METHOD FOR REDUCTION OF TULAREMIA PATHOGEN RESISTANCE TO CEPHALOSPORINS (VERSIONS) Russian patent published in 2017 - IPC A61K39/00 G01N33/00 

FIELD: medicine.

SUBSTANCE: invention is a method for reduction of tularemia pathogen resistance to cephalosporins, where a nonionic surfactant tween 80 is used as the drug in an amount of (0.5-1)% by means of which the permeability of the outer cells structures of the tularemia pathogen is increased. The studies are performed in vivo and in vitro. In the latter case, the disc-diffusion method and the serial dilution method are used, after which the studies results are evaluated according to the implemented methods. For the disc-diffusion method, Muller-Hinton agar plates are prepared in two rows, one of which is injected with (0.5-1)% tween 80, then the bacteria of the investigated tularemia strains are seeded onto the agar surface from a pre-prepared suspension containing 10⁸ mcl/ml. After the suspension is absorbed into the agar, discs with cephalosporin antibiotics are placed on its surface, and no discs are placed on the control plates with and without tween, the crops are incubated for 24-48 hours at 37°C, the results evaluation is carried out visually by formation of zones of bacterial growth inhibition, if the latter have a diameter of 25-40 mm, then their sensitivity to the antibiotic is confirmed. Zones of inhibition are absent on the plates with medium without tween 80. In addition, for the serial dilution method, two rows of plates with a Muller-Hinton culture medium are prepared, where a cephalosporin antibiotic is administered at various concentrations, namely 25, 50, 100, 250, 500 μg/ml of medium. In parallel, the same row of plates is prepared, with tween 80 is adding to the medium at a concentration (0.5-1)%, then 0.05 ml of the test strain from the bacterial suspension containing 108 mc/ml according to the optical turbidity standard of 10 units is seeded, Muller-Hinton medium without the antibiotic and Muller-Hinton medium containing (0.5-1)% of tween 80 without the antibiotic are used as a control, the cultures are incubated at 37°C for 24-48 hours, after that the growth of bacteria is taken into account by the value of the minimum suppressing concentration: the lower it is, the more sensitive is the strain of tularemia microbe to antibiotics with the obligatory bacterial growth on the control plates. In order to carry out the test in vivo, animals are infected subcutaneously with a previously prepared culture of tularemia pathogen in a volume of 0.1 ml containing 10³ mc/ml, then after 24 hours the animals are treated with ceftazidime in an amount of 12 mg/mouse diluted in a solution of (0.5-1)% between 80, the course is carried out for 7 days, and the results are evaluated by the effectiveness of therapy, percentage of surviving animals to the number of infected or average life expectancy of surviving animals. Moreover, preliminary preparation of the studied strains is carried out according to the following technology: bacterial suspension is prepared by concentration 10⁹ mc/ml according to the optical turbidity standard of 10 units, 10 times diluted with saline to 10⁸ mc/ml. In addition, preliminary preparation of the tularemia pathogen culture is carried out as follows: from the daily agar culture, a suspension is prepared in physiological saline corresponding to 1 billion mc/ml by the optical turbidity standard of 10 units, then titrated with 10-fold dilutions in saline to the concentration of 10⁴ mc/ml.

EFFECT: invention allows to increase the antibacterial activity of cephalosporins with respect to the tularemia germ in vitro and in vivo, using a non-toxic surfactant.

6 cl, 1 dwg, 3 tbl, 3 ex