Antimicrobial Susceptibility Testing

Summary

Antimicrobial susceptibility testing (AST) is performed by the growth of an isolate in the presence of a given antibiotic. There are a number of methods available for AST. In 2010 the NSSLRL changed from using disk diffusion to using the TREK Sensititre broth minimum inhibitory concentration (MIC) method for routine susceptibility testing of Salmonella and Shigella isolates. The NSSLRL changed it's interpretative criteria, on April 1st 2010, from those of the Clinical Laboratory Standards Institute (CLSI) to the European Committee on Antimicrobial Susceptibility Testing (EUCAST).

Purpose of examination

Antimicrobial susceptibility testing in the NSSLRL is used as an epidemiological marker for typing purposes.

Principle of examination

Disk diffusion

Antibiotic-impregnated paper disks are placed on the surface of an agar plate which has been seeded with the isolate being tested. If the organism is susceptible to the antibiotic tested its growth will be inhibited and a zone of inhibition will result around the antibiotic disk. The diameter of the zone of inhibition of growth is proportional to the MIC value. The zone size is measured in mm, the value is compared to the interpretive criteria developed by EUCAST. The isolate is assigned a sensitive, intermediate or resistant category for each antibiotic after comparison with the appropriate table.

Sensititre

Broth microdilution involves growth of bacterial isolates in broths with different concentrations of antibiotics. The lowest concentration of antibiotic that prevents growth is called the MIC. Broth microdilution methods are considered superior to disk diffusion as they provide a quantitative measurement of antibiotic resistance.

Specimen requirements

A pure culture of the isolate freshly grown on non-selective agar slope. 

Antibiotic codes and interpreting resistance patterns

The majority of published material (journals, reports etc) with reference to antibiotic resistance use a relatively standard set of abbreviations for antibiotics.  The NSSLRL uses a panel of 13 antibiotics for its sensitivity testing.  Concentrations of antibiotics are as per EUCAST standards.  The antibiotics and their abbreviations ( ) are as follows:

Ampicillin (A); Chlorampenicol (C); Streptomycin (S); Sulphonamides (Su); Tetracycline (T); Trimethoprim (Tm); Nalidixic acid (Na); Kanamycin (K); Ciprofloxacin (Cp); Ceftazidime (Cz); Gentamycin (Gm) and Cefotaxime (Ctx). 

Antibiotic resistance patterns are frequently reported in their abbreviated form, so for example the resistance pattern ACSSuT (also known as an antibiogram) indicates resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline.  Likewise TTm would indicate resistance to tetracycline and trimethoprim. 

Extended Spectrum b-Lactamase (ESBL) producing isolates

The CLSI have criteria for the identification of bacteria that are ESBL producers.  In testing for antimicrobial susceptibility one must be aware that there are two sets of criteria established for the interpretation of susceptibility to third generation cephalosporins, one for general interpretation and one for ESBL identification. A difference of 5mm in the zone diameter between the cefpodoxime disc (top) and the cefpodoxime clavulanic disc (bottom) indicates that this isolate is an ESBL producer. 

We have found that the use of cefpodoxime in our antimicrobial testing panel whilst less specific is more sensitive for the detection of ESBL production than ceftazidime (Morris, D, PhD Thesis, National University of Ireland, Galway, 2002).

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