Lactobacillus crispatus and molecular marker and use thereof

Abstract

Provided are a Lactobacillus crispatus and a molecular marker and use thereof. The molecular marker of the Lactobacillus crispatus has a nucleotide sequence selected from one of the following: (1) a nucleotide sequence shown in SEQ ID NO: 24; (2) a nucleotide sequence having at least 97%, 98%, 99% or more homology to the nucleotide sequence shown in SEQ ID NO: 24; and (3) a nucleotide sequence having one or more, such as 1, 2, 3, 4, 5, or more, nucleotide substitutions, deletions, or insertions in the nucleotide sequence shown in SEQ ID NO:24.

Description

Lactobacillus crispatus and its molecular markers and uses Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a Lactobacillus crispatus and a molecular marker and application thereof. Background Art

[0002] With economic development and rising living standards, the human disease landscape has undergone significant changes. The prevalence of female reproductive tract diseases has become increasingly prominent, with vaginitis being particularly prevalent, severely impacting women's quality of life and physical and mental health. Among vaginal infections, bacterial vaginosis (BV) is the most common. Epidemiological surveys show that the incidence of BV in my country ranges from 4.96% to 36.00%. The primary cause of this disease is a decrease in the dominant Lactobacillus bacteria that maintain the normal acidic vaginal environment, while increasing the abundance of various anaerobic microorganisms. The dominant Lactobacillus bacteria are replaced by Gardnerella vaginalis and mixed anaerobic bacteria. Gardnerella vaginalis is one of the most common pathogens of BV in women. The typical clinical feature of bacterial vaginosis is a foul-smelling, watery, gray vaginal discharge. Patients may also develop numerous complications due to the infection, including chronic cervicitis, pelvic inflammatory disease, endometritis, and even infertility. Vulvovaginal candidiasis (VVC), commonly known as candidal vaginitis, is a vaginal infection second only to bacterial vaginosis in incidence. The dominant organism is often Candida albicans. Studies have found that approximately 78% of women experience at least one episode of VVC in their lifetime, 65% experience three or fewer annual episodes, and 35% experience four or more annual episodes. VVC typically presents with vulvar itching, burning, painful urination, and a dreg-like vaginal discharge, often leading to a decline in patients' quality of life and a significant economic burden. The pathogenesis of VVC is complex and is considered a multifactorial disease, with vaginal microbial imbalance, estrogen levels, host susceptibility factors, genetic predisposition, and Candida morphology all contributing to its development. Imbalances in the vaginal microbiome are closely linked to the development of these two vaginal infections.

[0003] The vaginal microbiome differs from the microbiome of other parts of the body. The lower the diversity of a healthy vaginal microbiome, the better it maintains vaginal health. The female vagina is a closed body cavity, home to numerous bacteria. Because air is absent deep within the vagina, parasites are primarily facultative anaerobes and anaerobic bacteria. Over 50 species of microorganisms in a healthy woman's vagina are parasitic. Normally, the ratio of anaerobic to aerobic bacteria is approximately 10:1, with Lactobacillus species having the highest proportion, playing a crucial role in maintaining vaginal microecological balance. Under normal circumstances, Lactobacillus species coexist with other microorganisms in a state of microecological equilibrium. However, if various factors cause Lactobacillus species to lose their dominance and pathogenic and opportunistic pathogens to increase, corresponding changes in the enzyme profile disrupt the microecological balance, leading to disease. Lactobacillus species maintain vaginal microecological stability through multiple mechanisms: fermentation of intraepithelial glycogen to produce lactic acid, maintaining an acidic vaginal pH; competition with pathogens for adhesion to vaginal epithelial cells; production of the broad-spectrum antimicrobial factor H2O2; and stimulation of the immune system by Lactobacillus species and their metabolites, which inhibit the growth and reproduction of pathogens.

[0004] Currently, BV treatment primarily relies on Western medicine, often using antimicrobial agents such as antibiotics, including metronidazole, clindamycin, and tinidazole. However, the formation and persistence of pathogenic biofilms can lead to recurrence. According to the "Guidelines for the Diagnosis and Treatment of Bacterial Vaginosis," the recurrence rate for BV patients treated with oral metronidazole is 20% one month after treatment, 40% three months after treatment, and as high as 60% 12 months after treatment. By 12 months after treatment, 84% of patients had abnormal vaginal flora. Furthermore, long-term, high-dose antibiotic use not only increases pathogen resistance but also inhibits the growth of some vaginal flora, allowing the previously limited Candida albicans to proliferate, leading to vaginal flora disturbances. The role of live bacterial preparations in the treatment of BV remains controversial. Clinical studies have shown that the use of live bacterial preparations is primarily divided into two categories: conventional antibiotic treatment followed by probiotics or probiotics alone. Clinical trials of multiple combination therapies have shown inconsistent results, making it uncertain whether antibiotics combined with probiotics are effective in treating bacterial vaginosis. Other studies have shown that some patients fail to achieve satisfactory therapeutic effects using probiotic preparations alone. Therefore, there is still considerable controversy regarding the effectiveness of probiotics in treating BV, and extensive research is still needed. Currently, the only marketed probiotic for the treatment of bacterial vaginosis is the Lactobacillus Live Vaginal Capsule (Dingjunsheng) developed by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd. For the treatment of VVC, the most commonly used clinical medications are azoles and polyenes, such as diazepam suppositories, which are often used to treat candidal vaginosis. However, with the long-term use of clinical antifungal drugs, Candida albicans undergoes phenotypic changes, virulence factor mutations, and an increase in non-albicans Candida infections, posing challenges to the therapeutic efficacy of traditional antifungal drugs. In the treatment of various types of VVC patients, Lactobacillus can be used as an adjunctive therapy in combination with antibacterial drugs such as azoles, with some efficacy. However, compared with conventional antifungal treatment, there is currently insufficient evidence to demonstrate that the use of Lactobacillus preparations alone is effective in treating VVC.

[0005] Therefore, there is an urgent need for a lactobacillus that has good cell adhesion and the ability to inhibit the growth of multiple pathogens and inhibit biofilm formation, thereby being able to treat vaginal infections. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0007] Therefore, the inventors isolated a strain from the vaginal secretions of a healthy woman of childbearing age in Zhejiang Province. After identification, the strain was Lactobacillus crispatus (Lactobacillus crispatus HY1467). Experimental findings showed that the Lactobacillus crispatus has advantageous cell adhesion, enabling it to adhere to vaginal epithelial cells, forming a microecological barrier to prevent pathogen colonization or competition for epithelial cell receptors. Experiments also showed that the Lactobacillus crispatus has a strong ability to adhere to Hela cells and produce lactic acid, which can lower the pH and inhibit the reproduction of pathogens. Therefore, the Lactobacillus crispatus has the potential to maintain a weakly acidic environment in the vagina, improve host mucosal immunity and anti-infection, thereby preventing vaginal infections and treating inflammation.

[0008] In view of this, in the first aspect of the present invention, the present invention proposes a molecular marker for Lactobacillus crispatus. According to an embodiment of the present invention, the molecular marker has a nucleotide sequence selected from one of the following: (1) a nucleotide sequence shown in SEQ ID NO: 24; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 24; (3) a nucleotide sequence having one or more, such as 1, 2, 3, 4, 5 or more nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO: 24. Therefore, the molecular marker of the present invention can not only quickly and easily screen for the target Lactobacillus crispatus, but also can highly specifically identify the target Lactobacillus crispatus, eliminating the interference of other strains.

[0009] According to an embodiment of the present invention, the molecular marker has a nucleotide sequence as shown in SEQ ID NO:24.

