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Examples of Antibiotic Sensitivity Testing Methods

1. DILUTION METHODS


The Broth dilution method involves subjecting the isolate to a series of concentrations of antimicrobial agents in a broth environment.   Microdilution testing uses about 0.05 to 0.1 ml total broth volume and can be conveniently performed in a microtiter format.  Macrodilution testing uses broth volumes at about 1.0 ml in standard test tubes.  For both of these broth dilution methods, the lowest concentration at which the isolate is completely inhibited (as evidenced by the absence of visible bacterial growth) is recorded as the minimal inhibitory concentration or MIC.  The MIC is thus the minumum concentration of the antibiotic that will inhibit this particular isolate.  The test is only valid if the positive control shows growth and the negative control shows no growth. 

A procedure similar to broth dilution is agar dilution.  Agar dilution method follows the principle of establishing the lowest concentration of the serially diluted antibiotic concentration at which bacterial growth is still inhibited. 

 

2-23-DISK-DIFFUSION.jpgOn this agar plate, a bacterial isolate is tested for resistance to each of twelve different antibiotics.  The clear zones around each disc are the zones of inhibition that indicate the extent of the test organism’s inability to survive in the presence of the test antibiotic. (A)The disk shows a large zone of inhibition; whereas (B) shows no zone of inhibition, indicating resistance of the isolate to the test antibiotic  

Presence of zone of inhibition is not automatically interpreted as susceptibility to the antibiotic; the zone width has to be measured and compared against a reference standard which contains measurement ranges and their equivalent qualitative categories of susceptible, intermediately susceptible or resistant.

2-24-DISK-DIFFUSION-CLOSE-U.jpgFor example, this E.coli isolate on the right has a zone of inhibition of 10.1mm around ampicillin (AM); since the zone diameter interpretation chart is as follows:
Resistant: 13mm or less
Intermediate: 14-16 mm
Susceptible: 17 mm or more
This particular E.coli isolate is read as resistant to ampicillin.

2. DISK DIFFUSION METHOD


Because of convenience, efficiency and cost, the disk diffusion method is probably the most widely used method for determining antimicrobial resistance in private veterinary clinics.  

A growth medium, usually Mueller-Hinton agar, is first evenly seeded throughout the plate with the isolate of interest that has been diluted at a standard concentration (approximately 1 to 2 x 108 colony forming units per ml).  Commercially prepared disks, each of which are pre-impregnated with a standard concentration of a particular antibiotic, are then evenly dispensed and lightly pressed onto the agar surface.  The test antibiotic immediately begins to diffuse outward from the disks, creating a gradient of antibiotic concentration in the agar such that the highest concentration is found close to the disk with decreasing concentrations further away from the disk. After an overnight incubation, the bacterial growth around each disc is observed.  If the test isolate is susceptible to a particular antibiotic, a clear area of “no growth” will be observed around that particular disk. 

The zone around an antibiotic disk that has no growth is referred to as the zone of inhibition since this approximates the minimum antibiotic concentration sufficient to prevent growth of the test isolate.  This zone is then measured in mm and compared to a standard interpretation chart used to categorize the isolate as susceptible, intermediately susceptible or resistant.  MIC measurement cannot be determined from this qualitative test, which simply classifies the isolate as susceptible, intermediate or resistant.

 

 

3. E-TEST


E-test (AB Biodisk, Solna, Sweden) is a commercially available test that utilizes a plastic test strip impregnated with a gradually decreasing concentration of a particular antibiotic.  The strip also displays a numerical scale that corresponds to the antibiotic concentration contained therein. This method provides for a convenient quantitative test of antibiotic resistance of a clinical isolate.  However, a separate strip is needed for each antibiotic, and therefore the cost of this method can be high. 

4. AUTOMATED ANTIMICROBIAL SUSCEPTIBILITY TESTING SYSTEMS


Several commercial systems have been developed that provide conveniently prepared and formatted microdilution panels as well as instrumentation and automated reading of plates.  These methods are intended to reduce technical errors and lengthy preparation times.

