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

Abstract

We investigated the use of high-content aminoglycoside disks for determining Enterococcus faecalis susceptibility to aminoglycoside-penicillin synergy. The susceptibility of the organisms to synergy was established by 24-h time-kill studies performed with streptomycin, kanamycin, amikacin, gentamicin, and tobramycin, alone and in combination with penicillin. A total of 20 isolates that were susceptible to all drug combinations and 20 strains that were resistant to each aminoglycoside-penicillin combination were selected for testing against high-content disks. Disk-agar diffusion was performed on Mueller-Hinton agar, with and without 5% sheep blood, by using disks that contained either 300 or 2,000 ,g of streptomycin and either 120 or 2,000 ,g of kanamycin, amikacin, tobramycin, or gentamicin. Zone size results obtained for each aminoglycoside, except amikacin, could be used to differentiate between synergy-susceptible and-resistant isolates. No overlap occurred between the zone sizes of susceptible and resistant strains. Susceptibility to amikacin-penicillin synergy could reliably be tested with kanamycin, but not amikacin, disks. When the disks containing 120 ,g were tested, a narrow zone size range of 6 to 7 mm could be used to identify all resistant strains. In contrast, when the disks containing 2,000 ,ug were used, the zone size ranges for resistant isolates varied widely with the aminoglycoside being tested. The presence of blood in the medium did not appreciably affect the disk test results. To detect resistance to every aminoglycoside-penicillin combination that may be considered for therapy, E. faecalis isolates need to be tested against a maximum of three different high-content disks (i.e., streptomycin, gentamicin, kanamycin). The disk-agar diffusion test performed with high-content aminoglycoside disks can provide laboratories with a convenient and reliable method for detecting E. faecalis

Factors influencing determination of high-level aminoglycoside resistance in Enterococcus faecalis

“…There was complete agreement between disk diffusion and standard agar screen results, which is consistent with previously published studies (10,13,15). Although most resistant strains showed no inhibition zone, some resistant and susceptible isolates may give inhibition zones at or near the 10-mm breakpoint.…”

“…(ii) High-content disk diffusion. Disks containing 120 ,ug of gentamicin and 300 ,ug of streptomycin were prepared at the UC Medical Center as described previously (13). The disks were used with Mueller-Hinton agar plates (Becton Dickinson Microbiology Systems) to perform disk diffusion testing by recommended guidelines (7).…”

“…Test methods that have been evaluated include agar screen (12,15), broth microdilution (2,15,17,18,23), broth macrodilution (12,15,21), and disk diffusion (9,10,13,15). Comparisons of results reported from these different studies indicate that the accuracy of the screen may vary with the method being used.…”

The life and times of the Enterococcus.

“…We streaked a heavy unquantitated inoculum onto brain heart infusion agar with 1,000 tig of gentamicin per ml and .17 MHA 198 Disks were 6 mm in diameter. b HLR was defined as a MIC of >2,000 ,ug/ml, as resistance to synergism, as resistant by one of several other techniques, or a combination of these (177,180,198 2,000 pLg of streptomycin per ml and found that all strains that grew, even with only a few colonies, had HLR as confirmed by MIC. For those strains that did not grow, antibiotic MICs were <1,000 pLg of gentamicin per ml or <2,000 Vag of streptomycin per ml (135, 152; unpublished observations).…”

Comparison of 30 μg and 120 μg gentamicin disks for the prediction of gentamicin resistance in Enterococcus faecalis

“…However, lack of synergy both in vivo and in vitro is seen when E. faecalis strains express high-level (MIC 2 2000 mg/l) aminoglycoside resistance, a characteristic which is occurring with increasing frequency worldwide (4,6,8,12). Laboratory detection of high-level E. faecalis resistance is therefore of importance and can readily be performed using the disk diffusion method with high potency (gentamicin 120 pg) disks (14,15,16,19). The 10 pg gentamicin disk used for routine susceptibility testing in many countries cannot predict high-level resistance since strains with normal low-level resistance often fail to produce zones of inhibition (14,15).…”