KR100400411B1 - Viable bacterial formula for the treatment of vaginosis - Google Patents

Abstract

The present invention relates to a probiotic for treating vaginitis and a method for treating vaginitis, and to isolate L. crispatus KLB46 strain having excellent antibacterial activity in the vagina after isolating lactobacilli in the vagina of Korean women. Probiotics for the treatment of vaginitis using L. crispatus KLB46, which has the effect of treating bacterial, atrophic and trichomoniasis vaginitis as a result of investigating its characteristics and confirming the presence of harmful infectious virus by applying a virus test and applying it directly to the clinic. And providing a method of treating vaginitis using the therapeutic agent.

Description

Probiotic for treating vaginitis {Viable bacterial formula for the treatment of vaginosis}

The present invention relates to a probiotic for treating vaginitis and a method for treating vaginitis. More specifically, the present invention isolates lactic acid bacilli in the vagina of healthy Korean women, and selects and identifies strains with excellent growth inhibitory activity of pathogenic microorganisms from the isolated strains to produce probiotics for treating vaginitis and use them. To treat vaginitis.

Vaginitis accounts for 20-30% of gynecological outpatients, the most common disease among women of childbearing potential. The causative agents include E. coli, Tricomonas vaginalis , Candida albicans , Chlamydia and Haemophilus. ( Hemophilus vaginalis ), Herpes virus and Gardnerella sp., And vaginitis is often caused by a single or mixed infection of these bacteria.

Normal strains that live inside the vagina of women include lactobacilli, streptococcus (group β-hemolytic sreptococcus), coliform bacteria, and fungi. It maintains the normal range of pH 4.0-5.0 and prevents the growth of harmful microorganisms.

Bacterial vagivosis (BV) generally accounts for more than 50% of diseases of the female genital tract that present persistent chronic infections (Sobel, 1990). Bacterial vaginitis develops, causing changes in the vaginal flora of the vagina, resulting in the release of many amines, especially trimethylamine, odors, low acidity (pH> 5.0), and a reduction in Lactobacillus spp. A prominent lesion is the presence of Lactobacillus spp. In the normal flora of the dominant bacterium, which has increased the number of anaerobic bacteria ( Gardenerella vaginalis ), resulting in a fishy amine odor (Eschenbach et al. 1989; Pavlova et al. 1997). Vaginal secretions are homogeneous in appearance, unlike flocculent secretions from candida vaginitis, and unlike candidiasis and trichomoniasis, itching does not occur. The causative organism for BV is a bit controversial. Gardenerella vaginalis was isolated in 98% of women with bacterial vaginosis, but in normal vaginas without these symptoms, a small number of normal flora (68%) was also found. Totten et al., 1982). Under conditions of high concentrations of gardnerella, the number of lactobacilli is significantly reduced compared to normal quality. Normal vaginal flora has an average pH value of 4.0, consisting almost of Lactobacillus spp. (Hil and Embil, 1986. Bartlett and polk, 1984).

In general, such bacterial vaginitis is treated in the clinic by oral administration of antibiotics or by direct administration of an ointment or vaginal vaginal injection into the vagina (Blackwell et al. 1983). Imidazole and nitroimidazole derivatives Other types of medicaments that are often used and used include nitrofurfuryl derivatives and various antibiotics. These active antibiotic components can be formulated for oral or topical administration, while metronidazole is often administered by oral route, but mixed infections are intended for topical administration, in particular pessary forms containing two or more active ingredients. It is suitable to However, in the case of treatment with antibiotics, antibiotics affect the growth of pathogenic microorganisms that cause vaginitis, as well as the growth of normal bacterial flora in the vagina, especially lactobacillus, which reduces the number, thus destroying the bacterial flora in the normal vagina. Can be generated. In addition, treatment of vaginitis caused by the inability to use antibiotics freely in special situations, such as pregnant women and patients with hypersensitivity to certain antibiotics, is a difficult problem for clinicians. In addition, when antibiotics are administered due to systemic or local infections, the number of lactic acid bacilli in the vagina decreases, and various bacteria such as fungi, which cause vaginitis, proliferate and cause vaginitis.

