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Susceptibility Testing

Susceptibility Testing - the goal of antimicrobial susceptibility testing is to predict the in vivo success or failure of antimicrobial therapy. Tests are completed to measure the growth response of an organism to a specific antimicrobial. Automated antimicrobial susceptibility testing is performed using the Microscan system which is based on broth microdilution. The results are used to guide antimicrobial therapy and should always be combined with clinical experience and status of the patient in selecting appropriate antimicrobial therapy.

The results obtained from the Microscan system are based on the minimum inhibitory concentration (MIC). The MIC is defined as defined as the lowest concentration of the antimicrobial that completely inhibits growth of the organism. The MICs are established and published by the Clinical Laboratory Standards Institute (CLSI) and incorporate the organism and antimicrobial. For most organisms and antimicrobials, the MICs are reported as susceptible (S), intermediate (I) or resistant (R).

Susceptible (S) - implies that the isolate are inhibited by the usual achievable antimicrobial concentration when the recommended dose is used for the site of infection.

Intermediate (I) - implies that the isolate may or may not be inhibited by the usual achievable antimicrobial concentration when the recommended dose is used for the site of infection.

Resistant (R) - implies that the isolate will not be inhibited by the usual achievable antimicrobial concentration even when the recommended dose is used for the site of infection.

Figure 1 - demonstrates the broth dilution test in which bacteria are inoculated into tubes containing culture broth and varying concentration of the tested antimicrobial. The tubes are incubated to allow organism growth. The MIC is the lowest concentration of the antimicrobial that inhibits the growth of the organism. In the example, the MIC would be read as 8 mcg/mL.

Figure 1: MIC Example

Etest - refers to an agar based method that uses a predefined, stable gradient of 15 antimicrobial concentrations of decreasing concentrations of a plastic strip used to determine the MIC of antimicrobials. The Etest is mainly used for organisms for which there are no established CLSI breakpoints or for organisms that are not approved for use with the Microscan system. It is rapid and easy-to use and the MIC is read at the point where the organism crosses over the strip.

Figure 2 - demonstrates the Etest

Figure 2: Etest demonstration

Extended-spectrum beta-lactamase (ESBL) testing - ESBLs are enzymes produced in some Gram-negative organisms that confer resistance to penicillins, extended-spectrum cephalosporins and aztreonam. ESBLs are most commonly recognized in Klebsiella species and Escherichia coli (E. coli), but have recently been detected in a variety of Enterobacteriaceae and non-Enterobacteriaceae, including Pseudomonas species. Resistance mediated by ESBLs can be difficult to detect depending on the antimicrobial tested. Currently, CLSI recommends screening for ESBL activity using aztreonam, cefpodoxime, ceftazidime, cefotaxime and/or ceftriaxone. The presence of ESBL activity should be confirmed by testing the isolate with cefotaxime and cefotaxime plus clavulanic acid and ceftazidime and ceftazidiime plus clavulanic acid. If the disk containing cefotaxime (or ceftazidime)/clavulanic acid is 5mm in diameter greater than either cefotaxime (or ceftazidime) alone, it is considered a positive test. If an isolate is confirmed as an ESBL producer, all penicillins, cephalosporins and aztreonam are reported as resistant.

Figure 3 - ESBL test

Carbapenamase testing - infections with carbapenem-resistant Enterobacteriaceae (CRE) are emerging as an important challenge in healthcare settings. Currently, carbapenem-resistant Klebsiella species is the most commonly encountered in the United States, but the prevalence in other Enterobacteriaecae species is increasing. The gene that encodes the resistance enzyme is carried on a mobile piece of genetic material (plasmid) which increases the risk for dissemination. A difficult in detecting CRE is the fact that some strains that harbor the resistance enzyme have MICs that are elevated but still within the susceptible range for carbapenems. CLSI published a recommendation that carbapenem-susceptible Enterobacteriaceae with elevated MICs be tested for the presence of carbapenemases using the modified Hodge Test (MHT). The MHT is a phenotypic test used to detect carbapenemases in isolates demonstrating elevated but susceptible carbapenem MICs. Carbapenemase production is detected by the MHT when the test isolate produces the enzyme and allows growth of a carbapenem susceptible strain towards a carbapenem disk. The result is a characteristic cloverleaf-like indentation.

Figure 4 - Modified Hodge Test (MHT)

Figure 4: Modified Hodge Test (MHT)

Inducible clindamycin resistance - refers to a test performed on Staphylococcus species that test resistant to erythromycin and susceptible to clindamycin using routine antimicrobial susceptibility test methods. Resistance to macrolides can occur via two different mechanisms with the resulting phenotypes illustrated in the table.

Mechanism Determinant Erythromycin Clindamycin
Efflux msrA R S
Ribosome alteration erm R S
erm R R
msrA = macrolide/streptogramin resistance
erm = erythromycin ribosome methylase; confers resistance to lincosamide (clindamycin), macrolides (azithromycin, clarithromycin and erythromycin) and streptogramins (quinupristin/dalfopristin)

Inducible clindamycin resistance can be detected with a simple disk test, referred to as the "D test". For this test, an erythromycin disk is placed 15 mm to 26 mm from a clindamycin disk in a standard disk diffusion test. After incubation, a flattening of the zone in the area between the disks where both antimicrobials have diffused indicates that the organism has inducible clindamycin resistance and clindamycin should not be used during therapy.

Figure 5- Inducible clindamycin test ("D test")

Figure 5: Inducible clindamycin test (D test)