Antibiotic Sensitivity Testing - Establishing the Minimum Inhibitory Concentration

MICs can be determined by agar or broth dilution techniques by following the reference standards established by various authorities such as the Clinical and Laboratory Standards Institute (CLSI, USA), British Society for Antimicrobial Chemotherapy (BSAC, UK), AFFSAPS (France), Deutsches Institut f ür Normung e.V. (DIN, Germany) & ISC/WHO.

The broth dilution method depends upon inoculation at a specific inoculum density of broth media (in tubes or microtitre plates) containing antibiotics at varying levels - usually doubling dilutions are used and after incubation, turbidity is recorded either visually or with an automated reader, and the breakpoint concentration established. Microtitre plates or ready-to-use strips are commercially available with antibiotics ready prepared in the wells. A variation on this approach is the agar dilution method where a small volume of suspension is inoculated onto agar containing a particular concentration of antibiotic, when the inoculum has dried the plate is incubated and again examined for zones of growth.

Disk diffusion or the Kirby-Bauer test is one of the classic microbiology techniques and it is still very commonly used. A suspension of the isolate is prepared to a particular McFarland standard, then spread evenly onto an appropriate agar (such as Mueller-Hinton or for a more defined media Iso-Sensitest™ agar) in a petri dish, disks impregnated with various defined concentrations of different antibiotics are placed onto the surface of the agar. A multichannel disk dispenser can speed up placement of the disks. After incubation, a clear circular zone of no growth in the immediate vicinity of a disk indicates susceptibility to that antimicrobial. Using reference tables the size of zone can be related to the MIC and results recorded as whether the organism is susceptible (S), intermediately susceptible (I), or resistant (R) to that antibiotic.

There are a number of critical steps in this approach, such as which medium is used; depth and moisture content of the agar in the plate; incubation conditions; accurate inoculum density; disks must be firmly placed in contact with the agar surface otherwise the diffusion rate will not be correct.

A variation on this approach is to use a strip impregnated along its length, with a gradient of different concentrations of antimicrobial, after incubation this creates an ellipse shaped zone of no growth, where the ellipse meets the strip, the MIC can be read from the concentration markings on the strip. These are easy to read, no tables need to be referenced to get an MIC value and the test requires less manipulations, as one strip will cover the whole concentration range. These again can be manually or instrument read.

Whichever method is used, the result provides a key cutoff point which equates to the minimum inhibitory concentration of antibiotic for that test isolate, and methods initially require a pure culture to be prepared, which may take 1-2 days.

Pre-prepared antibiotic discs with full QC documentation provided by the manufacturer maintain reproducibility and considerably increases assay reliability. Discs should always be manufactured to an acceptable specification e.g. FDA, WHO, DIN. The DIN standard has the tightest range with antibiotic concentrations within 90%-125% of that stated.

Disk diffusion and broth dilution techniques can be semi-automated by using image analysers to read zones or turbidity readers for the broths, these give a more objective result and can come with software for automatically interpreting results.

For laboratories conducting a large number of assays there are fully automated systems which are widely used, these normally combine identification with sensitivity testing and as the whole test is set up and read automatically not only is the workload reduced but also the result is less subjective, more reproducible. Results are usually faster, with same day results possible as the instruments monitor growth by taking continuous readings and base results on growth kinetics. Whilst automated systems have many advantages, they can be less flexible in terms of the choice of antibiotics available, consumables costs are usually higher and equipment costs need to be met whether by outright purchase, leasing or reagent rental deals, together with service and maintenance charges.

For both the semi-automated zone readers and the fully automated ID and susceptibility systems, the data collected can be assessed by expert or smart software systems for interpretation, highlighting unusual anomalous results, suggesting other possible antibiotics to try and can be exported to other LIMS systems for further reporting.