[0010] In a second aspect of the present invention, the present invention provides a Lactobacillus crispatus According to an embodiment of the present invention, the Lactobacillus crispatus comprises the molecular marker described in the first aspect.

[0011] According to an embodiment of the present invention, the Lactobacillus crispatus was deposited in the General Microbiology Center of the China Culture Collection Administration on February 7, 2023, with a deposit number of CGMCC No. 26503.

[0012] According to an embodiment of the present invention, the 16S rDNA of the Lactobacillus crispatus has a nucleotide sequence that is one of the following: (1) a nucleotide sequence as shown in SEQ ID NO: 1; (2) a nucleotide sequence having at least 99.8%, 99.9% or higher homology with the nucleotide sequence shown in SEQ ID NO: 1; (3) a nucleotide sequence having one or more, for example, 1, 2, 3, 4, 5 or more nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO: 1.

[0013] According to an embodiment of the present invention, the 16S rDNA of the Lactobacillus crispatus has the nucleotide sequence shown in SEQ ID NO: 1.

[0014] In a third aspect of the present invention, the present invention proposes a Lactobacillus crispatus (Lactobacillus crispatus HY1467). According to an embodiment of the present invention, the Lactobacillus crispatus was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on February 7, 2023, with a deposit number of CGMCC No. 26503, and the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. As mentioned above, the inventors isolated and obtained the strain from the vaginal secretions of a healthy woman of childbearing age in Zhejiang Province. After further identification of the biochemical characteristics of the strain, it was found that the bacteria can produce a large amount of lactic acid, thereby having the ability to maintain the acidic pH value of the vagina. Furthermore, the strain exhibits a strong inhibitory effect against the pathogenic bacteria Gardnerella vaginalis (GV), Escherichia coli (EC), and Staphylococcus aureus (SA). Experiments have shown that the strain can inhibit the formation of biofilms of Candida albicans (CA), thereby inhibiting the growth of Candida albicans. It also exhibits a certain inhibitory effect against Atopobium vaginalis (FV) and Prevotella bivia (PB). Therefore, this strain has the potential to maintain a weakly acidic environment within the vagina, enhancing host mucosal immunity and anti-infection, thereby preventing vaginal infection and treating inflammation.

[0015] According to an embodiment of the present invention, the 16S rDNA of the Lactobacillus crispatus has the nucleotide sequence shown in SEQ ID NO: 1.

[0016] In a fourth aspect, the present invention provides a Lactobacillus crispatus. According to an embodiment of the present invention, the 16S rDNA of the Lactobacillus crispatus has a nucleotide sequence that is one of the following: (1) the nucleotide sequence shown in SEQ ID NO: 1; (2) a nucleotide sequence having at least 99.8%, 99.9% or higher homology with the nucleotide sequence shown in SEQ ID NO: 1; or (3) a nucleotide sequence having one or more, for example, 1, 2, 3, 4, 5 or more, nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO: 1. The Lactobacillus crispatus described in the present invention can simultaneously exert a strong inhibitory effect on the pathogenic bacteria Gardnerella vaginalis (GV), Escherichia coli (EC), and Staphylococcus aureus (SA). Experiments have found that the bacterium can inhibit the formation of Candida albicans (CA) biofilms, thereby inhibiting the growth of Candida albicans. It also has a certain inhibitory effect on Atopobium vaginalis (FV) and Prevotella bivia (PB). Therefore, the Lactobacillus crispatus has the potential to maintain a weakly acidic environment in the vagina, enhance host mucosal immunity and anti-infection, and thus has the ability to prevent vaginal infections and treat inflammation.

[0017] According to an embodiment of the present invention, the 16S rDNA gene sequence of the Lactobacillus crispatus comprises the nucleotide sequence shown in SEQ ID NO: 1.

[0018] According to an embodiment of the present invention, the microorganism of the second aspect, the third aspect or the fourth aspect further includes at least one of the following additional technical features:

[0019] According to an embodiment of the present invention, the gene sequence of the Lactobacillus crispatus further comprises a nucleotide sequence selected from the following: (1) a nucleotide sequence as shown in SEQ ID NO: 24; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology to the nucleotide sequence as shown in SEQ ID NO: 24; (3) a nucleotide sequence having one or more, for example, 1, 2, 3, 4, 5 or more nucleotide substitutions, deletions or insertions in the nucleotide sequence as shown in SEQ ID NO: 24. Therefore, the microorganism of the present invention can be specifically identified by the above nucleotide sequence.

[0020] According to an embodiment of the present invention, the gene sequence of the Lactobacillus crispatus further comprises a nucleotide sequence as shown in SEQ ID NO: 24.

[0021] According to a specific embodiment of the present invention, the Lactobacillus crispatus can produce at least 9.0-10.0 g / L of lactic acid under effective culture.

[0022] According to a specific embodiment of the present invention, the Lactobacillus crispatus can produce at least 9.1 g / L, 9.2 g / L, 9.3 g / L, 9.4 g / L, 9.5 g / L, 9.6 g / L, 9.7 g / L, 9.8 g / L, 9.9 g / L, and 10.0 g / L of lactic acid under effective culture.

[0023] According to a specific embodiment of the present invention, the Lactobacillus crispatus has an inhibition rate of at least 94-97% against GV.

[0024] According to a specific embodiment of the present invention, the Lactobacillus crispatus has an inhibition rate of at least 94%, 95%, 96%, or 97% against GV.

[0025] According to a specific embodiment of the present invention, the Lactobacillus crispatus has an inhibition rate of at least 58-61% against PB.

[0026] According to a specific embodiment of the present invention, the Lactobacillus crispatus has an inhibition rate of at least 58%, 59%, 60%, or 61% against PB.

[0027] According to a specific embodiment of the present invention, the Lactobacillus crispatus has an inhibition rate of at least 57-60% against FV.

[0028] According to a specific embodiment of the present invention, the Lactobacillus crispatus has an inhibition rate of at least 57%, 58%, 59%, or 60% against FV.

[0029] According to a specific embodiment of the present invention, the Lactobacillus crispatus has an inhibition rate of at least 78-82% against EC.

[0030] According to a specific embodiment of the present invention, the Lactobacillus crispatus has an inhibition rate of at least 78%, 79%, 80%, 81%, or 82% against EC.

[0031] According to a specific embodiment of the present invention, the Lactobacillus crispatus has an inhibition rate of at least 70-75% against SA.

[0032] According to a specific embodiment of the present invention, the Lactobacillus crispatus has an inhibition rate of at least 70%, 71%, 72%, 73%, 74%, or 75% against SA.

[0033] According to a specific embodiment of the present invention, the Lactobacillus crispatus has an inhibition rate of at least 45-50% against CA.

[0034] According to a specific embodiment of the present invention, the Lactobacillus crispatus has an inhibition rate of at least 45%, 46%, 47%, 48%, 49%, or 50% against CA.

[0035] In the fifth aspect of the present invention, the present invention proposes a primer set for detecting the Lactobacillus crispatus described in the second, third or fourth aspects. According to an embodiment of the present invention, the primer set includes a forward primer and a reverse primer; the forward primer has the nucleotide sequence shown in SEQ ID NO:2; and the reverse primer has the nucleotide sequence shown in SEQ ID NO:3. The primer set described in the present invention can highly match the DNA sequence of the Lactobacillus crispatus described in the second, third or fourth aspects, and can only produce specific amplification in the Lactobacillus crispatus. This enables the primers to accurately and reliably identify the Lactobacillus crispatus, eliminating interference from other non-target strains.

