RU2569150C1 - MYCOPLASMA (Mycoplasma hominis) INHIBITOR PRODUCER - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, microbiology. The strain Trichoderma harzianum Rifai VKPM (Russian National Collection of Industrial Microorganisms) F-180 is applicable as Mycoplasma hominis inhibitor producer for treating mycoplasma infections.

EFFECT: invention enables inhibiting growth of Mycoplasma hominis.

1 tbl

Description

The invention relates to medicine, biochemistry, pharmacology and may find application in biotechnology, microbiology.

Mycoplasma hominis is a conditionally pathogenic microorganism, lives in the urogenital tract (UGT) of a person, and, according to some authors, is involved in the development of chorioamnionitis, intrauterine pathology of the fetus, the development of bacterial vaginosis and other pathological conditions of UGT. Cases of the isolation of this mycoplasma from a brain abscess, with pneumonia, with pericarditis, endocarditis, wound infection (Rakovskaya I.V. Mycoplasmas - pathogens of human mycoplasma infections. In the book. Guide to medical microbiology / Edited by A.S. Labinskaya and Dr. M .: BINOM, 2010, p. 964-993).

For the treatment of mycoplasma infections, broad-spectrum antibiotics are used: most often tetracycline antibiotics (dioxicycline, minocycline) and macrolides (josamycin, clindomycin, midecamycin, roxithromycin). However, it is known that many strains of mycoplasmas that live in the urogenital tract are resistant to tetracyclines and macrolides. In such cases, it is recommended to use quinolones: levofloxacin, ofloxacin, pefloxacin, ciprofloxancin. But at present, patients resistant to quinolones are isolated from patients. It was also shown that the effectiveness of antibiotics depends on the state of the immune system. For patients with immunodeficiency, it is recommended to administer intravenous antibiotics and be sure to determine the sensitivity of the allocated mycoplasma to them. Prolonged therapy is often required. In this regard, leading mycoplasmologists believe that there are currently no radical remedies for the treatment of mycoplasma infections (Baseman JB, Tully JG Mycoplasmas: Sophisticated, Reemerging, and Burdened by Their Notoriety. Emerging Infect. Diseases. 1997, Vol 3, No. 1, P. 21-32.).

Known strain Trichoderma harzianum Rifai VKPM F-180 (patent RU 2493247). There is no information on the use of the strain as a producer of the growth inhibitor Mycoplasma hominis.

The technical result of the invention is the inhibition of the growth of Mycoplasma hominis.

The technical result is achieved by the fact that the strain Trichoderma harzianum Rifai is used as a producer of the growth inhibitor Mycoplasma hominis. The strain was deposited in the All-Russian collection of industrial microorganisms under the number F-180 (Moscow, Dorozhny 1-y passage, building 1 Institute of Genetics and Selection, 1987).

Morphological and biochemical characteristics of the strain.

The strain Trichoderma harzianum Rifai on wort-agar medium forms fast-growing colonies. The fungal mycelium is colorless, septate, prostrate. On the 4th - 5th day of growth, tufts with conidiophores appear, pillow-shaped, initially white, with a yellow or dark green color over time. Bottle phialides / 9-12µ /, located in whorls of 3 or more. Conidia glued into the head form on each phialid. The conidia of the fungus are round, smooth, small / 3-5µ /, in transmitted light pale green, and in the mass - dark.

On Chapek’s agar medium, the colonies of the producer strain are rapidly growing, but slightly spore-bearing. The strain grows well on the Chapek medium, but the best growth is observed on the wort-agar medium. Agar does not dilute gelatin, milk does not peptone. Aerobic mushroom. Temperature optimum + 28- + 29 ° C. On potato-dextrose agar, a decrease in growth intensity, the formation of aerial mycelium and sporulation are observed. Optimum conditions for the growth of the fungus at pH 4-6, however, can grow at pH from 1.5 to 9.

Trichoderma harzianum Rifai equally assimilates both ammonium and nitric acid salts. The best source of nitrogen from organic compounds is peptone. It grows well on media with asparagine and with glutamic acid. The best sources of carbon are xylose, glucose, sucrose, lactose, galactose, mannitol, and starch. On media with these sugars, intense growth is observed. Alcohols are poorly absorbed - methyl, ethyl, dulcite. It well assimilates wheat bran as a carbon source. The inoculum of the fungus grown on a medium with wheat bran gives a positive result when used in fermentation on media of different compositions.

The strain Trichoderma harzianum Rifai has L-lysine-α-oxidase activity. The activity of L-lysine-α-oxidase in the Trichoderma harzianum Rifai culture fluid was calculated by the growth of H ₂ O ₂ , the amount of which was determined by a spectrophotometric orthodiazidine micromethod. The essence of the method is the interaction of all formed in the reaction of H ₂ O ₂ with O-dianisidine hydrochloride. The incubation mixture contained 20 μg of peroxidase, 250 μg of O-dianisidine hydrochloride and 0.1-0.5 mg of protein in 1 ml of the final volume. After 20 minutes of incubation in a thermostat at a temperature of + 37 ° C, the samples were cooled to + 4 ° C. The optical density of the colored solutions of the experimental and control (without substrate) samples was measured on an SF-16 spectrophotometer at 540 nm against the second control sample (without peroxidase).

The activity of L-lysine-α-oxidase was calculated by the growth of H ₂ O ₂ and expressed per 1 ml of culture fluid.

