RU2118364C1 - Strain bacillus subtilis tpaxc showing resistance to tetracycline, rifampicin, ampicillin, chloramphenicol, streptomycin and exhibiting antibacterial activity with respect to pathogenic microorganism species - Google Patents

Abstract

FIELD: medicinal microbiology. SUBSTANCE: invention reports the strain Bacillus subtilis TPAXC-KM-117 that shows inhibiting activity with respect to pathogenic microorganisms and shows multiple drug resistance. Strain is resistant to tetracycline, rifampicin, ampicillin, chloramphenicol and streptomycin. This ensures to construct on its base antibiotic-resistant probiotic for treatment and prophylaxis of infectious diseases in combination with simultaneous antibiotic therapy. EFFECT: strain indicated above. 2 tbl, 2 ex

Description

 The invention relates to medical microbiology and relates to the production of genetically modified variants of probiotics while maintaining inhibitory properties to Bacillus anthracis, Vibrio cholerae, Yersinia pestis, Pseudomonas (Burkcholderia) pseudomallei. The strain is assigned the number KM-117 and it is deposited in the Russian collection of pathogenic bacteria "Microbe".

 In recent years, probiotics have been widely used in the treatment of infectious diseases - drugs that include live microorganisms (natural inhabitants of the human intestinal tract) or saprophytes that live in the external environment. Unlike antibiotics, they are completely harmless, environmentally friendly and have no contraindications for clinical use.

 The use of microorganisms from the genus Bacillus (more often B. subtilis) for the construction of probiotics led to the creation of some effective drugs (bactisubtil, bacterin-SL, biosporin, sporobacterin, sporolact, bificol, etc.), which are used in the USA, Italy, Yugoslavia, Germany , France, China, Ukraine, and relatively recently in Russia (Smirnov V.V., Reznik S.R., Vyunitskaya V.A. et al. "Modern ideas about the mechanisms of therapeutic and prophylactic action of probiotics from bacteria of the genus Bacillus", Microbiol. Journal, 1993, vol. 55, No. 4, pp. 92-112; Reznik S.R., Vyunitskaya V .A., Afonskaya SV, Smirnov VV "Serological response of a microorganism to pathogenic bacteria under the influence of probiotics from bacilli", Mikrobiol. Journal, 1993, v. 55, No. 5, pp. 81-83).

 The possibility of creating new probiotics, improving existing ones, as well as their wider use for the treatment and prevention of infectious diseases (including especially dangerous ones), dysbiosis and other disorders of the gastrointestinal tract is an urgent task.

 The instructions for use of "Sporobacterin", approved by the decisions of the Committee of Medical Immunobiological Preparations of the Pharmacommittee of Russia of 10.15.92, and deputy. The chief of the General Directorate of Veterinary Medicine on July 31, 1992, mentions that since the bacteria of the hay bacillus strain are highly sensitive to antibiotics, "the simultaneous administration of antibiotics and other antibacterial drugs, especially by mouth, dramatically reduces the effectiveness of sporobacterin."

The aim of the invention is to obtain a strain of B.subtilis with multidrug resistance (Rif ^r , Ap ^r , Str ^r , Tc ^r , Cm ^r ) while maintaining inhibitory properties against B.anthracis (anthrax pathogen), V. cholerae (various types of pathogen cholera), Y. pestis (the causative agent of the plague), P. pseudomallei (the causative agent of melioidosis), as well as the cultural-morphological and biochemical properties characteristic of the B. protubacterin 534 prototype strain, which is the basis of the Bakoren firm methodacterin preparation, Orenburg.

 To obtain antibiotic-resistant variants of B.subtilis using selection methods (Brown V. "Genetics of Microorganisms". Publishing House "Nauka", M., 1968, pp. 134-139) on gradient plates and using various recombination processes (Prozorov A. A. "Genetic transformation and transfection." Publishing House "Science", M., 1980, pp. 124-129; Battisti L., Green BD, Thorne CB Mating system for transfer of plasmids among Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis J. Bacteriology, 1985, V. 162, N 2. P. 543-550). The B. subtilis 534 strain with antimicrobial effect against pathogenic microflora was used as the initial (recipient) one. Marker strains of microorganisms were used as donor cultures for the implementation of recombination processes (Table 1).

 Preliminarily on Z-agar (Luria medium) with doses of 2.5; 5; The level of sensitivity (resistance) to the ten most commonly used antibiotics currently used in clinics: ampicillin (Ap) was determined for the 10 μg / ml antibiotic in B. subtilis 534 strain. benzylpenicillin (Bp); tetracycline (Tc); streptomycin (Str); chloramphenicol (Cm); erythromycin (Em); rifampicin (Rif); kanamycin (Km); ristomycin (Ris); carbinicillin (Car).

 In the strain B.subtilis 534, single cells of the bacillus population resistant to 10 μg / ml Str. B. subtilis 534 was sensitive to other antibiotics.

