KR101057053B1 - Culture medium for promoting the growth of Mycobacterium tuberculosis and culture method using the same - Google Patents

Abstract

The present invention relates to a medium for culturing Mycobacterium tuberculosis, and a method for producing the same, and a method for culturing Mycobacterium tuberculosis using the same. More specifically, Mycobacterium tuberculosis culture medium of the present invention is a tuberculosis cell wall component obtained from a mass of Mycobacterium tuberculosis cells and Including lysozyme, it is to promote the growth of Mycobacterium tuberculosis. The culture medium of the present invention promotes the culture of Mycobacterium tuberculosis, thereby culturing Mycobacterium tuberculosis more quickly, thereby enabling a rapid diagnosis of tuberculosis infection, thereby facilitating the treatment of tuberculosis quickly and by selecting carriers. It is effective to prevent the transmission.

Medium, Mycobacterium tuberculosis, Lysozyme, Mycobacterium

Description

Culture medium for promoting the growth of Mycobacterium tuberculosis and culture method using the same {Media for M. tuberculosis -growth enhancer and the Cultivation Method

The present invention relates to a Mycobacterium tuberculosis culture medium for promoting the growth of Mycobacterium tuberculosis and a method for culturing Mycobacterium tuberculosis using the same. It relates to a culture method used.

Infectious diseases of pathogenic mycobacteria, including Mycobacterium tuberculosis and leprosy, have been the most serious bacterial diseases to date. Tuberculosis caused by Mycobacterium tuberculosis alone Tuberculosis is one of the world's top ten causes of death, and it is the most infectious disease of international importance. Today, about two billion people, one third of the world's population, are infected with it. Korea ranks first among the 30 OECD member countries with tuberculosis mortality, killing 5.5 per 100,000 population (average 7.4 deaths per day) (2005 death statistics), 3.9 times higher than Japan and 23.3 times higher than the US. Among the infectious diseases of pathogenic mycobacterium, tuberculosis accounts for more than 80% of the domestic infectious diseases, and the single disease is so serious that it is within the fifth of domestic death factors.

Although the mortality rate from tuberculosis has decreased significantly as a result of the National Tuberculosis Management Program over the past 50 years, the number of new patients has slowed down in the 2000s, and the number of tuberculosis patients has been increasing gradually over the past three years. These reasons need to be considered in many aspects, but due to antibiotic-dependent treatments, the spread of MDR-TB, increased immunosuppressive diseases and treatments (e.g., organ transplantation), rapid growth of the elderly population and the influx of foreigners, and international travel Tuberculosis can be a social problem at any time. Moreover, because global warming is expected to change the pattern of infectious diseases, mycobacterial infections, including tuberculosis in Africa, Southeast Asia, and Brazil where the climate is widespread in the (sub) tropical region, need to be recognized and socially prepared. .

Until now, the method to confirm tuberculosis is to identify M. tuberculosis by sputum and other samples, culture and identify by PCR method. M. tuberculosis, however, takes about 4 weeks to incubate even under good conditions. In latent TB, TB bacteria are very difficult to culture in vitro. Although broth medium supplemented with OADC as 7H9 and supplement developed by BD company is widely used for culturing mycobacterium tuberculosis, this medium is expensive and takes more than 4 weeks even if the medium is used. The country also relies on imports of these badges. Recently developed BD company's Mycobacterial Growth Indicator Tube (MGIT) is an automated system that promotes the cultivation of Mycobacterium tuberculosis clinically because the sensitivity of the sensor is higher than that of the medium. In addition, the MGIT system is based on the 7H9 broth and is very expensive, so there is a limit to apply it to all clinical trials.

However, despite the desperate need for a medium-adding component or a new combination of media that can rapidly cultivate Mycobacterium tuberculosis, no media has been developed so far to specifically promote the growth of Mycobacterium tuberculosis using its components. .

In this regard, the researchers found that the cell wall components of Mycobacterium tuberculosis are very different from those of ordinary bacteria, and that carbohydrates are absolutely necessary as a proliferation energy source for the media components. Using a lysozyme that is structurally similar to the resuscitation-promoting factor related to the rearrangement of the cell wall in the growth of Mycobacterium tuberculosis, it develops a media component that specifically promotes the growth of Mycobacterium tuberculosis, and cultures containing such media component The present invention was completed by confirming that the growth of Mycobacterium tuberculosis in the culture of the Mycobacterium tuberculosis bacteria was much better than that of the general media.

