RU2564577C1 - STRAIN OF Trichoderma citrinoviride Bissett - PRODUCER OF ANTIBIOTICS-PEPTAIBOLS WITH ANTIFUNGAL AND ANTIBACTERIAL ACTIVITY - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of Trichoderma citrinoviride RNCIM F-1228 is a producer of membrane-active antibiotics-peptaibols having antifungal and antibacterial activity. The strain was obtained by selection from a strain of Trichoderma citrinoviride TV4-1. The values of the diameter of the inhibition zone of growth of pathogenic fungi Candida albicans ATCC 2091 and Aspergillus niger INA 00760 with extracts of the culture fluid of the proposed strain are respectively 15±2 mm 25±2 mm. The value of the diameter of the inhibition zone of growth of methicillin-resistant strain of Staphylococcus aureus FDA 209R with the extract of the culture fluid of the proposed strain is 35±2 mm.

EFFECT: improvement of the strain properties.

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Description

The invention relates to the field of medical microbiology and biotechnology, microscopic fungi - producers of antibiotic compounds for the production of drugs.

Against the background of a general decrease in the body's immune defense, an increase in the incidence of mycoses is observed [Blyth C., Hale K., Palasanthiran P., O'Brien T, Bennett M. Antifungal therapy in infants and children with proven, probable or suspected invasive fungal infections // Cochrane Database Syst Rev. 2010. - P. 213-220] and infections caused by resistant forms of pathogenic bacteria [Antoniev A.A. Examination of disability in skin diseases / A.A. Anthonyev. - M. - 1982. - 224 p.]. Cases of mixed infectious lesions are quite frequent, which requires the simultaneous use of antifungal and antibacterial drugs [Ruiz-Camps, Cuenca-Estrella Antifungals for systemic use // Enferm Infecc Microbiol Clin. 2009.V. 27 (6). - P. 353-362 .; Chagla Z., Salvadori M., Sontrop JM, John M., Hussain Z., Chagla Y., Warshawsky B., Achiam C., Thompson B. Genetically typed community-acquired methicillin-resistant Staphylococcus aureus in a Canadian hospital // Can J Infect Dis Med Microbiol. 2009. - 20 (3): e113-114]. The development of fungal and bacterial infections in immunoscopic and oncological patients is especially dangerous [Runke M. Fungal infections in immunocompromised patients (Epidemiology, diagnosis, therapy, prevention) Problems of medical mycology. - 2000. - No. 1. - S. 4-16; V.M. Gelfond Infectious complications in cancer patients / Practical Oncology, T. 10, No. 3 - 2009, p. 141-146]. Therefore, the search for microbial producers with the ability to synthesize compounds with a wide spectrum of action - antifungal and antibacterial, including against resistant forms of bacteria, is relevant.

Known strains of microscopic fungi Penicillium chrysogenum, Cephalosporium acrenomium (Acremonium chrysogenum), Aspergillus fumigatus, Penicillium griseofulvum, P. nigricans - producers of antibiotics against pathogenic bacteria or fungi and protozoa [Egorov, N.S. Fundamentals of the doctrine of antibiotics / N.S. Egorov // M .: Publishing house of Moscow State University. - 2004. - 512 s .; Industrial Microbiology: Textbook. allowance for universities on special. "Microbiology" and "Biology" / Z.A. Arkadyev, A.M. Bezborodov, I.N. Blokhin et al .; under the editorship of N.S. Egorova. M .: Higher. Shk., 1989. 688 p.].

Known strains of MG-97 T. asperellum having fungicidal activity against phytopathogens of the genus Fusarium, Alternaria [Patent RU No. 2171580. The strain of the fungus Trichoderma sp.MG-97, used to protect coniferous seedlings from fusarium infections. / Gromovykh T.I., Shmarlovskaya S.V., Tyulpanova V.A., Gromovykh B.C. Application No. 99126556/13 of 12/17/1999 Publ. 08/10/2001, Bull. No. 22. 2001] and T. lignorum T13-82, which is active against phytopathogenic fungi and bacteria [RU Patent No. 2121793. Stepanova EV, Sternshis MV, Repin V.E. Trichoderma lignorum strain used against phytopathogenic fungi and bacteria. Publ. 07/14/1998 g].