[0036] In a sixth aspect, the present invention provides a microbial preparation. According to embodiments of the present invention, the microbial preparation comprises the Lactobacillus crispatus described in the second, third, or fourth aspects. As previously mentioned, the inventors of the present invention have isolated a new Lactobacillus crispatus that exhibits a strong inhibitory effect against the pathogenic bacteria GV, EC, SA, and CA. Therefore, this Lactobacillus crispatus can be formulated into a microbial preparation for convenient patient administration.

[0037] According to an embodiment of the present invention, the microbial preparation further includes a pharmaceutically acceptable carrier or excipient.

[0038] In the seventh aspect of the present invention, the present invention provides a single-dose formulation. According to an embodiment of the present invention, comprising 1×10 6 ~1×10 10 The CFU Lactobacillus crispatus described in the second, third or fourth aspects is used as an active ingredient.

[0039] According to an embodiment of the present invention, comprising 10 6 CFU, 10 7 CFU, 10 8 CFU, 10 9 CFU, 10 10 The CFU Lactobacillus crispatus described in the second, third or fourth aspects is used as an active ingredient.

[0040] According to an embodiment of the present invention, including 1×10 6 ~1×10 9 The CFU Lactobacillus crispatus described in the second, third or fourth aspects is used as an active ingredient.

[0041] In the eighth aspect of the present invention, the present invention proposes the use of the Lactobacillus crispatus described in the second, third or fourth aspect, the microbial preparation described in the sixth aspect or the single-dose preparation described in the seventh aspect in the preparation of a medicament for treating and / or preventing vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection.

[0042] In the ninth aspect of the present invention, the present invention proposes the use of the Lactobacillus crispatus described in the second, third or fourth aspect, the microbial preparation described in the sixth aspect or the single-dose preparation described in the seventh aspect in the preparation of a drug for inhibiting vaginal pathogens.

[0043] According to an embodiment of the present invention, the use described in the eighth aspect or the ninth aspect further includes at least one of the following additional technical features:

[0044] According to an embodiment of the present invention, the vaginal pathogenic bacteria are selected from at least one of Gardnerella vaginalis (GV), Escherichia coli (EC), Staphylococcus aureus (SA), Fannyhessea vaginae (FV), Prevotella bivia (PB), and Candida albicans (CA).

[0045] According to an embodiment of the present invention, the vaginal pathogenic bacteria is selected from at least one of Gardnerella vaginalis (GV), Escherichia coli (EC), Staphylococcus aureus (SA), and Candida albicans (CA).

[0046] According to an embodiment of the present invention, the related diseases caused by vaginal pathogenic bacteria infection include at least one of bacterial vaginosis, aerobic vaginitis, vulvovaginal candidiasis, Trichomonas vaginitis, mixed vaginitis, HPV infection, gonorrhea, chlamydia infection, urinary tract infection, and pelvic inflammatory disease.

[0047] In the tenth aspect of the present invention, the present invention proposes the use of the Lactobacillus crispatus described in the second, third or fourth aspect, the microbial preparation described in the sixth aspect or the single-dose preparation described in the seventh aspect in the preparation of a product for regulating the balance of vaginal flora.

[0048] According to an embodiment of the present invention, the product includes at least one of a medicine and an external health care product.

[0049] In an eleventh aspect of the present invention, the present invention provides a molecular marker for Lactobacillus crispatus according to the third aspect or the fourth aspect. According to an embodiment of the present invention, the molecular marker comprises a nucleotide sequence selected from one of the following: (1) a nucleotide sequence as shown in SEQ ID NO: 24; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence as shown in SEQ ID NO: 24; (3) a nucleotide sequence having one or more, for example, 1, 2, 3, 4, 5 or more, nucleotide substitutions, deletions or insertions in the nucleotide sequence as shown in SEQ ID NO: 24.

[0050] According to an embodiment of the present invention, the molecular marker has a nucleotide sequence as shown in SEQ ID NO:24.

[0051] According to an embodiment of the present invention, the primers for molecular markers include the primer set described in the fifth aspect.

[0052] In a twelfth aspect of the present invention, a method for detecting the Lactobacillus crispatus described in the second, third, or fourth aspects is provided. According to an embodiment of the present invention, the method comprises: using the primer set described in the fifth aspect to amplify the DNA of the strain to be detected, so as to obtain the Lactobacillus crispatus described in the second, third, or fourth aspects. As described above, the primer set described in the fifth aspect is a specific primer for the Lactobacillus crispatus described in the present invention, is highly compatible with the DNA sequence of the Lactobacillus crispatus described in the second, third, or fourth aspects, and produces specific amplification only in the Lactobacillus crispatus strain. Using the method of the present invention, the Lactobacillus crispatus described in the present invention can be efficiently and conveniently identified and detected.

[0053] According to an embodiment of the present invention, the product of the amplification treatment comprises the molecular marker described in the first or eleventh aspect, indicating that the strain to be detected is the Lactobacillus crispatus described in the second, third, or fourth aspect. The nucleotide sequence of the molecular marker described in the first or eleventh aspect can be amplified using the primer set described in the fifth aspect, and the molecular marker described in the first or eleventh aspect is a specific fragment marker for the Lactobacillus crispatus described in the present invention, providing a reliable tool and basis for identifying and detecting the Lactobacillus crispatus described in the present invention.

[0054] According to an embodiment of the present invention, the method for detecting the Lactobacillus crispatus described in the second, third, or fourth aspects comprises the following steps: extracting a DNA sample from the strain to be detected, performing PCR amplification on the DNA sample using the primer set described in the fifth aspect, and then comparing the amplification result with the molecular marker described in the first or eleventh aspect, thereby identifying whether the strain to be detected is the Lactobacillus crispatus described in the second, third, or fourth aspects. The method of the present invention can efficiently and conveniently identify and detect the Lactobacillus crispatus of the present invention.

[0055] In the thirteenth aspect of the present invention, the present invention proposes use of the molecular marker described in the first aspect or the eleventh aspect in identifying and detecting the Lactobacillus crispatus described in the second aspect, the third aspect or the fourth aspect.

[0056] In the fourteenth aspect of the present invention, the present invention proposes the use of the Lactobacillus crispatus described in the second, third or fourth aspect, the microbial preparation described in the sixth aspect or the single-dose preparation described in the seventh aspect in inhibiting the growth of vaginal pathogens.

[0057] In the fifteenth aspect of the present invention, the present invention proposes the use of the Lactobacillus crispatus described in the second, third or fourth aspect, the microbial preparation described in the sixth aspect or the single-dose preparation described in the seventh aspect in the treatment and / or prevention of vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection.

[0058] According to an embodiment of the present invention, the vaginal pathogenic bacteria is at least one selected from the group consisting of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Atopobium vaginalis, Prevotella diversifolia, and Candida albicans.

[0059] According to an embodiment of the present invention, the related diseases caused by vaginal pathogenic bacteria infection include at least one of bacterial vaginosis, aerobic vaginitis, vulvovaginal candidiasis, Trichomonas vaginitis, mixed vaginitis, HPV infection, gonorrhea, chlamydia infection, urinary tract infection, and pelvic inflammatory disease.

[0060] In a sixteenth aspect, the present invention provides a method for inhibiting the growth of vaginal pathogens. According to an embodiment of the present invention, the method comprises: contacting at least one of the Lactobacillus crispatus described in the second, third, or fourth aspects, the microbial preparation described in the sixth aspect, or the single-dose preparation described in the seventh aspect with the vaginal pathogens.