Therefore, the development of a new yeast infection treatment that can overcome the problem of using antibiotics is very important. In the present invention, as an alternative to overcome the side effects caused by the use of antibiotics in the treatment of vaginitis, Lactobacillus spp. Was isolated in the vagina of healthy Korean women, and administered by intravenous as a probiotic to harmful vagina We have developed a method to treat vaginitis by restoring normal destroyed flora. The L. crispatus KLB46 strain of the present invention was tested for various viruses harmful to humans, and thus, no virus was detected. Therefore, the strain of the present invention can be directly injected into the human body as a probiotic, and normal strains can be applied to harmful species in the vagina. Relatively healing can cure vaginitis and improve vaginal condition.

Accordingly, it is an object of the present invention to provide a strain L. crispatus KLB46 which can be used as a probiotic for treating vaginitis due to its microbial growth inhibitory activity. Another object of the present invention to provide a probiotic for treating vaginitis containing the L. crispatus KLB46. Another object of the present invention to provide a method for treating vaginitis by using the L. crispatus KLB46 having a microbial antimicrobial activity.

The object of the present invention is to isolate and identify strains excellent in antimicrobial activity by isolating lactobacilli inhabiting the dominant bacteria in the vagina of healthy Korean women, and to analyze the base sequence of 16s rDNA for classification of the strains After investigating the characteristics, the virus test, which is a condition for administration with a probiotic, was applied, and then directly applied to a clinic to confirm the vaginitis treatment effect of the strain L. crispatus KLB46 of the present invention.

Hereinafter, the configuration of the present invention will be described in detail.

Figure 1 shows the nucleotide sequence of 16s rDNA of L. crispatus KLB46 of the present invention having antimicrobial activity.

Figure 2 shows the results of the HPV (Human Papillomavirus) test for the present invention L. crispatus KLB46.

Figure 3 shows the results of the CMV (Cytomegalovirus) test for the present invention L. crispatus KLB46.

Figure 4 shows the results of the Herpes Simplex Virus (HSV) test for the present invention L. crispatus KLB46.

Figure 5 shows the results of the Human Immunodeficiency Virus (HIV) test for L. crispatus KLB46.

FIG. 6 is a photograph taken at 400-fold magnification after collecting a portion of epithelial cells of a patient with bacterial vaginitis (a) and a state (b) after treatment with the present invention L. crispatus KLB46 and staining with a dye. to be.

The present invention comprises the steps of selecting and identifying the strain L. crispatus KLB46 of the present invention having the best growth inhibitory ability against vaginal microorganisms from about 100 lactobacilli isolated from the vagina of Korean women; Sequencing the 16s rDNA of the identified strain and investigating its properties; In order to examine the availability of the L. crispatus KLB46 strain as a probiotic, a virus test such as HPV (Human Papillomavirus), CMV (Cytomegalovirus), HSV (Herpes Simplex Virus), and HIV (Human Immunodeficien -cy Virus) was performed. Comprising steps to conduct and clinical trials to determine the effectiveness of the yeast infection treatment of L. crispatus KLB46 strain of the present invention.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1 Excellent microorganism growth inhibition L. crispatus Identification and Characterization of KLB46 Strains

First step. L. crispatus Identification and Analysis of Sequence of KLB46

The strains with the highest microbial growth inhibition ability were selected from 100 lactic acid bacillus isolates from the vagina of Korean women.

A sterile swab was inserted into the vagina of a healthy adult female subject and buried in the vaginal secretions, and then inoculated onto selective Rogosa SL agar plate (Difco) to isolate the lactobacillus. agar plate) was incubated for 48 hours at 37 ℃ in a candle jar substituted with CO ₂ . Selective Rogosa SL agar is known to grow only lactobacillus . Thus, colonies growing on this medium could first be identified as lactobacillus, observed cell morphology under a microscope, and confirmed positive results from Gram test, and Catalase test (catalase test) was confirmed to be negative.

Microorganisms were identified using the PCR-RFLP method, and the nucleotide sequences of the 16S rDNA of L. crispatus KLB46 were analyzed to confirm the identified results. The strains used in the PCR-RFLP method are known to be inhabited by the seven kinds of Lactobacillus spp. ( L. gasseri ATCC 9857, L. fermentum ATCC 23271, L. plantarum ATCC 14917, L. casei subsp.casei ATCC 393, L. rhamnosus ATCC 7469, L. jensenii ATCC 25258 , L. crispatus ATCC 33820 , L. acidophilus KCTC 3164,) and L. delbrueckii ATCC 15808.

