, Ukraine Strain deposited in the State collection of pathogenic microorganisms and cell cultures "GKM-Obolensk". Certificate of Deposit No. 349 dated 12.03.2012. The strain is assigned a registration number F-l 1. The strain is characterized by the following features.

Cultural and morphological characteristics of the strain: the Colony at a standard environment of čapek at 22°C grow quickly, reaching the edges of the Petri dishes for 5 days. The mycelium colorless, creeping, cobwebby. Sporulation appears on S day -12 growth in shallow convex merging the pads 2-4 mm in diameter, spaced evenly or in concentric zones, often just on the edge of the colony. Color gray-green, bluish-gray to lead-gray, sometimes dirty-green or dark green. Reverse side of colony colourless. The pigment in the environment is not allocated. The exudate is usually absent. It's faint mushroom. Hyphae colorless, smooth, 2-8 µm in diameter. Immersed mycelium thicker, up to 8-12 µm wide, with swellings�and and anastomoses. Chlamydospores are usually abundant, terminal or intercalary, round, oval or ovoid, colorless, smooth, 8-15 µm in diameter. Conidiophores are unpainted, smooth, straight, thick 18 µm in diameter. Branched at intervals, branches short and thick, are located at 2-4, sometimes one at a time, moving at right angles to coniditons. The tip of canadianese ends with a long, sinuous, branched, sometimes curled end sterile appendage having a smooth surface. Fieldy are located on the short branchlets with dense whorls of 5-8, rarely one or two. Ovate, broad and short, with short elongated neck, swollen in the middle of 3,5-5,5×2,5-4,5 µm. Conidia are gray-green, aggregated in slimy heads, elliptical, rather large, 3,5-5,7×2,2 -3,3 µm, smooth. On wort-agar colony forming more abundant aerial mycelium, later start sporozoite except pads appears sporulation in aerial mycelium.

Biochemical characteristics of the strain

The sources of carbon: good uses glucose, glycerol, mannitol. Can grow on media containing galactose, lactose, sucrose, starch. Not able to digest cellulose and chitin. The sources of nitrogen: assimilates ammonium nitrogen and organic nitrogen of amino acids. Restores nitrates.

Optimal us�ovia and the composition of the medium for culturing

The fungus grows in the range of pH values from 2.5 to 9.5. The optimum pH value of 5.5 to 6.5. In more acidic conditions (pH of 2.5-5.0) and more alkaline (pH of 7.0 to 9.5) changes of morphological characteristics of culture: delayed sporulation. Dense nutrient medium: Czapek agar, Sabouraud agar, wort agar. Aerobe. The optimum temperature for the growth of a culture of the fungus 22-25°C, the duration of cultivation to mass sporulation - 5-7 days, depending on the medium composition and temperature of cultivation.

Antagonistic properties

The strain of the fungus Trichoderma hamatum F-11 has an antagonistic activity against gram-positive and gram-negative bacteria.

Method, conditions and the composition of media for long term storage

1. In the dehydrated state to the ion-exchange resin, storage at a temperature of (4-8)°C, Peratallada after 5 years.

2. Under mineral oil on Czapek agar, stored at a temperature of (4-8)°C, Peratallada 1 year.

3. In liquid nitrogen, the shelf life is unlimited.

4. In lyophilized form, the storing temperature (4-8)°C, not less than five years.

Example 1. Cultivation of a strain of Trichoderma hamatum F-11 on a solid nutrient medium and evaluation of its antimicrobial spectrum

To determine the antimicrobial spectrum of activity of fungal culture of T. hamatum F-l 1 used the method of blocks, based on the ability of substances of diffunt�to adjust to the thickness of the agar and delay the growth of the test objects, in the zone of diffusion.

The strain of the fungus T. hamatum F-11 grown on nutrient dense modified mycological medium of the following composition (g/l): peptone and 5.0; yeast extract - 5,0; agar - 15,0; glucose - 10,0; maltose-10, NaNO3To 2.0; KCl - 0.5; The FeSO4×7H2O - 0,01; MgSO4×7H2O - 0,5; K2HPO4To 1.0; gentamicin - µg/ml.

The strain is grown within 5 -1 3 days in a thermostat at a temperature (24+0,5)°C. Then agar with the test culture of Trichoderma hamatum F-l 1 mirror drill cut blocks with a diameter of 5 mm and placed on seeded and dried lawns of the test cultures.

To prepare the lawns of the test objects used daily culture obtained from the Museum GNORPM that are grown in Petri dishes with medium timing (hydrolysate of fish meal). Empirically choose the concentration of microorganisms to obtain a Petri-dish solid, but not thick lawn. Standardization is carried out on a scale McFarlanda use the turbidity standards. Suspension DSA test-crop density of 106cells/ml were seeded on Petri dishes with a diameter of 90 mm, 0.2 ml, rubbed with a spatula to obtain a homogeneous lawn and left for 30 minutes for complete absorption in agar. In parallel using the method of deep sowing. In Petri dishes pre-poured into 10 ml of nutrient medium belt and left overnight in an incubator (for the dash�key to contamination). The following day, in the molten agar - 100 ml add 2 ml of suspension daily test culture density of 108cells/ml, mixed thoroughly and poured in 10 ml Petri dishes. Drives a culture of the fungus was placed on the surface of the second layer agar solidifies.

Each experiment was performed in three replicates. The activity of the strain was determined by the average value of the diameter of the zone of inhibition of growth of test cultures. The diameters of zones of inhibition of growth of test organisms around the blocks was measured with a ruler.