The present invention has been made to solve the above problems, the present invention is a tuberculosis cell wall component extracted from the Mycobacterium tuberculosis cells obtained by mass culturing Mycobacterium tuberculosis, and lysozyme that is structurally similar to the resuscitation-promoting factor and has a hydrolytic effect It is to provide a medium component that can promote the culture of Mycobacterium tuberculosis. In another aspect, the present invention is to provide a method for culturing Mycobacterium tuberculosis more quickly and simply by culturing Mycobacterium tuberculosis in a culture medium containing a medium component that promotes the culture of Mycobacterium tuberculosis.

In order to achieve the above object, the present invention provides Mycobacterium tuberculosis culture medium and its preparation method, and a method for culturing Mycobacterium tuberculosis using the same. More specifically, the present invention relates to a culture medium that promotes the growth of Mycobacterium tuberculosis by incorporating Mycobacterium tuberculosis cell wall components and lysozyme obtained from mass cultured Mycobacterium tuberculosis cells.

Hereinafter, the present invention will be described in detail.

According to the first aspect of the present invention, the medium for promoting the culture of Mycobacterium tuberculosis according to the present invention is characterized in that it comprises the Mycobacterium tuberculosis cell wall component obtained from the cells of mass cultured Mycobacterium tuberculosis. Preferably, the lysozyme further comprises.

Preferably, in the present invention, the cell wall component may include carbohydrates and further include lipids or lipoproteins. Mycobacterium tuberculosis has a very thick layer of lipids and carbohydrates on the cell wall, compared to other common bacteria, and metabolism of these lipids and carbohydrates (eg LAM, peptidoglycan). The process consumes more energy. In addition, the cell wall components of each mycobacteria are known to be different according to their species, which means that the cell wall components produced by each mycobacteria are different. In particular, the genes involved in the production and metabolism of fatty acids involved in cell wall production are more than four times more genes involved than E. coli , which usually replicates rapidly. In addition, the lipid may include, but is not limited to, for example, phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, and postidyl. Choline (phosphotidylcholine) or phophotidylethanolamine, tuberculostearic acid, arachidonic acid, C18-C31 fatty acids with or without double bonds and with or without double bonds Phospholipids or components of phospholipids such as C18-C18 fatty acid esters. The lipid protein includes a peptidoglycan constituting a cell wall of a bacterium, and the carbohydrate includes carbon and hydroxyl groups.

In the present invention, preferably, the concentration of the cell wall component is 50 to 100 μg / ml. The culture of Mycobacterium tuberculosis is promoted depending on the cell wall component of Mycobacterium tuberculosis. The concentration of the cell wall fraction of Mycobacterium tuberculosis is promoted in a concentration-dependent manner up to 100 μg / ml but less than 100 μg / ml. Accordingly, when diagnosing the infection of tuberculosis, it is preferable to carry out within the above range because the sensitivity to 50-100 μg / ml is high.

In the present invention, more preferably, the concentration of the lysozyme is characterized in that 10 ~ 100 μg / ml.

In general, the 7H9 broth medium, which is used for the cultivation of Mycobacterium tuberculosis, consists mainly of inorganic compounds and carbohydrates, and still promotes or selectively cultures the growth of Mycobacterium tubercurosis and slow-growing Mycobacteria. No medium has been developed. Recent reports indicate that mycobacterium tubercurosis is involved in dormant (a resting or non-replicating phase) in a stressed environment and resuscitation, an anti-dormancy factor, to regrow in the resting phase. It is known that a -promoting factor (Rpf) is required and that the rearrangement of peptidoglycans should be preceded. Rpf has a structural domain very similar to lysozyme and is known to degrade peptidoglycans. This fact suggests that hydrolysis of peptidoglycan is required for the reactivation of resting tuberculosis bacteria, and this mechanism alters the mechanical properties and structure of the cell wall, thereby signaling the anti-restor signal by the differentiation or degradation products of the cells. It is estimated that the tuberculosis bacteria are prevented from reactivating or falling into the resting phase.

Therefore, the lysozyme added in the present invention is to prevent the tuberculosis bacteria from being in the resting phase of the tuberculosis bacteria to be better cultured. When the concentration of the lysozyme is 10 μg / ml or less, the hydrolysis of the peptidoglycan does not occur, and if it is 100 μg / ml or more, it is preferable to add lysozyme within the above range because the cell wall is excessively degraded.

