RU2224017C2 - Strain lactobacillus plantarum dsm 11918 and its using for prophylaxis and/or treatment of urogenital infections - Google Patents

Abstract

FIELD: biotechnology, medicine, microbiology. SUBSTANCE: strain Lactobacillus plantarum LB 931 is placed for storage in German microorganisms storage, Braunschweig, Germany at deposit number DSM 11918. The strain LB 931 is designated for treatment and/or prophylaxis of urogenital infections. Addition of LB 931 to pharmaceutical compositions and individual care subjects, such as female hygiene subjects, diapers and hygienic napkins show some advantages. The strain elicits the enhanced stability, tolerates prolonged storage easily at room temperature and retains high cells viability and adhesion to vaginal epithelium cells. EFFECT: valuable medicinal properties of strain. 8 cl, 4 dwg, 8 tbl, 7ex

Description

 The invention relates to a new strain of Lactobacillus, which has valuable pharmaceutical characteristics. This invention also relates to pharmaceutical compositions and products for personal care, including this strain, as well as to the use of this strain for the prevention of genitourinary infections.

BACKGROUND OF THE INVENTION
(All quotes in the description below are incorporated by reference)
The common bacterial flora in the urogenital region consists of a complex ecosystem comprising more than 50 different types of bacteria (Hill et al., Scand. J. Urol. Nephrol. 1984; 86 (suppl.): 23-29). In the normal flora, bacteria belonging to the genus Lactobacillus (LB) predominate, which are gram-positive sticks adapted to the environment in the vagina of fertile women (capable of childbearing). These bacteria also contribute to maintaining a specific environment and ecological balance in the vagina.

 In addition to the complex system of interaction between the numerous bacterial flora of the vagina and the rest of the genitourinary area, it is necessary to consider various physical conditions that can affect the growth and adhesive properties of bacteria. Some LVS strains inhibit the growth of potentially pathogenic bacteria through various mechanisms. The metabolism of LB results in the formation of organic acids, mainly lactic and acetic acids, which contribute to a decrease in the pH of the vaginal fluid, which is undesirable for many other species. LBs can also produce soluble substances that directly inhibit the growth of potentially pathogenic bacteria and yeast. They can also produce hydrogen peroxide, which is toxic to bacteria that have lost the enzyme catalase, such as gram-negative anaerobic bacilli and Enterobacteriacae. These inhibition characteristics can vary greatly between different LB strains (Hooton et al., JAMA 1990; 265: 64-69).

 The weakness of the natural defense system can allow potentially pathogenic microorganisms to cause clinical infections, for example, due to drug treatment, poor personal care, or changes in the microflora of the skin or mucous membranes. LB prevails in the normal vaginal flora, and the pH of the environment is below 4.5. Yeast and enterobacteria are present in limited numbers or absent (Redondo-Lopez et al., Rev. Inf. Dis. 1987; 12: 856-872). Changes in the vaginal bacterial flora can occur in connection with various pathogenic conditions. In the vagina and mouth of the urethra of women suffering from a relapse of urinary tract infections, there is an increased number of enterobacteria, and the flora of their urogenital system contains a reduced amount of lactobacilli (Marrie et al., J. Clin. Microbiol, 1976, 8, 67-72). It is also known that the incidence of infections increases with the antibiotic treatment of other infections (Stamey, Rev. Inf. Dis. 1987: 9 (suppl. 2): 195-208; Reid et al., Curr. Opin. Inf. Dis. 1991 ; 4: 37-41). In addition, it has been demonstrated that children with a history of frequent cases of antibiotic treatment are more prone to acquired urinary tract infections (Marild et al., Ped. Inf. Dis. 1990; 22: 43-47).

 With bacterial vaginosis, the amount of LB decreases, and the pH increases. In addition, the species Bacterioides, Gardnerella vaginalis and Mobiluncus (Redondo-Lopez, supra) predominate. Vaginitis associated with an increased number of enterobacteria is often a significant problem in connection with antibiotic treatment. The usual oral administration of penicillin leads to the accumulation of this substance in the vaginal fluid (Sjoberg et al., Obstet. Gynecol. 1990; 75: 18-21), which is accompanied by the colonization of enterobacteria and yeast (Sjoberg et al., Gynecol. Obstet. Invest. 1992. ; 33: 42-46). Studies in monkeys (Masas fascicularis) showed that vaginal administration of amoxicillin impairs the ability of the natural bacterial flora to inhibit the colonization of E. coli pathogenic for the urinary tract (Herthelius et al., Infection 1988; 16: 263-266).