The cultivation conditions of the strain Trichoderma harzianum Rifai.

Fermentation of the strain was carried out on the equipment of the Experimental technological installation of IBPM RAS named after G.K. Scriabin (Pushchino city).

Used a BIOR-01 type fermenter manufactured by OKB TBM, Kirishi, with a volume of 100 l with a fill factor of 0.6. The fermenter is equipped with a magnetic stirrer, fine filters, air, temperature and pH sensors.

The nutrient medium was prepared directly in the apparatus. To do this, 60 l of tap water was poured into the apparatus, 1% of wheat bran was added, a stimulant - 0.1%, 1.3% ammonium sulfate, the pH value of 5.8-6.0 was set with a 10% HCl solution. Wheat bran and the stimulator were pre-soaked in 10 liters of water for 4 hours, sterilized in an autoclave for 1 hour at t ° + 125 ° C. Then the prepared fermenter was seeded with seed from flasks. Sowing dose of at least 5%. The cultivation was carried out at a temperature of + 26 ° C, air flow throughout the fermentation 30 l per minute, the speed of rotation of the mixer 200 rpm. The pH during the growth process was adjusted to 6.5. The duration of cultivation was 94-98 hours, the final pH values from 5.3 to 7.5.

At the end of the fermentation, the culture fluid was sent to the pre-treatment section, where the mycelium of the fungus was separated by filtration under vacuum on a suction filter. The weight of the obtained biomass was 3.5 kg. The resulting native solution was subjected to additional separation on an OSB separator at 9000 rpm for one hour at a temperature of + (2-4) ° C. Then the native solution in a volume of 55 l, obtained after separation of the biomass, was concentrated to 1.5 l (concentrate).

The activity of the obtained Trichoderma harzianum Rifai culture fluid concentrate was 0.54 U / ml.

The work used the type of microorganism of the class Mollicutes:

Mycoplasma hominis (Mh). The strain M.hominis H-34 was obtained in 1967 from the collection of Dr. Lemke R.M. (Lister Inst. Preventive medicine, London S.W.I.). The strain is stored and maintained in the laboratory of mycoplasmas and L-forms of bacteria FSBI NIIEM named after honorary academician N.F. Gamalei.

To grow Mycoplasma hominis strain, we used a broth (Difco PPLOBroth, Becton, Dickinson, USA) with 20% horse serum, 2% fresh yeast extract, 1% arginine or 1% glucose (depending on the nutritional needs of mycoplasma species) and 0.005% indicator phenol red. For titration of cultures, serial ten-fold dilutions of the sample were made in physiological saline with seeding from all dilutions on 0.3% agar (BBL Mycoplasma Agar Base, Becton, Dickinson, USA) with the same additives as in the broth. Three days later, the number of colonies in the last two tubes from a series of dilutions was counted and the average number of colonies was calculated. The crop titer was expressed by the average number of colonies grown, multiplied by the cultivation of the culture. Cultures with the previously established caption were taken into experience.

The experiments to determine the sensitivity of mycoplasmas to the concentrate of the culture fluid Trichoderma harzianum Rifai were carried out according to the following scheme. 1 ml of mycoplasma broth culture with a known titer was added to the tubes, then combined with 1 ml of culture fluid with 0.54 U / ml L-lysine-α-oxidase activity in dilutions of 1:10 and 1: 100 and 0.1 ml of mycoplasma culture with a famous caption. The mixtures were kept for 45 min at room temperature, then they were poured with a liquid nutrient medium and cultivated for 3 days, observing the growth rate and intensity by the color change of the culture medium. The titer of mycoplasma cells in the experimental and control tube was determined as described above.

It can be seen from the table that in the presence of the culture fluid of the producer of L-lysine-α-oxidase, the growth rate of Mycoplasma hominis, detected by the color change of the medium, was reduced, especially at a low inoculum dose. When determining the titer of CFU / ml, complete growth inhibition was observed upon contact of mycoplasma cells with undiluted trichoderma culture fluid with an activity of L-lysine-α-oxidase 0.54-0.56 U / ml even at a high inoculum dose. Tenfold dilution of the original trichoderma culture fluid also inhibited the growth of mycoplasmas, but to a lesser extent - by 3 Lg. When using a lower inoculum dose of mycoplasmas, growth was also completely suppressed by contact with the producer culture fluid

enzyme. The use of ten-fold dilution in experimental tubes led to the growth of only single colonies.

The titer of CFU / ml of Mh cultures was lower in comparison with the control by 3 Lg and 1 Lg (respectively) at a high inoculum dose of the culture and different concentrations of the culture fluid of the producer of L-lysine-α-oxidase. At a lower inoculum dose and different concentrations of the culture fluid of the studied strain producing the enzyme, the CFU titer of the grown culture differed by 3 and 2 Lg, respectively.

Thus, as a result of the studies, the obtained data indicate that the Trichoderma harzianum Rifai F-180 culture fluid is able to inhibit the growth of Mycoplasma hominis, which is clearly manifested after preliminary contact of mycoplasmas and trichoderma culture fluid. The degree of growth inhibition depends on the inoculum dose of mycoplasmas and the concentration of trichoderma culture fluid used.

Claims (1)

The use of the Trichoderma harzianum Rifai strain VKPM F-180 as a producer of the Mycoplasma hominis mycoplasma inhibitor.