From the data in table.1 data, a strain of the fungus Trichoderma hamatum F-l 1 has antimicrobial effects on pathogenic microorganisms of different taxonomic groups.

Example 2. Cultivation of a strain of Trichoderma hamatum F-11 in flasks in liquid culture medium of different composition

The pre-culture is enlivened by seeding tubes with a dense nutrient medium (Czapek agar, Sabouraud agar, wort agar). Sown shoals grown in a thermostat at a temperature of 22-25°C for 5-7 days before mass sporulation depending on the medium composition and temperature of cultivation. For cultivation of strain Trichodermahamatum F-l 1 in flasks using nutrient medium of the two compositions (g/l):

Wednesday No. 1

soy flour 30,0
yeast extract8,0
potassium phosphate odnozameshchenny3,0
drinking water before1 l
pH6,3±0,2

Wednesday No. 2

molasses30,0
urea0,5
magnesium sulfate0,3
potassium phosphate odnozameshchenny3,0
sodium citrate trehzameshchenny (sodium citrate)0,4
drinking water before1 l
pH6,3±0,2

Seeding a nutrient medium is prepared from a 3-day inoculum. To obtain the inoculum on Wednesday placed the agar block with lawn fungus grown on the environment of čapek+. For seed culture medium using an inoculum of 5 ml. the Cultivation is carried out in katalozhnyh flasks V=750 ml, containing 100 ml of medium at 25°C on m�resonant rocking with the speed of rotation of the platform 220 rpm for 72 hours. In the process of growth of a culture of control the absence of extraneous microflora, pH, productivity, morphological state of culture. As an indicator of productivity of strain of the fungus uses the value of dry weight of fungal biomass. As a result of cultivation of a strain of the fungus get a culture fluid with the following values of dry weight of fungal biomass (tab.2).

The results showed that the state of development of culture of Trichoderma hamatum F-l 1 on environment No. 1> and no 2 the same. But in terms of productivity (dry weight of biomass of the fungus) medium No. 1 is much superior environment No. 2 on all phases of culture growth. Therefore, for the cultivation of a strain of the fungus Trichoderma hamatum F-l 1 recommended environment No. 1.

Example 3. Obtaining fungal substance from the strain of Trichoderma hamatum F-11 and tested for activity against anthrax

To obtain fungal substance strain Trichoderma hamatum F-l 1 are cultured in the medium No. 1 according to the method described in example 2. Obtained by cultivation of the producer strain culture fluid was centrifuged at 6000 rpm for 20 min. In the result, get centrifugation of the culture liquid centrate (FMC), which is used to determine the antibacterial activity against the pathogen Siberian I�you (vaccine strain of Bacillus anthracis, STI-1). Characteristics of the strain are presented in table 3.

As a nutrient base for the cultivation of the vaccine strain is an enzymatic hydrolysate of meat or riboksina flour. To a nutrient base containing 100-120 mg % of amine nitrogen, contribute microbiological agar at the rate of 15 g/l, the pH was adjusted to 7.2 to 7.3, poured into vials and sterilized 12-20 minutes in an autoclave at 0.5 ATM. Before applying in agar nutrient medium is added polymyxin b at a concentration of 100 μg/cm3to prevent the growth of foreign microflora. The medium is poured into Petri dishes with a layer thickness of 2.0-2.5 mm.

Vaccine strain B. anthracis STI-1 cultured for 1-2 days in a thermostat at a temperature (24±0,5)°C. For seeding a nutrient medium for bacterial culture empirically choose the concentration of the microorganism to obtain a Petri-dish solid, but not thick lawn. Standardization is carried out on a scale McFarlanda using turbidity standards. The suspension of the daily culture density of 106cells/ml plated onto Petri dishes with a diameter of 90 mm, 0.2 ml, triturated with a spatula to obtain a homogeneous lawn and leave for 30 minutes for complete absorption in agar. Cups with a lawn of bacterial culture put in a thermostat and incubated for 1-2 days at a temperature of (24±0,5)°C.

To determine�Oia antibacterial activity have two doses of FMC (10 and 100 µl), which bring in 4 replications in the wells of 5 mm diameter on the lawn of the test culture of the vaccine strain of Bacillus anthracis STI-1. In this case, applying the following scale of activity: high activity - diameter of zone of inhibition 21-30 mm or more; moderate activity zone diameter 10-20 mm: low activity - the diameter of 6-9 mm.

For more informative and reliable data regarding activity of the strain Trichoderma hamatum F-11 is used for comparison FMC other strains of the most widespread natural fungus Trichoderma asperellum. (PL.4).

Installed, at doses of 10 and 100 ál of the high activity of the FMC noted only for the strain of Trichoderma hamatum F-11 respectively 23 to 25 and 35-45 mm. For FMC strains of T. asperellum F-1290 high activity (respectively 28-45 and 22-30 mm) is observed only at the dose of 100 µl. FMC strain of T. asperellum F-7 at a dose of 100 µl provides moderate activity. Therefore, FMC strain of the fungus Trichoderma hamatum F-11 has a high antibacterial activity at different doses its use against anthrax microbe Bacillus anthracis STI-1.

Thus, the strain of micromycetes Trichoderma hamatum F-11 has antibacterial activity against the causative agent of anthrax and can be recommended as the product of a biological preparation.

Strain of micromycetes Trichoderma hamatum with antibacterial�th activity against the anthrax Bacillus anthracis and deposited in the State collection of pathogenic microorganisms and cell cultures "GKM - Obolensk", registration number F-11.



 

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