DE2930394A1 - Determn. of antibiotic concn. in a medium - by observing change in adenosine concn. - Google Patents

Abstract

Determination of antibiotic (I) levels in a medium comprises measuring the initial increase in adenosine levels of a test culture caused by metabolic changes induced by (I). Pref. the change in ATP concn. in the organism is measured by the firefly bioluminescence system. Patic. the method is used to measure gentamycin or other aminoglycoside or toxic antibiotics in sera or other body fluids, or to measure (I) contents of milk, meats, and other foods. Rapid, simple and quantitative method for measuring (I) concn. and for assessing sensitivity of moc microbes to (I) or for determining min. inhibitory concns. Only simple instrumentation is needed.

Description

Method for determining the antibiotic level in a medium

The invention relates to a method for determining the antibiotic level in a medium by means of an antibiotic test in which the initial increase the concentration of ATP in bacterial samples as a result of the presence of the antibiotic Substance is determined.

Antibiotic levels are currently measured to determine antibiotic pick-up in the serum of a patient and to control the antibiotic content of food, to protect people from being overly exposed to antibiotics are, and in the case of milk, to ensure that the milk is suitable for fermentation processes (for the production of cheese, yogurt and the like.) Can be used. Antibiotic levels or antibiotic levels are usually measured using a bioassay (biological Detection method) in which a special strain of microorganisms is present an unknown sample is grown. The concentration of antibiotics in the sample is assessed on the basis of the degree of growth inhibition of the organism when used known concentrations of the antibiotic as standards. Such tests will for example with Klebsiella edwardsii var. atlantae (NCTC 10896) or E. coli (LUI4> fur Gentamycin carried out.

Another method of determining antibiotic levels (antibiotic levels) is the so-called high pressure liquid chromatography, but this method is used not generally used because such a device is used in hospital laboratories is seldom available and due to the fact that this procedure is used for routine examinations is too slow.

A third method of determining serum antibiotic levels consists in seeing the decrease in ATP concentration after 2 hours of incubation determined at 370C in the culture of a test organism compared to the detected Antibiotic sensitive (see M.J. Harber and A.W. Asscher, "A new method for antibiotic assay based on measurement of bacterial adenosine triphosphate using the firefly bioluminescent system "in" J. Microbiol. Chemother. ", 2, 35-41, 1977; L. Nilsson, H. Höjer, S. Ansehn and A. Thore, "A rapid seaiautomated bioassay of gentamicin based on luciferase assay of bacterial adenosine triphosphate "in '! Scand. J. Infect.

Dis. ", 9 232-236, 1977; and A. Thore, L. Nilsson, H. Höjer, S. See and L. Bröte, "Effects of ampicillin on intracellular levels of adenosine triphosphate in bacterial cultures related to antibiotic susceptibility "in" Acta Path. Microbiol. Scand.

Sect. B. ",, 161-166, 1977).

In these procedures there is a drop in the intracellular ATP level (Content) after 2 to 3 hours of incubation in correlation to the concentration of the antibiotic in the sample.

The ATP values of the sample are compared with those of known ones Standards compared to calculate the antibiotic levels in the samples. This method works well with so-called bacteriocidal antibiotics that kill bacteria while with so-called bacteriostatic antibiotics, the bacteria are not killed, but can only be prevented from multiplying. In the latter case, the bacterial cells can their protective cell wall does not form and the ATP can escape from the cells into the growth medium step out. This extracellular ATP is in the process by incubating the Samples with an ATPase enzyme (e.g. potato apyrase) destroys which ATP breaks down (splits). Before extraction and determination of intracellular ATP AlPase activity is increased by heating, by strong mineral acids or by other means Way destroyed.

Regarding the antibiotic level or antibiotic content in milk and other foods it should be noted that the antibiotics in the milk and in the other foods when animals are used to treat microbial infections, such as cattle mastitis (udder inflammation) are treated with it or if that Animal feed contains antibiotics as a preventive medicine.

