False susceptibility of enterococci to aminoglycosides with blood-enriched Mueller-Hinton agar for disk susceptibility testing.

Disk diffusion susceptibility tests for enterococci are frequently modified by adding 5% sheep blood (SB) to Mueller-Hinton agar; the performance standards from the National Committee for Clinical Laboratory Standards sanction this addition. Susceptibility testing of aminoglycoside antibiotics is not recommended for enterococci; in actual practice, however, some laboratories do include aminoglycoside antibiotics routinely, and others may test upon request or in selected situations. In examining 50 clinical isolates of enterococci, SB-enriched Mueller-Hinton agar frequently gave enlarged zone sizes that falsely indicated susceptibility (72% for gentamicin and tobramycin), with the average increase in zone size being 6.3 and 7.6 mm, respectively. Comparison agar dilution MICs demonstrated uniform resistance, with or without added SB. The effect was shown to be caused by heme in concentrations as low as 0.03 micrograms/ml, which, when combined with aminoglycoside antibiotics, caused a synergistic growth inhibition of the enterococci, resulting in larger aminoglycoside antibiotic zones. We postulate that the heme effect is related to a catalytic cleavage of intracellular H2O2 and resultant lipid peroxidation. No other organism or antimicrobial agent tested demonstrated a similar effect, although other investigators have shown a similar phenomenon with the broad-spectrum cephalosporins. Because enterococci grow well and give accurate susceptibility results on Mueller-Hinton agar without SB supplementation and because of the spectrum of definable problems with a number of antimicrobial agents, we recommend that enterococci routinely be tested without SB.