CS277248B6 - Method of quantitative evaluation of the effect of antibacterial agents "in vitro" - Google Patents

Abstract

Under appropriate growth conditions, the growth and reproduction of the control and bacterial cultures affected by various concentrations of antimicrobial substances are compared in parallel. For quantitative evaluation of the viability and growth properties of bacteria, a water-soluble salt of a tetrazolium dye is added to the culture medium, preferably 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in a final concentration of 0.005 to 5.0 g/l, preferably 0.25 g/l, the reduction of the added tetrazolium dye takes place in all cultures in parallel and for a precisely defined period of time, preferably 15 minutes, after which the reaction is terminated and the water-soluble colored product is solubilized by adding a mixture of detergents and an organic solvent, the amount of the formed colored product is quantitatively determined spectrophotometrically, preferably in the region of its absorption maximum, and the concentration of the antimicrobial substance necessary to achieve the desired bacteriostatic or bactericidal effect on the culture of microorganisms under investigation is determined based on the amount of the formed colored product.

Description

1 CS 277248 B6

BACKGROUND OF THE INVENTION The present invention relates to a process for "in vitro" evaluation of antibacterial activity by measuring the quantitative variation and multiplication of bacteria by a spectrophotometric method in which the color product produced by the living organisms depends directly on their number and metabolic activity.

In vitro evaluation of the number, growth and reproduction of bacteria is often used in assessing the efficacy of antimicrobials. One of the basic requirements for such a provision is its speed and accuracy as well as simplicity.

To quickly determine the number, growth and reproduction of bacteria, several techniques have been developed to date: 1. Direct mechanical or electronic particle counting (Pringle, JR Mor, JR: Methods for monitoring the growth of yeastcultures and for dealing with the dumping problem. Biol., XI, 1975, 153-168). 2. Photometric measurement of turbidimetry, but also light scattering (nephelometry) passing through a bacterial suspension (Koch, AL: Theory of Angular Dependence of Light Scatter by Bacteria and similar-sized biological objects. J. Theor. Biol. , 18, 1968, 133-156). 3. Measurement of electrical impedance changes in culture of growing microorganisms (Ur. A., Brown, D.F.J .: Rapid detection of bacterial activity using impedance measurement. J. Biomed. Eng., 9,1974, 18-20, U.S. Patent 4,528,270). 4. Microcaloric measurement in culture of growing microorganisms (Binford, JS, Binford, LS, Adler, P .: A microscopic microscopic calorimetry of antibiotic sensitivity testing. Am. J. Clin. Pathol., 59, 1973, 86-94) . 5. Measurement of pH change due to glucose fermentation in unbuffered culture medium (U.S. Pat. No. 215861); 6. Measurement of live bacteria produced by ATP (Karl, DM: Cellular nucleotide measurements and applications in micro-binary ecology, Microbiol. Rev., 44, 1980) , 739-796). 7. Radiometric methods (De Blanc, J.J., Charache, P., Wagner, H.N .: Automatic radiometric measurement of antibiotic effect on bacterial growth. Antimicrob. Agents Chemother., 2, 1972, 360-366). 8. Quantitative measurement of the amount of enzymes released by growing bacteria in the culture medium by fluorogenic substrates (Philips, I., Waren, C., Waterworth, PM: Determination of antibiotic sensitivities by Sensititre system. J. Clin. Pathol., 31, 1978, 531 -535).

The methods according to the examples have several drawbacks, which are mainly due to the necessity to use a suspension of microorganisms with a density of up to 3 x 10 8 / ml (procedures 2.4), the complexity of the procedure and the labor of the method (1, 4, 6), the requirement for complex evaluation CS 277248 B6 2, the need to work with radioactive substances (7) and a complicated distinction between the degree of active response from the background and non-specific artefacts (8).

