KR100280366B1 - New strain having resistance to rifampicin and various antibiotics, enterococcus faescalis br2 - Google Patents

Abstract

The present invention relates to a novel variant, Enterococcus faecalis BR2 (Accession No. KFCC-11075), which is resistant to rifampicin and various antibiotics, and, unlike the parent strain of enterococcus faecalis BR2, It is resistant to various antibiotics at the same time and has the same characteristics as the strains of the parent strain, which is effective in the treatment of intestinal diseases, suppression and prevention of myoclosis in patients with anti-tuberculosis drugs or antibiotics. When added, it has an effect of preventing disease and improving meat quality.

Description

Enterococcus faecalis (JR2), a new variant resistant to rifampicin and various antibiotics {NEW STRAIN HAVING RESISTANCE TO RIFAMPICIN AND VARIOUS ANTIBIOTICS, ENTEROCOCCUS FAESCALIS BR2}

The present invention relates to a novel strain that is resistant to rifampicin and various antibiotics, Enterococcus faecalis BR2 lactic acid bacteria, and more specifically, to induce tolerance by mutating the strain of Enterococcus faecalis BIO, Korea The Enterococcus faecalis BR2 of Accession No. KFCC-11075 deposited December 17, 1998 with the spawn association.

For a long time, lactic acid bactericides have been used for the purpose of treating and preventing intestinal diseases, and several reports have been reported. For example, since reports of the treatment of lactic acid bacteria in infants with diarrhea have shown therapeutic effects (RO Clock, JAMA Vol. 61, P.164, 1913), 70% of patients suffering from diarrhea have been treated. (LM Gornpertz, et al., JAMA Vol. 80, P.90, 1923), treatment of diarrhea with lactic acid bacteria, and the concentration of lactic acid bacteria in the colon and its clinical results are closely related Lactic acid bacteria, such as LF Rettger, et al., Lactobacillus acidophilus and its therapeutic applications, New Haven, Yale University Press, 1935, have a direct and indirect nutritional effect on the intestine and promote the regeneration of intestinal mucosa. It is said to have a preventive effect.

On the other hand, in patients with long-term anti-tuberculosis drugs or antibiotics, various tuberculosis drugs, such as antibiotics or rifampicin, can destroy the balance of normal intestinal flora, causing intestinal diseases such as diarrhea due to poor absorption, indigestion and myoclosis. Many side effects can occur. Therefore, when taking anti-tuberculosis drugs or antibiotics, it is recommended to use a formal probiotic in combination to prevent these side effects.

Therefore, the conditions of formal probiotics used for this purpose should not be easily destroyed by anti-tuberculosis or antibiotics, such as rifampicin and various antibiotics.

The development of such resistant strains is described in Korean Patent Publication No. 98-5232, which discloses a novel strain that is resistant to rifampicin and fluoroquinolone antibiotics as a variant of Bifidobacterium bifidum . One variant strain is Bifidobacterium bifidum OFR9 (KCTC 8640P).

According to the patent publication, the growth of Shigella dysenteriae ( Shigella dysenteriae , ATCC 9752) in a medium in which B. bifidum OFR9 culture medium and BL-liquid medium are mixed to adjust the pH to 4.92 It has been shown to be suppressed.

Regarding the development of another resistant heterotypic strain, microbiol. Immunol. Vol. 21 (2), 85-97, 1997) induces the resistance of Enterococcus faecalis strains. The data reported that resistance was induced as follows.

The mutant strains according to the paper showed that the resistance to rifampicin was less than 11 μg / ml, as the inventors analyzed by themselves.

Therefore, resistance was not induced in rifampicin by induction of resistance using a successive transfer culture using the above technique.

In order to induce the resistance of rifampicin and various antibiotics, the present inventors continue to pass the passage by an extended passaging method to induce drug resistance, and the purely isolated bacteria were suspended in a buffer containing NTG for 30 minutes, followed by centrifugation. Mutation of bacteria Well, by separating the mutated effective bacteria from the medium containing rifampicin, it was confirmed that the interesting fact that it is possible to make a microorganism resistant to rifampicin and various antibiotics, and completed the present invention.

Accordingly, it is an object of the present invention to provide novel strains resistant to rifampicin and various antibiotics.

Still another object of the present invention is to provide a lactic acid bacteria preparation comprising Enterococcus faecalis BR2.

Still another object of the present invention is to provide an animal feed comprising Enterococcus faecalis BR2.

In order to achieve the above technical problem, the present invention is characterized by providing a new variant strain Enterococcus faecalis BR2 (KFCC-11075) resistant to rifampicin and various antibiotics.

Hereinafter, the present invention will be described in detail.

Isolation identification and resistance induction of the novel mutant strain E. faecalis BR2 according to the present invention were as follows.

The parent strain used in the present invention was separated from the Enterococcus faecalis BIO, a lactic acid bacterium developed by the applicant Boryeong Pharmaceutical Co., Ltd. for the production of lactic acid bacteria, and stored in 2% lactose agar medium.

