RU2148639C1 - Strain if bacterium bifidobacterium breve p2 for preparing bacterial preparations normalizing microflora in vagina microbiocenosis disorder - Google Patents

Abstract

FIELD: biotechnology, medicine, gynecology. SUBSTANCE: strain is isolated from vagina of health woman of reproductive age and able to inhibit opportunistic microorganisms effectively both in vitro and in vivo. Strain produces acids and shows expressed adhesion to vaginal epitheliocytes. Strain Bifidobacterium breve P2 is deposited in TSMPM "VNIIgenetics" institute at N Ac-1579. Strain is nonpathogenic and apyretic for humans and animals, it normalizes microflora of genital tract in women. Strain is ready for using in obstetrics-gynecological practice as preparation-probiotic. Strain provides correction of dysbiotic disorders of vaginal microflora, inhibits the growth of opportunistic microorganism in vitro effectively. Invention can be used as preparation-probiotic for correction of microbiocenosis of genital tract in women. EFFECT: strain indicated above, high effectiveness of correction. 3 tbl, 3 ex

Description

 The present invention relates to medical microbiology, in particular to obstetric and gynecological practice, in particular, can be used in the manufacture of therapeutic agents for the normalization of microflora in cases of vaginal microbiocenosis.

 It is known that studies in the field of normalizing disorders of the vaginal microbiocenosis are concentrated primarily on the development of probiotic preparations based on lactobacilli. These bacteria are the main, but not the only component of the indigenous "protective" microflora of the urogenital tract, which probably explains the insufficient clinical effect of the use of lactobacilli monopreparations (Baerheim A. et al. Vaginal application in the prophylaxis reccurent lower urinary tract in women. Scandinavian Journal of Primary Health Care. 1994. vol. 12. p. 239-243).

 It is also known that commercially available bacterial preparations used to correct vaginal microflora do not meet the requirements for an effective probiotic preparation (Viki L. Hughes et al. Microbiologic Characteristics of Lactobacillus Products Used for Colonization of the Vagina. Obstetrics and Gynecology. 1990. . vol. 75. p. 244-248), or require the appointment of long-term (up to 1 year) courses of treatment to ensure clinical effect (Hilton E. et al. Annals of Internal Medicine. American College of Physicians. 1992. 116: 353- 357).

 There are also known attempts by some domestic researchers to use commercial preparations for sanitizing the vaginal cavity in pregnant and gynecological patients. An obvious disadvantage of industrially produced preparations containing intestinal strains of bifidobacteria is their low adhesion to vaginal epithelial cells, while the presence of this ability is considered as a necessary condition for engraftment of bacteria in this ecological niche (Reid G. et. Al. Implantation of Lactobaccillus casei var rhamnosus into vagina. Lancet. 1994. p. 1229).

 There are no data on the development and use of probiotic preparations based on indigenous vaginal bifidobacteria in the treatment of nonspecific bacterial vaginosis.

 The aim of the invention is to obtain a strain of bifidobacteria used in the production of a bacterial preparation to normalize violations of the vaginal microbiocenosis.

 This goal was achieved in that a strain of Bifidobacterium breve P2 was isolated from the vagina of a healthy woman of reproductive age, which has increased adhesive activity against vaginal epithelial cells, significant antagonistic activity and acid-forming function, and natural resistance to several antibiotics.

 The strain is deposited in the Central Museum of Industrial Microorganisms of the Institute "VNII Genetics" under the number Ac-1570 and is characterized by the following properties.

 Cultural and morphological properties.

 When cultured in a column of 0.75% agar, Blaurock forms colonies in the form of buckwheat grains or discs up to 0.5 - 1 mm in diameter for 2 days. When cultured in liquid medium, Blaurock under anaerobic conditions forms a loose precipitate and uniform turbidity after 24 hours. On a dense medium, Blaurock under anaerobic conditions grows in the form of S-type colonies, round with smooth edges, 2 to 3 mm in diameter, opaque, shiny, do not form pigment; the profile of the colonies is convex, easily removed by a loop from the surface of a dense medium.