[0061] In its seventeenth aspect, the present invention provides a method for treating and / or preventing vaginal pathogen infection or diseases caused by vaginal pathogen infection. According to an embodiment of the present invention, the method comprises administering to a patient a pharmaceutically acceptable dose of at least one of the Lactobacillus crispatus described in the second, third, or fourth aspects, the microbial preparation described in the sixth aspect, or the single-dose preparation described in the seventh aspect.

[0062] According to an embodiment of the present invention, the vaginal pathogenic bacteria is at least one selected from the group consisting of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Atopobium vaginalis, Prevotella diversifolia, and Candida albicans.

[0063] According to an embodiment of the present invention, the related diseases caused by vaginal pathogenic bacteria infection include at least one of bacterial vaginosis, aerobic vaginitis, vulvovaginal candidiasis, Trichomonas vaginitis, mixed vaginitis, HPV infection, gonorrhea, chlamydia infection, urinary tract infection, and pelvic inflammatory disease.

[0064] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0066] FIG1 is a colony morphology diagram of Lactobacillus crispatus HY1467 according to an embodiment of the present invention;

[0067] FIG2 is a bacterial morphology diagram of Lactobacillus crispatus HY1467 according to an embodiment of the present invention;

[0068] FIG3 is an electrophoretic pattern of PCR amplification of four Lactobacillus crispatus strains using primer 12-p1 according to an embodiment of the present invention, wherein M is a marker, 1 uses the supernatant after lysis of HY1467 as a template, 2 uses the supernatant after lysis of HY00744 as a template, 3 uses the supernatant after lysis of HY05276 as a template, and 4 uses the supernatant after lysis of HY00939 as a template;

[0069] FIG4 is an electrophoretic pattern of PCR amplification of four Lactobacillus crispatus strains using primer 4-p3 according to an embodiment of the present invention, wherein M is a marker, 1 uses the supernatant after lysis of HY1467 as a template, 2 uses the supernatant after lysis of HY00744 as a template, 3 uses the supernatant after lysis of HY05276 as a template, and 4 uses the supernatant after lysis of HY00939 as a template;

[0070] FIG5 is a graph showing the results of a hemolysis experiment using Lactobacillus crispatus HY1467 according to an embodiment of the present invention;

[0071] FIG6 is a diagram showing the results of a BV efficacy animal experiment according to an embodiment of the present invention;

[0072] FIG. 7 is a diagram showing the results of an animal experiment on the efficacy of VVC according to an embodiment of the present invention. DETAILED DESCRIPTION

[0073] The following describes the embodiments of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0074] Definition of terms

[0075] In the present invention, the term "Lactobacillus crispatus" or "L. crispatus" generally refers to a species of the genus Lactobacillus. This species is generally distinguished from other lactobacilli based on the polynucleotide sequence of the ribosomal 16S rDNA gene.

[0076] In the present invention, the term "effective culture conditions" generally refers to the environment in which Lactobacillus crispatus is placed or exposed to promote the growth of the bacteria. Therefore, the term generally refers to the culture medium, temperature, atmospheric conditions, substrate, agitation conditions, etc. that can affect the growth of the bacteria.

[0077] In the present invention, the term "antibiotic-sensitive" refers to a bacterium's weak resistance to antibiotics, where a small amount of antibiotics can affect its normal growth. According to an embodiment of the present invention, the Lactobacillus crispatus HY1467 is sensitive to clindamycin and cefuroxime antibiotics.

[0078] In the present invention, the term "vagina" generally refers to the vaginal area or surrounding area, including the labia, vulva, cervix, uterus, fallopian tubes, ovaries, urethra, bladder, anus and rectum, including their mucosal tissues.

[0079] In the present invention, the terms "disease" or "disorder" are used interchangeably and generally refer to any change in the state of the body or certain organs that prevents or disrupts the performance of functions and / or causes symptoms such as discomfort, dysfunction, suffering or even death in the person suffering from the disease or being exposed to it.

[0080] In the present invention, the term "pathogenic" (e.g., "pathogenic bacteria") generally refers to substances, microorganisms, or conditions that can cause disease. In some situations, pathogens also include microorganisms (e.g., bacteria) that are associated with a disease or condition, but for which a causal relationship (e.g., a direct causal relationship) has not yet been established or is yet to be established. In some embodiments, microorganisms that are not pathogens and can be symbionts can cause or be associated with disease or dysbiosis, depending on various factors (e.g., the immune status of the site, the abundance of microbial taxa, etc.). Such microorganisms are referred to as "pathogenic organisms."

[0081] In the present invention, the terms "vaginal flora" or "vaginal microbiota" are used interchangeably and generally refer to the microorganisms that colonize the vagina.

[0082] In the present invention, the term "inhibit" generally refers to the process of inhibiting or hindering the growth and reproduction of bacteria and their activity.

[0083] In the present invention, the term "isolated" as applied to a nucleic acid or protein generally means that the nucleic acid or protein is substantially free of other cellular components with which it is naturally associated. For example, it can be in a homogeneous state and can be in dry solution or in aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

[0084] In the present invention, the term "isolated" as applied to bacteria generally refers to bacteria that have been (1) separated from at least some of the components with which they were associated when originally produced (whether in nature or in an experimental setting), and / or (2) artificially produced, prepared, purified and / or manufactured, for example, using artificial culture conditions, such as (but not limited to) culturing on plates and / or in fermenters. Isolated bacteria include those that have been cultured, even if such cultures are not monocultures. Isolated bacteria can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the other components with which they were originally associated. In embodiments, the isolated bacteria are greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or greater than about 99%. According to an embodiment of the present invention, the bacterial population provided by the present invention includes isolated bacteria.

[0085] In the present invention, the term "pharmaceutically acceptable carrier" generally refers to a substance that facilitates administration of an active agent to a subject and absorption by the subject and that can be included in the Lactobacillus crispatus of the present invention without causing significant adverse toxicological effects on the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline solution, lactated Ringer's solution, ordinary sucrose, ordinary glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, spices, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and dyes. Such preparations can be sterilized, and those skilled in the art will recognize that other pharmaceutical carriers can also be used in the present invention.

[0086] In the present invention, the term "CFU (Colony-Forming Units)" generally refers to the total number of microorganisms such as bacteria, fungi, yeast, etc. in a product, and is usually used for calculation of viable bacteria.

[0087] In the present invention, the term "CFU / dose" means the amount of bacteria present in a food or dietary supplement / drug provided to a subject per day or per time. For example, in certain embodiments, the amount of Lactobacillus crispatus in the food or dietary supplement is 10 6 to 10 10 The amount of CFU / dose present (e.g. 10 6 to 10 9 CFU / dose). In this embodiment, if Lactobacillus crispatus is administered in a food product (e.g., in a solid beverage, yogurt), the food product (e.g., solid beverage, yogurt) provided to the subject each day or each time may contain about 10 6 to 10 10 Of course, alternatively, this amount of bacteria can be divided into multiple administrations, so long as the total amount of Lactobacillus crispatus received by the subject in any particular time (e.g., per 24-hour period) is from about 10 6 to about 10 10 CFU of bacteria, i.e. Lactobacillus crispatus in food products or dietary supplements meeting the above requirements, is 10 6 to 10 10 The amount of CFU / dose present (e.g. 10 6 to 10 9 CFU / dose).