After creating a liquid medium that contains a step by step by increasing the concentration of each antibiotic in an Erlenmeyer flask way, by incubation at a suitable temperature and inoculated with Enterococcus faecalis (E. faecalis) separate from above of the development of the bacteria stable maximum density vitro Lactobacillus was collected and purified purely. The purely isolated Enterococcus faecalis was continuously passaged by an extension passage to induce resistance.

At this time, 10-20 times in streptomycin-gradient plate, 20-30 times in chloramphenicol-gradient plate, 20-30 times in tetracycline-gradient plate, 10-20 times in erythromycin-gradient plate, aminobenzyl penicillin-gradient Plates were passaged 10-20 times to induce various antibiotic resistances.

The antibiotic-induced bacteria were separated from the pure water, suspended in a buffer containing NTG, centrifuged to collect the cells, washed in the same buffer, and resuspended. After 2 to 4 hours, rifampicin was purely separated from agar medium containing 50 to 400 μg, and the isolated bacteria were passaged 10 to 30 times in a rifampicin-gradient plate by an extended passaging method, and then separated and preserved.

The novel mutant lactic acid bacterium according to the present invention, which has been treated as described above, is named Enterococcus faecalis BR2 and deposited on December 17, 1998 at the Korea Bacterial Association Microorganism Conservation Center, Korea Patent Bacterial Depositary Organization, and deposited no. KFCC. 11075 was given.

The characteristics of Enterococcus faecalis BR2, a novel strain according to the present invention, and the parent strain Enterococcus faecalis BIO, a known strain, are summarized and summarized in Table 2 below.

The novel mutants according to the present invention have a strong resistance (360 μg / ml) to rifampicin, which has been treated with extend passage method and NTG, and 700 μg / ml for streptomycin, 100 μg / ml for chloramphenicol and 5 μg / ml for tetracycline. , 1,000 μg / ml for erythromycin, 100 μg / ml for aminobenzyl penicillin, 360 μg / ml for rifampicin, 150 μg / ml for penicillin G, 150 μg / ml for cephalexin, 30 μg / ml for gentamycin, 50 μg / ml for lincomycin It was confirmed that resistance of 2,500 μg / ml was induced to kanamycin.

In another aspect, the present invention is to provide a lactic acid bacterium preparation characterized by containing the novel Enterococcus faecalis BR2 (KFCC-11075). Gong.

The lactic acid bacterium containing the novel lactic acid bacterium Enterococcus faecalis BR2 according to the present invention is a variety of excipients prepared by separating and drying the Enterococcus faecalis BR2 according to the present invention by pure separation. It can be prepared in a conventional formulation such as a liquid or a tablet together with the perfume, various additives.

At this time, the dose may be added or decreased depending on the antibiotic dose, but it is usually preferable to administer about 30 ~ 150 mg 2-3 times a day based on the lactic acid bacteria.

Enterococcus faecalis BR2, a novel lactic acid bacterium according to the present invention, was found to be highly resistant to antibiotics as compared to conventional lactic acid bacteria as a result of animal experiments.

The present invention is also characterized by providing an animal feed characterized by containing the novel Enterococcus faecalis BR2.

Animal feed containing the novel Enterococcus faecalis BR2 according to the present invention is 0.01 to 1% by weight of Enterococcus faecalis BR2 lactic acid bacteria with respect to feed containing conventional antibiotics It can be prepared by mixing.

Animal feed containing E. faecalis BR2, a novel lactic acid bacterium, according to the present invention, has been found to have an effect of weight gain and quality improvement compared to pigs fed only feed containing conventional antibiotics. Appeared to be.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples Is provided to help the understanding of the present invention is not limited thereto.

Example 1: Strain Separation

Enterococcus faecalis BIO, a lactic acid bacterium developed by Boryung Pharmaceutical Co., Ltd. for the production of lactic acid bacteria, was isolated and purified, containing 1% meat extract, 1% peptone, 0.05% NaCl, 0.22% yeast extract, and 0.1% calcium carbonate. The plated in 2% lactose agar plate medium was incubated for 48 hours in a 37 ℃ incubator. Single colonies were isolated from the colonies of the resulting cultures and stored.

Example 2 Induction of Antibiotic Resistance

In an Erlenmeyer flask containing 425 ml of 2% lactose liquid medium prepared in advance to contain each antibiotic concentration stepwise, inoculated with Enterococcus faecalis BIO isolated in Example 1 at 37 ° C. The lactic acid bacteria were collected from the test tube of the highest concentration where the growth of the bacteria was stable for 3 days.

This strain was stored after isolation of resistant strains in 2% lactose agar medium containing 30 μg of aminobenzyl penicillin. Purely isolated Enterococcus faecalis was passaged continuously by extension passage to induce resistance.

20 times on streptomycin-gradient plate, 25 times on chloramphenicol-gradient plate, 30 times on tetracycline-gradient plate, erythromycin-gradient play Antibiotic resistance was induced by 16 passages in a rat and 10 passages in an aminobenzyl penicillin-gradient plate.