Most automated antimicrobial susceptibility testing systems provide automated inoculation, reading and interpretation.  These systems have the advantage of being rapid (some results can be generated within hours) and convenient, but one major limitation for most laboratories is the cost entailed in initial purchase, operation and maintenance of the machinery.  Some examples of these include: Vitek System (bioMerieux, France), Walk-Away System (Dade International, Sacramento, Calif.), Sensititre ARIS (Trek Diagnostic Systems, East Grinstead, UK), Avantage Test System (Abbott Laboratories,  Irving, Texas), Micronaut (Merlin, Bornheim-Hesel, Germany), Phoenix  (BD Biosciences, Maryland) and many more. 

 

 

5. MECHANISM-SPECIFIC TESTS


Resistance may also be established through tests that directly detect the presence of a particular resistance mechanism.  For example, beta lactamase detection can be accomplished using an assay such as the chromogenic cephalosporinase test (Cefinase disk by BD Microbiology Systems, Cockeysville, MD and BBL DrySlide Nitrocefin, Becton Dickinson, Sparks, MD) and detection for chloramphenicol modifying enzyme chloramphenicol acetyltransferase (CAT) may utilize commercial colorimetric assays such as a CAT reagent kit (Remel, Lenexa, Kansas).

 

 

6. GENOTYPIC METHODS


Since resistance traits are genetically encoded, we can sometimes test for the specific genes that confer antibiotic resistance.  However, although nucleic acid-based detections systems are generally rapid and sensitive, it is important to remember that the presence of a resistance gene does not necessarily equate to treatment failure, because resistance is also dependent on the mode and level of expression of these genes11.

Some of the most common molecular techniques utilized for antimicrobial resistance detection are as follows

Polymerase chain reaction (PCR) is one of the most commonly used molecular techniques for detecting certain DNA sequences of interest.  This involves several cycles of denaturation of sample DNA, annealing of specific primers to the target sequence (if present), and the extension of this sequence as facilitated by a thermostable polymerase leading to replication of a duplicate DNA sequence, in an exponential manner, to a point which will be visibly detectable by gel electrophoresis with the aid of a DNA-intercalating chemical which fluoresces under UV light.

DNA hybridization. This is based on the fact that the DNA pyrimidines (cytosine and thymidine) specifically pair up with purines (guanine and adenine; or uracil for RNA).  Therefore, a labeled probe with a known specific sequence can pair up with opened or denatured DNA from the test sample, as long as their sequences complement each other.  If this “hybridization” occurs, the probe labels this with a detectable radioactive isotope, antigenic substrate, enzyme or chemiluminescent compound.  Whereas if no target sequence is present or the isolate does not have the specific gene of interest, no attachment of probes will occur, and therefore no signals will be detected.  

Modifications of PCR and DNA hybridization.  With these basic principles, several modifications have been introduced which further improve the sensitivity and specificity of these standard procedures.  Examples of such development were the use of 5’-fluorescence-labeled oligonucleotides, the development of molecular beacons, development of DNA arrays and DNA chips, among many others. 


 

 

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Organizations Publishing

AST STANDARDS28

 

United States: Clinical and Laboratory Standards institute (CLSI; formerly NCCLS)

www.clsi.org

 

European Union: European Committee of Antimicrobial Susceptibility Testing (EUCAST)

www.eucast.org

 

European Union: Office International des Èpizooties (OIE)

www.oie.int

 

United Kingdom: British Society for Antimicrobial Chemotherapy

www.bsac.org.uk

 

Germany: Deutsches Institut für Normung

www.beuth.de  

 

France: Société Française de Microbiologie

www.sfm.assoc.fr

 

Sweden: Swedish Reference Group for antibiotics

www.srga.org

 

Australia: CDS disk diffusion method

www.med.unsw.edu.au/pathology-cds