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

Abstract

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 ,ug/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 H202 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.

Multilaboratory evaluation of screening methods for detection of high-level aminoglycoside resistance in enterococci. National Committee for Clinical Laboratory Standards Study Group on Enterococci

“…As has been seen by others (16,32), MHA supplemented with sheep blood resulted in larger zones of inhibition than those obtained in unsupplemented agar, a condition which resulted in more false susceptibility, especially for streptomycin. A higher breakpoint could not be used because overlap among the susceptible and resistant strains would have resulted in the opposite condition, i.e., a high percentage of false-positive results for resistance to streptomycin.…”

“…In 1984, Pfaller et al (27) confirmed that disks containing the usual 10-g amounts of gentamicin and streptomycin could not distinguish susceptible strains from those with HLR. Jenkins et al (16) showed that when MHA is supplemented with sheep blood, zones of inhibition with 10-g aminoglycoside disks are about 6 mm larger than those when unsupplemented medium is used.…”

Use of the National Committee for Clinical Laboratory Standards Guidelines for Disk Diffusion Susceptibility Testing in New York State Laboratories

“…except that 31.6% of laboratories selected Mueller-Hinton agar supplemented with sheep blood as the test medium. Mueller-Hinton agar containing 5% sheep blood was considered acceptable in 1979 and 1983, but NCCLS subsequently recommended Mueller-Hinton agar without supplements when studies demonstrated that the blood in the agar interacted with aminoglycoside disks to yield false-positive results for susceptibility (16). The continued use of this medium by some laboratories raises concerns that outdated practices still occur in clinical laboratories.…”

High-content aminoglycoside disks for determining aminoglycoside-penicillin synergy against Enterococcus faecalis

“…zone sizes were correlated with the results obtained by the time-kill studies. In addition, because it has been shown in other studies (4,13) that the presence of blood can affect the in vitro susceptibility of enterococci to aminoglycosides, disk tests were performed on Mueller-Hinton agar with and without 5% sheep blood.…”