During pregnancy, the composition of the vaginal flora can affect the incidence of the uterus and the baby. The presence of group B streptococcus (GBS) in the fecal and vaginal flora is common (up to 30% of all pregnant women). These bacteria usually do not constitute a threat to women's health. However, GBS can cause serious infections in newborns. In these cases, bacteria are transferred vertically from mother to baby before or during childbirth. Other bacteria can also be transmitted in this way and cause infections in children. There is a strong association between bacterial vaginosis and preterm birth (Martius et al. Arch. Gynecol. Obstet. 1990; 247: 1-13). The mechanisms of this phenomenon are not known. It has been shown that changes in the vaginal flora in the direction of the prevalence of gram-negative species increase the amount of the phospholipase A ₂ enzyme, which in turn can initiate the synthesis of prostaglandins, starting with arachidonic acid (Bejar et al., Obstet. Gynecol. 1981; 57: 479-482 ) The vaginal flora also produces large amounts of endotoxin (Sjoberg et al., Obstet. Gynecol. 1991; 77: 265-266), which can induce endogenous synthesis of prostaglandins (Romero et al., Obstet. Gynecol. 1989; 73: 31-34) possibly mediated by interleukins.

Theoretically, the positive characteristics of LB explain their use in commercial preparations intended to supplement and enhance the vaginal flora. Success was variable, and often the available drugs contained much lower amounts of LB than was established. In addition, some preparations were contaminated (Hughes et al., Obstet. Gynecol. 1990; 75 (2): 244-248). To supplement and improve the normal bacterial flora in the genitourinary area by adding LB, it is necessary to carefully select the bacterial strain that will be used. A suitable strain of LB must meet the following criteria:
1. Strain LB should produce large amounts of soluble substances that can inhibit the growth of enterobacteria, group B streptococci, staphylococci and yeast.

 2. Strain LB must be able to be transferred to the skin and mucous surfaces of the genitourinary area.

 3. Strain LB should be capable of adhesion on the mucous surfaces of the genitourinary area.

 4. The strain LB must be able to tolerate storage for a long period of time, and there must be the ability to induce the strain in various types of drugs.

 5. The strain LB must be able to maintain its viability and characteristics in commercial products or preparations when used.

 6. The strain LB should not be sensitive to spermicidal preparations containing nonoxynol-9.

 7. Strain LB should be isolated from the urogenital tract of women.

 8. Strain LB must allow the existence of human urogenital LB flora.

 Therefore, there is a need for strains that satisfy these requirements.

SUMMARY OF THE INVENTION
A new strain of Lactobacillus plantarum was designated, designated LB931, which satisfies the requirements listed above. This strain is stored at Deutsche Sammlung von Mikroorganismen (German Microorganism Collection), Braunschweig, Germany. He was assigned the deposit number DSM11918. Accordingly, LB931 can be used to treat and / or prevent genitourinary infections. The inclusion of LB931 in pharmaceutical compositions and in personal care products, such as diapers and sanitary napkins, has advantages.

Definitions
As used herein, the term “LB” refers to bacteria of the genus Lactobacillus.

 As used herein, the term urogenital area refers to the perineum, urethra, and vagina.

 As used herein, the term “absorbent article” refers to products suitable for the absorption of body fluids, such as blood or urine. Examples of such products are feminine hygiene products, incontinence pads, and diapers.

 As used herein, the term “GBS” refers to group B streptococcus.

 As used herein, the term “lactic acid bacteria” refers to bacteria producing lactic acid, such as bacteria belonging to the genera Lactobacillus and Lactococcus.