Because the uncontrolled use of antibiotics in food can lead to the formation of mute microbes that are resistant to antibiotics cause allergy reactions in humans or target microbes prevent growth in food processing (e.g. in cheese making, yogurt fermentation and the like), strict regulations exist in most countries regarding the antibiotic levels or contents in milk, meat and other foods.

In the usual tests, the inhibitory effect of the antibiotics on the Growth of a specific bacterial strain compared to the detected one Antibiotic sensitive, measured.

In some procedures, a liquid culture medium is used which is inoculated with a specific strain of bacteria. The growth of bacteria is monitored on the basis of the increase in the turbidity or the change in color of one pH indicator dye as a result of the metabolic products of the growing bacteria. The growth or lack of growth will be familiar with that of a group Standard antibiotic samples compared.

A second method is to make a semi-solid medium evenly inoculate with the test bacteria and place an absorbent paper disk on the surface to lay. The samples and antibiotic standards are then removed from the paper disks absorb. During the incubation, the antibiotic agents diffuse in the agar medium and prevent the bacteria from growing around the disc. Ever higher the antibiotic level (antibiotic content) in the sample or in the standard the wider the clear zone around the disk. The antibiotic concentration This sample is determined by comparing the diameter of the clear zone that is free of bacteria by those of known standards. The one for this.

Bacterial strains used in tests are compared to that detected (certain) antibiotic highly sensitive and these strains grow very quickly, if they can be incubated at elevated temperatures up to 650C. To such Strains include the species of Bacillus stearothermophilus var. Calidolactoris, Pseudomonas thermophilus 101, Bacillus subtilis ATCC 6633 and the like. The growth these organisms can occur within 2 to 4 hours of onset the Incubation can be observed visually.

A third method for determining the concentration of antibiotics in milk is based on the change in the pH value of the growth medium. A pH indicator dye in the medium changes its color with decreasing pH value as a result of the acidic metabolic products, generated by growing bacteria. In this procedure, the sample added to the medium containing the pH indicator and inoculated with the test bacteria, that are sensitive to the detected antibiotic agent. Samples are used for 2 to 3.5 hours at elevated temperatures, usually at 650C, incubated. After incubation, the color of the sample is indicated with a color card and compared to a control sample.

Samples in which the original color has changed will show suggest that antibiotics in the sample prevented the bacteria from growing. This Test is semi-quantitative. The sensitivity is about 0.005 international Units (IU) of penicillin per ml of milk.

A fourth method for the determination or detection of penicillin in milk relies on the growth inhibition of bacteria spores which are prevented from doing so will grow in the presence of penicillin in the samples in sterilized milk. Bacterial growth is monitored by determining the bioluminescence of ATP (cf. D.C. Westhoff and T. Engler, Detection of penicillin in milk by bioluminescence " in "J. Milk Food Technol.", jE, 537-539, 1975). In this procedure, milk samples are used with 1 9; or 5 levels of Bacillus subtilis ATCC 6633 spores inoculated. the ATP concentration is measured 2 or 3 hours afterwards and the values of the samples be with a Control sample and standards compared. The sensitivity is 0.03 IU for a 1% inoculum and 0.01 IU (international units) for a 5% inoculum after 3 hours of incubation.

The determination of ATP by light box bioluminescence is an on known procedure. Bacteria have a relatively constant concentration of ATP on, so that the bioluminescence determination of ATP has so far been used for rapid determinations of viable bacteria in different types of samples, for example in Urine samples. Determining the growth of the bacterial culture with ATP was also used to determine antibiotic levels in serum and milk applied. The principle of this procedure is the inhibiting effect of antibiotics to determine bacterial growth compared to control samples without antibiotics. The determination of the growth in this method is based on the Measurement of total ATP or intracellular ATP. In this process, the bacterial culture Incubate for at least 2 to 3 hours to allow the effect of the antibiotics to quantize. It was suggested that in the presence of antibiotics bacteria Release ATP to the growth medium or the sample solution (see A. Thore, L. Nilsson, H.

