JP2001270830A - Urinary tract infection prevention / treatment agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a preventive and therapeutic agent for urinary tract infection having an excellent preventive and therapeutic effect on infections in the urinary tract. SOLUTION: The preventive / therapeutic agent for urinary tract infection containing Lactobacillus casei as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a preventive and therapeutic agent for urinary tract infection used in the prevention and treatment of infectious diseases in the urinary tract, in particular, complicated urinary tract infections.

[0002]

BACKGROUND OF THE INVENTION Urinary tract bacterial infections are chronic infections caused by the attachment and growth of bacteria to the urinary tract. Urinary tract infections are classified into simple urinary tract infections in which there is no obvious underlying disease or abnormality in the urinary tract, and complex urinary tract infections in which there is an underlying urinary tract infection, which causes infection. You. Until around 1950, Staphylococcus genus was the main causative organism of urinary tract infections. However, the recent spread of antibiotics in the clinic has reduced the number of gram-positive bacteria, and is now Escherichia coli.
oli) (hereinafter E. coli). Pseudomonas aeruginosa (Pseudomona
s aeruginosa), Candida albi
Cases caused by opportunistic infection-causing bacteria (drug-resistant bacteria) such as cans) are also increasing.

[0003] The infection kinetics of causative bacteria in urinary tract infections are as follows:
It is divided into luminal (ascending urinary) and endogenous (hematogenic), the majority of which is ascending urinary. When the causative bacteria adheres to the external urethral opening: 1. It rises along the urethral wall with the power of the bacteria itself. Multiply the urethral bladder reflux that occurs at the end of urination,
3. It reaches the bladder by friction of the urethra, etc. Therefore, the bacteria attach to the bladder epithelial mucin layer and epithelial cells and multiply, and the infection worsens into the ureter and renal pelvis. However, in a healthy person, even if the causative bacteria attach to the external urethral orifice and further ascend to the bladder and adhere to the epithelium, then 1. 1. bladder epithelial mucin layer and red blood cell aggregates; 2. infected cells; 3. Polynuclear leukocytes that have phagocytosed the causative organism; It is eliminated from the body by urination in a state of being coated with urine trace substances (receptor substances such as fibronectin, IgA, etc.).

[0004] Therefore, in patients with urinary tract infection, underlying diseases or conditions lacking the above-mentioned exclusion mechanism have been recognized as prerequisite factors for infection. This includes systemic factors such as metabolic disorders, innate immunodeficiency, administration of immunosuppressants, radiation and local factors such as the urinary tract (the pelvic-ureteral junction, the ureter bladder junction, the bladder neck, Urethra) stenosis, stones,
Trauma, catheters, tumors and the like. In particular, it is considered that the latter local factor often promotes infection by the causative bacteria.

[0005] As an animal model of urinary tract infection, a model in which E. coli is transurethrally injected into a mouse bladder by operating a catheter has been reported. However, the method of transmitting E. coli to animals with normal urinary tract is established due to the pathogenicity of the causative bacteria rather than the underlying disease of the host, which is regarded as a clinical problem Such a model can be used as a model for acute urinary tract infection, but cannot be used as an evaluation model for complicated (chronic) urinary tract infection.

For this reason, a method capable of evaluating the effect on complicated (chronic) urinary tract infections is desired, and it is possible to find an effective means for the prevention and treatment of complicated (chronic) urinary tract infections. Is desired.

[0007] Antibiotics are generally administered for urinary tract infections, but the use of antibiotics exerts a good eradication effect on the target pathogenic bacteria, but on the other hand, It is feared that secondary infection caused by bacteria resistant to the antibiotics provided in the above may occur.

On the other hand, recently, the effect of lactic acid bacteria on urinary tract infection has also been reported. For example, Hilton, E., Rind
os, P and Isenberg, HD (1995) .Lactobacillus GG
vaginal Suppositories and Vaginitis. J. Clin. Micr
obiol. 33, 1443. and Bruce, AW and Reid, G. (1
988) .Intravaginal Instillation of lactobacillifor
Prevention of Recurrent Urinary Tract Infection.
In Can. J. Microbiol. 34, 339-343.
Attempts to prevent urinary tract infections by injecting lactic acid bacteria into the vagina have been reported. Reid, G., Chan, R.
CY, Bruce, AW and Costerton, JW (1985) .Preven
tion of Urinary Tract Infection inRats with an Ind
igenous Lactobacillus casei strain.Can.J.Microb
iol. 34, 339-343. report that urinary tract infection could be prevented in animal models by injecting lactic acid bacteria into the bladder. However, this does not include: 1. a complex urinary tract infection model that mimics the underlying disease of the urinary tract is not used; 2. detailed data on the protective effects of the infection are not shown; 3. other lactic acid bacteria And the effect is not compared.

