GB2026693A - Determination of Antibiotic Levels in a Medium - Google Patents

Abstract

A method of measuring the antibiotic level in a medium and which is particularly applicable to the rapid determination of antibiotic levels in serum and to the rapid testing of antibiotic levels in milk, meat and animal feed, is based on measurement of the initial increase of total or extracellular adenosine triphosphate (ATP) levels in the samples in the presence of antimicrobial agents.

Description

SPECIFICATION Determination of Antibiotic Levels in a Medium Antibiotic levels are measured presently for determining of antibiotic levels in the serum of a patient and to control antibiotic levels of foodstuff to protect human beings from excessive exposure to antibiotics, and in the case of milk to verify that the milk can be used for fermentation processes (for cheese, yogurt, etc.). Antibiotic levels are measured normally by means of bioassay where a particular strain-of micro-organism is grown in the presence of an unknown sample. The concentration of antibiotic in the sample is judged from the degree of growth inhibition of the organism using known concentrations of the antibiotic as standards. Such tests are performed, e.g. with Klebsiella edwardsii var. atlantae (NCTC 10896) or E. coliLU74 for gentamicin.Another method for determining antibiotic levels is so called high pressure liquid chromatography but this method is not generally applied due to the rareness of such equipment in hospital laboratories and the fact that this technique is slow for routine application.

A third method for determining the levels of antibiotics in serum is the measurement of the decrease in the ATP concentration after two hour incubation at 370C in culture of a test organism susceptible toward the assayed antibiotic (Harber, M. J. and A. W. Asscher. A new method for antibiotic assay based on measurement of bacterial adenosine triphosphate using the firefly bioluminescent system. J. Microbiol. Chemother. 3: 35-41, 1977; Nilsson, L., H. Haver, S. Ansehn and A. Thore. A rapid semi-automated bioassay of gentamicin based on luciferase assay of bacterial adenosine triphosphate. Scand. J. Infect. Dis. 9:232-236, 1977; and Thore, A., L. Nilsson, H. Hover, S.

Ansehn and L. Bröte. Effects of ampicillin on intracellular levels of adenosine triphosphate in bacterial cultures related to antibiotic susceptibility. Acta Path. Microbiol. Scand. Sect. B., 85:161-166, 1977).

In these methods the drop of intracellular ATP level after 2-3 hour incubation is correlated to the concentration of the antibiotic in the sample. ATP values of the samples are compared to those of known standards to calculate the levels of antibiotic 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 prevented from multiplying. In the latter case, the bacterial cells cannot produce their protective cell wail and ATP can leak out of the cells into the growth medium. This extracellular ATP is destroyed in the method by means of incubating samples with an ATPase enzyme (such as potato apyrase) that break down ATP. Before extraction and measurement of intracellular ATP, the ATPase activity is destroyed by means of heating, strong mineral acids or other means.

As to antibiotic levels in milk and other foodstuff, antibiotics enter milk and other foodstuff when animals are treated for microbial infections, such as bovine mastitis (inflammation of udder) or when the animal feed contains antibiotics as preventive medicine. Since uncontrolled use of antibiotics in food may produce strains of microbes that develop resistance toward antibiotics, cause allergic reactions in human beings or prevent beneficial microbes from growing in food processing (in cheese making, yogurt fermentation, etc.), most countries have strict controls on the levels of antibiotics in milk, meat and other foodstuff. The current tests apply the inhibition effect of antibiotics on the growth of a specific strain of bacteria which is susceptible toward the assayed antibiotic. Some methods utilize a liquid culture medium which is inoculated with a specific strain of bacteria.The growth of bacteria is monitored by means of increasing turbidity or by the change in the colour of a pH indicator dye due to the metabolic products of the growing bacteria. The growth or absence of growth is compared to that of a set of known antibiotic standard samples.

Second method is to inoculate a semisolid medium uniformly with the test bacteria and place an absorbent paper disc on the surface. The samples and antibiotic standards are then let to absorb on the paper discs. During the incubation the antibiotic agents diffuse into the agar medium and prevent the bacteria from growing around the disc. The higher the antibiotic level is in the sample or the standard, the wider the clear zone around the disc. The concentration of antibiotic in the sample is estimated by comparing the diameter of the clear zone void of bacteria to those of known standards. The strains of bacteria used for these tests are highly susceptible to the assayed antibiotic and these strains grow very rapidly as they can be incubated at elevated temperatures up to 650C.Such strains include species of Bacillus stearothermophilus var. calidolactoris, Pseudomonas thermophilus 101, Bacillus subtilis ATCC 6633, etc.

