Anthrax vaccine strain sti-pr-4 spread spectrum antibiotic-resistance

 

(57) Abstract:

The invention relates to medical Microbiology and can be used to construct antibiotic-resistant vaccines used for the prevention of anthrax infection on the background of antibiotic therapy. The anthrax strain STI-PR-4 resistant to modern means of emergency prophylaxis: doxycycline, pefloxacin, rifampicin, ampicillin, nalidixic acid and is not inferior in terms of immunogenicity strain STI-1, on the basis of which he received. 6 table.

The invention relates to medical Microbiology and the receipt of genetically modified variants of the anthrax vaccine strain spread spectrum antibiotic resistance and the retention of the main biological properties of strain STI-1.

Known anthrax vaccine, polyantibiotic-resistant strain STI-PR-3 (Buravceva N. P., Funtikova I.e., Alapin N. M. Immunogenic properties of the anthrax antibiotic-resistant vaccine STI-PR live dry // abstracts of the Interdepartmental scientific conference "Actual issues of prevention of especially dangerous infectious diseases", March 26-28, 1991 - Kirov, UEMOA effect is strain STI-AR (A. P. Pomeransev, Yu.V. Noskov, L. I. Marinin, A. V. Stepanov, L. G. 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 quinolone drugs series, and the strain STI-PR-3 and doxycycline. Currently, these antibiotics are used in the main drugs in an emergency nonspecific prevention of various infectious diseases, including anthrax.

The task of the invention to provide a new polyantibiotic-resistant strain-based anthrax vaccine strain STI-1 with resistance, especially to modern means of emergency prophylaxis: doxycycline and pefloxacin, as well as other antibiotics to which sensitive anthrax microbe: rifampicin and penicillin, and not inferior in terms of immunogenicity parent strain STI-1.

Resensi-1 under the control of the stability of this trait and polnorazmernoi gene coccinobaphi when several successive subcultures on solid nutrient medium in the absence of selective pressure.

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

Cultural properties.

Facultative aerobe. Optimum temperature for growth on agar and broth (361)oC, the optimum pH of the medium (7,40,2). On nutrient agar forms a colony R, less RO - and O-forms. The child does not form colonies. The broth gives flaky, easily broken precipitate in the form of a piece of cotton wool on the surface of the thin narrow wall film, the broth remains clear, on whey environments and in animals capsule does not form. Under cultivation on artificial nutrient media during the first two days of bacilli are found in the vegetative form, with the longer - pass in spore.

Morphological properties.

There are two morphological forms: vegetative backupsonline and spore. Spores stained by Zn, have an oval shape size (1,3-1,h,7-0,9) µm, pink with a red rim on the periphery.

Gram-positive bacilli in smears with liquid and solid nutrient media are in the form of long chains, in their connections are severed or slightly Vogue the Ute.

Toxigenic properties.

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

Biochemical properties.

Decomposes glucose, trehalose, maltose, mannose, Inositol, sucrose, starch, galactose.

Produces DNA-ABC, catalase.

As the source (recipient) was used strain of B. anthracis STI-1, currently in use for production of live dry anthrax vaccine.

Previously in strain STI-1 was determined level of sensitivity to a variety of the most used currently for the treatment of anthrax antibiotics: ampicillin (Amp); doxycycline (Dox); rifampicin (Rif); pefloxacin (Pef); nalidixic acid (Nal). The recipient strain revealed a single cell populations of bacilli resistant to 5 μg/ml Rif. To other antibiotics culture of strain STI-1 was sensitive.

Due to the complexity of direct receipt of a spontaneous mutant resistant to representative fluoroquinolone several antimicrobial drugs - pefloxacin, was originally a variant resistant to nalidixic acid. From the resulting variant (Nalr) re concentration (MIC) of more than 100 µg/ml).

In the next step of receiving polyantibiotic-resistant strain of the population (Nalr, Pef) option selected stable spontaneous mutant resistant to 50 µg/ml rifampicin. Resistance to the drug tetracycline - doxycycline was obtained by the method of conjugation of transposon Tn 916 from B. subtilis (Nalr, Pefr, Rifr) variant of the strain STI-1 using pefloksatsina as drug controlactive. The minimum inhibitory concentration (MIC) option on the doxycycline was 2.5 µg/ml.

For induction of resistance to ampicillin used seeding on solid nutrient medium (PPP) with increasing concentrations of the antibiotic (Nalr, Pefr, Rifr, Doxr) variant strain STI-1, while sowing dose of spore suspension to prevent false-positive results did not exceed 1107... 4107spores per Petri dish. IPC the resulting variant was 20 µg/ml, which obviously exceeds the achievable blood concentrations of this antibiotic.

Received option (STI-PR-4) when checking the soundness and stability of the determinants of coccinobaphi (pag, lef, cya) by polymerase chain reaction (PCR) was not different from the cauldron in the following examples.

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

From the presented data in table 1 shows 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. Antibiotic resistance and its stability after 5 transfers on solid nutrient medium (PPP) in the absence of selective pressure of antibiotics.

Presented in table 2 data shows that the levels of stability of the resulting strain is much higher than the original parent strain STI-1.

From the data presented in table 3, it is seen that the resulting strain after 5 transfers in the absence of selective pressure chemicals for clonal analysis fully preserved levels of antibiotic resistance.

Example 3. The definition of immunobiological properties of strain STI-PR-4.

Presented in the table 4 data show that strain STI-PR-4 maintained the high immunogenic properties of strain STI-1.

The data presented in table 5, show that subcutaneous administration of 250 million spores rabbits did not cause none of them rise mee experimental series strain STI-PR-4 harmless.

Example 4. Getting spore cultures of the anthrax vaccine strain STI-PR-4 way deep cultivation.

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

Thus, the resulting vaccine, polyantibiotic-resistant strain STI-PR-4 has multiple (Rifr, Ampr, Doxr, Pefr, Nalr) drug resistance that allows you to design on the basis of antibiotic-resistant vaccine drugs for the prevention of anthrax on the background of nonspecific emergency prevention.

Anthrax vaccine strain STI-PR-4 spread spectrum antibiotic resistance, resistant to modern means of emergency prophylaxis: doxycycline, pefloxacin, rifampicin, ampicillin, and nalidixic acid.

 

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SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

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