RU2122026C1 - Strain of bacillus subtilis tpac showing resistance to tetracycline, rifampicin, ampicillin, streptomycin and exhibiting antibacterial activity with respect to microorganism pathogenic species - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: strain B. subtilis TPAC-KM-116 shows inhibiting activity with respect to pathogenic species of microorganisms B. anthracis (anthracis pathogen), V. cholerae (different types of cholera pathogen), Y. pestis (plague pathogen), Ps. pseudomallei (meliodosis pathogen). Strain shows also multiple (Rif^r,Ap^r,Str^r,Tc^r) drug resistance. Strain ensures to construct on its basis high-active antibiotic-resistant probiotic that can be used for treatment and prophylaxis of patients with infectious diseases at simultaneous therapy with antibiotics. EFFECT: new strain indicated above, enhanced effectiveness of the strain. 2 tbl, 2 ex

Description

 The invention relates to medical microbiology and for the production of genetically modified variants of probiotics, while maintaining the inhibitory properties of Bacillus anthracis, Vibrio cholerae, Yersinia pestis, Pseudomonas (Burkcholderia) pseudomallei. The strain is assigned the number KM-116 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 animal 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, N 4, pp. 92-112; Reznik S.R., Vyunitskaya V. ., Afonskaya S. V., Smirnov VV "Serological response to microorganism pathogenic bacteria under the influence of probiotics bacilli", Mikrobiol. Journal, 1993, t.55, N 5, p. 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 Russian Pharmaceutical Committee dated 10/15/92 and the deputy head of the Main Veterinary Directorate of 07/31/92, mention that since the bacteria of the strain of hay bacillus are highly sensitive to antibiotics, then "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 ) while maintaining inhibitory properties against B.antgracis (anthrax pathogen), V. cholerae (various types of cholera pathogen), Y. pestis (the causative agent of the plague), P. pseudomallei (the causative agent of melioidosis), as well as cultural, morphological and biochemical properties characteristic of the strain of the prototype B.subtilis 534, which is the basis of the drug "sporobacterin" company "Bakoren", 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, p.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). As the initial (recipient) strain B.subtilis 534 was used with an antimicrobial effect against pathogenic microflora. Marker strains of microorganisms were used as donor cultures for the implementation of recombination processes (Table 1).

 Previously, on α-agar (Luria medium) with doses of 2.5; 5; The level of sensitivity (resistance) to various, currently most used in clinics, ten antibiotics: ampicillin (Ap) was determined for 10 μg / ml of the 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 B. subtilis 534 strain, single cells of the bacillus population resistant to 10 μg / ml Str. To other antibiotics B.subrilis 534 was sensitive.

With a high frequency, B.subtilis populations on gradient plates and Rif ^r variants are detected. Moreover, the resistance 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., 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 variant, TRAC-synthesis of β-lactamase is clearly inductive with 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 TRAC 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 ) 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 the B.subtilis TRAC culture grows; on the medium with St10, also B.subtilis TRAC and sometimes single colonies of B. subtilis 534, while on the L-agar without antibiotic both strains grow: B.subtilis TRAC and B.subtilis 534.

 On a medium with antibiotics Tc10, B. subtilis TRAC grows, while B. subtilis 534 is sensitive to TC10, i.e. no growth.

 Example 2. Determination of the antagonistic activity of B.subtilis TPAC 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 TRAC and B. subtilis 534 bacilli, plaques (3-4 mm in diameter) were inoculated with suspensions of test cultures on sectors of plates with BH1 (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 cover of each inverted plaque plate and then placed in a desiccator with a lid (chloroforming should take at least 2 hours, but can take a day 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% BH1 agar (3 ml per tube) and applied (rolled) onto the surface of BH1 agar with chloroformated plaques, but so that 0.5% BH1 agar does not get on the surface of the plaques, but only comes into contact with their edges. Cups with cultures of B.anthacis, 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 TRAC and B.subtilis 534 with respect to cultures of causative agents of especially dangerous infections are taken into account for 2 days in the zones of clearing (lysis) of lawns around B. subtilis plaques.

 In this example, the antagonistic activity of B.subtilis TRAC in relation to pathogenic species of microorganisms (Table 2) in the “delayed antagonism” test manifests itself as in B.subtilis 534.

Thus, the presented strain B.subtilis TRAC-KM-116 exhibits inhibitory activity against pathogenic microorganisms and has multiple (Rif ^r , Ap ^r , Str ^r , Tc ^r ) drug resistance, which makes it possible to design an antibiotic-resistant probiotic for it treatment and prevention of infectious diseases with simultaneous antibiotic therapy.

Claims (1)

 The strain Bacillus subtilis TRAC-KM 116 (Russian collection of pathogenic bacteria "Microbe"), resistant to tetracycline, rifampicin, ampicillin, streptomycin, which has antibacterial activity against pathogenic types of microorganisms.

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