The growth of these organisms can be visually observed within 2 to 4 hours after starting of incubation.

A third method of estimating the concentration in milk is based on the change of pH of the growth medium. A pH indicator dye in the medium changes colour with the lowering of pH due to acidic metabolites produced by growing bacteria. In this method the sample is added to the medium containing the pH indicator and inoculated with the test bacteria sensitive to the antibiotic agent assayed. Samples are incubated 2-3,5 hours at elevated temperatures, normally at 650C. After the incubation the colour of the samples are compared to a colour chart and a control. Samples where the original colour has changed indicate that antibiotics in the sample have prevented the bacteria from growing. This test is semiquantitative. The sensitivity is about 0.005 International Units of penicillin per millilitre milk.

A fourth method of testing penicillin in milk is based on the growth inhibition of bacterial spores from growing in sterilized milk in presence of penicillin in the samples. The growth of bacteria was monitored by means of bioluminescent measurement of ATP (D.C. Westoff and T. Engler. Detection of penicillin in milk by bioluminescence. J. Milk Food Technol. 38: 537-39, 1 975). In this method milk samples were inoculated with 1% or 5% levels of Bacillus subtilis ATCC 6633 spores. The concentration of ATP was measured after 2 or 3 hours and the values of samples compared to control and standards. The sensitivity was 0.03 with 1% inoculum and 0.01 International Units with 5% inoculum after 3 hours incubation.

Measurement of ATP with firefly bioluminescence is a well known method per se. Bacteria have a relative constant concentration of ATP, thus the bioluminscent measurement of ATP has been utilized for rapid estimations of viable bacteria in various types of samples, e.g. in urine samples. Likewise, the measurement of the growth of bacterial culture with ATP has been utilized in determination of antibiotic levels in serum and milk. The principle of these methods is to estimate the inhibitory effect of antibiotics on the growth of bacteria as compared to control samples without antibiotics. Estimation of growth in these methods is based on the measurement of total ATP or intracellular ATP. In these methods the bacterial culture requires 2-3 hours of incubation at the minimum in order to quantitize the effect of the antibiotics.It has been reported that in the presence of antibiotics bacteria release ATP to the growth medium or sample solution. (Thore, A., L. Nilsson, H. Höjer, S. Ansehn and L. Bröte.

Effects of ampicillin on intracellular levels of adenosine triphosphate in bacterial cultures related to antibiotic susceptibility. Acta Path.Microbiol.Scand.Sect. B 85: 161--166, 1977). This phenomenon is more pronounced with bacteristatic antibiotics which will not kill bacteria but prevent the protective cell wall from developing and thus prevent bacteria from multiplying, than with bacteriocidal antibiotics that kill bacteria.

It is the object of the invention to provide an improved method for antibiotic testing which is rapid, sensitive and quantative and is thus advantageous over the present methods.

According to the invention there is provided a method of measuring antibiotic levels in a medium by means of measuring the initial increase in the adenosine of a test microbial culture due to a change of the metabolic activity of test organism in the presence of the said antibiotic substance.

The invention relates to a method where the measurement of extracellular ATP or total ATP is used to estimate the antibiotic levels in serum, milk and other samples or the antibiotic susceptibility as well as minimum inhibitory concentration of antibiotics.

The method according to the invention is based on the fact that when bacteria or other microbial cells are exposed to antibiotics their cell wall is damaged or its permeability is altered so much that ATP and other relatively small molecules can leak out of the cells. As the turnover of ATP in bacterial cells is less than one second (Chapman, A. G. 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 Microbial Physiology (Rose, A. H. and D. W. Tempest, Eds.). Viol. 15. Academic Press, London and New York, 1 977) great quantities of ATP is produced by cells over time.If even a small percentage of ATP leaks out of the cell, the extracellular ATP concentration can in few minutes exceed that of intracellular 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 of bacteria increases under the stress by antibiotics. Therefore, the initial increase in intracellular ATP can be utilized for the estimation of antibiotic levels or the antibiotics susceptibility in various applications.

Principle and examples of utilization of initial increase in extracellular ATP are given below.

