Antibiotic disk susceptibility assays

To prepare for this assay, the medium is typically inoculated either by adding 

Another reason for careful interpretation of the disk-diffusion assay is that it is subject to false-positive results that can be misinterpreted as antibiotic activity. For example, physical characteristics of the extract (viscosity, pH, etc.) can generate small zones of growth inhibition when bacteria are inoculated directly onto the surface of the agar plate. In addition, we have observed that some primary metabolites can inhibit growth when tested at high concentrations. It is also possible that simple molecules, or extract degradation products, can exhibit mild antibiotic properties. For these reasons, it is important that replicate extracts are tested and that small zones of inhibition are interpreted with caution. It is also important to clearly state the concentrations tested, even if naturally occurring concentrations are not known, so that activities can be reproduced and evaluated at a later time. 

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Figure 1.1. Representation of a typical disk susceptibility assay. A paper disk impregnated with a suspected antibiotic compound is applied to an agar plate seeded with a test microorganism and the area of inhibited microbial growth is indicated by a cleared zone surrounding the disk.

It is very important to select promptly the most effective antibiotic for successful therapy of infectious diseases, and wound and post-surgical infections. The duration of standard microbiology assays applied in clinical practice exceeds 3-5 days since preliminary isolation of the pathogen from the clinical sample is required. In the present study we optimized a rapid bio luminescent antibiotic susceptibility assay based on comparison of bacterial ATP concentrations (bioluminescent signals) in a control (aliquot of the sample, free of the antibiotic) and probe (aliquot of the sample, supplied with antibiotic examined) after short-time incubation. For validation of the proposed assay, bacteria strains isolated from clinical samples were analyzed in parallel by the Bioluminescent Assay and Standard Microbiology Assay - Disk Method or Serial Dilutions Method. 

Bioluminescent Assay (BA) of antibiotic susceptibility. Bacteria stains were isolated from clinical sample and pure cultures of pathogens were obtained. Suspension of the each pure culture in saline was prepared and bacteria titer was established by the McFarland density standard. Bacteria suspension was diluted with rich nutritive media 102-104 times. Aliquots (0.2 or 1 mL) were pipetted into the cells of multidish supplied with disks of antibiotics examined (probes) and free of antibiotic (control). The multidish was incubated at 37 °C up to 12 h. After incubation 0.02 mL of probes and control were transferred using multi-channel pipette into the cells of 96-well strip plate filled with 0.18 mL of DMSO. The content of cells was mixed well and bacterial ATP-extracts were obtained. 

Standard Microbiology Assay of antibiotic susceptibility. Bacteria suspensions in saline ( 108 cell/mL) were assayed by Disk Method (DM) or Serial Dilutions Method (SDM) using automated analyzer Vitek (bio Meriuex, France). 

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