 The term "CFU" refers to colony forming units.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a new strain of Lactobacillus plantarum designated LB931 (DSM11918). This bacterial strain is useful for the prevention and / or treatment of genitourinary infections, as it inhibits the growth of a large number of pathogenic microorganisms. This strain is stable and easily tolerates long periods of storage at room temperature. Accordingly, products containing LB931 have a long shelf life. This strain can be easily transferred to human skin and vaginal epithelium. LB931 is resistant to therapeutic concentrations of certain antibiotics and spermicidal substances.

 The inhibition characteristics of LB931 were investigated. Examples of bacterial species that are successfully inhibited are Escherichia, Klebsiella, Proteus, Staphylococcus and Streptococcus group B. Therefore, LB931 is useful for the treatment and / or prevention of infections caused by these microorganisms.

 As noted above, the present invention also provides a number of pharmaceutical compositions, preferably suitable for external administration, including LB931 along with pharmaceutically or physiologically acceptable carriers, excipients and / or diluents. Typically, such carriers should be non-toxic to recipients at the applicable doses and concentrations. Typically, in preparing such compositions, a combination of a therapeutic agent with buffers, thickeners, gelling agents such as glycerin, polyethylene glycol, etc. is used. Antioxidants such as ascorbic acid, low molecular weight polypeptides (less than 10 residues), proteins, carbohydrates including glucose, sucrose and dextrins, and other stabilizers and excipients may be included. The pharmaceutical compositions may be in the form of ointments, creams, liquid solutions, suppositories or capsules.

 The present invention also relates to absorbent articles comprising LB931. Such products include a permeable outer layer, which is intended to be in close contact with the wearer's skin, preferably a liquid-impervious backing layer, which is designed to be remotely located from the wearer in use, and an absorbent structure located between the outer layer and the backing layer. In some cases, between the outer layer and the absorbent structure, an additional layer may be located in the form of, for example, cotton wool or the like. Microorganisms exhibiting antagonistic properties can be located in different parts of the absorbent article, for example, in the outer layer, in the absorbent structure of the absorbent article, between two layers of the absorbent article, in a loose product of the absorbent article, or in some other way.

The present invention will now be described with reference to the accompanying figures, in which:
FIG. 1 is a diagram showing the stability of the LB931 frozen-dried state at room temperature (+22 ^{° C.} ) and at +6 ^° C.;
FIG. 2 is a diagram showing the stability of an LB931 embedded in an absorbent article;
FIG. 3 and 4 show the amount of LB931 transferred to the mouth of the urethra and perineal skin of young girls after applying panty liners including LB931.

The present invention will now be described with reference to the following examples:
Example 1: Isolation and Identification of Lactobacillus plantarum, strain LB931
Bacterial samples were taken from healthy women. Bacterial strains were isolated from these samples and these strains were analyzed based on their ability to inhibit the growth of enterobacteria (data not shown). The best strain isolated from a healthy pregnant woman was classified as Lactobacillus plantarum according to the API 50 CH test kit (API systems, BioMerieux, FR), with a predominance of LB931. This strain was then identified by DNA analysis using SDS-PAGE (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) on a BCCM / LMG device (Belgium) as Lactobacillus plantarum-pentosus-paraplantarum.

EXAMPLE 2: Inhibitory ability of strain LB931
The purpose of this experiment was to illustrate the ability of strain LB931 to inhibit the growth of other bacteria. LB931 was grown in MRS medium (Merck, DE) at 37 ^{° C.} in 5% C0 ₂ overnight. One ml containing 10 ⁸ bacteria was added to 25 ml of molten 2% agar in MRS medium. The mixture was poured into a Petri dish, allowed to solidify and incubated as described above for 24 hours. Another 25 ml of agar-M17 (Merck, DE) was poured onto the surface of the first layer and the plates were left at room temperature for 4 hours. the same agar plates not containing LB931, which were used as a control.

Indicator bacteria were grown separately in a TY medium (Holm et al., APMIS 1967; 69, 264) at 37 ^° C in air. Cultures were transferred to a Bertani pan from 25 compartments, each compartment containing 0.25 ml (10 ^e bacteria / ml). Bacteria were transferred from each of these trays and fixed onto agar plates containing Lactobacillus using the Steer steel replicator pin (Steers et al., J. Antibiot. Chemother. 1979, 9, 307). These plates were incubated at 37 ^{° C.} overnight. Dies were read and installed a) if indicator bacteria grow; or b) the growth of indicator bacteria is inhibited; or c) no growth of indicator bacteria is observed. The pH of each of the dies was also determined.