PCR primers for amplification of 16S rDNA were fD1 (5'-AGAGTTTGATCCTGGCTCAG-3 ') and rD1 (5'-AAGGAGGTGATCCAGCC-3'). The primer has a sequence complementary to the integrity of the 16S rDNA in many microorganisms, enabling the amplification of almost all bacteria 16S rDNA.

The PCR reaction composition of 50 µl of total volume was Lactobacillus spp. 2 μL of DNA samples, 150 μM of dNTPs each, 0.1 μM of primers each, and 1.25 U of Taq DNA polymerase (Takara). The PCR reaction solution was preheated at 95 ° C. for 5 minutes in a thermal cycler, followed by 35 denaturation 94 ° C. for 1 minute, annealing at 60 ° C. for 1 minute, and extension. After 72 ° C., 1 minute, the final extension was amplified by 72 ° C. for 8 minutes. After PCR, 16S rDNA amplified product of Lactobacillus spp. Was electrophoresed on 120% agarose gel (agarose gel, Sigma) using 1% (w / v) agarose gel (Aigrose gel, Sigma), and the result was confirmed. Was treated with 5U restriction enzyme at a final volume of 20 μl. Enzymes used are Alu I, Cfo I, Dde I, Msp I, Hae III, Hinf, Taq I (Takara) and the like. Restriction enzyme-treated DNA was confirmed by electrophoresis on 2% agarose gel, and electrophoresis was performed at 100 V and 80 mA. After electrophoresis, the gel was dyed using an ethidium bromide (EtBr) solution (1 mg / mL), and then irradiated with ultraviolet light and photographed.

DNA profiles were recorded by dichotomy (ie 1 = common band, 0 = independent band) and then genetic correlation coefficients were calculated. RFLP results were calculated by the above equation, and the degree of similarity was worked through NTSYS-pc (numerical taxonomy system of multivariate statistical programs), and a dendrogram was created to examine the similarity between strains. In this way, Latobacillus KLB 46 was found to be the most similar to crispatus, and DNA sequencing was performed for more accurate identification. Using two primer sets F1 (5'-AGAGTTTGATCCTGGCTCAG-3) and R1 (5'-TCTACGCATTCCACCGCTAG-3 '), and F2 (5'-GTGCCAGCAGCCGCGG-3') and R2 (5'-GGGTTGCGCTCGTTG-3 ') After PCR, sequencing was performed to synthesize the results. This sequencing work was performed by Dr. Lin Tao's team (University of illinois, Chicago). Genes that have already been published using data from the NationalCenter for Biotechnological Information (http://www.ncbi.nlm.nih.gov// BLAST /), which are available via the Internet to identify specific species through the obtained sequencing results. Compared to the sequence.

As a result, the strain was found to be Lactobacillus crispatus (L. crispatus) , the base sequence of the 16S rDNA is as shown in FIG. The analyzed 16S rDNA sequence of FIG. 1 was registered as AF 243167 in GenBank of the National Center for Biotechnolgical Information.

The strain of the present invention, which was identified as Lactobacillus crispatus , was named Lactobacillus crispatus KLB46, and was deposited on September 8, 2000, at the Gene Bank in the Biotechnology Research Institute Microbial Deposit Center (KCTC 0860BP).

Second step. The identified strain L. crispatus Characteristics of KLB46

The antimicrobial activity of the identified strain L. crispatus KLB46 was examined. In order to examine the production of antibiotics in isolated Lactobacillus spp., Direct and differed techniques were modified. Lactobacillus delbrueckii subsp, lactis ATCC 15808, E. coli K12 were used as indicator strains, and L. acidophilus KCTC 3164 was used as a control strain. After incubating the isolated Lactobacillus spp. For 12 hours in MRS broth, inoculated on a solid MRS agar (agar) and then incubated overnight at 37 ℃, anaerobic conditions (anaerobic conditions). When colonies were formed on the plate, the solid MRS agar was covered with 5 mL of soft MRS agar (0.8%) mixed with 1% of the indicator strain. Plates were incubated for 24 hours according to the condition of the indicator strain. Several strains with well-retained ring were selected and dropped into the MRS agar plate by 1 μl, and the above experiment was repeated.