 In smears, the cells are arranged in the form of "Chinese characters", they look like straight or branched sticks with Y-shaped bifurcations at one or both ends, sometimes they form thickenings at the ends. Fixed, smeared randomly in the smear. Gram-positive, sometimes granularity is revealed inside the cells. They do not form a dispute.

 Physiological properties.

Obligate anaerobic; grows in a high agar column in vitro, in test tubes with liquid medium and on Petri dishes with dense medium under anaerobic conditions. When cultured in a liquid medium under anaerobic conditions 18–20 hours after plating, 1.0 • 10 ⁸ - 5.0 • 10 ⁸ live microbial cells grow in 1 ml of medium. The maximum growth at 37.5 ^o C.

 Biochemical signs.

 Synthesizes the enzyme fructose-6-phosphate phosphoketalase.

 It ferments glucose, mannitol, lactose, sucrose, maltose, salicin, manose, raffinose, sorbitol. Hydrolysis of esculin.

 Does not ferment xylose, arabinose, glycerol, celobiosis, melysitosis, rhamnose, trehalose.

 Does not form indole and catalase, do not reduce nitrates. Does not thin the gelatin.

Acid formation when grown in Blaurock medium after 72 hours - 66.9 ^o (according to Turner).

 Sensitivity to antibiotics is presented in table 1.

 Laboratory testing showed the non-pathogenicity of the Bifidobacterium breve P2 strain for experimental animals (mice), as well as for humans (in experiments on volunteers).

 The use of the strain is illustrated by the following examples.

 Example 1. The study of the adhesive ability of Bifidobacterium breve P2 on vaginal epithelial cells of healthy women.

Vaginal epithelium was obtained from three healthy women, centrifuged at 420 g for 10 min, the pellet was resuspended in McIvaine's buffer (pH 5.4), based on 0.1 N C ₆ H ₈ O ₇ and 0.2 N NaH ₂ PO ₄ , and centrifuged again. Centrifugation was repeated five times. Then a suspension of epithelial cells with a concentration of 1.0 • 10 ⁵ cells / ml was prepared.

A 16-hour culture of bifidobacteria was centrifuged twice at 1700 g for 10 minutes in the same buffer as the epithelial cells. A suspension of bacteria was prepared at a concentration of 1.0 • 10 ⁸ mt / ml. Suspensions of bifidobacteria and epithelial cells were mixed in equal volumes (0.5 ml) in test tubes. The mixture was incubated at +37 ^o C for 2 hours (with constant slight horizontal stirring). Then the mixture of bifidobacteria and epithelial cells was centrifuged at 420 g for 5 min five times. The precipitate was resuspended in 1 ml of buffer, a smear was prepared on a glass slide, dried in air, fixed in methanol for 15 minutes and stained with Gram. After that, the total number of bifidobacteria attached to the surface of 100 epithelial cells was counted. As a control, B. bifidum 791 strain, isolated from the feces of a healthy adult and used for the preparation of "Sour-milk bifidumbacterin", was used.

 The adhesive activity of bifidobacteria was evaluated by the average number of bacteria attached to one epithelial cell.

 It was found that the number of bifidobacteria of the strain Bifidobacterium breve P2, adhered to 1 epithelial cell, was 27.3 ± 1.2, and the number of bifidobacteria of the B. bifidum 791 strain of intestinal origin was 2.6 ± 0.2 (see Table 2).

 Thus, the strain Bifidobacterium breve P2 has a higher adhesive ability to vaginal epithelial cells with the possibility of longer and more stable engraftment in the female genital tract than the strain of intestinal origin.

 Example 2. The study of the antagonistic activity of a strain of Bifidobacterium breve P2 in vitro.

