RU2542390C1 - NUTRIENT MEDIUM FOR DETERMINING ANTIBIOTIC SUSCEPTIBILITY OF Legionella pneumophila - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: nutrient medium comprises enzyme-autolyzate of cattle spleen, monosubstituted potassium phosphate, disubstituted potassium phosphate, potassium gluconate, mesoinosite, soluble starch, gelatin, L-cysteine, iron (III) sodium salt, high-grade ink and distilled water in a predetermined ratio of components.

EFFECT: invention enables to improve the accuracy of determining of antibiotic susceptibility of cultures of legionellosis causative agent.

3 tbl 8 ex

Description

The present invention relates to medical microbiology, namely to obtain a dense nutrient medium with which it is possible to determine the antibiotic sensitivity of Legionella pathogen cultures - Legionella pneumophila.

A common medium for determining the antibiotic sensitivity of the vast majority of pathogenic bacterial species is Muller-Hinton medium (The Oxoid Manual, 1976, p. 193), containing, g / l :. agar Difco - 10; beef meat infusion - 300; casein hydrolyzate - 17.5; soluble starch - 1.5; distilled water up to 1 liter at pH 7.4.

However, legionella on this medium, due to the nature of their metabolism, do not grow well even with various additives, therefore Muller-Hinton medium is not used to study the antibiotic sensitivity of legionella.

For the prototype, a nutrient medium was selected for the cultivation and isolation of Legionella BCUEA medium (see WHO. Legionella and prevention of legionellesis. WC 200. WHO. 2007), which has the following composition, g / l: yeast extract - 10.0; Difco agar 13.0; coal Norit N - 2,0; ACES - 10; alpha-ketoglutaric acid - 1.0; L-cysteine - 0.4; iron pyrophosphate - 0.25; glycine - 3.0; cycloheximide - 80.0-100.0 mg / l; vancomycin - 1.0 mg / l; polymyxin B 80-100 thousand units / l; distilled water up to 1 liter at pH 7.1.

The disadvantage of the prototype is that legionella are very sensitive to peroxide compounds and the growth of single virulent pathogen cells in this medium is possible only if the medium has a highly active adsorbent that blocks the action of free radicals - activated carbon prepared by special technology.

However, coal strongly adsorbs many currently used antibiotics, such as ofloxacin, ciprofloxacin, rifampicin, clindamycin, etc. (see article K. Nilsen, JV Bangsborg, N. Hoiby Susceptibility of Legionella to five antibiotics and development of resistance by exposure to erythromycin, ciprofloxacin and rifampicin. Diagn. Microbiol. Infect. Dis., 36 (2000), 43-48).

Therefore, for this reason, the results characterizing the antibiotic susceptibility of Legionella cultures obtained in this medium are unreliable, since the indicators of Legionella inhibition in most cases are artificially underestimated.

The technical task of the invention was to create a new nutrient medium with a high degree of accuracy in determining the antibiotic sensitivity of Legionella pneumophila.

This object is achieved by the fact that in the nutrient medium for determining the antibiotic sensitivity of Legionella pneumophila, including microbiological agar and L-cysteine, EDTA iron (III) sodium salt is additionally introduced, growth stimulants are monosubstituted potassium phosphate and disubstituted potassium phosphate, and mesosinosis as osmoprotectants and potassium gluconate, while the enzyme-autolysate of cattle spleen and edible gelatin were used as a nutrient base in the medium, in the following ratio of components, g / l:

Bovine spleen enzymeautolysate 6-8.0 Microbiological agar 14.0-16.0 Potassium phosphate monosubstituted 1.5-1.7 Potassium Phosphate Disubstituted 3.3-3.6 Potassium gluconate 2.4-2.6 g Instant starch 2.4-2.6 Gelatin 2.4-2.6 Mesoinositis 2.4-2.6 L-cysteine 0.3-0.5 EDTA iron (III) sodium salt 0.026-0.028 High quality mascara 4-6 ml Distilled water at pH 7.1 to 1 liter

The nutrient medium is prepared as follows. The starting materials for the proposed environment (designated B-2): microbiological agar 14.0-16.0; L-cysteine 0.3-0.5 and soluble starch 2.4-2.6; fermentoautolysate of bovine spleen 6.0-8.0 and edible gelatin 2.4-2.6; a mixture of phosphate salts is used as a buffer: potassium phosphate monosubstituted (KH ₂ PO ₄ ) 1.5-1.7 and potassium phosphate disubstituted (K ₂ HPO ₄ ) 3.3-3.6. Mesoinositol 2.4-2.6 and potassium gluconate 2.4-2.6 were selected as osmoprotectors, and the EDTA complex iron (III) sodium salt 0.026-0.028 was used as a source of iron. To give the medium a dark shade, on which the zones of inhibition of the growing Legionella culture are more clearly visualized, use 4-6 ml high-quality finely divided sterile mascara.

