PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART II. ANTIMICROBIAL AGENTS

CHAPTER 13. PRINCIPLES OF METHODS FOR DETERMINING THE ACTIVITY OF ANTIBIOTICS AND OTHER CHEMOTHERAPEUTIC AGENTS

The antimicrobial activity of a preparation refers to its ability to kill microorganisms or inhibit their growth. Accordingly, a distinction is made between the bactericidal and bacteriostatic effects of a substance. For antifungal agents, one speaks of fungicidal and fungistatic activity [28].

The activity of Antibiotics and other antimicrobial agents is determined:

a) during production (producer activity, product quality control);

b) when studying the pharmacokinetics of a drug in animals or humans;

c) when selecting a drug for Chemotherapy and monitoring its course.

In this context, the antibiotic may be present in the culture fluid, exist as a chemically pure substance, be formulated into various dosage forms, or be contained in biological fluids (Blood, urine, etc.). In each case, the appropriate Methods must be selected. Traditionally, microbiological assays are used to determine activity; alongside these, enzymatic, immunological, and chromatographic methods utilizing automated equipment and computer technology are also employed.

Each method involves The Use of a reference standard substance for comparison or for constructing a calibration curve. The accuracy of the method is achieved by performing the required number of replicate determinations and statistically Processing their results.

13.1 Microbiological Methods

To express biological activity quantitatively, METABOLISM/2.html">THE CONCEPT OF the unit of activity (UA) has been introduced. The unit of antibiotic activity is defined as the minimum amount of a substance that inhibits the growth of a standard microorganism strain under strictly specified conditions. The UA represents the activity of a strictly

defined weight of the substance adopted as a standard. For most antibiotics, 1 UA corresponds to 1 mcg of the substance. One milligram of benzylpenicillin sodium salt contains 1,670 UA, oxytetracycline contains 925 UA, and nystatin contains at least 4,000 UA. As a test culture, a non-pathogenic or opportunistic microorganism exhibiting the highest sensitivity to the given substance is typically used. To evaluate THE SPECTRUM OF activity of an antibiotic, various microorganisms, including pathogens, are employed. These strains are maintained under specific conditions and continuously monitored for their morphological and physiological properties.

Meat-peptone broth or meat-peptone Agar is generally used as a nutrient medium suitable for most test cultures. For the cultivation of Yeasts and Fungi, 1% glucose is added to these media, or Sabouraud medium is used. When determining activity by the agar diffusion method, base-layer agar (Water agar) is used. Its composition is: agar-agar — 1.5–2.0 g, phosphate buffer with pH 6.8–7.0 — 100 ml.

13.2 Determination of Antibiotic Activity by the Serial Dilution Method

The serial titration method can be performed in various volumes of medium (from 1 to 10 ml) under aseptic conditions, using sterile pipettes for each reaction ingredient. Titration can be carried out in Solid and liquid media. When titrating in liquid media, a strictly defined volume of nutrient medium is poured into a series of test tubes. A specific amount of the antibiotic solution is added to the first tube, mixed, and then a certain volume of the mixture from the first tube is transferred to the second, mixed, and the same volume of the mixture is transferred from the second to the third, and so on. An equal volume of the mixture is discarded from the last tube containing the antibiotic so that the volume of liquid is identical in all tubes. The tube containing no antibiotic serves as the control. Thereafter, an equal volume of the test culture suspension is added to all tubes containing the serially diluted antibiotic and to the control tube. The test tube rack is shaken and placed in an incubator at 37°C for 18–20 hours.

The test microbe suspension is prepared in an isotonic (0.85%) sodium chloride solution, with mandatory comparison against turbidity standards. When titrating antibacterial antibiotics, the microbial load typically amounts to 2.5×10⁵ microbial Cells per 1 ml of the antibiotic solution in nutrient broth. When Yeast is used as the test culture, the microbial load is 4×10⁶ cells per 1 ml.

The serial dilution method in solid media has the advantage that contaminating microbes are easily detected here and essentially do not alter the overall titration results, whereas in liquid media, the entire experiment may be ruined due to the entry into the tubes of even single cells of extraneous resistant microorganisms. This method is also used when WORKING WITH MICROORGANISMS that do not grow on conventional liquid media, such as Mycobacterium tuberculosis, which are grown on a medium containing coagulated serum. First, a series of serial dilutions of the antibiotic is prepared, and then 1 ml of each dilution is added to a test tube containing 4 ml of melted and cooled to 45–50°C agar medium. The tubes are then slanted until the agar solidifies, and the test microbe suspension is inoculated onto The surface of the solid medium using a loop.