[0009]

Under such circumstances, it has been desired to develop a safer and more effective therapeutic agent for urinary tract infections, particularly for complicated urinary tract infections, with a high therapeutic effect.

[0010]

Means for Solving the Problems The present inventors have developed a highly accurate method for evaluating urinary tract-infected mice by inducing chronic local urinary tract infection by chemically treating the inside of the mouse bladder. When Lactobacillus casei, especially Lactobacillus casei strain YIT9029, has been shown to be effective in preventing and treating urinary tract infections, especially for complicated urinary tract infections. In addition, they have found that they are excellent in safety and recoverability, and completed the present invention.

[0011] That is, the invention according to claim 1 of the present invention relates to a preventive and therapeutic agent for urinary tract infection containing Lactobacillus casei as an active ingredient.

Further, the invention according to claim 2 of the present invention is characterized in that Lactobacillus casei is Lactobacillus casei YIT9.
The present invention relates to the agent for preventing or treating urinary tract infection according to claim 1, which is a 029 strain.

[0013] The invention according to claim 3 of the present invention relates to the agent for preventing or treating urinary tract infection according to claim 1 or 2, wherein Lactobacillus casei is contained as a viable bacterium. .

Further, the invention according to claim 4 of the present invention relates to the agent for preventing or treating urinary tract infection according to claims 1 to 3, wherein the urinary tract infection is complicated urinary tract infection. is there.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Lactobacillus casei contained as an active ingredient of the prophylactic / therapeutic agent for urinary tract infection of the present invention comprises:
Lactobacillus belonging to the genus Lactobacillus. In the present invention, various strains classified as Lactobacillus casei can be effectively used, and in particular, Lactobacillus casei YIT902
By using 9 strains (FERM BP-1366), a particularly excellent effect of preventing and treating urinary tract infection can be obtained.

The prophylactic / therapeutic agent for urinary tract infection of the present invention must contain the above-mentioned Lactobacillus casei as an active ingredient. However, it does not prevent the other ingredients from being contained. Auxiliaries and the like may be added.
The Lactobacillus casei may be contained in either a live or dead state, but is preferably contained in a live state.

The dosage of the agent for preventing or treating urinary tract infection of the present invention varies depending on the administration method, the age, weight and condition of the patient, but is 100 mg to 10 g per day for an adult patient. The degree can be administered directly or orally to the affected area.

[0018]

【Example】Creation of a chronic urinary tract infection model  Animals: Female C3H / HeN (SLC) SPF female, 8 weeks old, per group
6 animals were used. Rearing is performed with MF feed and water ad libitum.
Was. Preparation of infectious bacterial solution: Dispensed from the Department of Urology, Nagoya University
E., a urine isolate from a patient with a severe urinary tract infection coli YU strain
Used as infectious bacteria. This strain makes contact with bladder epithelial cells.
Type reported to be involved in urinary tract infection
I have I-fimbriae and P-fimbriae
I checked it in advance. E. coli in tryptoose liquid medium
(Difco) at 37 ° C. for 14 hours with shaking. Cultured cells
Wash 3 times with sterile saline at 4 ℃, 3,500 rpm, 5 minutes
After purification, 5.0 × 107 (Colony Formation Units / ml, below CF
U / ml) (hereinafter referred to as E. coli bacterial solution).

Preparation of urinary tract infection model: After intravenously indwelling catheter (BECTON DICKINSON) was inserted into the bladder of a mouse under Nembutal anesthesia, 100 μl of 0.1N-HCl (Wako Pure Chemical) was injected. . Then after 45 seconds 0.1N-KOH
Immediately after injecting 100 μl of (Wako Pure Chemical), PBS pH 7.
The inside of the bladder was washed by injecting 200 μl of 2. Twenty-four hours after washing, 20 μl of the above E. coli bacterial solution was injected into the bladder.

Test Example 1 (Pre-addition of Lactobacillus) Preparation of Lactobacillus Administered Bacterial Solution: Lactobacillus casei Y
IT9029 (YIT9029), Lactobacillus fermentum YIT0159 (YIT0159) or Lactobacillus plantarum YIT0102 (YIT0102)
The cells were cultured in an RS liquid medium (Difco) at 37 ° C. for 48 hours with carbon dioxide gas. The cultured cells were centrifuged and washed three times with sterile physiological saline at 4 ° C., 3,500 rpm for 5 minutes, and adjusted to 5.0 × 10 ⁹ CFU / ml.
Further, an administration bacterial solution of 5.0 × 10 ¹⁰ or 5.0 × 10 ⁸ (CFU / ml) was prepared.

