CS226174B2 - Method of increasing sensitivity of bacteria to antibiotics - Google Patents

Abstract

A nitrogen-containing polysaccharide, which is obtainable by extracting mycelia of basidiomycetes belonging to the genus Coriolus of Polyporaceae with an aqueous solvent, is effectively used in promoting drug sensitivity of bacteria resistant to antibiotics.

Description

The invention relates to the use of nitrogenous polysaccharide to increase the sensitivity of an antibiotic in bacteria that have become antibiotic resistant.

Progress in the field of chemotherapeutic techniques, in particular the introduction of numerous antibiotics, has led to a drastic reduction in bacterial infectious diseases such as tuberculosis, dysentery, etc., and their contribution to general health is beyond discussion. However, these modern successes have prompted the rapid development of so-called drug-resistant bacteria that resist existing chemotherapeutic agents. The situation is all the worse because many multiple-resistant bacteria, ie bacteria resistant to two or more types of drugs, have evolved from important microbes such as Mycobacteria, Shigella, Staphylococci and others, and these new strains pose a serious problem in their chemotherapy. of infectious diseases.

For these reasons, antibiotic preparations usually have a short lifetime in practice, and drug manufacturers are forced to market new and new types of antibiotics at great cost. This can lead to huge losses that cannot be overlooked in terms of social economy.

In view of the above situation, extensive studies have been conducted on drug resistant bacteria, which have resulted in the finding that the use of a specific nitrogen polysaccharide obtained by the method described in Czechoslovak Patent No. 219380 in combination with an antibiotic can result in a significant increase in susceptibility of bacteria. that have made these antibiotics resistant. It has also been found that this nitrogenous polysaccharide is not only able to increase the effectiveness of an antibiotic against resistant bacteria, but also extends the effectiveness of the antibiotic, thus allowing the long-term use of the antibiotics without altering their effect.

It is therefore an object of the present invention to re-use the aforementioned nitrogenous polysaccharide to enhance non-antibiotic susceptibility in baateas that have become non-antibiotic resistant, as well as to increase the potency of the antibiotics against bacteria, thereby broadening the area of antibiotic action.

The invention is described in detail below.

Brief description of the drawings: ob] ?. Fig. 1 represents the infrared absorption spectrum of the nitrogenous polysaccharide (PN) used in the sense of the invention; Fig. 2 represents the proton nuclear moggotic resonance absorption spectrum (NMR) of this substance; and Figs. 3-6 are graphs illustrating the effects of the invention.

The dicalcium polysaccharide, which is used as an active ingredient of the agent of the invention, is obtained by extracting the myiolea basidiooycet belonging to the genus Coriolus from the Polyporeceae family with an aqueous solvent. The term myyeeia basidiomycet belonging to the genus Ccoiolus of the family Polyporeeeae ^н is based on the classification given by the Color Illustratoon of Fungi of Japan by Rokuya Imazeki and Tsugio Hongo (Hoikusha Pub. Cco).

The process for producing the nitrogenous polysaccharide experienced as the active slogs of the present invention is described briefly below.

Myclia besidiomycetes belonging to the genus Ccoiolus, which are used as the starting maaerial for the extraction process, can be prepared naturally or by artificial kiiutivation. In the case of artificial cultivation of a fungus using a liquid medium, it is possible to use as a starting material all of the cultivation product including not only the produced soap, but also the liquid medium eluate after cultivation. The starting oattaial thus obtained is extracted with an aqueous solvent and the extract is subjected to a suitable treatment, for example, filtration, neutralization, filtrate concentration, dialysis, salting, etc., to remove low-recurrence (less than 5,000 molecular weight) from the extract and dried. and e to obtain a pulverulent substance. The pulverulent material thus obtained consists of a nitrogenous polysaccharide and can be used as active ingredients according to the present invention.

The nitrogenous polysaccharide obtained as described above has the following properties:

Physico-chemical properties

1. General

The substance has a powder form and a brown color, does not have a certain melting point and carbon monoxide when heated. It is insoluble in organic solvents such as pyridine, chloroform, biosane, hexane, etc., but is water soluble.

