RU2173342C1 - Anthrax vaccine strain sti-pr-4 with broadened spectrum of resistance to antibiotics - Google Patents

Abstract

FIELD: medicinal microbiology. SUBSTANCE: anthrax strain STI-PR-4 is resistant to the modern agents of emergency prophylaxis, namely to doxycyclin, pefloxacin, rifampicin, ampicillin, nalidixic acid, and shows identical values of immunogenicity with the strain STI-1 which is the parent strain. Invention can be used for constructing vaccine preparations exhibiting resistance to antibiotics and used for prophylaxis of anthrax infection on the background of antibiotic therapy. EFFECT: enhanced resistance of strain. 6 tbl, 4 ex

Description

 The invention relates to medical microbiology and relates to the production of genetically modified variants of the anthrax vaccine strain with an extended spectrum of antibiotic resistance and preservation of the basic biological properties of the STI-1 strain.

 The known anthrax vaccine polyantibiotic-resistant strain STI-PR-3 (Buravtseva N.P., Funtikova T.N., Nelyapin N.M. Immunogenic properties of the anthrax antibiotic-resistant vaccine STI-PR live dry // Abstracts to the Interdepartmental Scientific Conference "Actual issues of the prevention of especially dangerous infectious diseases", March 26-28, 1991 - Kirov, 1991. - P.45-46), resistant to penicillins and rifampicin.

 The closest in characteristics and achieved effect is the STI-AR strain (AP Pomeransev, Yu.V. Noskov, LI Marinin, AV Stepanov, LG Podunova. Anthrax profilacsis by antibiotic resestant strain STI-AR combination with urgent antibiotic therapy. // International Workshop on Anthrax, 19-21 September, 1995.- Winchester, England, 1995. - P.58), which is resistant to ampicillin, rifampicin, doxycycline, chloramphenicol, macrolides. Cultural and morphological properties, spore-forming ability, reactogenicity and immunogenicity remained at the level of the parent strain.

 A disadvantage of the known strains is the lack of resistance to the drugs of the quinolone series, and the strain STI-PR-3 and to doxycycline. Currently, these antibiotics are used as the main drugs for emergency non-specific prophylaxis of various infectious diseases, including anthrax.

 The objective of the invention is to obtain a new polyantibiotic-resistant strain based on the anthrax vaccine strain STI-1, which has resistance, primarily to modern means of emergency prevention: doxycycline and pefloxacin, as well as other antibiotics, which are sensitive to anthrax microbe: rifampicin and penicillin, inferior in terms of immunogenicity to the parent strain STI-1.

 The solution to the problem is achieved by sequentially expanding the spectrum of antibiotic resistance of the culture of strain STI-1 under the control of the stability of this trait and the full size of the toxin formation genes with several successive transfers in a dense nutrient medium in the absence of selective pressure.

 The strain is characterized by the following cultural-morphological, immunobiological and biochemical characteristics.

 Cultural properties.

Optional aerob. The optimum growth temperature on agar and broth (36 ± 1) ^o C, the optimum pH of the medium (7.4 ± 0.2). On nutrient agar forms colonies of R, less often RO- and O-forms. It does not form daughter colonies. On the broth, it gives a flocculent, easily breaking sediment in the form of a piece of cotton wool, on the surface - a thin narrow parietal film, the broth remains transparent, on serum media and in the animal organism it does not form capsules. When cultured on artificial nutrient media during the first two days, the bacilli are in a vegetative form, and if they are longer, they become spore-like.

 Morphological properties.

 It exists in two morphological forms: vegetative - capsuleless and spore-like. Spores painted according to Ziehl-Nielsen have an oval shape (1.3-1.5x0.7-0.9) microns in size, pink in color with a red rim on the periphery.

 Gram-positive sticks in smears from liquid and solid nutrient media are arranged in the form of long chains, at the junction they look chopped off or slightly concave, resembling a bamboo cane. The size of the sticks: length 3-8, sometimes up to 10 microns, width - 1-1.5 microns. Flagella do not have.

 Toxigenic properties.

 With subcutaneous administration of 250 million spores to white mice, the death of animals can reach 70% or more within 2-7 days.

 Biochemical properties.

 It decomposes glucose, trehalose, maltose, mannose, inositol, sucrose, starch, galactose.

 Produces DNAase, catalase.