All of the above components, except for L-cysteine and EDTA iron (III) sodium salt, are added to the container and up to 1 liter of cold distilled water is added. The mixture is heated in a steam sterilizer and boiled for 30 minutes at 100 ° C with thorough stirring. Then the liquid is autoclaved at 0.5 atm for 20 minutes. The result is a base that is stored at a temperature of (2-8) ° C in the refrigerator for 2 years.

If it is necessary to prepare a working medium, the base is melted and the required amount of the remaining two additives is introduced into it: L-cysteine and EDTA iron (III) sodium salt. The melted medium (45 ° C) is poured into plastic Petri dishes with a diameter of 90 mm in a volume of 30 ml and with a layer thickness of 0.7 cm. The cups are stored in an airtight bag at a refrigerator temperature for 1-2 months.

The main biological indicators of the proposed B-2 medium are determined in comparison with the best BCYEAά medium for legionella cultivation. Initially, it is characterized by its sensitivity, the time of germination of the colonies of legionella, as well as the effectiveness of the medium (the size of the grown colonies and the degree of development of the lawn culture) - examples 1, 2, 3, 4 and 5.

After that, using the same VSUEA medium for comparison, they determine the principal suitability of B-2 medium for determining the antibiotic sensitivity of legionella cultures using antibiotic discs (Example 6), strips with gradations of antibiotics (Example 7) and using the method of serial dilutions of antibiotics in the medium (example 8). In the experiments, virulent and avirulent strains of legionella were used, obtained from the Museum of Living Cultures of the Rostov-on-Don Antiplague Institute:

1) L. pneumophila Philadelphia ATCC 33152, 1 serogroup, LD50 - 4 10 ⁵ m.k. for nautical. guinea pigs (virulent)

2) the same, but avirulent strain

3) L. pneumophila Blumington ATCC 33155, 3 serogroup (avirulent)

4) L. pneumophila Chicago ATCC 332 215, 6 serogroup (avirulent)

Example 1

To prepare the medium, the ingredients are used in the following minimum concentrations, g / l: cattle spleen enzyme-autolysate - 5.0; microbiological agar - 12.0; potassium phosphate monosubstituted - 1.0; disubstituted potassium phosphate - 3.0; potassium gluconate - 2.0; soluble starch - 2.0; gelatin - 2.0; mesoinositis - 2.0; L-cysteine - 0.1; EDTA iron (III) sodium salt - 0.01; high-quality mascara - 3 ml; distilled water up to 1 liter, pH 7.1.

The composition of the comparison medium VSUEAά similar to the above (see prototype).

We used the standard method for determining the sensitivity of nutrient media for legionella. Used a typical strain of Legionella - Legionella pneumophyila Philadelphia - 1 - virulent strain, LD 50 - 4 10 ⁵ MK for guinea pigs and three avirulent strains. Inoculum preparation: cultures are grown for 2 days on BCUEA medium at a temperature of 35 ° C, then washed off with potassium phosphate buffer (pH 7.1).

After this, a suspension of cells with a concentration of 1 billion m.k. / ml is prepared, which is diluted to a working concentration of 10 ³ m.k. / ml and seeded in plates of 0.1 ml, i.e. 100 mk Crops are incubated for three days at 35 ° C, the number of grown colonies is calculated, the time of their appearance, the degree of typical morphology of legionella colonies are determined, and their diameter is compared on different media at different incubation times. Crops were performed three times; average values were used for comparison. The statistical significance of similarities and differences in values is determined using the Chi-square test.

As a result, it was found that the sensitivity of both media was statistically different: from 100 seeded m. legionella of all four strains on the third day of incubation on medium B-2 of the composition of example 1 grew to 20 colonies of the pathogen, on the control medium - 50-60 typical colonies of legionella (p - 0.05, Chi-square 6.0), i.e. . the degree of sensitivity of the first medium is insufficient. Similar differences were also recorded in the time colonies appeared on the media — in all cases, on the proposed medium they were recorded with a stereo microscope on the fifth day of incubation, visually on the sixth — in the form of small dewy colonies up to 1 mm in diameter, stereomicroscopically with typical fine-grained and finely greenish glow On the prototype medium stereomicroscopically, legionella colonies were registered on the second day, visually on the third, which meets the standards. The effectiveness of the VSUEA medium was much better than the B-2 medium of Example 1, i.e. the volume of colonies on the 5th day of growth on average in the first medium was 4-5 mm; on the second, the maximum diameter of the colonies on the 6th day of growth was 2-3 mm. The growth of the culture lawn on the prototype medium when inoculating 0.1 ml of suspension of Legionella culture of all the tested strains after a day of incubation at 35 ° C was sufficient for its visual registration; on the proposed one, it was extremely weak.

