RU2678447C2 - Method for researching increase of antibiotic sensitivity of conditionally-pathogenic microflora in vitro with milk-foundation fodder additive containing streptococcus termophilus th-4 microorganism culture - Google Patents

Abstract

FIELD: microbiology.SUBSTANCE: invention relates to veterinary microbiology and can be used in laboratory practice in the study of the antibiotic susceptibility of microorganisms. Method of research increase in antibiotic sensitivity of conditionally pathogenic microflora in vitro with a lactic feed additive containing a Streptococcus termophilus TH-4 microorganism culture, includes the use of the supernatant of a lactic acid feed additive containing Streptococcus termophilus TH-4 microorganism culture, obtained in different periods of cultivation – at 1, 10, 20 and 30 days, then adding 0.5 ml of the supernatant of the lactic acid feed additive to 0.5 ml of the BCH medium, adding 0.5 ml of the BCH of the daily culture of one of the strains E. fecalis 200, St. albus ATCC 25923, E. coli ATCC 25222, Pr. vulgaris 192, Kl. pneumonia 71 of conditionally pathogenic microflora, the resulting solution is incubated for 18–24 h at T=37.0±0.5 °C, then carrying out the determination of the antibiotic sensitivity of microorganisms by disc diffusion method on MPA with 24 types of antibacterial drugs.EFFECT: method allows to expand the arsenal of methods for increasing the antibiotic sensitivity of microorganisms in vitro.1 cl, 2 tbl, 2 ex

Description

The invention relates to veterinary microbiology and can be used in laboratory practice in the study of antibiotic sensitivity of microorganisms.

The well-known "Drug for the prevention and treatment of diseases of the gastrointestinal tract" ToxibioVit "" (RF patent No. 2475254, CL A61K 35/74 of 12/13/2011) and the method of its use, including the introduction of substances that inhibit the development of pathogenic microflora, stimulating enzyme activity and antioxidant protection, as well as having a wide range of trace elements and stimulating the development of beneficial microorganisms, namely, metabolites of the microorganisms Bacillus subtilis, Bacillus licheniformis, Halobacterium halobium immobilized on an active carrier, representing a mixture of glauconite with cell walls of brewing yeast Saccharomyces cerevisiae.

The disadvantages of this method are the lack of data on the growth of antibiotic sensitivity to a specific type of antibacterial substance, as well as the lack of data on the growth of indicators of growth inhibition zones of the test culture before and after contact with the test supernatant (supernatant) of the probiotic culture.

Evaluation of the antibiotic sensitivity of microorganisms was carried out in accordance with the guidelines “Determining the sensitivity of microorganisms to antibacterial drugs" (MUK No. 4.2.1890-04, 2004).

The essence of the invention lies in the fact that the proposed method uses the supernatant of Streptococcus termophilus TH-4, obtained at different times of cultivation and introduced into the composition of the lactic acid feed additive.

The objective of the invention is the expansion of the arsenal of methods for increasing the antibiotic sensitivity of microorganisms in vitro.

This task is achieved in that in a method of increasing the antibiotic sensitivity of opportunistic microflora in vitro with a lactic acid feed additive containing a culture of microorganisms Streptococcus termophilus TH-4, comprising the use of a supernatant with a milk acid feed additive containing a culture of microorganisms Streptococcus termophilus TH-4 obtained at different times of different times cultivation.

According to the proposed method, to 0.5 ml of MPB medium (meat-peptone broth), add 0.5 ml of the supernatant of a milk-acid feed additive, after which 0.5 ml of MPB of a daily culture of opportunistic microflora is added, the resulting solution is incubated 18-24 h at T = 37.0 ± 0.5 ° C, after which they determine the antibiotic sensitivity of microorganisms on MPA (meat-peptone agar) with 24 types of antibacterial drugs by the disk diffusion method.

Lactic acid feed additive includes: milk, strain Streptococcus termophilus TH-4 containing 11 * 10 ⁸ CFU / 1 ml at pH = 3.41, manufactured according to TU 9224-001-014185347608.

Example 1. Analysis of changes in the zone of growth inhibition of microorganisms, when determining antibiotic sensitivity after contact with a lactic acid feed supplement containing a culture of microorganisms of different cultivation periods, showed different sensitivity in the studied representatives of conditionally pathogenic microflora. Thus, the greatest increase in the antibiotic sensitivity of microorganisms is observed after cultivation with ten and thirty day cultures (Table 1).

P <0.05

The greatest increase in antibiotic sensitivity (by 92%) was observed in E. fecalis 200 after contact with a ten-day culture of lactic acid feed supplement Streptococcus termophilus TH-4.

Thus, an increase in the antibiotic sensitivity of E. fecalis 200, St. albus ATCC 25923, E. coli ATCC 25222, Pr vulgaris 192, Kl. pneumonia 71 after contact with the supernatant of Streptococcus termophilus TH-4 lactic feed additive on the first day of cultivation was from 16.1 to 50%, on the tenth day from 25.7 to 92%, on the twentieth day from 22.2 to 33% and on the thirtieth days from 50 to 74.2%, respectively.

Example 2. It was found that throughout the experiment, the supernatant of the lactic acid feed supplement Streptococcus termophilus TH-4 of different cultivation periods, causes a steady increase in the number of antibacterial drugs to which the studied strains of opportunistic microflora are sensitive. In addition, the increase in the period of incubation of the probiotic coincides with a decrease in the number of low-sensitivity preparations in the strains of opportunistic microflora (Table 2).

Note: the number of antibacterial drugs to which the microflora is stable is U (growth inhibition zone less than 10 mm), insensitive to M (growth inhibition zone less than 15 mm), sensitive - H (growth inhibition zone more than 15 mm).

Claims (1)

A method of studying the increase in the antibiotic sensitivity of opportunistic microflora in vitro with a lactic acid feed additive containing a culture of Streptococcus termophilus TH-4 microorganisms, comprising the use of a supernatant of a lactic acid feed additive containing a culture of Streptococcus termophilus TH-4 microorganisms obtained at different cultivation times, by 1 Day 20 and 30, after which 0.5 ml of lactic acid feed supplement supernatant is added to 0.5 ml of MPB medium, then 0.5 ml of MPB daily culture of one of the strains of E. fecalis 200, St. albus ATCC 25923, E. coli ATCC 25222, Pr. vulgaris 192, Kl. pneumonia 71 of conditionally pathogenic microflora, the resulting solution is incubated for 18-24 hours at T = 37.0 ± 0.5 ° C, then the antibiotic sensitivity of microorganisms is determined by the disk diffusion method on MPA with 24 types of antibacterial drugs.