Hörer, S. Ansehn and L. Bröte, "Effects of ampicillin on intracellular levels of adenosine triphosphate in bacterial cultures related to antibiotics susceptibility " in "Acta Path.

Microbiol. Scand. Sect. B ",, 161-166, 1977). This phenomenon is With bacteriostatic antibiotics, the bacteria do not kill, but develop prevent the protective cell wall and thus prevent the bacteria from multiplying, more pronounced than with bacteriocidal antibiotics that kill bacteria.

The aim of the present invention is to provide an improved method for the determination or detection of antibiotics to be developed quickly and sensitively and is quantitative and is therefore superior to the previously known methods.

The invention relates to a method for detection or determination the antibiotic level or antibiotic content in a medium, which is characterized by this is that one can see the initial increase in adenosine content of a test microbe culture as a result of a change in the metabolic activity of the test organism in the presence the antibiotic substance determined.

The invention also relates to a method in which the determination (Measurement) of extracellular ATP or total ATP is used to measure antibiotic levels in serum, milk and other samples or the antibiotic sensitivity as well as the to determine or estimate the minimum inhibitory concentration of the antibiotics.

According to a preferred embodiment, the invention relates to the improvement of the antibiotic detection or the antibiotic determination to two different areas of application, to which it is not limited. According to In a first embodiment it relates to a rapid determination of antibiotic levels in serum and according to a second embodiment, it relates to a rapid one Determination or rapid detection of antibiotic levels in milk, meat, Animal feed, food and the like. Both methods are based on the determination of the initial increase in adenosine triphosphate (ATP) levels in the samples in the presence of antimicrobial agents. The results can determined either as an increase in total ATP or as an increase in extracellular ATP will.

The inventive method is based on the fact that then, when bacteria or other microbe cells are exposed to antibiotics, their Cell wall is destroyed or its permeability is changed so much that ATP and other related small molecules can leak out of the cells. Because sales of ATP in bacterial cells is less than 1 second (cf. A.G. Chopman and D.E. Atkinson, "Adenine nucleotide concentrations and turnover rates, their correlation with biological activity in bacteria and yeast ", pp. 253-306, in" Advances in Microbiol Physiology, "Volume 15, Academic Press, London and New York, 1977, A.H. Rose and D.W. Tempest, Eds.), Large amounts of ATP are formed in the cells over time. Self if only a small percentage of ATP leaks out of the cell, the concentration may decrease of extracellular ATP that of intracellular ATP within a few minutes Exceed ATP. It also appears that in the presence of antibiotics the intracellular ATP increases during the first hour of incubation. This indicates that the metabolic activity the bacteria increase under exposure to antibiotics. That's why the initial increase in intracellular ATP to determine antibiotic levels or contents or the sensitivity to antibiotics for different purposes be exploited.

The following are the principle and application examples for the initial Increase in extracellular ATP described.

A) Method of determining the antibiotic levels in a sample with extracellular ATP During the test to determine the gentamycin level in the serum was carried out, extracellular levels rose ATP with increasing gentamycin concentration in the sample, while the contents of intracellular ATP and total ATP during the first three hours of incubation decrease (see Fig. 1 of the accompanying drawings). The test was as follows carried out: 1.) 50 to 200 l of an overnight culture of gentamycin-specific Bacteria such as E. coli (LU14) are pipetted into sterile test tubes (106 up to 10 cells); 2.) 200 to 1000 μl of sterile nutrient broth is added to each tube; 3.) Then 10 to 100 Sl samples (serum, gentamycin standard or other gentamycin samples) admitted; 4.) the sample is incubated for 60 to 120 minutes at 37 ° C; 5.) 10 up to 200 μl of the sample are pipetted into a measuring cell and placed in a photometer the free ATP is determined using the Leuchtköfer luciferin luciferase reagent. When determining gentamycin in serum samples, these are first 30 minutes heated to 56 C for a long time in order to destroy the bactericidal effect of the serum itself. The extracellular ATP content increases over time (see the following table I) and the measurement can take between 10 and 120 minutes, preferably between 60 and 120 minutes.