To detect the bactericidal effect of the preparation, subcultures are made onto fresh nutrient medium from all tubes where Microbial growth was not visually observed. For persistent antimicrobial substances that adsorb onto microbial cells and inhibit their growth even in fresh nutrient medium, appropriate neutralizers are applied.

13.3 Determination of Antibiotic Activity by the Agar Diffusion Method

This method is more precise than the serial dilution method and is therefore more frequently used in practice. However, the diffusion method has certain limitations. Criteria for evaluating data obtained with fast-growing microorganisms are not applicable to slow-growing strains. Therefore, if a slow-growing microorganism must be used as the test culture, the dilution method is applied, or test conditions are optimized in special experiments. The same applies to slowly diffusing antibiotics.

Nutrient agar mixed with the test culture is poured into Petri dishes. The amount of the latter is taken at The rate of 20 million cells per 1 ml of medium. After the agar solidifies, cylinders are placed on its surface, into which 0.1 ml of the test antibiotic solution is added in parallel with the standard solution. Instead of cylinders, wells made in the agar using a special device can be used. Afterward, the dishes are placed in an incubator at 37°C for 16–18 hours. Upon completion of this time, the sizes of the test microbe growth inhibition zones are measured.

The calculation of antibiotic activity based on the sizes of the inhibition zones can be performed using V. S. Dmitrieva's calculation tables (State Pharmacopoeia) or standard curves.

Determination of microbial sensitivity to antibiotics by the disk method is carried out primarily to evaluate the effectiveness of antibiotics in clinical settings. Clinical material or a microbial culture isolated from a patient is inoculated onto the surface of nutrient agar as a confluent lawn, and paper disks impregnated with an antibiotic solution (commercial samples containing specific concentrations are used) are applied. After incubation at 37°C for the time required for the growth of the isolated pathogen, the diameter of the growth inhibition zone is determined. The obtained values are compared with the growth inhibition zone sizes specified in the instructions supplied with the disks, after which the isolated microorganisms are classified as susceptible, intermediate, or resistant. Before introducing any antimicrobial preparation to the market, the manufacturer is required to determine its spectrum of activity against thousands of strains of various microorganisms, taking into account that the pharmacokinetic Properties of the compound must maintain serum concentrations 2 to 4 times higher than the minimum inhibitory concentration. When isolating specific pathogens, defined sets of disks are used (Table 25).

Class="center">Table 25. Disk sets for determining the susceptibility of certain microorganisms

13.4 Enzymatic Methods

13.4.1 Accelerated METHOD FOR DETERMINING Microbial Susceptibility to Antibiotics

Pour 15 ml of nutrient agar into a Petri dish. Once the agar solidifies, apply a mixture of 4 ml of the same agar, 1 ml of the test culture suspension prepared According to the standard of 1 billion cells per 1 ml, and 1 ml of a 0.2% aqueous solution of 2,6-dichlorophenolindophenol (pH 7.2-7.3). Clinical material can be used instead of the test culture. Then, place antibiotic-impregnated disks onto the solidified bright blue agar, and incubate the dishes at 37 °C. After 2-4 hours, evaluate the results by the diameter of the blue zones where growth is absent. Microbes resistant to the antibiotic reduce the dye, decolorizing it or transforming it into a yellow color.

This dye inhibits the growth of staphylococci; therefore, when working with this microorganism, 2-3 ml of the indicator solution should be poured onto the surface of the dish only after incubating the dish with the disks in the thermostat. Drain the excess indicator after 5-7 minutes and read the results.

13.4.2 Determination of the Ability of Microorganisms to Produce β-Lactamase

The RESISTANCE OF MICROORGANISMS to penicillin may be associated with their ability to produce the enzyme lactamase, which inactivates this antibiotic.

Add 0.5 ml of a overnight broth culture of a standard penicillin-sensitive strain of Staphylococcus aureus and 20 ml of nutrient agar melted and cooled to 45 °C into a Petri dish. Mix quickly and leave until the agar solidifies. Afterwards, place a paper disk containing penicillin in the center of the medium's surface. Inoculate the test cultures radially toward the disk using a loop. Incubate the cultures at 37 °C for 24 hours. The ability of bacteria to produce β-lactamase is judged by the presence of growth of the standard staphylococcal strain around the test culture.