In the present invention, the Mycobacterium is characterized by belonging to the Mycobacterium Species (Mycobacterium sp.). The tuberculosis bacterium is a bacterium that causes tuberculosis in humans, and the tuberculosis bacterium is not limited thereto but is collected from sputum, blood, tissues, and the like. The Mycobacterium tuberculosis is not limited thereto, but more preferably, the Mycobacterium spp. May include Mycobacterium tubercurosis , Mycobacterium paratuberosis , Mycobacterium reprae , and non-tuberculosis mycobacterium. It may be selected from the group consisting of (NTM: Nontuberculous mycobacterium) or atypical Mycobacterium other than tuberculosis (MOTT).

According to the second aspect of the present invention,

The present invention relates to a method for culturing Mycobacterium tuberculosis in the culture medium of Mycobacterium tuberculosis described above. Cultivation of Mycobacterium tuberculosis is promoted by culturing in the culture medium, and the present invention can cultivate Mycobacterium tuberculosis within a period of 10 days to 20 days, whereas the normal tuberculosis culture period is 30 days.

Cell wall fractions or cell wall fractions and lysozyme can be treated from the cells of Mycobacterium tuberculosis cultured in a general medium in order to prepare the medium of the present invention, but the general medium suitable in the present invention is not limited thereto. Egg-based media such as 7H9, 7H10, 7H11, 7H12B, Sauton's medium, Dubos medium, mWR medium or Lowenstein-Jensen medium.

When culturing tuberculosis bacteria according to the present invention as described above, it is possible to cultivate tuberculosis bacteria more quickly by culturing the tuberculosis bacteria, and to make early diagnosis of tuberculosis infection and to quickly and easily treat tuberculosis. In addition, the spread of tuberculosis can be prevented by screening carriers.

On the other hand, there is a method of diagnosing tuberculosis infection by diagnosing tuberculosis by a doctor's clinical judgment that combines chest radiography or other evidence, even if tuberculosis bacteria are not identified in the lesion. There are antimicrobial staining, antimicrobial culture and identification, molecular biological methods, immunological methods, and drug sensitivity test methods. Therefore, in the medium of the present invention, tuberculosis bacteria isolated from samples such as sputum, blood, tissue, and the like can be cultured and identified by PCR to diagnose tuberculosis infection. In addition, it is possible to diagnose whether tuberculosis is infected using a diagnostic kit containing a medium for promoting the culture of Mycobacterium tuberculosis. As a method for detecting tuberculosis infection by the kit, tuberculosis bacteria cultured from the medium, which are known in the art, may be detected by staining, immunological methods, and drug sensitivity test methods. The diagnostic kit includes a culture medium for promoting the culture of Mycobacterium tuberculosis, thereby facilitating the cultivation of Mycobacterium tuberculosis and shortening the confirmation time for the infection of Mycobacterium tuberculosis.

According to the third aspect of the present invention,

The present invention relates to a method for producing a culture medium of Mycobacterium tuberculosis described above. The method according to the third aspect is a) step of culturing the tuberculosis bacteria to produce an initial culture solution, step b) of collecting the tuberculosis cells in the initial culture medium to obtain cell wall fractions from the tuberculosis cells, separating the cell wall components from the cell wall fractions C) and d) adding the cell wall component and the lysozyme to the general medium.

In one embodiment of the present invention, in order to pulverize the collected tuberculosis cells and to better isolate the components constituting the tuberculosis cells, the cell wall fractions were obtained by autoclaving the collected tuberculosis cells and centrifugation after ultrasonic grinding, and the cell wall The components were separated by resuspending the cell wall fractions, ultrasonically pulverized and resuspended to recover the suspended matter. In order to more efficiently obtain carbohydrates and lipids or lipoprotein components from the cell wall components, the cell wall fractions are resuspended to recover the suspended matter.

In the present invention, preferably the concentration of the cell wall component is characterized in that 50 ~ 100 μg / ml, the concentration of the lysozyme is characterized in that 10 ~ 100 μg / ml.

As described above, the Mycobacterium tuberculosis culture medium of the present invention as described above includes fractions containing the cell wall components of Mycobacterium tuberculosis bacteria obtained from the cells of mass cultured Mycobacterium tuberculosis. Through this, it is possible to promptly diagnose whether the tuberculosis is infected and the drug susceptibility test to facilitate the treatment of tuberculosis and prevent tuberculosis.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are intended to better understand the present invention and are not intended to limit the invention thereby.