2. Infrared absorption spectrum

Infrared absorption spectrum of the substance shows absorption in the vicinity of and at 3600 and even 3200 ад ^"1 2920 and even 2900 ад" ¹ 1660 and even 1610 ад "" 1460 cm ^"1, 1410 сф" ^1, 1360 cm “ ¹ ,

230 ад "'ад1150''1080ад" ^{1 and} 1060 and 990 cm ^"1, 925 cm' ^{1890 cm '1840 cm' 1.}

755 cm ^-1 and 7 ⁰ 5 cm -1. ^J sou patterned atoorpce fl-neck ^ug Lukane in the ar ^ ^ ^ when part of the network.

89 ⁰ cm ^-1 and other atoar ^ a ^d -vaza ^bg lukaou inc. ⁸⁴⁰ cm ”.

3. Elimeniary analysis

The composition of the material, expressed by eloenear analysis, comprises 42 to 46% carbon, 5.3 to 7.0% hydrogen, and 0.5 to 8.0% nitrogen. The rest is oxygen.

4. Optical rotation

Optical rpm ^ACI ness retracts substance-specific data Renate otáčivooti ® is ^0? 50 degrees.

5. Color reaction

The substance provides a positive reaction with phenol and sulfuric acid, anthrone and sulfuric acid, and a ninhydrin reaction. A positive response to these color reactions shows that the active ingredients used according to the invention consist of carbohydrate and protein. In order to know the carbohydrate composition of the nitrogenous polysaccharide, the sample was hydrolyzed with a methanolic hydrochloric acid solution and, after triethylsilylation, carried out in a conventional manner, subjected to gas chromatography.

The result shows that the substance consists only of glucose and also contains mannose, galactose, xylose and fucose. Analysis of the amino acid protein part of the nitrogenous polysaccharide showed that the protein part of the substance consists of aspartic acid, threonine, lilac, glutamic acid, proline, glycine, alanine, cystine, valine, meeionin, isoleucine, leucine, tyrosine, thylanine, phenylaine, phenylaine alginine. They are predominantly aspartic acid, threonine, glutamic acid, glycine, alanine, valine and leucine and account for more than 70% of the total molar amine-4-carboxylic acid.

6. Proton nihclear magnetic resonance spectrum (NMR)

NMR absorption spectroscopy was performed to determine the ratio of mm 2 by carbohydrate to protein by part of the nitrogenous polysaccharide as well as its carbohydrate bonds.

The NMR absorption spectrum of the sample was measured at 100 MHz using heavy water as solvent and 2,2-dimethyl-2-silanopentao-5-sulfonic acid sodium salt (D.S.S) as internal standard. As shown in Figure 2, the absorption at 0.9 ± 0.2 to 1.2 ± 0.2 ppm, 2.0 ± 0.2 ppm, 4.5 ± 0.2 ppm, 4.7 ± 0.2 ppm, 5.0 and 0.2 ppm, and 5.4 ± 0.2 ppm, and at 3.0 to 4.4 ppm, a wide absorption band is seen. The protein portion of the tulle is monitored, provided that the region of 0.5 to 2.5 ppm belongs to the proton intensity of the protein portion and the region of 2.5 to 6.0 ppm belongs to the proton intensity of the carbohydrate: The protein portion is less than 40%. Assuming that the 4.4 to 4.9 ppm region belongs to the β-bonds of the saccharide and the 4.9 to 5.4 region belongs to the bonds, it was found that their ratio (g / α) ranged from 85/15 to 40/60.

7. Mooocular hmoonost

The monococcal hmoonoot of the nitrogenous polysaccharide of the invention, as measured by traceotrifuration, ranged from 5,000 to 300,000.

Alkuu.in toxicity

It was truncated in 4 to 5 weeks old ICR-JCL mice weighing 21 to 24 g and in 4 to 5 weeks old Donryu rats weighing 100 to 150 g. The substance dissolved in saline saline was administered by the following four routes : intravenous, hypertensive, oral iodine and oral. General symptoms, mortality and change in body weight of test animals were monitored for seven days; it was then killed and dissected. As shown in Table 1 below, the result was that no death was observed in either the rat or the rat, nor after the highest dose. Determination of LD 50 was virtually impossible since none of the animals died during the experiment.