Populations of B. subtilis on gradient plates and Rif ^r variants are detected with high frequency. Moreover, the stability of 10 μg / ml of an antibiotic in the blood (cerebrospinal fluid) is extremely rarely cumulated (Navashin S.M., Fomina I.P. Rational antibiotic therapy. M., Medicine, 1982, p. 495).

 Ampicillin resistance (i.e. the synthesis of β-lactamase) in B.subtilis was obtained using genetic recombination processes and has a number of features characteristic of resistance to this antibiotic - in the B.subtilis TPAXC variant, β-lactamase synthesis is clearly inductive with a severity mass effect ", i.e. sowing a small number of spores or vegetative cells of this variant on a medium containing 5-10 μg / ml ampicillin cannot grow on such a medium due to the small pool. At the same time, plating the fresh bacterial mass (vegetative cells) of this B.subtilis variant onto a medium containing even more than 10 μg / ml ampicillin gives an increase in cells with a clear phenotypic manifestation.

 When constructing probiotics with the required properties, a promising strain should combine antagonistic activity with respect to a given set of pathogenic cultures with sufficient resistance to adverse environmental conditions (in this case, antibiotic resistance).

 Example 1. Testing a genetically labeled strain of B.subtilis TPAXC for the manifestation of antibiotic resistance in comparison with the original strain prototype B.subtilis 534.

Previously, both B.subtilis cultures were plated in L-broth tubes (Difco company). Bacillus cultures grow at 37 ^o C during the night (12-18 hours). Then the broth culture with a bacteriological loop is plated (streaks) on L-agar sectors with antibiotics (Rif ^r , Ap ^r , Str ^r , Tc ^r , Cm ^r ) at 10 μg / ml and without them. Cultivation of crops on L-agar at 35-37 ^o C is carried out for 3 days. The results of the growth of the test and control cultures on media with various antibiotics are taken into account after 48-72 hours.

 On Ap10 and Rif10 antibiotic media, only B.subtilis TPAXC culture grows; on the medium with St10, also B. subtilis TPAXC and sometimes single colonies of B. subtilis 534, while on the L-agar without antibiotic both strains grow: B. subtilis TPAXC and B. subtilis 534. Only B grows on the medium with Tc10 .subtilis TPAXC.

 On the L medium with Cm10, only the B.subtilis TPAXC variant grows, and the initial B.subtilis 534 variant does not grow in the presence of this antibiotic.

 Example 2. Determination of the antagonistic activity of B.subtilis TPAXC bacilli and B.subtilis 534 prototype against pathogenic microorganisms (Table 2) was carried out using the “delayed antagonism” test (Konstantinov G. Lysozyme. Determination of lysozyme activity // Experimental Microbiology / Under Edited by S. Byrdarov. - Sofia: Medicine and Physical Education, 1965, p. 327), modified by us.

To determine the antibacterial activity of B.subtilis TPAXC and B. subtilis 534 bacilli, plaques (3-4 mm in diameter) were inoculated with suspensions of test cultures on the sector of plates with BHI (firm Difco) agar (the number of seeded plates should correspond to the number of indicator cultures , i.e. 6). Plaques were grown at 37 ^o C for 48-72 hours (their sizes increase to 15-25 mm in diameter). Plates with grown plaques were treated with chloroform vapors; for this, 15-20 drops of chloroform were added to the lid of each inverted plaque plate and then they were transferred to a desiccator with a lid (chloroforming should take at least 2 hours, but can take 24 hours or more). Then the cups were removed from the desiccator, ventilated from chloroform vapor.

0.1 ml daily broth culture 1-6 (Table 2) was seeded in molten and cooled to 45 ^° C. 0.5% BHI agar (3 ml per tube) and applied (rolled) onto the surface of BHI agar with chloroformated plaques, but so that 0.5% BHI agar does not get on the surface of the plaques, but only comes into contact with their edges. Cups with cultures of B. anthracis, V. cholerae, P. pseudomallei were placed in a thermostat for 24 hours at 37 ^o C, and Y. pestis EV - at 28 ^o C.

 The results of "delayed antagonism" of B.subtilis TPAXC and B.subtilis 534 with respect to cultures of causative agents of especially dangerous infections are taken into account for 2 days along the zones of clearing (lysis) of lawns around B.subtilis plaques.

 In this example, the antagonistic activity of B.subtilis TPAXC with respect to pathogenic microorganisms (Table 2) in the “delayed antagonism” test appears in the same way as in B.subtilis 534.

Thus, the presented strain B.subtilis TPAXC-KM-117 exhibits inhibitory activity against pathogenic species of microorganisms and has multiple (Rif ^r , Ap ^r , Str ^r , Tc ^r , Cm ^r ) drug resistance, which makes it possible to construct on its basis antibiotic-resistant probiotic for the treatment and prevention of infectious diseases with simultaneous antibiotic therapy.

Claims (1)

 \ \\ 1 The strain Bacillus subtilis TRAX-KM 117 (Russian collection of pathogenic bacteria "Microbe"), resistant to tetracycline, rifampicin, ampicillin, chloramphenicol, streptomycin, which has antibacterial activity against pathogenic types of microorganisms.

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