[0088] In the present invention, the term "treating and / or preventing" includes not only treating and / or preventing a disease, but also generally includes preventing the onset of the disease, slowing or reversing the progression of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated therewith, and / or preventing further increase in the severity of the disease and / or any symptoms associated therewith, and preventing, reducing or reversing any physiological damage caused by the disease and any pharmacological effects generally beneficial to the patient being treated. It is not necessary for the Lactobacillus crispatus of the present invention to achieve a complete cure or eradication of any symptoms or manifestations of the disease in order for it to constitute a viable therapeutic agent. As recognized in the relevant art, drugs used as therapeutic agents can reduce the severity of a given disease state, but do not need to eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, a prophylactically administered treatment does not need to be completely effective in preventing the onset of the condition in order for it to constitute a viable prophylactic agent. Simply reducing the effects of the disease in a subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect) or reducing the likelihood of the disease occurring or worsening is sufficient.

[0089] In the present invention, the term "about" generally refers to a variation within the range of about 0.5%-10% above or below the specified value, for example, a variation within the range of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% above or below the specified value.

[0090] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0091] Example 1 Screening and Isolation of Lactobacillus crispatus

[0092] (1) Collect samples

[0093] The sources of samples are naturally fermented foods with regional characteristics, vaginal secretion samples collected by hospital gynecologists from volunteers who meet the inclusion criteria, and human samples (feces and breast milk).

[0094] (2) Strain isolation

[0095] The sample was serially diluted tenfold with physiological saline, and appropriate dilution gradients were plated onto anaerobic blood plates (purchased from Huankai Microbiology). The plates were then incubated at 37°C in an anaerobic workstation for 48-72 hours. Individual colonies of varying morphology were streaked onto the plates for purification. Further incubation was performed, and pure cultures were selected for strain identification (16S rDNA sequencing). Through identification and screening, a strain of Lactobacillus crispatus was isolated from the vaginal secretion sample and named Lactobacillus crispatus HY1467.

[0096] After the strain species was determined, the pure culture (Lactobacillus crispatus HY1467) was inoculated into MRS broth medium (purchased from Qingdao Haibo) for expansion. When the strain grew to an appropriate concentration, sterile 50% glycerol solution was used to mix the liquid culture with the same volume and stored in a -80°C strain bank.

[0097] Example 2 Identification of Lactobacillus crispatus

[0098] (1) Colony characteristics

[0099] Dip the inoculating loop into the culture tube and streak onto an MRS plate. Incubate anaerobically at 37°C for 48 hours and observe the colony morphology. As shown in Figure 1, the strain forms round, milky white colonies with irregular edges and a central protrusion when cultured on the MRS plate.

[0100] (2) Staining microscopy

[0101] Use an inoculating loop to transfer one loop of sterile distilled water to a clean glass slide. Pick a single colony from the MRS plate, mix it with distilled water, and evenly spread it on the slide. Stain according to the instructions of the Gram stain kit (purchased from Qingdao Haibo), and observe the bacterial morphology under an electron microscope. As shown in Figure 2, the stained strain was observed under an optical microscope and was Gram-positive, with short rods.

[0102] (3) Biochemical identification and analysis

[0103] ① Lactobacillus culture: The target strain was inoculated into MRS broth medium (purchased from Qingdao Haibo), placed in an anaerobic incubator, and cultured at 37°C for 24 hours.

[0104] ② Preparation of bacterial suspension: centrifuge the fermentation liquid of the strain at 4000 rpm for 5 min in a centrifuge, remove the supernatant, wash the bacterial mud with physiological saline, centrifuge at 4000 rpm for 5 min, remove the supernatant, wash the bacterial mud with physiological saline again, centrifuge at 4000 rpm for 5 min, remove the supernatant, add physiological saline and the bacterial mud and mix evenly for later use.

[0105] ③1% sodium hippurate identification test: aspirate 50 μL of bacterial suspension, add it to 1% sodium hippurate identification tube, seal it with sealing film and incubate it in a 37°C water bath for 2 hours, then slowly add 200 μL of ninhydrin solution (3.5% ninhydrin solution: 0.175 g of hydrated ninhydrin, 2.5 mL of acetone, and 2.5 mL of butanol) along the wall of the tube. Do not shake it, place it in a 37°C water bath for 10 minutes and then read the results.

[0106] ④ Other identification experiments: Pipette 50 μL of bacterial suspension and add it to the biochemical identification tube (Note: After adding the bacterial solution, the liquid surface of the aesculin identification tube needs to be covered with sterile liquid paraffin), seal it with sealing film, place it in an anaerobic incubator, and incubate it at 37°C for 48 hours before interpreting the results. The interpretation results are shown in Table 1:

[0107] Table 1 Interpretation results of Lactobacillus crispatus identification tube Note: + represents positive; - represents negative; +w represents weak positive.

[0108] The results of biochemical identification showed that except for raffinose and 1% sodium hippurate, Lactobacillus crispatus could utilize the other nine carbon sources (esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, inulin and lactose).

[0109] (4) 16S rDNA identification

[0110] The above-mentioned Lactobacillus crispatus was amplified and sequenced by 16S rDNA, and a BLAST comparison was performed in the NCBI database after the sequence was obtained. The 16S comparison result showed that the above-mentioned strain was Lactobacillus crispatus (Lactobacillus crispatus), and was named Lactobacillus crispatus HY1467. The 16S rDNA gene sequence was determined as follows:

[0111] (5) Specific nucleotide sequence molecular markers

[0112] ① Specific primer design

[0113] a. Screening for specific nucleotide sequence fragments: The HY1467 strain was subjected to whole genome sequencing and genomic analysis, and compared with the genome sequences of Lactobacillus crispatus strains included in the NCBI database. By comparing the genome sequences of other Lactobacillus crispatus strains, the specific nucleotide sequence of Lactobacillus crispatus HY1467 was screened.

[0114] b. Primer Design: Design primers for the specific gene nucleotide sequences screened in step a. Design specific primer fragments with predicted product lengths of approximately 200 to 800 bp for the screened specific nucleotide sequences. Also design 10 pairs of primers for other specific nucleotide sequences across the entire genome as controls. See Table 2 for primer sequences:

[0115] Table 2 Primer list

[0116] c. Primer Screening: Template preparation was performed using Lactobacillus crispatus HY1467 and three strains of the same species as a control group (see Table 3 for specific strain information). Single colonies were picked from each strain in 50 μL of lysis buffer (TaKaRa), lysed at 80°C for 15 minutes, and centrifuged at 4000 rpm for 5 minutes. The supernatant was used as the template. After primer screening, 12-p1 was selected for PCR amplification. The PCR amplification system consisted of 12.5 μL of Taq enzyme, 1 μL of F, 1 μL of R, 1.5 μL of template, and ddHO to 25 μL. PCR reaction conditions are shown in Table 4. After the PCR experiment, 1.5 g of agarose was added to 100 mL of 1× TAE buffer and heated thoroughly to melt. 5 μL of Gel Red dye was added and the sample was poured onto a gel plate to solidify. 4 μL of the PCR product was subjected to agarose gel electrophoresis under the following conditions: voltage 130 V, current 400 mA, and time 35 minutes.

[0117] Table 3 Strain information

[0118] Note: The strains numbered HY00744, HY05276, and HY00939 in Table 3 were obtained by screening Lactobacillus crispatus from samples collected by the inventors according to Example 1. To verify the specificity of the above-mentioned specific nucleotide sequence to Lactobacillus crispatus HY1467, PCR amplification was performed on HY1467 and three strains of the same species using the above-mentioned primers.