Table 1

Animal Method of administration female “50 (mg / kg) males mouse Intravenous > 1 300 > 1 300 Subcutaneous > 5 000 > 5 000 Intraperitoneal > 5 000 > 5 000 Oral > 20 000 > 20 000 rat Intravenous > 600 > 600 Subcutaneous > 5 000 > 5 000 Intraperitoneal > 5 000 > 5 000 Oral > 20 000 > 20 000

We will now describe the effect of nitrogenous polysaccharide (hereafter simply referred to as the invention) on drug sensitivity in drug resistant bacteria that have acquired antibiotic resistance *

Antibiotics which, when combined with a compound of the invention, are effective against drug resistant bacteria are derived from fungi, bacteria, actinomycetes and the like, and typical examples of such antibiotics are listed below:

Penicillin G (abbreviated as PG)

Streptomycin (abbreviated as SM)

Kanamycin (abbreviated as KM)

Chloramphenicol (abbreviated as cp)

Tetracycline (abbreviated as TC)

Erythromycin (abbreviated as EM)

Aminobenzylpenicillin (abbreviated as ABPC)

Cephaloridine (abbreviated as CEN)

Colistin (abbreviated as CL)

The compound of the invention is effective in enhancing drug sensitivity in the following drug resistant bacteria, and not the following antibiotics:

Eschorichla coli

Streptococcus faecalis

Staphylococcus aureus

Enterobacter aerogenes

Salmonnella anteritidis

Shigella Sonnei

Klebsiella pneumoniae

Proteus mirabilie

Pseudomonas aeruginosa

The effect of the compound of the invention in enhancing drug sensitivity in drug resistant bacteria was demonstrated as follows.

The bacteria resistant to dvAm were obtained as described below by concentration gradient plate culture (see ChernooherraeuUica and ReeSstant Baateria, 1970, by Chikar Waaanabe, Asakura Shoten).

Agar plates with a concentration gradient ranging from 10 to 10 µl / ml were prepared and the bacteria of each test strain were inoculated by scoring or painting. These plates were maintained at 37 ° C for several days and the colony formed in the high concentration area was isolated and similarly inoculated onto the plates. This operation was repeated several times to obtain bacterial strains resistant to the infused drugs.

Drug sensitivity in resistant kau strains of parent strains (susceptible strains) was compared using a minimum concentration of in vitro growth (hereinafter abbreviated as MIC) according to the Japanese Society of Chemical Society standard method (see Chemooherapy Vol. 22, No. 6, 1126 (1974), MIC Determination). Method Reforma CommiOtte).

Two-fold systems of each drug were prepared and mixed with agar medium from the brine (Nippon Eiyo Kagaku Co., Ltd.) to form agar plates. Then, a loop of each strain that was cultured at 37 ° C in trypto-soy broth (Nippon Eiyo Kagaku Co., Ltd.) pp for 18 hours was coated on each plate and after cultivation for 18 hours at 37 ° C was not every plate was monitored for strain growth.

In order to show the MIC change for each resistant strain in the combined use of the compound of the invention and antibiotics (those that have lost their effect on bacterial strains), the same procedure was performed as above except for admixing an additional 10 to 1000 µg / ml of the compound into the systems and the MIC value was determined by the above method on agar plates.

The MIC was clearly reduced by less than half the value before or less by the addition of the compound of the invention. The amount of added substance according to the invention is more than 10 µg / ml, preferably more than 100 µg / ml. The pH of the medium was maintained at 7.2 ± 0.1 so that

The MIC was not affected by the pH of the environment. It was also found by agar dilution culture technology that the compound of the present invention, as such, does not have antibacterial activity against tesolated bacterial strains.

Therapeutic test of infectious diseases

The therapeutic test for infectious diseases was performed as follows.

1

Mice were inoculated intraperitoneally with 1x10 ° cells of bacteria resistant to drugs, and 1-3 hours pozdéjd was applied BU3 intrapertooneálně or orally 10-1 000 m & amp / kg (no mouse body weight) ^treats Va ^1, and ^{Z g /} kg latte ^p ccording to the invention, . V ^d Ennio observation was continued for seven days after administration in order to determine the number of survival · účinnooti for evaluating compounds of the invention. In this case, it was found that the effective dosage of the compound of the invention was preferably above 10 m @ 2 kg for intraperitoneal administration and above 100 m @ 2 kg for oral administration. It was higher than 40

It has been found that the compound of the invention is capable of enhancing the therapeutic effect not only in in vitro applications, but also in the case of thrombotic and test infectious diseases.