Known strains of the species Trichoderma asperellum and T. koningii synthesizing peptiboles that inhibit pathogenic yeast fungi and have antiprotozoal activity [Reino J.L. Secondary metabolites from species of the biocontrol agent Trichoderma I J.L. Reino, R.F. Guerriero, R. Hema`ndez -Gala`, I.G. Collado // Phytochem Rev. - 2008. - Vol. 7. - P. 89-123].

Closest to the proposed invention, which should be taken as the closest analogue, is a strain of Trichoderma citrinoviride 2963, which produces an antibiotic from the group of peptiboles that inhibits the growth of methicillin-resistant Staphylococcus aureus [Alimova F.K. Trichoderma Hypocrea (Fungi, Ascomycetes, Hypocreales): taxonomy and distribution / F.K. Alimova // Kazan: Kazan State. University. - 2005. - 264 s]. However, this strain does not have antimycotic activity against opportunistic and pathogenic mycelial and yeast fungi, as well as against pathogenic bacteria other than methicillin-resistant Staphylococcus aureus.

The objective of the present invention is to search for a strain of the microscopic fungus Trichoderma citrinoviride, producing antibiotics with high activity against bacteria, including resistant forms of staphylococcus, and to opportunistic and pathogenic mycelial and yeast fungi.

The technical result of the invention is a new strain of TYVI 4/11 VKPM F-1228 Trichoderma citrinoviride, which produces peptiboli with high antibacterial activity, including resistant staphylococci, and fungicidal activity - for opportunistic and pathogenic species of fungi. The strain is designed to create broad-spectrum antibiotics - antibacterial and antifungal.

The essence of the invention lies in the fact that a new strain of Trichoderma citrinoviride VKPM F-1228 is grown on Saburo liquid nutrient medium at 22-25 ° C for 10-14 days, fungal biomass is separated from the culture fluid by filtration, antibiotic compounds are extracted with ethyl acetate from the filtered culture fluid (when the ratio of extractant to culture liquid 3: 1), the obtained extracts are evaporated to dryness under vacuum at 42 ° C, the dry preparation is stored at 5 ° C. To determine the antibiotic activity, the standard disc method is used, which is impregnated with an extract (preparation) dissolved in a 60% aqueous ethanol solution and dried in air under sterile conditions. About the antibiotic activity of the drug is judged by the diameter of the zones of suppression by the disk of test strains of pathogenic and conditionally pathogenic bacteria and fungi [Egorov, N.S. Fundamentals of the doctrine of antibiotics / N.S. Egorov .// M.: Publishing House of Moscow State University. - 2004. - 512 p.]. A higher or comparable effect of the Trichoderma citrinoviride VKPM F-1228 preparation is obtained in comparison with standard disks with amphotericin B (NII Pasteur, 40 μg / ml) and ampicillin (NII Pasteur, 10 μg / ml). The presence of peptiboles in the preparation by mass spectrometry [T. Neuhof, R. Dieckmann, I. Druzhinina, C.P. Kubicek, N. von Do¨hren Intact-cell MALDI -TOF mass spectrometry analysis of peptaibol formation by the genus Trichoderma. Hypocrea: can molecular phylogeny of species predict peptaibol structures? // Microbiology. - 2007. - Vol. 153. - P. 3417-3437; N. Stoppacher, S. Zeilinger, M. Omann, P. Lassahn, A. Roitinger, R. Krska, R. Schuhmacher Characterization of the peptaibiome of the biocontrol fungus Trichoderma atroviride by liquid chromatography / tandem mass spectrometry // Rapid Commun. Mass Spectrom. 2008; 22: 1889-1898].

Collection cultures of pathogenic mycelial and yeast microscopic fungi Aspergillus niger INA 00760, Candida albicans ATCC 2091, C.tropicales INA 00763, conditionally pathogenic micromycetes - A. oryzae 1K, A. niger 2K, A. fumigatus 4K, A. terreus 4K, A. fisheri 3K, A. flavus 7K, A.ustus 8K and test cultures of gram-positive bacteria strains - Bacillus subtilis ATCC 6633, B. mycoides 537, Micrococcus luteus NCTC 8340, methicillin-resistant strain Staphylococcus aureus FDA 209P, Bacillus , gram-negative bacteria - Escherichia coli ATCC 25922, Comamonas terrigena ATCC 8461.

