Susceptibility Testing

Routine Testing (Kirby-Bauer)

Since enterococcal susceptibility to antimicrobial agents is unpredictable, the site of infection and/or the significance of a particular isolate determines which antimicrobials should be included in susceptibility testing. Drugs to which enterococci are intrinsically resistant, such as cephalosporins, oxacillin, trimethoprim-sulfamethoxazole (TMP-SMX) (in vivo resistance), clindamycin and aminoglycosides (when used as monotherapy), should not be tested. On the other hand, susceptibility to penicillin or ampicillin and vancomycin should be determined routinely. For urine isolates, fluoroquinolones, nitrofurantoin and fosfomycin may be added. Using the Kirby-Bauer technique, a 16 mm or less zone diameter of inhibition around ampicillin 10 |lg disks and a zone of 14 mm or less around penicillin 10 unit disks are considered resistant. A vancomycin (30 g disk) zone of inhibition of 14 mm or less is reported as resistant, 15-16 mm as intermediate and 17 mm or more as susceptible (plates should be held for a full 24-h period). It is recommended that any haze or colonies within the zone should be taken into account and that an MIC test be performed for strains with intermediate-susceptibility zones if vancomycin is to be used for treatment (Swenson etal., 1992).

Agar Dilution MICs

For agar dilution testing, interpretative criteria of the National Committee for Clinical Laboratory Standards (NCCLS) for ampicillin and penicillin are the following: MIC < 8 |g/ml, susceptible; MIC > 16 |g/ml, resistant (NCCLS, 2003). For vancomycin, MICs of <4 |g/ml and 8-16 |g/ml are considered susceptible and intermediate, respectively (the latter are considered resistant in some countries), whereas an MIC of vancomycin of >32 |g/ml is considered resistant (NCCLS, 2003). For the screening of VRE, NCCLS recommends the use of a BHI agar with 6 |g/ml of vancomycin and an inoculum of 1-10 |l of a 0.5 McFarland standard suspension. Growth after 24 h of incubation at 35 °C is interpreted as resistant.

Aminoglycoside HLR and ¡P-Lactamase Screening

For serious infections, particularly endocarditis and possibly for meningitis and deep-seated infections in immunocompromised patients, HLR to aminoglycosides and P-lactamase testing should be performed. HLR to gentamicin indicates resistance to synergism with all currently available aminoglycosides except streptomycin and, occasionally, arbekacin. MICs of arbekacin (available in Japan) are variable for strains with HLR to gentamicin. High-level aminoglycoside resistance can be detected by agar or single-tube broth screening with 500 | g/ml of gentamicin. For streptomycin, 2000 | g/ml and 1000 | g/ml is used for agar and broth screening, respectively. Disks containing 300 |g of streptomycin and 120 |g of gentamicin may also be used to predict synergy or the lack of synergy. E-Test also demonstrates concordance in the detection of HLR to aminoglycosides among enterococci when compared with agar dilution screening. Broth microdilution systems appear reliable for HLR to gentamicin, but they have missed some strains with HLR to streptomycin in the past. New versions of automated systems for susceptibility testing of enterococci display a high degree of correlation with standard methods, including VRE strains and strains with HLR to aminoglycosides (d'Azevedo etal., 2001; Ligozzi et al., 2002).

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