[0119] Table 4 PCR reaction conditions

[0120] Among the primers designed for specific nucleotide sequences, PCR amplification experiments were performed using 12-p1 as a primer. Only HY1467 had a single amplified band, and the band was clear and high in concentration, with relatively few primer dimers, while HY00744, HY05276, and HY00939 all had no amplified bands, as detected by agarose gel electrophoresis. Figure 3 shows a pattern of amplified bands in the control bacteria group when using primers designed for other nucleotide sequences to perform PCR amplification experiments (using 4-p3 as an example, agarose gel electrophoresis detection pattern is shown in Figure 4). Therefore, this indicates that primer 12-p1 is a molecular marker primer for HY1467, and the amplified product produced by this primer is a molecular marker for Lactobacillus crispatus HY1467. The amplified product of the 12-p1 primer is 558bp in size, and its nucleotide sequence is shown in SEQ ID NO:24. Thus, it was determined that the nucleotide sequence shown in SEQ ID NO:24 was part of the specific nucleotide sequence screened in step a, further verifying that primer 12-p1 can be used as a specific primer for Lactobacillus crispatus HY1467, and the nucleotide shown in SEQ ID NO:24 is a specific nucleotide sequence marker for Lactobacillus crispatus HY1467. This provides a reliable tool and basis for identifying and detecting Lactobacillus crispatus HY1467.

[0121] d. During experiments, the inventors discovered that when sequencing multiple Lactobacillus crispatus HY1467 samples using the 12-p1 primer, the amplified product sequences differed from the nucleotide sequence set forth in SEQ ID NO:24 at individual bases. However, compared to the control bacteria, all samples showed a single, distinct, and high-concentration amplified band, with relatively few primer dimers. Therefore, after multiple sequencing and alignments, the homology range between the molecular marker of Lactobacillus crispatus HY1467 and the nucleotide sequence set forth in SEQ ID NO:24 was determined. Specifically, the molecular marker of Lactobacillus crispatus HY1467 possesses a nucleotide sequence that is at least 97%, 98%, 99%, or higher in homology to the nucleotide sequence set forth in SEQ ID NO:24.

[0122] Example 3 Bacterial characteristics

[0123] (1) Antibiotic sensitivity test

[0124] Lactobacillus crispatus HY1467 was cultured in MRS broth, and the culture solution was evenly spread on MRS plates. After the solution was absorbed and dried, antibiotic susceptibility paper was applied and incubated anaerobically at 37°C for 48 hours. The diameter of the inhibition zone was measured using a vernier caliper. The antibiotic susceptibility of the strain was determined by the ring diameter on the antibiotic susceptibility zone. The results are shown in Table 5. The strain was resistant to metronidazole, norfloxacin, ofloxacin, ciprofloxacin, and kanamycin, but sensitive to clindamycin and cefuroxime.

[0125] Table 5 Antibiotic sensitivity test results Note: S: sensitive (15-20mm high sensitivity; >20mm extremely sensitive) I: intermediate (10-14mm moderately sensitive) R: resistant (<10mm insensitive)

[0126] (2) Toxicity test

[0127] ①Hemolysis test

[0128] The bacterial suspension from the cryopreservation tube was streaked onto an anaerobic blood plate. The plate was then incubated anaerobically at 37°C for 48 hours, and the color change of the blood plate surrounding the colonies was observed. The results of the hemolysis test are shown in Figure 5. Small, off-white colonies appeared in the culture medium surrounding the colonies, and no hemolytic rings appeared around the colonies, indicating that Lactobacillus crispatus HY1467 is gamma-hemolytic, meaning it is non-hemolytic.

[0129] ②Toxicity test in mice

[0130] Five mice weighing 18-22 g were used, and each mouse was orally gavaged with 0.5 ml of fresh bacterial solution (no less than 1.0 × 10 9 CFU / 0.5 ml) once daily for three consecutive days. The mice were observed for survival and weight from day 1 to day 7. The experimental results showed that all mice survived and gained weight after oral gavage with fresh Lactobacillus crispatus HY1467.

[0131] (3) Determination of metabolite content

[0132] ①D-lactic acid detection

[0133] The D-lactic acid production of the lactobacillus supernatant was detected using a D-lactic acid detection kit (purchased from Sigma-Aldrich). The method used in this kit is as follows: D-lactic acid is oxidized by a specific D-lactate hydrogenase, producing a color reaction that is proportional to the D-lactic acid concentration, and the absorbance at 450 nm is measured.

[0134] ②L-lactic acid detection

[0135] The supernatant was filtered through a 0.22 μm sterile filter membrane and the L-lactic acid concentration was measured using a biosensor.

[0136] Lactobacillus crispatus HY1467 was cultured in MRS broth for 24 hours, and the results of D-lactic acid and L-lactic acid testing are shown in Table 6. These organic acids, D-lactic acid and L-lactic acid, are antibacterial substances that can compete with pathogens for nutrients and adhesion sites, enhancing host mucosal immunity and anti-infection capabilities. The D-lactic acid content of Lactobacillus crispatus HY1467 metabolites was 4.14 g / L, and the L-lactic acid content was 5.55 g / L. The total acid production of Lactobacillus crispatus HY1467 was 9.69 g / L, while that of DJS-Lactobacillus delbrueckii was only 5.48 g / L. Therefore, Lactobacillus crispatus HY1467 has the potential to enhance host mucosal immunity and anti-infection capabilities, thereby preventing vaginal infections and treating inflammation.

[0137] Table 6 Metabolite content determination results

[0138] Example 4 Application Function Analysis

[0139] (1) Antibacterial experiment

[0140] 1. Preparation of working bacterial solution: Lactobacillus was inoculated into MM medium (MRS broth modified medium, composed of: peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K2HPO4 2.0 g / L, ammonium citrate tribasic 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) at a 0.5% inoculum size and cultured in an anaerobic workstation. Six pathogenic bacteria (Gardnerella vaginalis ATCC14018, abbreviated as GV; Escherichia coli ATCC 25922, abbreviated as EC; Staphylococcus aureus ATCC 25923, abbreviated as SA; Fannyhessea vaginae CCUG 38953, abbreviated as FV; Prevotella bivia NCTC 11156, abbreviated as PB; Canidia albicans ATCC 10231, abbreviated as CA) were cultured in an adapted medium. After the target strains were cultured, the supernatant was centrifuged and filtered through 0.22 μm to obtain a cell-free supernatant, which was used immediately or stored in a -80°C refrigerator. OD values ​​were measured after the pathogens were cultured. 600 value, diluted to about OD 600 The value is 0.005 (the number of viable bacteria is maintained at 5.0×10 5 CFU / mL~5.0×10 6 CFU / mL) (WS / T 650-2019 Evaluation method for antibacterial and antibacterial effects).

[0141] ② Interaction: Take the same volume of supernatant and pathogenic bacteria solution and mix them evenly. Immediately take 100 μL of the mixed bacteria solution and place it into a blank 96-well plate to measure OD 600 The remaining culture medium was placed at 37°C and cultured anaerobically or aerobically according to the culture conditions of the pathogen. After 48 hours of culture, 100 μL of the mixed bacterial solution was taken and placed into a blank 96-well plate to measure the OD 600 A blank control group was set up, with two replicate wells for each sample, and the inhibition rate of Lactobacillus against pathogenic bacteria was calculated according to the following formula.

[0142] Antibacterial rate = (AB) / A*100%

[0143] A: OD increase in the positive control group (blank culture medium) within 48 hours 600 value;

[0144] B: OD increase in the experimental group within 48 hours 600 value.

[0145] Three different strains of the same bacterial species were selected for antibacterial activity testing, along with the positive drug Dingjunsheng and the selected Lactobacillus crispatus HY1467. The results, shown in Table 7, showed that this strain exhibited significant antibacterial activity against GV, PB, FV, EC, SA, and CA, with a significant advantage in GV inhibition compared to the positive control group or the control group with different strains of the same bacterial species.