< Example  1-Initial culture of Mycobacterium tuberculosis: Preparation of single cell suspension >

5 ml of 7H9-OADC by picking-up a single colony of Mycobacterium tuberculosis ( M. tuberculosis H37Rv, ATCC 27294) and K strain ( M. tuberculosis K-strain, Korean epidemic strain, Yonsei University College of Medicine) on 7H10-OADC agar plate The broth was inoculated and incubated at 37 ° C. for 30 days. Each cultured strain was centrifuged at 8,000 rpm for 30 minutes to collect only bacterial pellets, which were inoculated again in 100 ml of 7H9-OADC and incubated at 37 ° C for 30 days. The culture was removed in the same manner as above and washed three times with 10 mM PBS (pH 7.4) to collect only pellets. The collected pellets were homogenized into single bacterial cells, filtered through a 1.2 μm syringe filter and observed under a microscope to confirm the presence of more than 90% of single cells. The initial culture (Seedlot) of the tuberculosis bacteria thus made was dispensed 1ml each and stored at -80 ℃. Other mycobacterium also prepared the initial culture strain by the above method.

< Example  2-Mass culture and collection of cell wall fractions of Mycobacterium tuberculosis>

The conserved single cells of Mycobacterium tuberculosis produced by the method described in Example 1 were diffused into 7H10-OADC agar to perform colony counting. 100 μl of 10 ⁸ CFU / mL Mycobacterium tuberculosis was inoculated into a flask containing 35 ml of mWR (pH 6.0) broth medium (1 L at a time, approximately 33 flasks) and surface standing culture at 37 ° C. do. After incubation at 37 ° C. for about a month, centrifuge at 10,000 × g to collect only the cells. The collected cells are washed three times with 10 mM PBS (pH 7.4). About 10 g of cells are dispensed into 50 mL conical tubes and autoclaving at 121 ° C for 10 minutes to kill the cells. 10 g of cells were resuspended in 10 ml of 10 mM PBS (pH 7.4) and subjected to ultrasonic grinding (45% power, 2 min 30 sec, 6 times). After ultrasonic grinding, only the supernatant was collected by centrifugation at 3,000 rpm, which was again centrifuged at 27,000 xg for 30 minutes to collect only pellets. This pellet fraction is a cell wall component of Mycobacterium tuberculosis and contains a large amount of carbohydrates and lipids or lipoproteins.

The collected pellets were resuspended in 10 ml of 10 mM PBS (pH 7.4) and ultrasonically pulverized using a bath sonicator for 10 minutes to resuspend the pellets. This was named as a growth promoting factor of this experiment and it was confirmed by various experiments whether or not to promote the growth of Mycobacterium tuberculosis. In this experiment, we used the extract of M. bovis BCG belonging to the family of Mycobacterium tuberculosis as a control to identify species-specific growth promoters.

Growth promoters were divided into three groups as follows.

① cell wall fraction

② Cell Wall Fraction + Lysozyme 100㎍ / mL (High lysozyme, incubated at 37 ° C for 1 day)

③ Cell Wall Fraction + Lysozyme 10㎍ / mL (Low lysozyme, incubated at 37 ° C for 1 day)

< Example  3 - Growth factor Impact on promoting growth

In this example, in order to observe the growth of Mycobacterium tuberculosis in the cell wall fraction extracted by the method of Example 2, Mycobacterium tuberculosis was cultured with and without the growth promoter.

The initial culture solution of Mycobacterium tuberculosis K strain obtained in the above test method was divided by CFU concentration (10 ¹ ~ 10 ⁵ CFU / mL) and the control group (no cell wall fraction only) without adding anything and Factor A (1) with different growth factors. : 100, 1.0%), Factor B (1: 1000, 0.1%) was added to spot each 5μL in 7H10-OADC medium and incubated for 3 weeks at 37 ° C. As a result, referring to Figure 1, in the control (control) it was confirmed that colonies were not observed in 10 ¹ and 10 ² when cultured for 3 weeks, but both growth factor A and Factor B growth growth factor was added. It was also confirmed that the growth rate of Mycobacterium tuberculosis was accelerated in a concentration dependent manner (Factor A and Factor B).

In cultured conclusion, the addition of growth-promoting factors in conditions of incubation time, such as Mycobacterium tuberculosis was promote the growth of M. tuberculosis compared to the control similar to the colony size of 10 ² CFU 10 ⁴ CFU level of the control group, Factor A containing 1% Was confirmed (observed about 100-fold growth effect). This means that it promotes the growth of Mycobacterium tuberculosis more effectively in the presence of a lower concentration of bacteria than a high concentration of bacteria. Referring to FIG. 2, the same results were obtained when the above experiment was repeated.