Thus, the medium prepared according to example 1 does not satisfy the requirements for the quality of diagnostic culture media for Legionella pneumophila, and cannot be used to determine its antibiotic sensitivity.

Example 2

To prepare the medium, the above-mentioned ingredients were used in the following concentrations, g / l: cattle spleen enzyme-autolysate - 6.0; microbiological agar - 14.0; potassium phosphate monosubstituted - 1.3; potassium phosphate disubstituted - 3.2; potassium gluconate - 2.3; soluble starch - 2.3; gelatin - 2.3; mesoinositis - 2.3; L-cysteine - 0.3; EDTA iron (III) sodium salt - 0.025; high-quality mascara - 4 ml; distilled water up to 1 liter, pH 7.1.

The methods for determining and comparing the sensitivity, germination time and effectiveness of the proposed nutrient medium B-2 and VSUEAά were identical to those described in example 1.

Comparison results: from seeded 100 mk virulent and avirulent strains of legionella on both media grew on the third day for 50-60 typical colonies (p - 0.05, Chi-square - 0.31), the period of formation of colonies - 2 days when observed with a stereo microscope and 3 days with visual observation . But according to the growth efficiency of the colonies on the 5th day of incubation, the VSUEA medium was slightly better prepared according to Example 2. At the same time, the severity of the culture lawn on the proposed medium was sufficient for visual registration on the 2nd day of growth. Thus, the composition of the medium prepared according to example 2, provides the growth of legionella to the extent that this medium was considered as consistent with generally accepted WHO standards.

Example 3

To prepare the medium, the above-mentioned ingredients were used in the following concentrations, g / l: cattle spleen enzyme-autolysate - 7.0; microbiological agar - 15.0; potassium phosphate monosubstituted - 1.6; potassium phosphate disubstituted - 3.4; potassium gluconate - 2.5; soluble starch - 2.5; gelatin - 2.5; mesoinositis - 2.5; L-cysteine - 0.4; EDTA iron (III) sodium salt - 0.027; high-quality mascara - 5 ml; distilled water up to 1 liter, pH 7.1.

The methodology for the comparative study of the sensitivity and effectiveness of both media was consistent with the above. The results obtained (the growth of 45-65 typical colonies of all tested strains on both media, the time for colonies to appear is 2-3 days when incubated at 35 ° C, the sufficient growth efficiency of the colonies and lawns of the cultures with some excess of these indicators on the comparison medium) indicate that the medium taken in this composition fully complies with the requirements for the reference diagnostic nutrient medium BCYEAά used for the cultivation and isolation of Legionella pneumophila. The test medium in the specified composition according to the listed characteristics can be used to detect antibiotic sensitivity of legionella cultures and for their usual cultivation.

Example 4

To prepare the medium, the above-mentioned ingredients were used in the following concentrations, g / l: cattle spleen enzyme-autolysate - 8.0; microbiological agar - 16.0; potassium phosphate monosubstituted - 1.7; potassium phosphate disubstituted - 3.6; potassium gluconate - 2.6; soluble starch - 2.6; gelatin - 2.6; mesoinositis - 2.6; L-cysteine - 0.5; EDTA iron (III) sodium salt - 0.028; high-quality mascara - 6 ml; distilled water up to 1 liter, pH 7.1.

The methodology for the comparative study of the above indicators of both environments corresponded to those described. The results obtained (the growth of 50-65 typical colonies of all tested strains on both media, the time for colonies to appear is 2-3 days when incubated at 35 ° C, the sufficient growth efficiency of the colonies and lawns of the cultures with some excess of these indicators on the comparison medium) indicate that the medium taken in this composition fully complies with the requirements for the reference diagnostic nutrient medium BCYEAά used for the cultivation and isolation of legionella. The test medium in this composition according to the above characteristics can be used to detect antibiotic susceptibility of Legionella pneumophila cultures and for their normal cultivation.

Example 5

The medium in this example was prepared with the maximum concentrations of the ingredients, g / l: cattle spleen enzyme-autolysate - 9.0; microbiological agar - 17.0; potassium phosphate monosubstituted - 1.8; disubstituted potassium phosphate - 4.0; potassium gluconate - 2.8; soluble starch - 2.8; gelatin - 3.0; mesoinositis - 3.0; L-cysteine - 0.8; EDTA iron (III) sodium salt - 0.03; high-quality mascara - 10 ml; distilled water up to 1 liter, pH 7.1.