The compound of the invention also has very low acute toxicity and can be administered in various ways, for example by intraperitoneal injection, dermal administration, oral administration and intrarectal administration. The agent of the invention may be mixed with an anti-neon during drug preparation or may be administered by itself.

When the substance is formulated into tablets, granules, powder, wafers and the like for oral administration, the composition of such a preparation may contain ingredients of the type commonly used in the preparation of medicaments such as binders, fillers, fillers, glidants, disintegrants, wetting agents etc. according to the invention, it is used in oral liquid formulations, it can be formulated as an ingested liquid medicament such as a shaking mixture, a suspension, an emulsion, a syrup, etc., or a dry product can be prepared which dissolves just before use . Such liquid preparations may also contain additives and / or preservatives of the type commonly used for medicinal products.

The injections may contain additives such as a stabilizer, a buffer, a preservative, an isotonizing agent, etc., and may be administered in unit dose ampoules or in multi-dose containers.

The above preparations may also be prepared in the form of an aqueous solution, suspension, solution or emulsion in an oily or aqueous vehicle, while the active ingredient may be in powder form that is dissolved in an aqueous vehicle, for example sterile pyrogen-free water, just prior to use. Where the substance is used in the form of a maati or rubbed lubricant, it may comprise an oil or fat base, an emulsion base, a water-soluble base, a preservative and the like.

The invention is further described by means of embodiments which further illustrate the significant effects of the invention.

Examples

Example 1

Preparation of nitrogenous polysaccharide

A liquid culture medium of the following composition was prepared:

peptone5 g yeast extract3 g primary potassium phosphate 0.3. g secondary potassium phosphate0,3 g magnesium sulphate crystalline 0,3 g glucose55 g water5 Шг pH6,0

An aliquot of 150 ml of this broth was placed in 100 Erlermayer lira flasks, and after closing each flask with a cotton plug, the liquid broth was sterilized for 30 minutes at 120 ° C and then inoculated on slanted agar separately by cultured myelium in the usual manner. Coriolus versicolor (Fr.) strain Quel.CM-105 (Ferm. Res. Inat. Accession number Ferm-P-2416), followed by stationary cultivation at 25-27 ° C.

The culture (culture broth) thus obtained was dried in a double drum tumbler chamber to give 451 g of dry product. 150 g of this dry product was triturated and extracted with 0.1 N sodium hydroxide at 95-98 ° C and under normal pressure for 3 hours using a stainless steel extractor. The alkaline extract solution thus obtained was neutralized and filtered, and the filtrate was concentrated to 500 ml. This concentrated solution was sealed into a cellophane film and dialyzed in running water for 90 hours.

The obtained dialysate was further concentrated under reduced pressure and spray dried to give 19.5 g of a powdered product.

In order to test the properties of this powdered product, its sample was subjected to elemental analysis using a CJ-H-Correr MT-2 analyzer (Yanagimoto Seisakujo Co., Ltd.) to find 42.1% carbon, 6.9% hydrogen and 5.8% nitrogen. Stanoven :! Specific rotation was performed in a 0.25% aqueous sample solution using the YAIUCO-OR-50 model (Yanagimoto Seisakujo Co., Ltd.). The specific rotation of this product was + 12 °.

To know the composition of the carbohydrate component of the product, 10 mg of the sample was added to 3% methanolic hydrogen chloride for 16 hours at 100 ° C by methanolysis. The reaction mixture was filtered after neutralization of the hydrochloric acid with strontium carbonate at room temperature and the filtrate was concentrated and evaporated to dryness. The solid product was then dissolved in 0.5 ml of pyridine, and 0.2 ml of hexamethyllisilazane and 0.3 ml of trimethylhiol was added, the mixture was allowed to stand at room temperature for 30 minutes to effect triethyl.

The product was then dissolved in chloroform and after removal of excess reagent and dewatering, the filtrate was evaporated to dryness. This product was then dissolved in carbon tetrachloride and subjected to gas chromatography. 70.5% glucose, 3.8% gelactose, 10.3% mannose, 5.4% xylose and 10.0% fucose were found.