 The mutual influence test results are shown in Table I.

 The results show that Lactobacillus plantarum, LB931, inhibits or prevents the growth of a large number of bacterial strains and generally does not affect other strains of Lactobacillus.

 EXAMPLE 3: The ability of LB931 to maintain viability in various preparations a) LB931, dissolved in a suspension of equal parts skim milk and 0.9% NaCl.

 LB931 was dissolved in skim milk containing 0.9% NaCl. Dissolved bacteria were then incubated at different temperatures. The number of bacteria was continuously recorded by cell counting. The results are shown below in table II.

The results demonstrate that LB931 is stable in a mixture of skim milk and NaCl for one month at + 4 ^o C.

b) Preparations LB931 in skim milk were dried from a frozen state in accordance with standard methods. The resulting powder was stored in Petri dishes at room temperature and at +6 ^o C. The number of bacteria was determined after 7 and 25 days, respectively. The results are shown below in table III.

The number of bacteria in the dried powder was also determined every four weeks up to 68 weeks. The results are shown in FIG. 1. From this figure it is obvious that LB931 is stable both at room temperature and at +6 ^o C for 22 weeks. After a year at +6 ^o With more than 10 ⁵ CFU / mg LB931 are viable.

c) The ability of LB931 to survive in synthetic urine, pH 6.6, was determined. Synthetic urine contained mono- and divalent cations and anions, as well as urea, and was prepared as described in Geigy, Scientific Tables, vol. 2, 8: th ed. 1981 p. 53. A culture medium for microorganisms was added to sterile synthetic urine. The nutrient medium was prepared in accordance with the data on the composition of the Hook medium and FSA medium. 10 ³ bacteria LB931 was added to 1 ml of synthetic urine and the samples were incubated for 18 h at 32 ^° C. After incubation, the number of bacteria was> 10 ⁵ / ml. LB931 is capable of survival and growth in synthetic urine.

 d) Investigated the ability of LB931 to survive on an absorbent article (panty liners). A suspension of LB931 (150 μl) was added to the absorbent product, and then this product was stored in an impermeable packaging for 9 months. The results are shown in FIG. 2. A large number of bacteria survived for seven months.

 e) Finally, the characteristics of LB931 were tested during the growth and storage period. LB931 was cultured in MRS medium and every third day for three months a new sowing was performed. After that, the initial sample LB931 and the last culture were compared in the API test, PGFE and in the mutual influence test. Two samples were identical in all tests. This shows that LB931 is very stable after storage and several crops in a nutrient medium.

Example 4: Transfer of LB931 to the skin of the perineum and mouth of the urethra of women
To study the transfer of LB931 to the perineum using panty liners, the following studies were performed. The subjects tested were women from 12 to 60 years old, and the tests were performed at an appropriate time between menstrual periods. The tested products were made from traditional panty liners, including a liquid-permeable outer layer, a liquid-impervious lower lining layer, and between them an absorbent layer; from 100-200 g / m ² chemical pulp. A suspension of LB931 bacteria was sprayed onto the absorbent side of the test product in an amount of 10 ⁹ colony forming units per product.

 In order to determine the presence of LB931 in the perineum of 13 test subjects, a so-called smear test was performed. Bacteria were harvested by rubbing a sterile cotton-tipped stick, which was dipped in a sterile sodium chloride solution over a defined area of the skin. The presence of LB931 and other LBs on the perineal skin and at the mouth of the urethra was determined. Such tests were performed among test subjects in order to establish a control sample. After that, test subjects wore panty liners for 5 hours in the morning. Panty liners were removed and the presence of added lactic acid bacteria and natural lactic acid bacteria, respectively, was determined again immediately after removal of the panty liners. This sample was designated as sample 1. After another 4-5 hours, the following sample was taken and designated as sample 2. The type of lactic acid bacteria was determined using Rogosa agar with vancomycin for LB931, and Rogosa agar plates incubated anaerobically for other LB . The following determinations were made using API (BioMerieux, FR) and PFGE (Pulsed Field Gradient Electrophoresis Gel Electrophoresis). The results are shown in table IV. LB931 is found on the perineal skin and at the mouth of the urethra in all women after using panty liners on which LB931 was sprayed.