The methodology for the comparative study of the above indicators of both environments was as described (see example 1). The results: growth of up to 20 typical colonies of all tested strains on the test medium with significantly better growth on the comparison medium (50-60 colonies with a significant statistical difference of P - 00.5 Chi-square - 8.0), longer periods of colony appearance on the environment of this composition - for 4-5 days when incubated at 35 ° C. Conclusion: the insufficient growth efficiency of the colonies and lawns of cultures on this medium indicate that the medium taken in the present composition does not meet the requirements for the reference diagnostic nutrient medium BCYEAά used for cultivation and isolation of legionella. The test medium in this composition, according to the above characteristics, cannot be used to detect the antibiotic sensitivity of legionella cultures and for their usual cultivation.

Example 6

Confirmation of the suitability of the proposed medium in determining the antibiotic sensitivity of legionella cultures by the disk method in comparison with the VSUEAά medium

Prepare Wednesday for the recipe of example 3 and Wednesday VSUEA according to the commonly used recipe. The first medium is dark in color, the other is black. The volume of media in Petri dishes is 30 ml. One-day cultures of the indicated Legionella strains are prepared, which are plated on agar plates of both media with 0.1 ml of suspension of Legionella cells (4 × 10 ⁹ m.k. each), they are distributed over the surface and the agar is dried. Then, discs with antibiotics are most often used on the agar, most often used to treat legionellosis: rifampicin, ofloxacin, clindamycin, erythromycin, doxycycline, moxifloxacin, azithromycin, levofloxacin and ciprofloxacin (the antibiotic content in the disk is 1-1.25 μg). No more than three discs are placed on each cup. Crops are incubated for 48 hours at 35 ° C; the first count is carried out after 24 hours, measuring the zones of inhibition of culture growth and comparing their diameters on both media. On the second day, these indicators are measured again. The results of the comparative test in determining the atibiotic sensitivity of legionella cultures are presented in table 1.

As can be seen from table 1, the zones of inhibition of the growth of lawn of Legionella cultures of all tested strains around eight antibiotic disks (rifampicin, ofloxacin, clindamycin, erythromycin, moxycycline, levofloxacin and ciprofloxacin) on the medium proposed are 2-4 times statistically more reliable (with an average Chi-square = 6.3) and exceed similar zones obtained on the BCYEAά control medium, which is explained by the adsorbing effect of activated carbon in the latter. A small zone of inhibition of Legionella growth around a doxycycline disk depends on the relatively weak sensitivity of Legionella to this agent, accompanied by a complete absence of an adsorbing effect. Thus, on the proposed medium, sensitivity to legionella antibiotics is more fully and accurately detected than on BCYEAά medium.

Example 7

This example presents the results of a comparative test of the medium taken in the optimal composition (similar to Example 3) and BCYEAά medium for determining the antibiotic sensitivity of legionella cultures using strips containing the concentration gradient of the antibiotic erythromycin and levofloxacin (manufactured by Oxoid, E - test). Legionella cultures are prepared as described in Example 6. Strips are applied to the sown lawn of the crop in an amount of two per cup. The results are taken into account after a day of incubation at 35 ° C; on the other days, the results are repeated. If the culture was sensitive to the antibiotic being tested, a racket-shaped zone of inhibition of Legionella culture growth was formed around the strip, the lower edge of the inhibition zone corresponded to the MIC - the minimum inhibitory concentration. The results are presented in table 2.

As can be seen from table 2, the zone of inhibition of the growth of lawns of Legionella cultures on the medium proposed was about 5 times higher than the MIC on the control medium (mean Chi square = 6.3). Therefore, the results of determining the antibiotic sensitivity of legionella using strips are more accurate on the proposed environment.

Example 8

Determining the suitability of the proposed environment for determining the antibiotic sensitivity of legionella cultures by the method of serial dilutions of antibiotics in comparison with the VSUEA medium

Cups are prepared with the author's medium and VSUEAα, as indicated in the previous case, except that an antibiotic is introduced into the medium in stepwise increasing concentrations: 0.01, 0.05, 0.1, 0.25, 05, 0.75, 1 mcg / ml. The above legionella cultures are seeded on the surface of the agar with antibiotics in a dose - a large suspension loop with a concentration of 4 × 10 ⁹ m.k., forming a plating in the form of a disk with a diameter of 1 cm. Crops are dried and incubated under the same conditions as in the previous case. The incubation period is 2 days, accounting after 24 and 48 hours. The results are taken into account by the presence or absence of culture growth (see table 3). The indicators are compared for both environments.