A. Method of Measuring Antibiotic Levels in a Sample with Extracellular ATP During test carried out to determine the levels of gentamicin in serum the levels of extracellular ATP increased with increasing concentration of gentamicin in the sample while the intracellular and total ATP decreased during the first 3 hours of incubation (Figure 1): Test was performed as following: 1. 50-200 yl of overnight culture of gentamicin specific bacteria, such as E. coll(LU14) is pipetted to sterile test tubes (106--107) cells).

2. 200--1000 ul sterile nutrient broth is added to each tube.

3. 10--100 ul of sample (serum, gentamicin standard or other gentamicin sample) is then added.

4. The sample is incubated at 370C for 60--120 minutes.

5. 10-200 ,uI of sample is pipetted to a measuring cuvette and free ATP measured firefly luciferin-luciferase reagent in a photometer. When serum samples were measured for gentamicin these were first heated to 560C for 30 minutes to destroy the bacteriocidal effect of serum itself.

Extracellular ATP increases over time (Table 1) and measurement can be done between 10-120 minutes, but preferably between 60-120 minutes.

Table 1 Concentration of extracellular ATP as function of incubation time. Gentamicin concentration was 10 ng/cm3 of sample (see Figure 2).

Concentration of extracellularA TP Incubation time Relative Light Units 0 0.04 20 0.05 60 0.25 90 0.65 120 1.23 B. Measurement of Antibiotic Levels in Food Stuff with the Initial Increase of Total ATP THe method was tested with different concentrations of penicillin in milk using Streptococcus thermophilus 101 as test organism and 650C as incubation temperature.

The total amount of ATP increased rapidly in the samples containing penicillin (Figure 3) and the values of total ATP after 1 0-35 minutes incubation correlated with the concentratipn of penicillin (Figure 4).

Test was performed as following: 1. To 2.5 cm3 fresh milk, 1 cm3 overnight culture of Streptococcus thermophilus 101 or other bacteria, sensitive to tested antibiotics, is added.

2. The sample is incubated at 370C for 10-40 minutes, preferably for 20-30 minutes.

3. An aliquote of 100 yl is taken and ATP extracted from cells by boiling tris (0.02 M)-EDTA (0.002 M) buffer, mineral acids, etc. being known methods used for extracting ATP from bacteria, but preferably there is used 100 yI by Nucleotide Releasing Reagent for Microbial Cells (NRB, registered trade mark of Flu mac Systems AG, Basel.) and mixed through shaking by hand for five seconds, NRB release.

ATP within a few seconds and samples can be measured without any neutralizing, cooling or other further treatment. The sample is placed in a luminescent photometer and 10-1000 yI, preferably 50-200 ,uI firefly lucifern-luciferase reagent is injected and the light intensity as relative light units recorded.

4. A set of standards of antibiotic in milk, treated in the same way as the samples, is measured.

5. The concentration of antibiotics is calculated from the standard curve.

Figure 3 shows the behaviour of total ATP in the milk samples as a function of time. Due to the antibiotics present in the milk the ATP concentration in the bacterial inoculum increases rapidly during the incubation within 10 to 60 minutes as compared to the control sample not containing antibiotics.

The concentration of total ATP increases linearly with the increasing concentration of ATP when the samples are measured after 10-60 minutes of inoculation (Figure 3).

The present invention of utilizing the initial increase of free ATP for testing bacteriocidal antibiotic levels, and total or free ATP for testing bacteriostatic is a rapid, simple and quantitative method. The instrumentation required is also simple and inexpensive, thus the invention provides several advantages over conventional methods.

Claims (6)

Claims

1. A method of measuring antibiotic levels in a medium by means of measuring the initial increase in the adenosine level of a test microbial culture due to a change of the metabolic activity of test organism in the presence of the said antibiotic substance.

2. The method of Claim 1 wherein the change of adenosine triphosphate in the test organism is measured by means of firefly bioluminescence system.

3. The method of Claim 1 or Claim 2 wherein the antibiotic level is measured in serum or other body fluid.

4. The method of Claim 3 wherein the antibiotic is selected from gentamycin, other amifloglycoside and other toxic antibodies whose level in the body has to be monitored.

5. The method of Claim 1 or Claim 2 wherein the antibiotic level is measured in milk, meat and other food products.

6. A method as claimed in Claim 1, substantially as described.