The strain Trichoderma citrinoviride TYVI 4/11 was obtained as a monospore clone by selection for antimicrobial and antitumor activity from strain TV4-1, previously isolated from the mulch of the nursery of the Kaa-Khem forestry of the Republic of Tuva of the Russian Federation.

The strain Trichoderma citrinoviride is deposited in the All-Russian Collection of Industrial Microorganisms under the number VKPM F-1228.

Strain TYVI 4/11 VKPM F-1228 was identified as Trichoderma citrinoviride based on sequencing of ITS1 and ITS2 genome regions and cultural morphological characters.

The strain Trichoderma citrinoviride Bissett VKPM F-1228 belongs to mitospore fungi - anamorphs of ascomycetic affinity: the genus Hypocrea, the family Nurosgeaseae, order Hypocreales, class Sordariomycetes, Ascomycota (www.indexfungorum.org).

Cultural and morphological features

Macromorphological signs. On Chapek’s medium at 25 ° C, growth is fast, the colony edge is fringed, mycelium is colorless, creeping, cobwebby, cottony, sporulation color is bright - apple-green or greenish-yellow, forms a yellow pigment, exudate, limited to small droplets.

Micromorphology. The hyphae are colorless, smooth, 2-8 µm in diameter. Conidiophores unpainted, smooth, 2.5-5 µm in diameter. The main axis is long, branched irregularly, on the conididiophore one or two branches. The phialids are located directly on the conidiophore or on the branches, one at a time, less often whorls of two or three. Sporulation in large pads, conidia are small, smooth, 2.5-3.5 X 1.5-2.8 μm, forms chlamydospores.

For the cultivation of the strain, in addition to the екаapek dianostic medium, wort agar and Saburo medium are used.

Molecular genetic traits

The strain has the following sequence registered in the NCBI under the code KF928670:

The degree of similarity of the new strain TYVI 4/11 VKPM F-1228 with the collection strains of the species Trichoderma citrinoviride is 98.0%, which indicates, along with the cultural and morphological properties, its belonging to Trichoderma citrinoviride.

Physiological and biochemical characteristics

Aerob, optimal growth temperature 25-26 ° C, pH 5.5-5.8. The strain assimilates various readily available carbohydrates - mannose, raffinose, lactose and other, mineral and organic (peptone) sources of nitrogen, resistant to elevated concentrations of cadmium, nickel and lead in the medium. It grows well and spores on media containing wort and / or glucose.

The strain is stored on wort agar in test tubes at a temperature of 5-6 ° C with periodic reseeding (1 time in 6-12 months). For long-term storage, lyophilization and cryopreservation methods are used.

The strain is a non-pathogenic and non-toxic microorganism.

The invention is confirmed by the following examples

Example 1. Determination of the fungicidal activity of strain TYVI 4/11 VKPM F-1228 T. citrinoviride against strains of pathogenic and opportunistic fungi

As a seed, a 5-day culture obtained on wort agar is used. The strain is grown in Saburo liquid standard medium (300 ml) under stationary conditions in 500 ml flasks for 14 days at 25 ° C. The mycelium biomass and spores are separated by filtering the culture fluid through membrane filters on a Seitz funnel under vacuum. Antibiotic substances from the filtered culture fluid (KZh) and fungal biomass are extracted with ethyl acetate (at a ratio of extractant to culture fluid 3: 1). The obtained extracts are evaporated in vacuo on a Rotavapor-RBüchi rotary evaporator (Switzerland) to dryness at 42 ° C, dry residue - the preparation is stored at 4 ° C. To determine the antibiotic activity using the disk method [Egorov, 2004]. The disks are impregnated with a preparation dissolved in a 60% aqueous ethanol solution and dried in air under sterile conditions.

Collectible strains of pathogenic mycelial and yeast microscopic fungi Aspergillus niger INA 00760, Candida albicans ATCC 2091, C.tropicales INA 00763 and conditionally pathogenic micromycetes - A. oryzae 1K, A. niger 2K, A. fumigatus 4K, A. terreus 4K, A. fisheri 3K, A. flavus 7K, A.ustus 8K. The control is standard disks with amphotericin B (NII Pasteur, 40 μg / ml). The diameter of the zone of inhibition of growth of test cultures is estimated at 5-7 days. Petri dishes with yeast are incubated at 37 ° C in Saburo's medium, with pathogenic and opportunistic fungi, microscopic mushrooms at 28 ° C in Chapek's medium.