The hydrogen peroxide generating ability of the identified strain was confirmed. 4.3 g of Brucella agar base (Difco), 20 mg of benziden (Sigma), 1 mg of horseradish peroxidase (sigma), 0.5 g of hemin (hemin, bovine crystalline, Sigma) : Dissolve 50 mg in 1.0 mL of 1.0 N NaOH, mix with 100 mL of water to prepare a stock solution, use 1 mL), 0.1 g of Vitamin K1 (Sigma: Prepare a stock solution of 25 μl in 5 mL of 95% ethanol, and use 0.02 mL). 1 g of starch was dissolved in 100 mL of distilled water.

When isolated Lactobacillus spp. Was inoculated into the medium, grown for 2 to 3 days at 37 ° C. in anaerobic conditions, and exposed to air, the colony producing horseradish peroxidase and H ₂ O ₂ in the medium were Benzidene is oxidized to turn the colony black.

The following experiment was conducted to determine if the isolated Lactobacillus spp. Had cell surface hydrophobicity. Subcultured cells were incubated for 10 hours at 10mL in MRS broth and centrifuged for 10 minutes at 8,000 rpm. The supernatant was discarded and 5 mL of phosphate buffer (phosphate buffer, pH 7.0) was added, vortexed, and centrifuged at 8,000 rpm for 5 minutes. After washing one more time as described above, cells were collected, suspended in 5 mL of phosphate monomer, and placed in a test tube containing 5 mL of hexadecane (Sigma), and mixed well for 1 to 2 minutes using a vortex mixer. . After that, the result was confirmed after being left at room temperature for about 1 hour.

In order to be used as a treatment for bacterial vaginitis, the microbial antimicrobial activity of lactic acid bacilli should be high, and the cell surface adhesion should be excellent because it must adhere to the cell wall. The present invention L. crispatus KLB46 is used as an indicator strain (indicator strain) L. delbrueckii subsp. It has been shown to have strong microbial growth inhibitory ability against lactis (ATCC 15808) and to produce hydrogen peroxide (H ₂ O ₂ ), a microbial growth inhibitor. In addition, the cell surface adhesion ability turned out to be quite excellent.

The characteristics of L. crispatus KLB46 are shown in Table 1.

Various characteristics of L. crispatus KLB46 Antibacterial ability Hydrogen peroxide (H ₂ O ₂ ) Cell surface adhesion L. crispatus KLB46 good Slightly produce good

Third step. The present invention L. crispatus KLB46 Virus Test

In order to use the isolated L. crispatus KLB46 lactic acid strain of the present invention as a probiotic for the treatment of bacterial vaginosis, there should be no virus causing a harmful infection in the strain. Therefore, in order to confirm the safety of the present invention L. crispatus KLB46 lactic acid bacilli as a drug, virus tests such as HPV (Human Papillomavirus), CMV (Cytomegalovirus), HSV (Herpes Simplex Virus), and HIV (Human Immunodeficiency Virus) 2, 3, 4, and 5.

If the L. crispatus KLB46 sample contains the virus, the S column of FIG. 2 should have a band at a specific site such as the P column. As shown in FIGS. 2, 3, 4 and 5, the L. crispatus KLB46 sample did not show the same pattern as any band of the virus. Therefore, no virus was detected in the L. crispatus KLB46. It can be seen that. Since L. crispatus KLB46 does not contain any harmful viruses, it is expected that there will be no problems in the manufacture of pharmaceuticals in terms of safety.

Example 2: The present invention L. criapatus Clinical Trials and Results of Treatment of Bacterial Vaginitis and Atrophic Vaginitis with KLB 46

In order to examine the effects of the present invention L. crispatus KLB46 lactobacillus on the treatment of vaginitis such as bacterial vaginitis, atrophic vaginitis and trichomonas vaginitis. Patients supported by Ewha Womans University Mokdong Hospital were inoculated twice with L. crispatus KLB46 at weekly intervals. All the data below, including clinical trials and treatment findings, were conducted by Dr. Seung-Chul Kim, an obstetrics and gynecologist at Ewha Womans University Mokdong Hospital.Cultivated L. crispatus KLB46 was inoculated for 16 hours, the cells were centrifuged, washed twice in distilled water and distilled water was added again The number of cells in the inoculum was adjusted to 10 ⁹ or more / mL per 1 mL, and 1 mL was inoculated each time, and a more preferable concentration range showing an inoculation effect was 10 ⁹ -10 ¹² cells / mL (Table 2).