To determine the protein composition of the powdered product, the sample was subjected to a conventional amino acid analysis procedure. It was found by the following composition: 15% aspartic acid, 8% t-nemine, 6% lilac, 14% glutamic acid, 4% proline, 8% glycine, 10% bacon, 4% isolamine, 6% leucine, 1% tyrosine, 4% phthymol, 2% trisin, 2% lysine, 3% nitganine, 4% ammonia and 1% glucose, and a trace of histiitt.

Heavy water was used as the solvent for NMR absorption, while DSS was used as an internal standard, and post-correction values based on the assumed L-Jrenz curve were used to avoid any effect of residual light water in heavy water. Under these conditions, the ratio of saccharide to protein is determined provided that absorption at 0.5 to 2.5 ppm is due to the proton in the protein portion and absorption at 2.5 to 6.0 ppm is due to protons of the saccharide count. Obtained ratio of carbohydrate to protein tyl 89/11. tulle also determined the ratio (/ <г assuming that the absorption band at 4.4-4.9 ppm belongs to β-carbohydrate bonds and the absorption band at 4.9-6.0 ppm pelt to α-bonds. This tulle ratio 65 / 35.

Moeekoic thyroidity of tulle determined by tltractttrftugait and tulle performed by sedimentation equilibrium and synthetic bonding using an interference optical system under the following conditions: 0.3% sample concentration, solvent M / 10 potassium chloride, 'temperature 25 ° C' liquid column 1.7 mm, 22,000 rpm, measuring 5 hours.

Acquired average moocular tulle of 100,000. Test for the effect of drug sensitivity escalation in drug resistant bacetia that have become antibiotic resistant:

1. Preparation of drug resistant bacteria

Drug-resistant samples were obtained by culturing the above-mentioned agar plates with a concentration gradient using Staphllrcoccus aureus, Streptoccus faecaais, Escherichia coli, Salmonneia et al. The MIC values of the original battens and those that have developed drug resistance are summarized in Table 2 below.

Table 2

Bacteria

Antibiotics

Minimum MIC ithibus growth concentration (µg / ml)

Original km ^ n

Resistant strain

Staphylococcus aureus PC 0,025 12.5 CP 3.1 25 TC 0.8 25 EM 0.4 12.5 SM 1.6 100 · Streptococcus faecalis TC 0.4 25 Escherichia coli PC 12.5 100 ALIGN! SM 3.1 100 ALIGN! KM 6.3 200 CP 1.6 50 TC 3.1 25 CER 3.1 25 EM 100 < - ' Enterobecter aer ^ ogenes KM 3.1 100 < Salmonneia enneritidis PC 6.3 100 ALIGN! CP 0.8 12.5 SM 25 100 < TC 1.6 12.5 EM 100 < - Shigella sonnei KM 3.1 50 Klebbiella pneumoniae PC 6.3 100 < SM 1.6 100 ALIGN! CP 0.8 25 TC 1.6 25 me 12.5 100 < Proteus mirabblis ΑΒΡΟ 0.8 25 Pseudomonas eeroginosa CL 12.5 200

MIC PC values were also calculated assuming 1.677 (units) = 1 mg

MIC CL values were calculated from the assumption that 30,000 j (units) = 1 mg.

2 · Effect of drug sensitivity escalation in drug resistant bacteria

To see the change in MIC value of drug-resistant strains by concomitant use of the compound of the invention and various antibiotics (those used for resistant bacteria), the compound of the invention was added to the culture medium in amounts of 10 to 1000 µg / ml and MICs were obtained by agar plate method. The results are summarized in Table 3 below.

Table 3

Resistant bacterial strain

Drug (antibiotic)

Concentration of the substance according to the invention (yUg / ml)

Minimal concentration inhibiting growth (pg / ml) not added ** added *

Staphylococcus aureus

PC-resistant PC 1 000 12.5 1.6 CP-resistant CP 100 ALIGN! 25 6.3 TC-resistant TC 100 ALIGN! 25 3.2 EM-resistant EM 1 000 12.5 3.2 Streptococcus faecalis TC-resistant TC 100 ALIGN! 15 Dec 6.3 Escherichia coli SM-resistant SM 1 000 100 ALIGN! 25 KM-resistant KM 1 000 200 50 CP-resistant CP 100 ALIGN! 50 2,5 TC-resistant TC 100 ALIGN! 25 6.3 CER-reslstent CER 10 25 12.5 Enterobacter eerogenes KM-resistant KM 1 000 100 ALIGN! > 2,5 Selmonelle enteritidis CP-resistant CP 100 ALIGN! 12.5 3.2 TC-resistant TC 100 ALIGN! 12.5 6.3 Klebslelle pneumoniae SM-resistant SM 1 000 100 ALIGN! 25 CP-resistant CP 100 ALIGN! 25 6.3 TC-resistant TC 100 ALIGN! 25 > 2,5