Ethanol and methanol extracts from the mycelium of Trichoderma citrinoviride TYVI 4/11 VKPM F-1228 are obtained by extraction with methanol or ethanol for 24 hours (1 g of mycelium per 100 ml of extract) and it is established that they do not show antimycotic activity or is trace.

Extracts of the culture fluid of the strain Trichoderma citrinoviride TYVI 4/11 VKPM F-1228 have a wide range of fungicidal activity against pathogenic and conditionally pathogenic types of fungi. The values of the zone of growth inhibition of test cultures of C. albicans ATCC 2091, Candida tropicales INA 00763 and A. niger INA 00760 are 25 ± 2 mm, 28 ± 2 mm and 31 ± 2 mm, respectively. The strain inhibits the growth of all opportunistic test cultures, but is especially effective against A. fumigatus 4K, A. tereus 4K, A. ustus 8K. The antibiotic activity of the drug from the culture fluid citrinoviride TYVI 4/11 VKPM F-1228 in most cases exceeds the activity of amphotericin B (table 2).

Example 2

The experiment is carried out according to example 1, but the strain Trichoderma citrinoviride TYVI 4/11 VKPM F-1228 is cultivated for 10 days at 25 ° C.

Extracts of the culture fluid of the strain Trichoderma citrinoviride TYVI 4/11 VKPM F-1228 have a wide range of fungicidal activity against pathogenic and conditionally pathogenic types of fungi. The values of the growth inhibition zone of test cultures of pathogenic fungi C. albicans ATCC 2091 and A. niger INA 00760 are 15 ± 2 mm and 25 ± 2 mm, respectively. On the 10th day, the strain is not active against C. tropicales INA 00763.

Example 3

The experiment is carried out according to example 1, but Gram-positive bacteria strains — Bacillus subtilis ATCC 6633, B. mycoides 537, Micrococcus luteus NCTC 8340, methicillin-resistant Staphylococcus aureus strain FDA 209P, Bacillus coagulans 429, and gram-negative bacteria — Escherichia col , Comamonas terrigena ATCC 8461.

Test cultures of B. subtilis ATCC 6633 and B. mycoides 537 are grown on a medium prepared on Hottinger broth in a 1: 2 dilution of the following composition (g / l): glucose - 10.0; NaCl - 2.0; agar - 20.0. E. coli ATCC 25922, M. luteus NCTC 8340, S. aureus PDA 209P, terrigena ATCC 8461- on meat-peptone agar (MPA) (CJSC NICF, Russia). Daily cultures of bacteria are used for sowing. Bacteria are grown in tubes with beveled nutrient agar, then the cells are suspended in physiological saline to a turbidity of 0.5 according to McFarland standard (1.5 × 10 ⁸ CFU / ml) and used for 15 minutes. Bacilli, staphylococci and micrococci are cultured at 37 ° C, E. coli at 42 ° C, B. coagulans at 55 ° C. Inoculation of cultures on a cup is carried out by strokes in 3 directions at an angle of 60 °, each time turning the cup around its axis. The antibacterial effect of the extracts of the strain is evaluated by the accepted method using sterile paper disks soaked in extracts and dried under sterile conditions. The control are standard disks with ampicillin (NII Pasteur, 10 μg / ml). The diameter of the zone of inhibition of growth of test cultures is evaluated on the 2nd day of the disk [Egorov, 2006].

Ethanol and methanol extracts from the mycelium Trichoderma citrinoviride TYVI 4/11 VKPM F-1228 do not show antibacterial activity or it is trace.

The QOL extract of the proposed strain inhibits the growth of methicillin-resistant Staphylococcus aureus S.aureus FDA 209P, which causes dangerous "hospital" infections (Table 3). The size of the zone of inhibition of its growth is 35 mm from the extract in comparison with 15 mm from ampicillin. The strain also has higher antibiotic activity to B. subtilis ATCC 6633, B. mycoides 537 and C. rigena ATCC 8461 and is close to ampicillin to other test bacteria.

Example 4

The experiment is carried out according to example 3, but the cultivation of the proposed strain is carried out 10 days at 22 ° C.