Measurement of protective effect by animal model: 15 minutes after the chemical treatment, 20 μl of the bacterial solution of lactobacillus was injected into the bladder.
Injected. 24 hours after injection, E. coli bacteria solution is placed in the bladder
A 20 μl injection was performed, followed by dissection at 15 minutes, 1, 3, and 7 days, and the bladder and kidney were aseptically harvested and suspended in 5 ml of sterile saline. The number of E. coli and Lactobacillus in each organ suspension stirred and crushed with a Teflon homogenizer was confirmed using DHL and MRS plate agar medium (Difco), respectively. The results are shown in FIGS. 1, 2 and 3.

As shown in FIG. 1, chronic infection with E. coli occurred in the bladder from day 1 to day 7 after infection in the control group. In contrast, in the YIT9029-administered group, the number of infected bacteria was 1/100, 1/100, and 1/1000 on the 1, 4, and 7 days of infection, respectively, compared to the control group. In the kidney, the number of infected bacteria was less than 1/10 in the YIT9029 strain administration group compared to the control group. This confirmed that intravesical injection of the YIT9029 strain had a significant protective effect against chronic urinary tract infection of E. coli.

As shown in FIG. 2, the number of infected bacteria was 1/100 in the group administered with the YIT9029 strain as compared with the control group, and a significant protective effect against infection was observed. On the other hand, a decrease in the number of infectious bacteria was also observed in the group administered with the YIT0159 strain, but this was not a significant effect. In the YIT0102 strain administration group, no decrease in the number of infectious bacteria was observed. From this fact, the protective effect of lactobacilli was different between the strains (species), and it was confirmed that the YIT9029 strain had a particularly strong protective effect.

As shown in FIG. 3, 10 ⁸ and 10 ⁹ (CFU / mous
In the group that was administered YIT9029 shares of e), infection 4 day infection the number of bacteria is to 1/1000 of the control group, but significant protective effect against infections were observed, is a ^10-7-treated group was weakened protective effect . On the other hand, in the YIT0159-administered group, the number of infected bacteria was slightly reduced when any of the numbers of bacteria was administered, and no significant protective effect was observed. It was confirmed that the protective effect of the YIT9029 strain was affected by the number of bacteria administered. From the results shown in FIG. 1, FIG. 2 and FIG. 3, the system to which the Lactobacillus casei YIT9029 strain was administered had a clear protective effect against infection.

[0025]Test example 2  15 minutes after the chemical treatment, the YIT9029 strain was placed in the bladder.
20 μl was injected. 24 hours after injection Urinary tract infection
E. coli YU strain (Type I-fimbriae / P
-fimbriae; + / +) and SK strains (+/-), or
Inject 20 µl of the bacterial solution of a heron fecal isolate (-/-) into the bladder
did. On day 4 after injection, dissection was performed to eliminate E. coli in the bladder.
 For the number of YIT9029 strains, DHL and LLV
It was confirmed using a plate agar medium. Table 1 shows the results.

[0026]

【table 1】

[0027] In the control group the infection day 1, E. coli YU strains detected from the bladder, infections number of bacteria SK strains, and rabbit feces isolates, respectively ^{10 6 (CFU / Bladder),} 10 5, and
10 was ^five . In contrast, in the YIT9029 strain administration group, the number of infectious bacteria was 1/100 or less for all infectious bacteria. Regardless of the presence or absence of pili involved in adhesion to the urinary tract, a significant protective effect was observed against any infectious bacteria. No difference was found in the number of recovered lactobacilli among the infected bacteria. In the system to which Lactobacillus casei YIT9029 was administered, an excellent protective effect against various Escherichia coli having different pathogenicity was observed.

Test Example 3 (Addition after lactobacillus) 24 hours after the chemical treatment, E. coli bacterial solution was injected into the bladder.
20 μl was injected. 24 hours after injection, 1 day of YIT9029 strain solution
One injection, 20 μl, was injected into the bladder a total of 11 times. Dissected 12 days after infection with E.coli, E.c in bladder and kidney
oli and YIT9029 strains for DHL and DIT respectively
It was confirmed using an LLV plate agar medium. Table 2 shows the results.

[0029]

[Table 2]

In the control group, infectious bacteria were detected in the bladder in 6 out of 7 cases, and the number of infected bacteria in the detected individual was 10 ⁶ (CF
U / Bladder) level. In contrast, in the YIT9029 strain-administered group, only 2 out of 8 cases showed infectious bacteria and the number of bacteria was 1
0 was at ^4th level. Lactobacillus casei after urinary tract infection
When YIT9029 strain was injected into the bladder, a clear therapeutic effect was observed.