[0146] Table 7 Antibacterial test results Note: The strains numbered HY00744, HY00714, and HY01628 in Table 7 are Lactobacillus crispatus screened from samples collected by the inventors according to Example 1, in order to verify the inhibition rates of Lactobacillus crispatus HY1467 and three strains of the same species against different pathogens.

[0147] (2) Co-culture antibacterial experiment

[0148] 1) Interaction and co-culture experiment of Lactobacillus and CA

[0149] Lactobacillus culture: A glycerol tube of Lactobacillus crispatus was taken and inoculated into MM medium (MRS broth modified medium, composition: peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K2HPO4 2.0 g / L, ammonium citrate tribasic 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L), and the cultured bacterial solution was concentrated or diluted to about 1 × 10 9 CFU / mL, used as Lactobacillus working culture solution.

[0150] CA culture: Inoculate the CA glycerol tube into Sabouraud medium and culture aerobically at 37°C. Centrifuge the culture solution to remove the supernatant, and then adjust the concentration of the culture solution to about 1.0 × 10 7 CFU / mL, used as CA working bacterial solution.

[0151] 400 μL of each Lactobacillus and CA working culture solution were inoculated into 40 mL of MRS liquid medium. CA alone was inoculated as a blank control. Two replicates were performed in each group. The culture solution was gently shaken and incubated at 37°C under an anaerobic bench. Samples were taken 20 hours after incubation, and viable CA cells were counted using Candida albicans chromogenic medium (purchased from CHROMagar, France). The experimental results are shown in Table 8. Lactobacillus crispatus HY1467 had a certain inhibitory effect on the growth of CA.

[0152] Table 8 Antibacterial test results of co-culture of Lactobacillus and CA

[0153] 2) Lactobacillus and GV co-culture experiment

[0154] Lactobacillus culture: Take a glycerol tube of Lactobacillus crispatus and inoculate it into MM medium (MRS broth modified medium, composition: peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K2HPO4 2.0 g / L, ammonium citrate tribasic 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L), and concentrate or dilute the cultured bacterial solution to about 1 × 10 9 CFU / mL concentration is used as the Lactobacillus working culture solution.

[0155] GV culture: GV glycerol tubes were inoculated into BHI liquid medium containing 10% fetal bovine serum and cultured anaerobically at 37°C. The cultured bacterial solution was centrifuged to remove the supernatant, and then the bacterial solution concentration was adjusted to about 1.0 × 10 7 CFU / mL, used as GV working bacterial solution.

[0156] 400 μL of lactobacillus and GV working bacterial solutions were inoculated into 40 ml of BHI liquid medium containing 10% fetal bovine serum. GV was inoculated alone as a blank control group. Two replicates were placed in an anaerobic workstation for cultivation. Samples were taken 27 hours after cultivation and the viable count of GV was detected by fluorescence quantitative qPCR using GV-specific probe primers. After Lactobacillus crispatus and GV were co-cultured for 27 hours, the viable count of GV was detected. The experimental results are shown in Table 9. After Lactobacillus crispatus HY1467 was co-cultured with GV for 27 hours, the inhibition rate reached 88%, indicating that the strain had a strong inhibitory effect on the growth of GV.

[0157] Table 9 Antibacterial test results of co-culture of Lactobacillus and GV

[0158] (3) Biofilm removal experiment

[0159] 1) Lactobacillus removal of GV biofilm

[0160] After GV culture, the bacterial solution was adjusted to 1.0×10 7 CFU / mL were inoculated into 96-well plates in two groups, each with four replicates. The two groups were supplemented with equal volumes of blank MM liquid medium (MRS broth modified medium, consisting of: 10 g / L peptone, 5.0 g / L beef extract, 4.0 g / L yeast extract, 15 g / L glucose, 2.0 g / L K2HPO4, 1.0 g / L triammonium citrate, 2.5 g / L sodium acetate, 0.2 g / L magnesium sulfate, and 0.05 g / L manganese sulfate) and the supernatant of Lactobacillus crispatus HY1467. The plates were incubated at 37°C in an anaerobic workstation for 24 hours.

[0161] After the incubation period, the inhibitory effect of Lactobacillus crispatus HY1467 on GV biofilm formation was determined using a microplate crystal violet staining method. The experimental results, shown in Table 10, show that the supernatant of Lactobacillus crispatus HY1467 had a strong inhibitory effect on GV biofilm formation, reaching 90.9%. Therefore, it can effectively inhibit the growth of GV.

[0162] Table 10 Lactobacillus removal test results of GV biofilm

[0163] 2) Lactobacillus removal of CA biofilm

[0164] Inhibition of biofilm removal experiment: The bacterial solution after CA culture was adjusted to 1.0×10 7 CFU / mL, 100 μL was inoculated into 96-well plates, with two groups of four replicates each. Equal volumes of Sabouraud dextrose broth and Lactobacillus supernatant were added to each group. The plates were incubated at 37°C in an anaerobic workstation for 24 hours. After incubation, the inhibitory effect of Lactobacillus on CA biofilm formation was determined using crystal violet staining in microplates.

[0165] Biofilm removal experiment: adjust the bacterial solution after CA culture to 1.0×10 7CFU / mL, inoculated into 96-well plates, with four replicates in each group. The plates were incubated at 37°C in an anaerobic workstation for 24 hours. After 24 hours of incubation, the mixture was discarded and the wells were washed with sterile PBS. An equal volume of MM medium (MRS broth modified medium, composition: peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K2HPO4 2.0 g / L, ammonium citrate tribasic 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) and Lactobacillus supernatant were added to each group and incubated at 37°C for 24 hours. After incubation, the effect of the Lactobacillus supernatant on the removal of mature CA biofilms was determined using crystal violet staining in microplates.

[0166] The experimental results are shown in Table 11. Compared with the effect of clearing the formed CA biofilm, the Lactobacillus crispatus HY1467 has a stronger effect of inhibiting the formation of CA biofilm, with an inhibition rate of up to 75.2%.

[0167] Table 11 Results of the experiment on the removal of CA biofilm by lactobacilli

[0168] (4) Cell adhesion assay

[0169] ① Preparation of working bacterial solution: culture Lactobacillus and test the OD of bacterial solution 600 The cultured Lactobacillus culture solution was centrifuged at 4°C and 4000 rpm for 5 min, the supernatant was discarded, and the solution was washed three times with PBS. Finally, the Lactobacillus was resuspended in MEM complete medium (purchased from Zhongqiao Xinzhou). An appropriate amount of the resuspended solution was spread on a plate to detect the number of viable bacteria, which was L1.

[0170] ②Cell culture: HeLa cells were seeded in 24-well plates, with 1.5×10 cells per well. 5 The HeLa cells cultured in 24-well plates were washed with serum-free MEM medium (purchased from Zhongqiao Xinzhou) and then counted, recording C1.

[0171] ③ Interaction: Lactobacillus was inoculated into the cells at a bacteria-to-cell ratio of 100:1 and incubated at 37°C in a 5% CO2 environment for 1.5 hours. The supernatant after centrifugation was added to the cells as a blank control. After 1.5 hours, the culture medium in the wells was collected, and the cells in the wells were washed with MEM medium. 100 μL of trypsin was added to each well for digestion, and then MEM complete medium was added to terminate the reaction. The suspension was collected, and a portion was taken to count the number of cells (C2). The number of viable Lactobacillus bacteria adhering to the cells was determined by plate spreading (L2).

[0172] ④Calculate the adhesion number and adhesion rate: average Lactobacillus cell adhesion number = L2 / C2.

[0173] The single cell adhesion number of Lactobacillus crispatus HY1467 to Hela cells was 18.69 CFU, indicating that this strain has good adhesion or colonization characteristics to vaginal epithelial cells.