Shigelle sonnei

KM-resistant KM

000

Proteus mirabilis

ABPC-resistant ABPG

Pseudomonas aeruginose

CL-resistant CL

000

5012.5

2512.5

200100

Comment:

+ The substance of the invention was added to the culture medium ++ The substance of the invention was not added to the culture medium

Example 1 a d 2

Therapeutic test for infectious diseases was carried out five groups of masses male mice 'ICR-JCL (each · hmoonooti 22 TJG) _t where each group consisted of 10 masses, using p ^ i ^ i ^ (^ I1 (^ o ^ u The G-resistant strain of Staphylococcus aureus obtained in Example 1. The G-resistant strain, which was cultured on a tryptosoo agar broth (Nippon Eiyo Kageku Co. Ltd.) for 18 hours at 37 ° C, was suspended in a TIP ^0. ® New Broth (Nippon Eiyo Co. · Ltd ·), containing 5% hiotanotic mucilages and 0.25 nT of such suspension, was inoculated iotreperione at every Mass ·

Q

It was checked that the inoculum contained 5 characterized 10 ° cells per mš · Two hours after the incubation was Penly-DMN G and ^p LIKOV ^u n io ^t гeperiOone ^AL Oe at ^2, 5 x ¹⁰ U / kg (body hmooncoti ° ё1) and ⁵ x ¹⁰ U / kg, while ^la Units of invention: ^p Iskan ^and method ccording BC ^IKL range of 1 was also applied intreperiooneálně 100 mg / kg / body hmoonc> oti MSS) ·

Treated masses were monitored every day for seven days and the number of survivors of the vaccinated musks was evaluated. The results are shown in Fig. 3. These results have shown that nitrogenous polysaccharide (NP) is able to favorably influence the therapeutic effect of penicillin G to pe ^ clUn in vivo ·

Example 3 · '

A similar therapeutic test for infectious diseases was performed in five groups of male ICR-JCL 22 ± 1 g mice, each group consisting of 10 masses, using a tetracycline-resistant Escherichia coli strain and a compound of the invention prepared according to Example 1 · 0.25 ml suspension tiypto soy broth (see example 2 ·), containing 5% hmoonnotních Musina and bekkeeií resistant _{netracytllož>} prepared as in example 1, ·, it was vaccinated iotraperi0oneáloě each Mass · s

Inoculo contained 5x10 cells per mouse. Two hours after inoculation, tetracycline was administered orally at doses of 80 m ^ kg (body hmoonoot mass) and 160 m / kg, with concomitant oral administration of the compound of the invention at a dose of 100 oo / kg. Fig. 4 in the form of survival during sedoid period after application

Obviously, the combined use of a compound of the present invention results in a higher therapeutic effect than when using tetracycline alone, since the same effect can be derived both from the oral administration of the compound of the present invention, The compound according to the invention can be characterized in addition to its high activity

Q resistant Staphylococcus aureus was vaccinated in шюЫу! 5 x 10 cells / mouse of five groups (10 IFSI group) male mice ICR-JCL (hooonoot 22 ± 1 g) as a straight ^IKL ADU 2 us ^ Doval iotraper ^and tooθ ^much as ^s administration ² 5 * ¹ 0 * jAg of penicillin G and 10, 10 and 10,000 mg / kg of the compound according to the invention to four groups of experimental animals. Daily observation of these errors for 7 days after application provides the results shown in FIG.

1

Example 5

A similar test for therapeutic efficacy against infectious diseases was performed using the purified groups of male ICR-JCL male mice (10 mice per kaid group), analogously to the case of 3 inoculated ⁵ x W ⁸ tonnes tetracycline-resistant Escherichia coli mice.

