Use of Microorganisms and their Products in Assays

USE OF MICROORGANISMS AND THEIR PRODUCTS IN ASSAYS

Microorganisms have found widespread uses in bioassays for:

·                     determining the concentration of compounds (e.g. amino acids, vitamins and some antibiotics) in complex chemical mixtures or in body fluids

·                     diagnosing diseases

·                     testing chemicals for potential mutagenicity and carcinogenicity

·                     monitoring processes involving the use of immobilized enzymes

·                     sterility testing of antibiotics.

    A)  Antibiotic Bioassays

Although antibiotics may be assayed by a variety of methods, the following section will only take into consideration microbiological and radioenzymatic assays.

i)  Microbiological assays

In microbiological assays the response of a growing population of microorganisms to the antimicrobial agent under investigation is measured. The usual methods involve agar diffusion assays in which the drug diffuses into agar seeded with a susceptible microbial population producing a zone of growth inhibition.

In the commonest form of microbiological bioassay used today, samples to be assayed are applied in some form of reservoir (porcelain cup, paper disc or well) to a thin layer of agar seeded with indicator organism. The drug diffuses into the medium and after incubation a zone of growth inhibition forms, in this case as a circle around the reservoir. All other factors being constant, the diameter of the zone of inhibition is essentially related to the concentration of antibiotic in the reservoir.

During incubation the antibiotic diffuses from the reservoir and that part of the microbial population distant to the influence of the antibiotic increases by cell division. The edge of the area of microbial growth is formed when the minimum concentration of antibiotic that will inhibit the growth of the organism on the plate (critical concentration) reaches, for the first time, a population density too great for it to inhibit. The position of the zone edge is thus determined by the initial population density, the growth rate of the organism and the rate of diffusion of the antibiotic.

In situations where the likely concentration range of the tests will lie within a relatively narrow range (e.g. in determining potency of pharmaceutical preparations) and maximal precision is sought, then a Latin square design with tests and calibrators at two or three levels of concentration may be used. For example, an 8 × 8 Latin square can be used to assay three samples and one calibrator, or two samples and two calibrators at two concentrations each (over a twofold or fourfold range), with a coefficient of variation of around 3%. Using this technique, parallel dose–response lines should be obtained for the calibrators and the tests at the two dilutions (Figure 26.6). Using such a method, potency can be computed or determined from carefully prepared nomograms.

Conventional plate assays require several hours’ incubation and consequently the possibility of using rapid microbiological assay methods has been studied. Two such methods are:

•              Urease assay.

When Proteus mirabilis grows in a ureacontaining medium it hydrolyses the urea to ammonia and consequently raises the pH of the medium. This production of urease is inhibited by aminoglycoside antibiotics (inhibitors of protein synthesis). In practice, it is difficult to obtain reliable results by this method.

•              Luciferase assay.

In this technique, firefly luciferase (or similar enzyme) is used to measure small amounts of ATP in a bacterial culture, ATP levels being reduced by the inhibitory action of aminoglycoside antibiotics.

ii) Radioenzymatic (transferase) assays

Radioenzymatic assays depend on the fact that bacterial resistance to aminoglycosides, such as gentamicin, tobramycin, amikacin, netilmicin, streptomycin, spectinomycin, and chloramphenicol is frequently associated with the presence of specific enzymes (often coded for by transmissible plasmids), which either acetylate, adenylylate or phosphorylate the antibiotics, thereby rendering them inactive . Aminoglycosides may be susceptible to attack by aminoglycoside acetyltransferases (AAC), aminoglycoside adenylyltransferases (AAD) or aminoglycoside phosphotransferases (APH). Chloramphenicol is attacked by chloramphenicol acetyltransferases (CAT). Acetyltransferases attack susceptible amino groups and require acetyl coenzyme A, while AAD or APH enzymes attack susceptible hydroxyl groups and require ATP (or another nucleotide triphosphate).

Several AAC and AAD enzymes have been used for assays. The enzyme and the appropriate radio-labelled cofactor ([1-¹⁴C] acetyl coenzyme A, or [2-³H] ATP) are used to radiolabel the drug being assayed. The radiolabelled drug is separated from the reaction mixture after the reaction has been allowed to go to completion; the amount of radioactivity extracted is directly proportional to the amount of drug present. Aminoglycosides are usually separated by binding them to phosphocellulose paper, whereas chloramphenicol is usually extracted using an organic solvent.

These types of assay are rapid, taking approximately 2 hours, show good precision and are much more specific than microbiological assays.

B)  Vitamin And Amino Acid Bioassays

The principle of microbiological bioassays for growth factors such as vitamins and amino acids is quite simple. Unlike antibiotic assays that are based on studies of growth inhibition, these assays are based on growth exhibition. All that is required is a culture medium that is nutritionally adequate for the test microorganism in all essential growth factors except the one being assayed. If a range of limiting concentrations of the test substance is added, the growth of the test microorganism will be proportional to the amount added. A calibration curve of concentration of substance being assayed against some parameter of microbial growth, e.g. cell dry weight, optical density or acid production, can be plotted. One example of this is the assay for pyridoxine (vitamin B₆), which can be assayed using a pyridoxine-requiring mutant of the mould Neurospora. Using elegant study designs, it is possible to assay a variety of different growth factors with a single test organism simply by preparing a basal medium with different growth-limiting nutrients. Table 26.5 summarizes some of the vitamin and amino acid bioassays currently available. In practice however, high performance liquid chromatography (HPLC) has replaced bioassays as the method of choice for most amino acids and several B group vitamins.