< Example  4-of growth promoters Broth  Effect on Culture>

In this example, the resazurin microtitre assay (REMA) plate method experiment was performed to determine whether the extracted growth promoter is effective in broth culture. Addition of a growth promoter (1%, Factor A) and 1: 1000 (0.1%, Factor B) to the 7H9-OADC broth at a ratio of 1: 100 and Compared.

As a result, referring to Figure 3, even in broth cultivation, a growth promoting effect of about 100-fold was observed depending on the concentration of growth factors (Factor A: 10 ¹ , control: 10 ³ ).

< Example  5-Identification of species specific growth promoters>

Cell wall extracts of BCG were used for identification of species-specific growth promoters with lysozyme (Species-specific growth promoters) (FIG. 4). The group was composed of high concentrations of lysozyme (HL) and low concentrations of lysozyme (LL) in the same cell wall (1%, 1: 100 dilution in broth media). 10 μg / mL) was added and cultured.

As a result, the growth of Mycobacterium tuberculosis K strain showed significantly faster growth when the cell wall components of Mycobacterium tuberculosis K strain were specifically added than BCG extract. It was also confirmed that the addition of lysozyme significantly promoted the growth of Mycobacterium tuberculosis. As shown in Figure 4, the number of bacteria obtained in the culture on the 7th day of the control (control) was similar to the number of bacteria on the 4th day of the group with the growth promoter and lysozyme was confirmed to specifically promote the growth of Mycobacterium tuberculosis .

< Example  6 - Mycobacteria  Identification of species-specific growth promoters in phage>

The same experiment as in Example 5 was carried out using mycobacterial phage. That is, if the number of bacteria is high mycobacterial phage to dissolve the tuberculosis bacteria, so the growth is promoted so much dissolution to the mycobacterial phage. Based on this, the relative lysis unit (RLU) was measured using mycobacterial phage, and the growth rate of mycobacterium tuberculosis bacterium added to the growth promoter was much faster than that of the control.

Referring to FIG. 5, the growth rate on the 8th day of the control was confirmed to be very fast growth of 2.5 to 5 days of the tuberculosis bacteria added growth promoter and lysozyme.

Although the present invention has been described with reference to the examples, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

1 is a view showing a comparison of the growth rate of Mycobacterium tuberculosis K strain according to the growth promoting factor;

Figure 2 shows the comparison of the growth rate of Mycobacterium tuberculosis K strains according to the growth promoter by the same experiment;

3 is a view showing the effect of the growth promoter concentration;

4 is a graph showing species specific growth promoters; And

5 is a graph showing species specific growth promoting factors using Mycobactera phage.

Claims (10)

And a cell wall component and lysozyme isolated from the mycobacterium bacterium, wherein the cell wall component comprises carbohydrates and lipids or lipoproteins. delete The method according to claim 1, wherein the Mycobacterium fungus is Mycobacterium tubercurosis, Mycobacterium paratubulocysis, Mycobacterium leprae, Nontuberculous mycobacterium (NTM) Or atypical Mycobacterium tuberculosis (MOTT; Mycobacterium other than tuberculosis). The culture medium according to claim 1, wherein the concentration of the cell wall component is 50-100 μg / ml. According to claim 1, wherein the concentration of the lysozyme culture medium, characterized in that 10 ~ 100 μg / ml. A culture method for promoting the growth of Mycobacterium spp. By culturing the Mycobacterium spp. In a culture medium according to any one of claims 1 and 3 to 5. The method of claim 6, wherein the culturing of the mycobacterium bacteria is characterized in that the culture is made for 10 to 20 days. A) preparing an initial culture solution by culturing the Mycobacterium spp .; B) obtaining a cell wall fraction from the mycobacterium cells by collecting the mycobacterium cells from the initial culture solution; C) separating cell wall components including carbohydrates from the cell wall fraction and further comprising lipids or lipoproteins; And And d) adding the cell wall component and the lysozyme to the general medium including the carbohydrate and further comprising a lipid or a lipoprotein, to the growth medium of Mycobacterium spp. The method of claim 8, wherein the concentration of the cell wall component is 50 to 100 μg / ml. The method of claim 8, wherein the concentration of the lysozyme is characterized in that 10 ~ 100 μg / ml.

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