As can be seen from table 3, the minimum inhibitory concentration of antibiotics determined for Legionella pneumophila on the proposed medium is 5-10 times less than on the medium containing activated carbon.

Therefore, tests of two media for determining the antibiotic sensitivity of legionella using various methods, namely, using discs, strips and serial dilutions, confirmed that the B-2 medium works most accurately and reliably.

Thus, when eliminating a strong adsorbent - activated carbon, which binds a number of antibiotics, thereby lowering the sensitivity indices, the new medium makes it possible to accurately determine the antibiotic sensitivity of Legionella pneumophila without lowering the inhibition indices, which is of great importance in clinical practice in the treatment of legionesis.

Table 1 Legionella strains Wednesday The diameters of the zones of inhibition of Legionella lawn around discs with antibiotics (mm): erythromycin azithromycin clarithromycin doxycin levofloxacin moxifloxacin oflaxacin clindamycin rifampicin L. pneumophila Philadelphia - 1 (virulent), 1 serogroup BCYEAα thirty fifteen 22 8 28 twenty thirty 28 twenty B-2 38 twenty 36 10 0 35 40 40 40 L. pneumophila Philadelphia - 1 (avirulent), 1 serogroup BCYEAα 32 twenty 23 8 thirty twenty 31 25 thirty B-2 40 28 38 9 42 thirty 44 38 42 L. pneumophila bloomington (avirulent), 3 serogroup BCYEAα 28 16 twenty 8 26 21 thirty 26 19 B-2 36 22 38 10 38 33 41 38 40 L. pneumophila Chicago (avirulent), 6 serogroup BCYEAα thirty twenty 21 7 22 22 thirty 26 eighteen B-2 40 thirty 32 8 32 36 41 42 38

table 2 Legionella strains Wednesday Minimum Inhibitory Concentration (MIC) of Antibiotics Determined with Strips (E-Test) Erythromycin (mcg) Levofloxacin (mcg) L. pneumophila Philadelphia - 1 (virulent), 1 serogroup BCYEAα 0.5 0.12 B-2 0.1 0.01 L. pneumophila Philadelphia - 1 (avirulent), 1 serogroup BCYEAα 0.5 0.12 B-2 0.1 0.01 L. pneumophila bloomington (avirulent), 3 serogroup BCYEAα 0.4 0.15 B-2 0.1 0.01 L. pneumophila Chicago (avirulent), 6 serogroup BCYEAα 0.5 0.12 B-2 0.1 0.01

Table 3 Legionella strains Wednesday Minimum Inhibitory Concentration (MIC) of antibiotics as determined by the serial dilution method (μg): erythromycin doxycycline levofloxacin oflaxacin rifampicin L. pneumophila Philadelphia - 1 (virulent), 1 serogroup BCYEAα 0.5 0.5 0.1 0.25 0.25 B-2 0.1 0.25 0.01 0.01 0.01 L. pneumophila Philadelphia - 1 (avirulent), 1 serogroup BCYEAα 0.5 0.25 0.1 0.25 0.25 B-2 0.12 0.1 0.01 0.01 0.02 L. pneumophila bloomington (avirulent), 3 serogroup BCYEAα 0.4 0.25 0.1 0.25 0.25 B-2 0.1 0.1 0.01 0.01 0.01 L. pneumophila Chicago (avirulent), 6 serogroup BCYEAα 0.5 0.5 0.1 0.25 0.25 B-2 0.1 0.1 0.01 0.01 0.01

Claims (1)

Nutrient medium for determining the antibiotic sensitivity of Legionella pneumophila, including microbiological agar and L-cysteine, characterized in that EDTA iron (III) sodium salt is additionally added to the medium, growth stimulants are monosubstituted potassium phosphate and disubstituted potassium phosphate, and mesinositis as osmoprotectants and potassium gluconate, while the enzyme-autolysate of bovine spleen and gelatin gelatin were used as a nutrient base in the medium, in the following ratio of components, g / l:
Bovine spleen enzymeautolysate 6-8.0 Microbiological agar 14.0-16.0 Potassium phosphate monosubstituted 1.5-1.7 Potassium Phosphate Disubstituted 3.3-3.6 Potassium gluconate 2.4-2.6 g Instant starch 2.4-2.6 Gelatin 2.4-2.6 Mesoinositis 2.4-2.6 L-cysteine 0.3-0.5 EDTA iron (III) sodium salt 0.026-0.028 High quality mascara 4-6 ml Distilled water at pH 7.1 up to 1 l