[0174] (5) BV (bacterial vaginosis) animal model efficacy test

[0175] Healthy SPF-grade Balb / c female mice, 6-8 weeks old, were used for modeling. After adaptive culture, the animals were randomly divided into 8 groups: a model group (M), an experimental group (Lactobacillus crispatus HY1467), and a positive control group (DJS). Before inoculation with pathogenic bacteria, the animals in each group were pretreated with subcutaneous injection of estradiol benzoate injection, and then vaginally administered GV (20 μl) to establish the BV pathogen model. After successful modeling, the experimental group was vaginally administered Lactobacillus crispatus HY1467 solution (1×10 10 CFU / mL, 20 μL), and the positive control group was given an equal amount of Lactobacillus delbrueckii solution (Dingjunshengzhong strain, 1×10 10 CFU / mL, 20 μL). The model group received an equal volume of normal saline. After treatment, vaginal lavage was performed four times per animal with 50 μL of PBS. The fluid was collected, placed in a 1.5 mL Eppendorf tube, and frozen at -80°C. The GV load in the lavage fluid was measured using qPCR, and the differences in GV load in the lavage fluid were compared between the groups.

[0176] The experimental results are shown in Figure 6. The experimental results show that the use of Lactobacillus crispatus HY1467 to treat BV-infected animal models significantly reduced the GV content in the vagina, and the therapeutic effect was significantly better than that of the positive drug (DJS), indicating that this strain has a good therapeutic effect on BV in mice.

[0177] (6) VVC (vaginal candidiasis) animal model efficacy test

[0178] Healthy SPF-grade Balb / c female mice, 6-8 weeks old, were used for modeling. After adaptive culture, the animals were randomly divided into 8 groups: a model group (M), an experimental group (Lactobacillus crispatus HY1467), and a positive control group (Dapoxetine suppository). Each group received a subcutaneous injection of estradiol benzoate for pretreatment. CA (15 μL / mouse) was administered continuously from D0 to D2, and the experimental group received a vaginal administration of Lactobacillus crispatus HY1467 (5×10 β-lactamase) daily from D3 to D7. 9CFU / mL, 20 μL). A positive control group was given 30 mg of bisoprolol suppositories, and a model group was given an equal volume of normal saline (20 μL). Vaginal lavage was performed on day 8. The lavage fluid was diluted and spread on a Candida albicans identification medium (chromogenic medium). After incubation at 37°C for 48 hours, the number of green colonies on the culture plate was observed and counted. One-way analysis of variance was performed using GraphPad Prism 5 software, and P < 0.05 was considered statistically significant.

[0179] The experimental results are shown in Figure 7. The results show that compared with the model group, the content of pathogenic bacteria in the vaginal lavage fluid of the Lactobacillus crispatus HY1467 group was significantly reduced after treatment, which is equivalent to the therapeutic effect of the positive drug diazepam suppository, indicating that this strain has a good therapeutic effect on VVC in mice.

[0180] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0181] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A molecular marker of Lactobacillus crispatus, characterized in that, having a nucleotide sequence selected from one of the following: (1) the nucleotide sequence shown in SEQ ID NO:24; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO:24; (3) a nucleotide sequence having one or more, such as 1, 2, 3, 4, 5 or more nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO:

24.

2. The molecular marker according to claim 1, wherein The molecular marker has the nucleotide sequence shown in SEQ ID NO:

24.

3. A Lactobacillus crispatus, characterized in that, Comprising the molecular marker according to claim 1 or 2.

4. The Lactobacillus crispatus according to claim 3, characterized in that, Deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 7, 2023, with the deposit number CGMCC No. 26503.

5. The Lactobacillus crispatus according to claim 3, characterized in that, The 16S rDNA of the Lactobacillus crispatus has a nucleotide sequence selected from one of the following: (1) the nucleotide sequence shown in SEQ ID NO:1; (2) a nucleotide sequence having at least 99.8%, 99.9% or higher homology with the nucleotide sequence shown in SEQ ID NO:1; (3) a nucleotide sequence having one or more, such as 1, 2, 3, 4, 5 or more nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO:1; Preferably, the 16S rDNA gene sequence of the Lactobacillus crispatus comprises the nucleotide sequence shown in SEQ ID NO:

1.

6. A primer set for detecting Lactobacillus crispatus according to any one of claims 3-5, characterized in that, Including a forward primer and a reverse primer; The forward primer has the nucleotide sequence shown in SEQ ID NO:2; The reverse primer has the nucleotide sequence shown in SEQ ID NO:

3.

7. A microbial preparation, characterized in that, Comprising the Lactobacillus crispatus according to any one of claims 3-5.

8. The microbial preparation according to claim 7, wherein, Further comprising a pharmaceutically acceptable carrier or excipient.

9. A single-dose formulation, characterized in that, including 1×10 6 ~1×10 10 CFU Lactobacillus crispatus according to any one of claims 3-5 as an active ingredient.

10. Use of the Lactobacillus crispatus according to any one of claims 3-5, the microbial preparation according to claim 7 or 8, or the single-dose preparation according to claim 9 in the preparation of a drug for treating and / or preventing vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection.

11. Use of the Lactobacillus crispatus according to any one of claims 3-5, the microbial preparation according to claim 7 or 8, or the single-dose preparation according to claim 9 in the preparation of a drug for inhibiting vaginal pathogenic bacteria.

12. The use according to claim 10 or 11, characterized in that, The pathogenic bacteria are selected from at least one of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Atopobium vaginae, Prevotella bivia, and Candida albicans.

13. The use according to claim 10 or 11, characterized in that, The vaginal pathogenic bacteria are selected from at least one of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, and Candida albicans.

14. Use of the Lactobacillus crispatus according to any one of claims 3-5, the microbial preparation according to claim 7 or 8, or the single-dose preparation according to claim 9 in the preparation of a product for regulating vaginal flora balance.

15. A method for inhibiting the growth of vaginal pathogenic bacteria, characterized in that, Including: Contact at least one of Lactobacillus crispatus according to any one of claims 3-5, the microbial preparation according to claim 7 or 8, or the single-dose preparation according to claim 9 with the vaginal pathogenic bacteria.

16. A method for treating and / or preventing vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection, characterized in that, Comprising: Administering to a patient a pharmaceutically acceptable dose of at least one of Lactobacillus crispatus according to any one of claims 3-5, the microbial preparation according to claim 7 or 8, or the single-dose preparation according to claim 9.

17. The method according to claim 15 or 16, characterized in that, The vaginal pathogenic bacteria are selected from at least one of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Atopobium vaginae, Prevotella bivia, and Candida albicans.

18. A method for detecting Lactobacillus crispatus according to any one of claims 3-5, characterized in that, Comprising: Performing an amplification treatment on the DNA of the strain to be detected using the primer set according to claim 6, so as to obtain Lactobacillus crispatus according to any one of claims 3-5.

19. The method according to claim 18, characterized in that, The product of the amplification treatment has the molecular marker according to claim 1 or 2, which is an indication that the strain to be detected is Lactobacillus crispatus according to any one of claims 3-5.

20. The method according to claim 19, wherein Comprising the following steps: extracting a DNA sample from the strain to be detected, performing a PCR amplification treatment on the DNA sample using the primer set according to claim 6, and then comparing the amplification result with the molecular marker according to any one of claims 1-2, so as to identify whether the strain to be detected is Lactobacillus crispatus according to any one of claims 3-5.

21. Use of the molecular marker according to any one of claims 1-2 for identifying and detecting Lactobacillus crispatus according to any one of claims 3-5.

Images