Two hours after inoculation, tetracycline was administered at a dose of 80 m @ 2 kg of body mass in the body, and the compound used in Example 1 was orally administered at a dose of 0, 10, 100, 1000 and 100 respectively. 10,000 mg / kg. The sixth group of animals is 0оп0го1п1. The results from daily observations of the masses for 7 days are shown in Figure 6.

Example 6

Knen CM-151 Coriolus hirsutus (Fr.) Quel (Ferm. Fes. InsU, accession number Ferm-P 2711) was cultured as in Example 1, followed by extraction and purification. 17.5 g of a pulverulent substance is obtained which, according to determinations carried out analogously to Example 1, has the following properties:

1. Elemental analysis: 43.7% C, 6.4% H, 5.5% N,

2. Specific rotation: = + 30 °.

3. Carbohydrate composition: 79% glucose, 15% mannose, 2% xylose, 4% galactose and · traces of fructose.

4. Protein content: 33

5. Binding ratio in carbohydrate β / α: 71/29.

6. Average molecular h ^ c ^ ot ^ c ^^ t: 95 000 »

7. Protein composition: 15% aspartic acid, 9% threonine, 5% sarin,% glutamic acid, 6% proline, 9% glycine, 9% alenine, 7% valine, 1% methionine, 5% isoleucine, 6% leucine, 2% tryptophan, 4% phenylalanine, 2% lysine, 3% alginine, 2% ammonia, 1% N-glucosamine and traces of cystine, tyrosine and histidine.

The following experiment was then carried out with a compound of the invention having the above-mentioned properties. CChooamffnikol-resident Salmoinlla enteeitidis was obtained as in Example 1.

0.25 ml of a suspension of the above mentioned immunogenic bacteria in thyroid broth (see Example 2), containing 5% by weight of mucin, was vaccinated iotinitritally to five groups of mice, the group consisting of 10 mice. The inoculum contained 1 ^× 10 ⁸ cells / µl. Two ^to five hours after cet ionkulmc ^and rearward ^p ODAN iotrjpliitotl ^and LTE 50 mgAg chloramphenicol. Another group received 500 mg / kg of the compound of the invention. The survival rate of 5 days post-dosing in the chloramphoso alone group was 40%, while in the group where iotibiol was administered in conjunction with the compound of the invention, it was 80%.

Example 7.

Stil tnmšCntriliE ^^ ^{p-temperature} Klebbiella pnlumbntml · Tyl obtained according to Example 1 and 0 ', 25 ml of Tryptic Soy Broth (see Example 2), the content-enhancing ^ hmoOnontních 5% mucin and these bacteria were inoculated two groups iotгjpliinooláL.oě MMSI containing 10 mice in your Mn-1 and ^{10 to} Bune ® mouse. Three hours of this ^{yp t i} nn ulmci occiput .ntr j ^{p n} LII ooláltlě administered 100 mg / kg strepoomšcinu. In addition, a group of 120 mg / kg of the compound of the present invention obtained as described in Example 6 was administered to the higher group. The survival rate of 6 days after administration in the streptomycin alone group was 50%, but in the group where the compound of the invention was used with streptomycin, more than 80

Example 8

0.25 ml of a colistin-resistant Pseudomonas aeruginosa suspension, cultured as in Example 1 in trypto-soy broth (see Example 2) containing 5% by weight of tnucin, was inoculated intraperitoneally with two groups of male ICR-JCL male mice (body weight: 22 i 1 g, 10 mice per group) at 1 x ΙΟθ cells / mouse. Two hours after vaccination, 190 mg / kg (1 µg = 30 µl) of colistin was administered intraperitoneally. The compound of the invention, obtained as in Example 6, was also administered orally at a dose of 1500 mg / kg. Survival in the colistin alone group at 6 days post-administration was 40%, in the combination use group of the invention 70%.

Claims (1)

SUBJECT OF THE INVENTION Use of nitrogenous polysaccharide from culture fluid or mycelium of a Basidiomycetic fungus of the genus Coriolus of the family Polyporeceae for increasing the sensitivity of resistant bacteria selected from the group consisting of Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, Klebsiella pneumoniae, Shigella sonusecis, aerogenes, to antibiotics selected from the group consisting of streptomycin, chloramphenicol, penicillin G, kanamycin, aminobenzylpenicillin, tetracycline, erythromycin, cephaloridine and colistin.