 Example 5: Transfer of LB931 to the skin of the perineum and the mouth of the urethra in girls.

The study included thirteen young girls from 3 to 12 years old. Bacterial samples from the skin of the perineum and the mouth of the urethra were obtained first by dipping a cotton swab in MRS medium, then by carefully rubbing the sticks on the skin or epithelial surface. At the end, the swab was immersed in a tube containing MRS medium. Samples were obtained in accordance with the following scheme:
Sample 0: A control sample was obtained in the evening before the start of the study on the content of bacteria LB931. Evening, day 1; put on panty liners.

 Sample 1: Morning, Day 2. New panty liners were worn throughout the day.

 Sample 2: The sample was taken in the evening before the optional bath and before applying a new panty liner. Evening, day 2.

 Sample 3: Same procedure as for sample 1. Morning, day 3.

 Sample 4: Same procedure as for sample 2. Evening, day 3.

 Sample 5: Same procedure as for sample 1. Morning, day 4.

Sample 6; A sample was taken in the evening before an optional bath. Panty pads were not worn at night. Evening, day 4;
Sample 7: A sample was taken in the morning on day 5. Panty pads were not worn during the day.

 Sample 8: The last sample was taken in the evening on day 5.

 The results are shown in FIG. 3 (mouth of the urethra) and FIG. 4 (perineal skin). The results show that LB931 can be transferred from an absorbent article.

EXAMPLE 6: Sensitivity to antibiotics and spermicidal agents
MIC values for Lactobacillus plantarum LB931 were determined using the E-test (Brown et al. J. Antimicrob. Chemother. 1991; 27: 185-190). The results are shown in table V.

 Sensitivity to antibiotic substances was also determined using the SIR system. In table VI below, H stands for sensitivity and Y stands for stability.

 LB931 is sensitive to certain antibiotics that are usually prescribed for the treatment of urinary tract infections, but in addition, LB931 is resistant to, for example, nalidixic acid and norfloxacin. LB931 is also resistant to vancomycin.

MIC tests were also performed on the spermicidal agent tergitol. LB931 was grown on MRS agar in 5% CO ₂ , 37 ^° C for 48 hours. Bacteria were seeded in 3 ml of MRS medium and incubated for 10 hours under the same conditions as described above. 1.5% Cultures were re-plated in 3 ml of MRS medium and incubated under the same conditions for 18 hours. NP-9 Tergitol (Sigma, US), lot 47F0002, was diluted with MRS medium having a temperature of 37 ^° C (low viscosity), getting a 40% stock solution. Using this stock solution, 3 ml solutions were prepared having the following concentrations: 0%, 5%, 10%, 20%, 30% and 40%. 10 μl of the bacterial culture was added to each solution. The control solution was mixed and added to MRS agar plates. The plates were incubated in 5% CO ₂ at 37 ^{° C.} for 48 hours to determine cell density. The remaining solutions were incubated without stirring in 5% CO ₂ at 37 ^° C for 18 hours. A solution containing 30% and 40% NP-9 was diluted with MRS medium at 37 ^° C (low viscosity). All solutions were vigorously mixed; diluted with sterile 0.9% NaCl and added to MRS agar plates. The plates were incubated in 5% CO ₂ at 37 ^{° C.} for 48 hours to determine CFU / ml.

 The results are shown in table VII.

The introduced amount of LB931 was 1.0 • 10 ⁷ CFU.

 These results show that LB931 survives well in a solution of tergitol NP-9 with a concentration of up to 40%.