Table I Concentration of extracellular ATP as a function of incubation time; 3 the getamycin concentration was 10 ng / cm3 sample (cf. FIG. 2) incubation time (Min.) Concentration of extracellular ATP relative light units 0 0.04 20 0.05 60 0.25 90 0.65 120 1.23 B) Determination of antibiotic levels in food Hand of the Initial Increase in Total ATP The procedure has been varied with Penicillin concentrations in milk using Streptococcus thermophilus 101 was tested as a test organism and using an incubation temperature of 650C.

The total amount of ATP increased in the samples containing penicillin quickly to (see. Fig. 3) and the values for total ATP after 10-6is 35 minutes of incubation correlated with the penicillin concentration (cf. FIG. 4). The test was as follows performed: 1.) Add 1 cm3 of Streptococcus overnight culture to 2.5 cm3 of fresh milk thermophilus 101 or other bacteria sensitive to the antibiotics tested are, admitted; 2.) the sample is 10 to 40 minutes, preferably 20 to 30 minutes incubated long at 37 ° C; 3.) an aliquot of 100 rl becomes removed and the ATP is extracted from the cells with a boiling Tris (0.02M) EDTA (0.002 M) buffers, mineral acids and the like using known methods for extracting ATP from bacteria, but preferably 100 p1 of a nucleotide-releasing Reagent for microbial cells (NRB, registered trademark of Lumac Systems AG, Basel) and mixed by hand by shaking for 5 seconds, whereby NRB releases ATP within a few seconds, the samples can be used without neutralization, Cooling or other further treatment can be measured. The sample is in a Luminescence photometer is used and 10 to 1000 Eul, preferably 50 To 200, ul firefly luciferin luciferase reagent injected and the light intensity is recorded in the form of relative light units; 4.) a group of standards for antibiotics in milk that have been treated in the same way as the samples are is measured; 5.) the antibiotic concentration is taken from the standard curve calculated.

Fig. 3 shows the behavior of the total ATP in the milk samples as Function of time. Because of the antibiotics present in milk, the rate increases ATP concentration in the bacterial inoculum during incubation within 10 to 60 minutes on quickly compared to the control sample that did not have antibiotics contains.

The concentration of total ATP increases linearly with the increase Concentration of ATP when the samples after 60 to 10 minutes of the Inoculation be measured (see. Fig. 3).

The method of the invention, in which the initial increase in Free ATP content to determine the bactericidal antibiotic level or content and total or free ATP to determine bacteriostatic antibiotic levels and content is used, provides a quick, simple and quantitative Process. The equipment required for this is also simple and cheap, so that the method according to the invention has numerous advantages over the conventional one Procedure offers.

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Claims (5)

Patent pending method for determining antibiotic levels respectively. -Contents in a medium, as indicated by the fact that the initial increase in adenosine level or content of a test microbe culture due to a change in the metabolic activity of the test organism in the presence the antibiotic substance is determined. 2. The method according to claim 1, characterized in that the change the adenosine triphosphate (ATP) content in the test organism by means of the firefly bioluminescence system is determined. 3. The method according to claim 1 or 2, characterized in that the Antibiotic level in the serum or in another body fluid is determined. 4. The method according to claim 3, characterized in that it is the antibiotic around gentamicin, other aminoglycosides or other toxic antibiotics acts whose level in the body must be monitored. 5. The method according to any one of claims 1 to 4, characterized in that that the antibiotic level or content in milk, meat and other food products is determined.