 As an initial (recipient) strain, B.anthracis STI-1 was used, which is currently used to produce live dry anthrax vaccine.

 Previously, the level of sensitivity to various antibiotics most commonly used for the treatment of anthrax was determined for strain STI-1: ampicillin (Amp); doxycycline (Dox); rifampicin (Rif); pefloxacin (Pef); nalidixic acid (Nal). Single cells of the bacillus population resistant to 5 μg / ml Rif were detected in the recipient strain. The culture of strain STI-1 was sensitive to other antibiotics.

Due to the difficulty of directly obtaining a spontaneous mutant that is resistant to the representative of the fluoroquinolone series of antimicrobials, pefloxacin, a variant resistant to nalidixic acid was originally obtained. From the obtained variant (Nal ^r ) by reseeding on a solid nutrient medium, a variant resistant to pefloxacin (minimum inhibitory concentration (MIC) of more than 100 μg / ml) was obtained.

At the next stage of obtaining a polyantibiotic-resistant strain, a stable spontaneous mutant resistant to 50 μg / ml rifampicin was isolated from the population (Nal ^r , Pef) of the variant. Resistance to the tetracycline drug, doxycycline, was obtained by conjugation of the transposon Tn 916 from B.subtilis strain in (Nal ^r , Pef ^r , Rif ^r ) variant of the STI-1 strain using pefloxacin as a counter-selection drug. The minimum inhibitory concentration (MIC) of the doxycycline variant was 2.5 μg / ml.

To induce ampicillin resistance, we used seeding on a solid nutrient medium (PPS) with increasing concentrations of the antibiotic (Nal ^r , Pef ^r , Rif ^r , Dox ^r ) of the STI-1 strain variant, while the inoculum dose of the spore suspension did not exceed the appearance of false positives 1 • 10 ⁷ ... 4 • 10 ⁷ spores per petri dish. The MPC for the obtained variant was 20 μg / ml, which obviously exceeds the concentrations of this antibiotic that are achievable in the blood.

 The obtained variant (STI-PR-4) when checking the usefulness and stability of the determinants of toxin formation (pag, lef, cya) by the polymerase chain reaction (PCR) did not differ from the reference culture of the initial strain.

 The possibility of carrying out the claimed invention is shown by the following examples.

 Example 1. Description of the main biological properties of the strain STI-PR-4 in comparison with the original strain STI-1.

 From the data presented in table 1 it is seen that the main cultural and morphological properties of the obtained strain did not differ from the properties of the reference culture of the original strain.

 Example 2. Resistance to antibiotics and its stability after 5 transfers in a solid nutrient medium (PPS) in the absence of selective pressure of antibiotics.

 From the data presented in table 2, it is seen that the levels of resistance of the obtained strain are significantly higher than that of the original parent strain STI-1.

 From the data presented in table 3, it is seen that the obtained strain after 5 passages in the absence of selective pressure of chemotherapeutic agents during clone analysis completely preserved the antibiotic resistance levels.

 Example 3. Determination of immunobiological properties of the strain STI-PR-4.

 From the data presented in table 4, it is seen that the STI-PR-4 strain retained the high immunogenic properties of the STI-1 strain.

The data presented in table 5 show that subcutaneous administration of 250 million spores to rabbits did not cause any of them to rise in temperature by more than 1 ^o C and drop in body weight by more than 10% of the initial values. Thus, all experimental series of the strain STI-PR-4 are harmless.

 Example 4. Obtaining spore cultures of anthrax vaccine strain STI-PR-4 by the method of deep cultivation.

 From the data presented in table 6, it follows that the native culture of the genetically modified strain obtained by deep cultivation, the main quality indicators does not differ from the native cultures of the reference vaccine strain STI-1.

Thus, the obtained vaccine polyantibiotic-resistant strain STI-PR-4 has multiple (Rif ^r , Amp ^r , Dox ^r , Pef ^r , Nal ^r ) drug resistance, which makes it possible to construct antibiotic-resistant vaccine preparations for the prevention of anthrax on its basis against the background of non-specific emergency prevention.

Claims (1)

 Siberian-ulcer vaccine strain STI-PR-4 with an extended spectrum of antibiotic resistance, resistant to modern means of emergency prevention: doxycycline, pefloxacin, rifampicin, ampicillin and nalidixic acid.

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