Accurate and easy determination of minimum inhibitory concentration with M.I.C.Evaluators

The ability of bacteria to adapt in response to selective pressure is an increasing challenge for clinical microbiologists. While it cannot be denied that the discovery and mass production of antibiotics has been a significant advance in improving the health and life expectancy of humans and animals, it is ironic that the very use of such agents actively selects for the proliferation of resistant strains within the bacterial population. In fact, it was only four years after penicillin became widely available that resistant strains of Staphylococcus aureus began to appear and, unfortunately, similar developments have been witnessed with other organisms and many new compounds.

To further complicate matters, resistance is often easily transferable, both within and between species, making it difficult to predict accurately the response of bacteria to antibiotics. Some organisms, known as multidrugresistant strains, have even acquired resistance to a number of different antibiotic groups.

The role of the microbiology department, therefore, is not only to identify the organisms responsible for serious infections, but also to assess their susceptibility to a range of antibiotic agents and to provide information that allows clinicians to predict accurately the bacteriological response to, and clinical outcome of, antibiotic therapy. The administration of targeted antibiotic therapy, at the correct dose and at the earliest opportunity, will significantly improve the prognosis for the patient.

Susceptibility testing methods

Antimicrobial susceptibility testing (AST) is one of the most important tasks carried out in clinical microbiology laboratories today. Methods for performing this function have been refined and standardised over the years and there now exist a number of methods by which the susceptibility of organisms to antibiotics can be determined reliably.

Disc diffusion

The disc-diffusion method is one of the most widely used susceptibility testing methods worldwide. It is a simple and relatively low-cost technique with visual controls and complete flexibility in the compounds selected for testing. Antibiotic-impregnated paper discs are placed on pre-inoculated agar media, such as Oxoid Iso-Sensitest agar or Oxoid Mueller Hinton agar, which are designed to support the growth of a wide range of microorganisms but not to interfere with the activity of the antibiotic compounds.

During incubation, the antibiotics diffuse from the discs into the surrounding agar, forming a concentration gradient to which the test organism is exposed. The organism is categorised as sensitive, intermediate or resistant to the antibiotics tested according to the size of the zone of inhibition surrounding each disc (Fig 1).

As a visual technique, disc diffusion offers a number of advantages over some alternative methods:

• it gives a clear indication of inoculum level (a key factor for influencing results)

• contamination, which can confuse results in other methods, is readily visible on the plate

• b-lactamase activity is relatively easy to visualise

• interactions between antibiotics in adjacent discs, such as synergy or antagonism, are easily seen

• presence of resistant mutants in the inoculum can be detected.

In order to obtain accurate and reproducible results, however, it is important to standardise variables such as inoculum density, medium (eg pH, depth and composition), incubation (eg time, temperature and atmospheric conditions) and disc content. Guidelines for standardisation are provided by internationally recognised bodies such as the Clinical Laboratory Standards Institute (CLSI), the British Society for Antimicrobial Chemotherapy (BSAC) and Deutsches Institut für Normung (DIN).

Disc diffusion is a valuable and highly flexible method of susceptibility testing, but it is purely qualitative and there are certain clinical situations in which quantitative result may be required (see below). In addition, some compounds diffuse poorly through the medium and there is little correlation between the zones of inhibition and the minimum inhibitory concentration (MIC), causing some resistance mechanisms to go undetected (eg glycopeptide resistance). For these reasons, disc diffusion is often supplemented with another AST technique for specific problem situations.

Breakpoint testing

The breakpoint testing method is a popular technique in larger laboratories because of its suitability for multiple sample testing. It is performed by adding a defined concentration of antibiotic to an agar medium. The test organism is inoculated and, following incubation, is determined to be sensitive or resistant to that level of compound according to the growth (or lack of growth) observed. The ease of standardisation and automation of this method makes it ideal for multiple sample testing if a multipoint inoculator is used.

As the antibiotic is evenly distributed throughout the agar, this method does not present a problem for poorly diffusing compounds. However, it is difficult to detect contamination or to assess inoculum density, due to the small amount of sample that is present. Problems may also be encountered with compounds that are unstable in solution – requiring small batches of plates to be prepared as required – and the presence of a swarming Proteus sp. potentially can destroy multiple test results. The information provided by the breakpoint testing method is relatively limited (ie the result is ‘sensitive’ or ‘resistant’) and subtle changes in susceptibility may not be detected. Thus, other techniques are usually employed when more detailed information is required.