Test Example 4 (Urine leukocyte count and urine Myelope
Influence on roxidase (MPO) activity 15 minutes after the chemical treatment, the YIT9029 strain was introduced into the bladder.
20 μl was injected. Twenty-four hours after the injection, 20 μl of the bacterial solution of the E. coli YU strain was injected into the bladder. 1, 4 and 7 after injection
On the day, urine was collected and the number of leukocytes in the urine was measured using a hemocytometer. In addition, 100 μl of the collected urine and extraction buffer (Hexadecyltrimethylammonium bromide, 10 g / l; KH ₂ PO
₄ , 6.82 g / l; pH 6.0) was mixed and sonicated for 10 minutes in ice. After two freeze-thaw cycles, 4
50 µl of the supernatant centrifuged at 15,000 rpm for 10 minutes at ℃, and the reaction solution (O-dianisidine dihydrochloride, 167 mg / l; K
H ₂ PO ₄ , 6.82 g / l; Hydrogen peroxide, 40μl / l; pH
After mixing 150 μl of 6.0), the mixture was reacted at 25 ° C. for 5 minutes. Thereafter, 50 μl of 1N HCl was added to terminate the reaction, and the absorbance at 405 nm was measured. MPO activity in urine samples was derived from changes in absorbance of MPO solutions exhibiting known enzyme activity. FIG. 4 shows the results.

No increase in urine leukocyte count was observed by chemical treatment alone. One day after infection with E. coli, the control group showed a marked increase in white blood cell count, whereas YIT
Leukocyte increase was slight in the 9029 strain administration group. Infection 4-7
On the day, the white blood cell count of the control group was maintained high,
The leukocyte count in the YIT9029 strain administration group decreased.

The chemical treatment alone did not increase the urinary MPO activity. On the first day after E. coli infection, the control group showed an increase in MPO activity, while the YIT9029 strain administration group
The increase in MPO activity was slight. On days 4-7 of infection,
While the MPO activity was maintained at a high level, no change was observed in the MPO activity of the YIT9029 strain administration group. Therefore, when Lactobacillus casei strain YIT9029 is injected into the bladder, the increase in urinary leukocytes caused by infection with E. coli,
It was confirmed that the increase in MPO activity was suppressed.

[0034]

EFFECTS OF THE INVENTION According to the preventive and therapeutic agent for urinary tract infection of the present invention containing Lactobacillus casei as an active ingredient, excellent preventive and therapeutic effects on urinary tract infection can be obtained. In addition, Lactobacillus casei, which is an active ingredient of the agent for preventing or treating urinary tract infection of the present invention, is a strain used for lactic acid bacteria beverages and the like, and thus has no problem in safety at all. Furthermore, the Lactobacillus casei of the present invention, especially the Lactobacillus casei strain YIT9029, can be expected to have an excellent therapeutic effect on urinary tract infections caused by drug-resistant bacteria when used in combination with antibiotics.

[Brief description of the drawings]

FIG. 1 shows that casei YIT9029 strain has a protective effect against chronic urinary tract infection

FIG. 2 is a graph showing a comparison between the casei YIT9029 strain and other Lactobacillus infection-protecting effects.

FIG. 3 is a graph showing the effect of the number of bacteria administered to the casei YIT9029 strain on the protective effect against infection.

[FIG. 4] Administration of casei YIT9029 strain showed urinary leukocyte count and
Diagram showing the effect on MPO activity

Continued on the front page (72) Inventor Tomomi Suzuki 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (72) Inventor Masaaki Watanuki 1-1-1-19, Higashi-Shimbashi, Minato-ku, Tokyo Stock Company F-term of Yakult Honsha (reference) 4B065 AA26X AA30X AA93X BC07 BC50 CA44 4C087 AA01 AA02 BC57 NA05 NA14 ZA82 ZB35 ZC75

Claims (4)

[Claims] 1. A preventive and therapeutic agent for urinary tract infection comprising Lactobacillus casei as an active ingredient. 2. The preventive and therapeutic agent for urinary tract infection according to claim 1, wherein the Lactobacillus casei is Lactobacillus casei YIT9029 strain. 3. The preventive and therapeutic agent for urinary tract infection according to claim 1, wherein Lactobacillus casei is contained as a viable bacterium. 4. The preventive and therapeutic agent for urinary tract infection according to claim 1, wherein the urinary tract infection is complicated urinary tract infection.