13.4.3 Determination of the Ability of Microorganisms to Produce β-Lactamase

Urease Test

Aminoglycoside antibiotics, acting as Protein Synthesis Inhibitors, suppress urease production by Proteus mirabilis, which is used as a test Organism. A nutrient medium containing urea is dispensed into two rows of test tubes. A standard solution of the test antibiotic at specific concentrations is added to one row, and the test solution is added to the other. Inoculate the test tubes with P. mirabilis and, after 60-75 minutes of incubation, measure the pH using a potentiometer. The pH values in the test tubes containing the standard solution are used to construct a standard curve, which serves to determine the amount of antibiotic in the test solution.

Luciferase Test

The method is based on the ability of aminoglycoside antibiotics to inhibit ATP production by microbial cells. The luminescent method for determining ATP in the presence of luciferase is characterized by high sensitivity. The bacterial culture is introduced into test tubes containing the standard or test antibiotic solution, incubated for 90 minutes, after which the amount of ATP is determined using a specialized device—a luminometer.

13.4.4 Chromatographic Methods

High-Performance Liquid Chromatography (HPLC). The test solution is passed through a chromatographic Column packed with a support consisting of a finely dispersed hydrophobicized sorbent. The hydrophobic coating of the support varies depending on the Molecular Weight of the separated mixture.

When a micro-amount of the test solution is applied, amphiphilic substances are sorbed onto the surface of the support particles and displaced from it using an aprotic solvent gradient (water–acetonitrile system). Substances are separated according to their affinity for the sorbent (Structure/106.html">Hydrophobicity).

The efficiency of the analysis is determined by the micron-sized particles of the support, so the analysis time does not exceed 5-10 minutes.

The solution exiting the column is analyzed in a flow-through UV detector with simultaneous recording. THE POSITION OF the peak of the eluting substance is its calibration characteristic. The amount of substance in the applied sample is determined by an automatic integrator.

The advantages of the method include high speed of determination, accuracy, Specificity, and sensitivity.

Quantitative determination of Tetracycline by HPLC

Prepare solutions of the test sample and the tetracycline reference standard, and chromatograph them alternately on a liquid chromatograph with a UV detector, obtaining at least 5 chromatograms of each solution under strictly specified conditions of Temperature and Mobile phase flow rate. Calculate the amount of tetracycline in the sample using the formula:

X — amount of tetracycline in the test sample, in grams;

S_sample — mean value of the tetracycline peak areas calculated from the chromatograms of the test drug solution;

S_std — mean value of the tetracycline peak areas calculated from the chromatograms of the reference standard;

A — the content of tetracycline in the reference standard, in grams.

13.4.5 Spectrophotometric Methods

Quantitative determination of ampicillin by spectrophotometry is based on The formation of a colored complex when ampicillin is heated in a CuSO₄ solution. An accurately weighed sample of the antibiotic is dissolved in a copper sulfate buffer solution and kept in a water bath at 80°C for 30 min, then cooled rapidly. The optical density of the solution is measured using a spectrophotometer at a wavelength of 320 nm, with the unheated buffer solution of the test substance used as a reference.

In parallel, the optical density of the reference ampicillin standard solution, treated in the same manner as the test preparation, is measured.

The percentage content of ampicillin in the preparation (X) is calculated using the formula:

D₀ is the optical density of the test drug solution;

D_СТ is the optical density of the reference standard solution;

a is the weight of the ampicillin reference standard, in grams;

b is the content of ampicillin in the reference standard, in percentage.

13.4.6 Immunological Methods

Immunological methods rely on the use of specific Antibodies and therefore require extensive preliminary preparation (obtaining antibodies by immunizing animals with a specific antibiotic). The second component of the reaction is a labeled analogue identical to the analyte. The label can be radioactive, fluorescent, or enzymatic, which determines the method for recording the assay results. The outcome depends on the competitive binding to antibodies between the test drug and the labeled analogue: the greater the amount of the test drug present in the mixture, the less labeled analogue will bind to the antibodies. The antibodies are immobilized in the wells of a microplate, into which the test solution and the test system (the labeled analogue and the necessary detection reagent) are added sequentially. The advantages of immunological methods include their high specificity and sensitivity.