Automated broth microdilutions

The appeal of speed and automation has made rapid automated susceptibility testing increasingly popular around the world. Defined levels of antibiotic are stabilised in microtitre wells covering two or three concentrations, usually around the breakpoint where the test organisms either grow or are inhibited by the compound. Specimens are added in liquid medium, and bacterial growth is measured either by detecting turbidity or by the hydrolysis of fluorogenic substrates, depending on the system in use.

These systems offer the advantages of speed, reproducibility and automated reporting. The panels of agents available are extensive; however, the flexibility to change the content of a panel is limited and costly. Furthermore, the updating of panels and software with new compounds or amended breakpoints can be extremely time-consuming, due to validation and registration requirements.

Although the performance of automated broth microdilution systems generally correlates well with reference methods, there are some limitations with certain organism/antibiotic combinations, often relating to short incubation times. For example, inducible resistance mechanisms need time to express resistance before it can be detected. In addition, there is concern that broth dilution techniques are less likely to reflect the clinical situation, as bacteria in vivo will normally multiply on solid surfaces. Consequently, bacteria grown on solid media are more likely to behave similarly to bacteria in vivo. This may be significant for organisms such as Pseudomonas aeruginosa from cystic fibrosis patients. Colonies of this organism often produce a mucoid alginate on agar, but not in broth. This lack of alginate production in a broth can make the organism appear more susceptible than it is in reality, as compounds may be deterred in vivo by the exudate.

Minimum inhibitory concentration testing

The MIC is the minimum concentration of an antibiotic required to inhibit the growth of the test organism over a specified time (usually overnight, but this depends on the growth rate of the organism). Unlike other AST methods, MIC testing generates a quantitative result. This may be of greater value in certain clinical situations or in research settings, for example to:

• guide therapy when low-level resistance is suspected           

• guide therapy in critically ill patients with potentially fatal infections (eg septicaemia, meningitis, pneumonia and endocarditis) or in patients with inaccessible/deep-seated infections

• monitor the development of resistance during therapy

• confirm borderline/unusual results by other methods

• use as an epidemiological tool to validate empirical therapy

• determine in vitro activity of new antimicrobial agents.

Conventional methods for MIC determination are usually performed using a range of doubling dilutions incorporated in agar or broth microdilutions. In-house media preparation for these methods is tedious, time-consuming and subject to errors. Although products are available commercially for traditional MIC testing methodology, there is a more simple and convenient alternative, utilising an antibiotic gradient stabilised on a polymer strip.

Enter M.I.C.Evaluators

New Oxoid M.I.C.Evaluators (M.I.C.E.) combine the simplicity and ease of use of the diffusion method with the accuracy of an MIC test. Each polymer strip provides a gradient of stabilised antimicrobial agent, covering 15 doubling dilutions, to give an accurate MIC over the range 256 µg/mL to 0.016 µg/mL (specialist high- and low-level concentration strips are also available for some compounds).

The thickness of the M.I.C.E. strip makes it easy to handle as it is applied to a pre-inoculated agar plate (Fig 2). Furthermore, this helps to minimise the incidence of bubbles trapped under the strip and reduces slipping on the surface of the agar.

On application, the antimicrobial agent is released into the agar, forming a defined concentration gradient in the area around the strip. After appropriate incubation, a zone of inhibition will have formed around the M.I.C.E. strip. An accurate MIC is easily read as the value in the graduated box where the growth of the organism touches the strip (Fig 3). This box format of the scale reduces subjectivity in interpretation of the result.

Each M.I.C.E. strip is individually foil wrapped with desiccant to maintain its integrity until use. This ensures that the quality and performance of unused strips are preserved, and is particularly useful for storing strips in different locations. Once the sachet is peeled apart, the handle of the M.I.C.E. strip is presented for easy extraction/application (Fig 4). All M.I.C.E. strips are stored at 2–8°C and are supplied in durable, stackable boxes of 10 or 50 strips.

The accurate MIC value that is available using M.I.C. Evaluators is invaluable in the numerous clinical and research settings mentioned above. This method is simple, precise and easy to implement in any routine microbiology laboratory, without the need for investing in additional complicated equipment.

For further information and full details of the range available, contact the Oxoid office (tel +44 [0] 1256 841144, fax +44 [0] 1256 329728, email [email protected]) or visit the Oxoid website (www.oxoid.com).