The above-mentioned drawbacks are eliminated by the quantitative evaluation of the effect of antibacterial agents "in vitro" according to the invention, which consists in quantifying the changes in the number, growth and reproduction of live, metabolically active bacterial cells based on the reduction of the tetrazolium dye salt. In a reaction that occurs exclusively in living cells, there is an interaction between the tetrazolium salt and the cytochrome system of the bacterial cell, whereby the tetrazolium salt is reduced to the color product, the amount of which is proportional to the number and metabolic activity of the bacterial cells present. Advantages of the process according to the invention compared to the previous approaches include high sensitivity (the density of the bacterial suspension of 2 x 10 4 / ml is sufficient for the determination), the reaction proceeds exclusively in living cells, the minimum required reaction time is 1 within the metabolic activity of most bacteria up to 30 minutes. The method is simple and requires no separation steps, which, together with the spectrophotometric method of evaluation, create the right conditions for simple automation. The cost of the determination is financially lower than for comparable methods.

The possibilities of using the process of the invention are explained, for example, by the rapid determination of the susceptibility of the Escherichiacoli bacterium to the bacteriostatic antibiotic tetra-cyclin. (A) and bacteriocidal antibiotic with rapid onset colicin (B). Example A. Evaluation of antibacterial effect of tetracycline.

Pipette 100 µl of Muller-Hinton, 100 µl / well, with decreasing tetracycline concentrations (256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0 µg / ml) into individual wells of a 96-well flat bottom microtest plate. ). In this broth, a suspension of 18 hour culture of Escherichia coli bacteria at a density of 1x10 6 bacteria / ml is prepared. 100 µl of bacterial suspension is added to the antibiotic wells to give a final antibiotic concentration of 128, 64, 32, 16, 8, 4, 2, 1, 0.5 0.25 and 0 µg / ml. The control well contains only pure Mueller-Hinton broth instead of bacterial suspension. The plate is placed in a thermostat and incubated at 35 ° C for 120 minutes. After incubation, most wells are added; 20 µl of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide in 2.5 mg / ml saline and incubate for an additional 15 minutes in a 35 ° C thermostate. Then 50 µl of solubilization solution (20% SDS in 50% aqueous solution of Ν, β-dimethylformamide, pH adjusted with HCl and acetic acid to 4.7) is added to all wells. The amine content is mixed. and the plates are incubated. additional 5 minutes at 35 ° C. Measure the absorbance of each well using a spectrophotometer (ELISA; reader at 550 nm (specific absorbance) and at 630 nm (non-impact)

Claims (1)

Cical absorbance). The measured corrected absorbance values E E550-E630 ú are plotted in a linear plot versus the antibiotic concentration and an appropriate curve is constructed (Fig. 1). The efficacy of antibiotics on the tested bacterial strain is shifted from the acquired curve. Example B. Evaluation of Antibacterial Effect of Colistin. The procedure is the same as in Example A, only the antibioticmtetracycline is replaced by the antibiotic colistin. The result is the same as in Example A, whereby the decrease in absorbance to "zero" (Fig. 2) means the killing of bacteria in the test suspension. PATENT REQUIREMENTS A method of quantitatively assessing the effect of antibacterial "in vitro" agents in which Growth ConditionsParallel Comparison of Growth and Propagation of Control and Rhial Concentrations of Antimicrobial Affected by Bacterial Culture, characterized in that quantitative evaluation of bacterial growth properties is added to the culture medium with a water soluble salt of tetrazolium dye, preferably 3- (4,5-dimethylthiazol-2-yl) - 2,5-diphenyltetrazolium bromide in a final concentration of 0.005 to 5.0 g / l, preferably 0.25 g / l, reduction of the added tetrazolium dye takes place in all cultures in a parallel and precisely limited time, preferably 15 minutes after which the reaction is complete and the water-soluble color product is solubilized by adding a detergent / organic solvent mixture, the amount of color product formed is quantitatively determined spectrophotometrically, advantageously in the region of its absorption maximum, and according to the amount of color product formed, the concentration of antimicrobial agent required to achieve the desired bacteriostatic or bacteriocidal effect is determined. microorganisms. 1 drawing