Clinical Results of Treatment of Vaginitis with L. crispatus KLB46 Name (age) Clinical diagnosis How to treat Complaint Before Treatment Symptom changes after treatment LKJ (44) BV lactobacilli Cold, odor, itching Symptom disappearance (cure) PHJ (36) BV lactobacilli Cold, itching Symptom disappearance (cure) KSH (30) BV lactobacilli Cold, itching Symptom disappearance (cure) PHS (42) BV lactobacilli Cold, odor, itching Symptom relief BSJ (27) BV lactobacilli plusmetronidazole Cold, odor, itching Symptom disappearance (cure) OYM (39) BV lactobacilli plusmetronidazole Cold, odor Symptom disappearance (cure) BYS (42) BV lactobacilli plusmetronidazole Cold, odor Symptom disappearance (cure) SKJ (41) BV lactobacilli plusmetronidazole Cold, odor Symptom disappearance (cure) KEJ (40) TV lactobacilli Cold, itching Symptom relief KSY (52) AV lactobacilli Cold, burning, smell Symptom disappearance (cure) BYH (55) AV lactobacilli Cold, burning No symptom change LCL (57) AV lactobacilli Cold, itching Symptom relief KSH (63) AV lactobacilli plus estrogen vaginal tablet Cold, burning, itching Symptom relief NCS (64) AV lactobacilli plus estrogen vaginal tablet Cold, burning Symptom disappearance (cure) KHS (55) AV lactobacilli plus estrogen vaginal tablet Cold, burning, itching Symptom relief [Note] BV: bacterial vaginosis AV: atrophic vaginosis (atrophic vaginitis) TV: trichomonas vaginosis (trichomonas vaginitis)

As shown in Table 2, 7 of 8 patients with bacterial vaginosis disappeared and were found to be cured. The other 1 also improved. Metronidazole [250mg / tablet, Frasin oral (Hanil Pharmaceutical)] was orally administered three times a day for 7 days when metronidazole, an antimicrobial agent currently used to treat vaginitis, was administered with L. crispatus KLB46. In general, metronidazole is used in the above manner in the treatment of bacterial vaginitis. However, vaginitis often recurs when metronidazole alone is used because lactobacilli cannot settle in the vagina. Therefore, it can be seen that it is more advantageous to use vaginal lactic acid bacteria for the purpose of preventing recurrence clinically.

Patients with bacterial vaginitis were harvested 400 times after staining with epithelial cells in the vagina and some epithelial cells after treatment with the L. crispatus KLB46 strain of the present invention. As shown in FIG. 6, pathogenous coccus (Coccus) inhabits epithelial cells around the epithelial cells before treatment, but after treatment, lactobacilli is settled as dominant bacteria.

Than from those of patients suffering from atrophic vaginitis only one input L. crispatus KLB46 L. crispatus KLB46 jiljeong with estrogen [estrogen tablet, Menozole jiljeong (geunhwa constraints) stilbesterol 0.5mg] to sleep during the week one tablet once a day Insertion into the vagina gave better results. Atrophic vaginitis is one of the diseases caused by thinning of the vaginal wall due to estrogen deficiency. Since estrogen is produced mainly in women's ovaries, women who have undergone surgery to cut both ovaries or postmenopausal women whose ovaries are not functioning have estrogens. Lack of atrophic vaginitis occurs a lot. The results of these clinical trials showed that treatment with L. crispatus KLB46 showed good results not only for bacterial vaginitis but also for atrophic vaginitis with the addition of female hormone estrogen.

As apparent from the above examples, the present invention is a lactic acid bacterium excellent in pathogenic microbial growth inhibitory activity isolated from the vagina of healthy Korean women, which can be used as a probiotic for the treatment of bacterial vaginosis, atrophic vaginitis and trichomas vaginitis . Crispatus KLB46 strain and the strain, metronidazole and estrogen using the tablets to provide a method for treating vaginitis, it is a very useful invention for the national health promotion and pharmaceutical manufacturing industry.

Claims (6)

Lactobacillus crispatus KLB46 (KCTC 0860BP) having a therapeutic effect on bacterial vaginosis, atrophic vaginitis and trichomonas vaginitis occurring in the vagina of a woman and having 16S rDNA of SEQ ID NO: 1. A probiotic for the treatment of bacterial vaginitis, atrophic vaginitis or trichomonas vaginitis, comprising the Lactobacillus crispatus KLB46 (KCTC 0860BP) of claim 1. delete delete delete delete