 To determine the antagonistic activity of Bifidobacterium breve P2 in relation to Escherichia coli, Klebsiella ozaenae, Staphylococcus aureus. Streptococcus faecalis used the method of Muriana and Klaenhammer. Bactofoc agar (Gidrobiosis, Moscow) (1.5%) was used as a nutrient medium for the growth of bifidobacteria, and semi-liquid (0.75%) Brain Heart Infusion agar (BBL, USA) was used for indicator microorganisms.

When studying the antagonistic activity of bifidobacteria strains against Pseudomonas aeruginosa at the first stage, 3.5 ml of 1.5% agar based on Brain Heart Infusion medium was applied to plates with grown colonies of bifidobacteria. After the intermediate layer has solidified, 0.1 ml of a 12-hour culture of the indicator strain from a dilution of 10 ^{-1 was} applied to its surface and ground on the surface of the agar.

After an 18-hour incubation under aerobic conditions at 37 ^° C, the radii of the zones of growth inhibition of indicator strains around colonies of bifidobacteria were measured.

 The results of an experiment on the study of antagonistic activity in vitro showed that Bifidobacterium breve P2 actively inhibits the growth of Escherichia coli (radius of the growth inhibition zone is 4.3 ± 0.1 mm), Klebsiella ozaenae (9.6 ± 0.2 mm). Staphylococcus aureus (3.9 ± 0.1 mm), Streptococcus faecalis (2.7 ± 0.1 mm) and Pseudomonas aeruginosa (3.2 ± 0.2 mm) (see table 3).

 Thus, the Bifidobacterium breve P2 strain actively inhibits the growth of opportunistic microorganisms in vitro.

 Example 3. The study of the effect of a strain of Bifidobacterium breve P2 on the microflora of the female genital tract.

The lyophilized dried Bifidobacterium breve P2 strain was administered on a vaginum swab to a woman A., 27 years old, for 10 days at a dose of 10 ⁸ (before use, the lyophilisate was diluted in 5 ml of cooled boiled water). Bacteriological examination of the vaginal discharge was carried out before and after the use of microorganisms.

Analysis of bacteriological data revealed an increase in the level of lactobacilli (from <10 ² mt / g to 4.0 • 10 ² mt / g) and bifidobacteria (from <10 ² mt / g to 2.0 • 10 ² mt / g). At the same time, a decrease in the level of Bacteroides spp was noted. (from 2.8 • 10 ⁸ mt / g to 1.56 • 10 ⁷ mt / g) hemolysin-producing Streptococcus spp. from 1.4 • 10 ³ mt / g to <10 ² mt / g and hemolysin-producing Staphylococcus aureus from 2.36 • 10 ⁴ mt / g to <10 ² mt / g .

 Thus, the use of a strain of Bifidobacterium breve P2, isolated from the vagina of a healthy woman of reproductive age, causes positive changes in vaginal microflora in vivo, manifested by an increase in the level of bifidobacteria and one of the main representatives of the microflora of the genital tract - lactobacilli, and a decrease in the level of opportunistic microorganisms, which provides pronounced correction of dysbiotic disorders of vaginal microflora and restoration of the dominant position of lactic acid bacteria.

 Despite its apparent simplicity, the restoration of the microflora of the vaginal tract through the use of probiotic preparations is a complex microbiological and gynecological problem. Isolation from a vagina of a healthy woman, which is promising for the production of a strain of bifidobacteria, and the study of its properties is the result of long research work, which resulted in the first production of a strain of Bifidobacterium breve P2, which has high adhesive ability to vaginal epithelial cells, natural resistance to a number of antimicrobial agents, and antagonistic activity against opportunistic microorganisms not only in vitro, but also in vivo. All these properties make it possible to use the Bifidobacterium breve P2 strain as a probiotic drug in the clinic for the prevention and treatment of microflora disorders of the female genital tract.

 The strain Bifidobacterium breve P2 is completely ready for use.

Claims (1)

 The bacterial strain Bifidobacterium breve P2 (Central Museum of Industrial Microorganisms Institute VNII Genetics As-1570), used to prepare bacterial preparations that normalize the microflora in cases of vaginal microbiocenosis.

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