EXAMPLE 7: Adhesion of LB931 to Vaginal Epithelial Cells
a) Preparation of LB931 Suspension
LB931 was grown on MRS agar (in 5% CO ₂ , 37 ^° C; 48 h). The culture was introduced into 3 ml of medium (in 5% CO ₂ , 37 ^° C; 8 hours). 2% of the resulting culture was re-introduced in 10 ml of MRS medium (in 5% CO ₂ , 37 ^° C; 18 hours). The resulting culture was centrifuged for 8 min at 20 ^o C and at 2000 rpm in a removable rotor (820xg). The resulting cell pellet was washed in 5 ml of lactic acid buffer (10 mM lactic acid, pH 4.5, 0.15 M NaCl). The bacteria were diluted with lactic acid buffer so that the OD ₅₀₀ was approximately 1.0 (approximately 10 ⁸ CFU / ml).

b) Obtaining vaginal epithelial cells:
Vaginal epithelial cells were harvested using a sterile cotton swab and transferred into 4 ml of lactic acid buffer or PBS in a small tube. The contents of the tube were mixed and a cotton swab was removed. The tube was centrifuged at 700 rpm at 20 ^o C for 8 min in a removable rotor Jouan CR.-12 (≈ 100xg), and the resulting precipitate was washed in 3 ml of lactic acid buffer or PBS. Cells were counted in a hematocytometer and the concentration was adjusted to 10 ⁵ -10 ⁶ cells / ml using lactic acid buffer or PBS. 25 μl of the cell suspension was smeared on a microscope coverslip to control the washing procedure (see below).

c) Adhesive test
0.5 ml of LB931 suspension and 0.5 ml of cell suspension were quickly mixed in an 1.5 ml Eppendorf tube. A control sample was prepared by mixing 0.5 ml of cell suspension and 0.5 ml of buffer. The tubes were centrifuged at 20 ^o C and 2000 rpm (≈ 720xg), and then incubated for 1 h at 37 ^o C. After incubation, the tubes were centrifuged for 8 min at 20 ^o C and 700 rpm (≈ 90xg). Precipitation was washed in 1 ml of lactic acid buffer or PBS. At the end, the samples were suspended in 400-500 μl of lactic acid buffer or PBS.

d) Analysis
25 μl of the Suspended Precipitate was allowed to air dry on a microscope coverslip, then fixed and Gram stained. Of each sample, 50 epithelial cells were analyzed. The number of LB931 adhered to these cells was counted, and the results were divided into 5 groups (0-10, 11-30, 31-50, 51-100,> 100 bacteria / cell).

 The results are shown in table VIII.

 The results clearly demonstrate that LB931 bacteria adhere to the cells of the vaginal epithelium, regardless of when these cells were taken.

Claims (8)

1. The strain Lactobacillus plantarum DSM 11918 as an active ingredient in a pharmaceutical composition or adsorbent product used for the prevention and / or treatment of genitourinary infections, such as colonization of enterobacteria. 2. The strain of claim 1, wherein the absorbent article is a diaper, sanitary napkin, panty liners or incontinence pads. 3. A pharmaceutical composition for the prevention and / or treatment of genitourinary infections, such as colonization of enterobacteria, containing a culture of lactic acid bacteria Lactobacillus plantarum and pharmaceutically acceptable carrier and / or diluent, characterized in that it contains biomass of lactic acid bacteria strain Lactobacillus plantarum DSM 11918 in an amount from 1 · 10 ⁴ to 10 ¹¹ CFU / ml. 4. The pharmaceutical composition according to claim 3, where the amount of Lactobacillus plantarum DSM 11918 is from 1 · 10 ⁵ to 10 ⁹ CFU / ml 5. The pharmaceutical composition according to claim 3, which is a suspension, spray, gel, cream, powder, capsule or vaginal insert. 6. The pharmaceutical composition according to claim 3, where the specified pharmaceutically acceptable carrier is skim milk. 7. An absorbent product, such as a feminine hygiene product, diaper, sanitary napkin, panty liners or incontinence pads, characterized in that it contains lactobacillus plantarum DSM 11918 biomass of bacteria in an amount of 1 · 10 ⁴ up to 10 ¹¹ CFU / ml. 8. The absorbent product according to claim 7, where the amount of Lactobacillus plantarum DSM 11918 is from 1 · 10 ⁵ to 10 ⁹ CFU / ml

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