The QOL extract of the proposed strain inhibits the growth of methicillin-resistant Staphylococcus aureus S. aureus FDA 209P, which causes dangerous "hospital" infections (Table 4). The size of the zone of inhibition of its growth is 30 mm from the extract in comparison with 15 mm from ampicillin. The strain also exhibits higher antibiotic activity against B. coagulans 429, B. subtilis ATCC 6633, B. mycoides 537 and C, rigena ATCC 8461 and is close to ampicillin to other test bacteria.

Example 5. Isolation and identification of peptibole from ethyl acetate extracts of the culture fluid of the strain Trichoderma citrinoviride TYVI 4/11 VKPM F-1228

Thin layer chromatography is used to isolate the active fractions of peptibole from ethyl acetate extracts. Samples of extracts (10-15 μl) were applied onto DCAlufoilien Kieselgel-60 plates (Merck, Germany) 15 × 15 cm in size with a thin layer of silica gel and the chloroform: methanol (40:10) and ethyl acetate: methanol (35: 5) systems were placed ) Bioautography of antibiotic substances is carried out with the test organisms B. subtilis ATCC 6633 and A. niger INA 00760. The active fraction is removed with a spatula from a plate with TLC, then eluted in 60% ethanol, filtered through membrane filters and evaporated to dryness under vacuum at 42 ° C. then dissolved in 60% ethanol.

The molecular weights of the active compounds are determined on an Ultraflex II MALDI ToF / ToF Bruker Daltonics mass spectrometer (Germany) equipped with a 355 nm (Nd) UV laser in the mode of obtaining positive ions using a reflectron. 1 μl of a sample solution and 0.3 μl of a solution of 2.5-dihydroxybenzoic acid with a concentration of 10 mg / ml in 20% acetonitrile with 0.5% trifluoroacetic acid are mixed on the target, and the resulting mixture is dried in air.

The IR spectra of the compounds of the active fractions were obtained on a Nicolet iS10 IR Fourier spectrometer (Thermo Fisher Scientific, USA) using a DTGS detector, a KBg beam splitter, and a Smart Performer attachment equipped with a ZnSe crystal. The measurement is carried out at a resolution of 4 cm ^-1 ; spectral region 3000-650 cm ^-1 . The IR spectra of the compounds are processed using the OMNIC-7.0 program, and their interpretation is made according to Tarasevich [B. Tarasevich IR spectra of the main classes of organic compounds / BN. Tarasevich // Reference materials. Moscow State University, 2012. - 55 s].

The active components present in the fraction have the following (Yes) masses: 1701, 1712, 1714, 1724, 1726, 1728, 1730, 1736, 1742, 1743, 1749, 1752, 1754, 1756, 1766, 1768, 1770, 1784 (Fig. one).

The IR spectrum of the active fraction is characterized by the presence of the following absorption bands (ν _max , cm ^-1 ): a pronounced absorption band in the region of 3360-3370 cm ^-1 , which corresponds to stretching vibrations of the OH group, small peaks at 2854 cm ^-1 can be caused by vibrations aliphatic groups of CH ₂ ; peak at 1739 cm ^-1 corresponds to the stretching vibrations of C = O in the COOH from dicarboxylic acids band at 1600-1560 cm ^-1 corresponds to vibrations of CO ₂ from amino group, with absorption maximum at 1261 cm ^-1 can be caused by stretching vibrations of C -O of primary and secondary alcohol groups.

A comparison of the mass spectrum and IR spectra of the active fraction of the ethyl acetate extracts of the QOL of Trichoderma citrinoviride TYVI 4/11 BKHMF-1228 strain with those of the known compounds, as well as their antibacterial and fungicidal activity, indicates that the antibiotic compounds in the culture fluid extract belong to a group of membrane-active peptides - peptiboles [T. Neuhof, R. Dieckmann, I. Druzhinina, C.P. Kubicek, H. von Do¨hren Intact-cell MALDI - TOF mass spectrometry analysis of peptaibol formation by the genus Trichoderma. Hypocrea: can molecular phylogeny of species predict peptaibol structures? // Microbiology. - 2007. - Vol. 153. - P. 3417-3437].

FIG. 1. Mass spectrometric analysis of the T. citrinoviride TYVI 4/11 fraction having fungicidal activity.

FIG. 2. The IR spectrum of the active fraction of T. citrinoviride TYVI 4/11 with fungicidal activity.

Claims (1)

The strain of micromycete Trichoderma citrinoviride Bissett VKPM F-1228 is a producer of membrane-active antibiotics of peptibole with antifungal and antibacterial activity.

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