Microorganisms antibiotic assays

Microbiologically based assay systems invariably measure the active antibiotic(s) or forms of the antibiotic that can be inhibitory to microorganisms. Immunological assays can measure both the active antibiotic as well as microbiologically inactive species. 

R. R. Yocum T. A. Patterson, Microorganisms and Assays for the Identification of Antibiotics Acting on the Pantothenate Kinase Encoded by the coaX Gene. U.S. Patent 6,830,898, 14 December 2002. 

In recent years several reports have been published (24,25, 26) on microcalorimetric studies of the action of antibiotics on various microorganisms. The assay principle is that the total heat generated by the cells when fed an appropriate medium is compared with that registered when varying concentrations of the actual antibiotic are supplied. 

This assay, commonly referred to as the Charm Test, is based upon the affinity of antibiotics for specific sites on the cell wall of microorganisms and the irreversible binding of the antibiotic to these sites. By adding C-labelled or H-labelled antibiotic to a sample of milk, urine or the aqueous extract of tissues together followed by microbial binding sites and measuring the quantity of the labelled antibiotic that binds to the microbial sites, the antibiotic residue can be measured. 

For antimicrobial assays, there are several common methods employed. Due to its ease of operation, the most common method used is the disk diffusion method, which involves the application of a material onto a filter paper disk, and then the disk is placed onto solid medium previously seeded with the test microorganism of interest. Sometimes, the sample is dissolved in an appropriate solvent before application onto the paper disk. This method is very common in the evaluation of antibiotics and is the method adopted by the National Committee for Clinical Laboratory Standards (NCCLS). The method depends on the aqueous solubility of the antibiotics in order to facilitate diffusion through the solid medium. Essentials oils, however, are generally hydrophobic, do not readily diffuse through an aqueous medium and, therefore, the prevalence of false negatives or reduced activity might then be anticipated. 

The starting point in the technical development of a microbial inhibition assay is the sensitivity of the test microorganism to different antimicrobial compounds under different media conditions. Factors such as the pH and nutrient profile of the media can be varied to obtain the optimum performance. The sensitivity of MIAs may also be modulated by the addition of specific compounds to the test composition. For certain antimicrobial compounds an enhancement in sensitivity may be advantageous, whereas for others, a sensitivity decrease is required to avoid the number of false non-compliant results. For example, cysteine can reduce the overall sensitivity to the P-lactam class. Antifolates, such as ormethoprim or trimethoprim, are known to improve the sensitivity of the test organism to sulfonamide compounds. Sensitivity to sulfonamides can also be modulated by using the enzyme dihydropteroate synthetase that selectively inhibits the response of sulfonamides." Antibiotic receptors such as antibodies can also be employed to decrease the sensitivity of the test to specific compounds, as required. 

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