Example 3 Measurement of Minimum Growth Inhibition Concentration (MIC)

In Example 2, the lactic acid bacteria isolated purely were suspended in 2% lactose medium, prepared to have an absorbance of 0.3, and used as a microbial solution for measuring MIC (minimum growth inhibition concentration). First, antibiotic concentrations were diluted in steps so that the first test tube had the highest expected MIC concentration, and less than two dilutions. Stepwise diluted antibiotics are placed in pre-prepared 2% lactose agar medium (maintained at 45 ° C.) and poured into Petri dishes to harden. The prepared microbial solution was plated on a 2% lactose agar plate containing antibiotics by 0.1 ml, and the lactic acid bacteria which did not induce resistance as a control were also plated and compared. After 24 hours of incubation at 37 ° C., the concentration of antibiotics in the last plate without bacterial growth was defined as the minimum growth inhibition concentration (MIC). The results are shown in Table 3 below.

As shown in Table 3, the resistant strains induced in Example 2 were significantly increased resistance to various antibiotics than the parent strain, but rifampicin was found to decrease resistance.

Example 4 Rifampicin Resistant Mutant Isolation

The bacteria isolated from Example 2 were suspended in 0.1M sodium citrate buffer (pH 5.6) containing 150 μg / ml of NTG for 30 minutes, collected by centrifugation, and washed three times in the same buffer. After resuspension. After standing for 2 hours, 2% lactose agar medium containing 75 μg of rifampicin was isolated purely, and the separated bacteria were passaged 20 times on a rifampicin-gradient plate using an extension method, and then separated, and rifampicin 75 μg, aminobenzyl penicillin It was stored in a medium containing 30 μg.

The mutant strains obtained above and the mutant strains in which only antibiotics were extended by culturing without antibiotics were measured in the same manner as in Example 3, respectively. As a result, as shown in Table 4, mutants treated with NTG showed a 33-fold increase in rifampicin resistance than mutants not treated with NTG, and resistance to other antibiotics was also significantly increased.

Example 5 Preparation of Lactobacillus

After collecting the bacteria by physiological saline to the test tube cultured by the same method as in Example 4, the purely isolated strain cultured, 180 g of skim milk powder, 820 ml of purified water and 0.66 g of L-cysteine hydrochloride (pH 7.0) Place in a 250 ml Erlenmeyer flask containing the composition-containing medium and incubate at 37 ° C. for 72 hours.

The culture solution is added to an anaerobic culture tank containing 250 l of medium, mixed well, incubated at 37 ° C. for 48 hours, and then spray dried. The strain was at least 1 × ^{10 9} / g.

Example 6 Preparation of Lactic Acid Bacteria Formulations

6 g (lactic acid number 1x10 ⁹ / g) of lactic acid powder prepared in Example 5 was added 0.1 g of sodium saccharin and 2.5 g of hydroxypropyl cellulose, and then put an appropriate amount of corn starch and flavoring (lemon milk) to make 100 g. Lactobacillus formulations according to the invention were prepared by mixing.

As a result of administering 1 g of the lactic acid bacterium preparation prepared in Example 6 in combination with various antibiotics three times a day through an animal experiment using mice, the total number of resistant strains was similar to the number of strains in the normal mouse group, and slightly increased during the administration period. Also appeared. In addition, a control test of commercially available lactic acid bacteria formulations showed that the number of enteric lactic acid strains was increased.

Example 7 Preparation of Animal Feed

The lactobacillus prepared in Example 5 was added to the feed to which the existing antibiotic was added, and the experiment was performed for about 3 months. As an antibiotic, the amount of tetracycline 2.0 mg / lactic acid powder 1g / kg of feed, rifampicin 0.36 mg / lactic acid powder 1g / kg of food, kanamycin 2.5 mg / lactic acid powder 1g / kg of feed was used. For the test subjects, 74 pigs were divided into the control group and the test group, and then the average weight gain for three months, the daily weight gain, the feed efficiency (total weight / feed consumption), and the decrease in fat thickness were measured. Table 5 shows.

From the above results, enterococcus faecalis BR2, a novel lactic acid bacterium according to the present invention, was added to feed to prevent enteric disease that may occur when only antibiotics were added to the feed. This seems to show the effect of weight gain and meat quality of the animal.

Enterococcus faecalis BR2, a novel mutant lactic acid strain according to the present invention as described above, is a lactic acid strain having strong resistance to rifampicin and various antibiotics. By co-administration, it is possible to prevent side effects such as suppressing harmful bacteria in the intestine caused by lactic acid bacteria and diarrhea due to myoclosis, while maintaining the efficacy of anti-tuberculosis drugs and antibiotics. In addition, it is possible to obtain an immune enhancing effect expected as an effect of the lactic acid bacteria preparation.

In addition, when the novel mutant lactobacillus according to the present invention is added to the feed, the antibiotics added in the conventional feed prevents the meat from deteriorating and the meat quality deteriorates, thereby preventing the myoclosis, gaining the weight, and improving the meat quality. Is also expected.

Claims (3)

Enterococcus faecalis BR2 (KFCC-11075). A lactic acid bacterium preparation comprising Enterococcus faecalis BR2 (KFCC-11075). Animal feed comprising Enterococcus faecalis BR2 (KFCC-11075).