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

The MICs and MBCs of CI-934, ciprofloxacin, difloxacin (A-56619), A-56620, norfloxacin, enoxacin, amifloxacin, and coumermycin were determined for 43 clinical isolates of Enterococcus faecalis known to be resistant to penicillin-aminoglycoside synergy. Results were compared with those obtained for 37 synergysusceptible E. faecalis and 22 Enterococcus faecium strains. Although no substantial differences in quinolone activities were observed between synergy-resistant and -susceptible E. faecalis strains, CI-934 and ciprofloxacin were the drugs that demonstrated the greatest bactericidal activity against both types of E. faecalis. The MBCs of the other quinolones were generally within a single twofold dilution of the MICs, but their antienterococcal activity did not approach that of CI-934 or ciprofloxacin. The MBCs for 90% of the isolates of CI-934 for synergy-resistant and -susceptible E. faecalis strains were 1 and <0.5 ,ug/ml, respectively. The ciprofloxacin MBC for 90% of the E. faecalis strains tested was 1 ,ug/ml. For E. faecium isolates the CI-934 and ciprofloxacin MBCs for 90% of the isolates were 8 and 4 ,ug/ml, respectively. Time-kill assays performed with synergysusceptible enterococcal strains showed that the bactericidal activities of both CI-934 and ciprofloxacin were less than those of the penicillin-aminoglycoside combinations tested. However, against synergy-resistant isolates the activities of these two quinolones were comparable with and sometimes greater than those of penicillinaminoglycoside combinations.Aminoglycoside and penicillin (or ampicillin) combinations have been the most effective treatment for serious enterococcal infections (13,17,22). The synergistic bactericidal activity that these drugs demonstrate is particularly important for their use in the treatment of enterococcal endocarditis. However, by acquiring plasmids that encode for various aminoglycoside-modifying enzymes, Enterococcus faecalis isolates can exhibit high-level aminoglycoside resistance (MIC, -2,000 Fig/ml) and be refractory to antibiotic synergy (6,12,19). Infections caused by these resistant strains present serious therapeutic dilemmas (10), and their incidence at one medical center has been reported as high as 55% of all clinical enterococcal isolates (37). In addition, the therapeutic problems posed by the emergence of high-level aminoglycoside resistance have been complicated by the discovery of ,-lactamase-producing strains of E. faecalis (25). The resistance emerging among E. faecalis isolates as well as Enterococcus faecium resistance to antibiotics, which has been known for some time (20,23), indicates that the choice of antibiotics that may be selected for use against enterococci is extremely limited.Several of the recently developed fluoroquinolone antibiotics have demonstrated a broad spectrum and a high level of antibacterial activity (3,18,30,34), and certain of these drugs have shown good activity against some E. faecalis isolates (15, 18). However, thorough evaluations of fluoroquinolone activity aga...

The ability of six screening methods to detect high-level aminoglycoside resistance in enterococcal species other than Enterococcusfaecalis was investigated. The 85 Enterococcus isolates, which included 55 E. faecium, il E. gallinarum, 9 E. casseliflavus, 5E. raffinosus, 4E. avium, and 1 E. mundtii, were tested by using aminoglycoside-supplemented brain heart infusion agar (BHI), Remel EF Synergy Quad plates, high-content aminoglycoside diffusion disks, standard (prepared in-house) microdilution panels, Pasco MIC Gram Positive microdilution panels, and Vitek GPS-TA cards. When tested on BHI, 32 and 35 strains showed resistance to gentamicin and streptomycin, respectively. Resistance profiles obtained with Remel EF Synergy Quad plates were in complete agreement with those obtained on BHI. However, growth on Mueller-Hinton agar-based plates was not as heavy. Some isolates showed only weak growth and required 48 h for resistance to become evident, especially with swab inoculation of quadrants containing 2,000 ,ug of gentamicin per ml. Profiles obtained by use of the agar-based screens were used as the basis for evaluating the other methods. Disk diffusion showed complete agreement. No false resistance occurred by either microdilution method, but 48 h of incubation was needed for detection of some gentamicin-resistant isolates, and 14% of the streptomycinresistant strains were not detected by standard microdilution. The Vitek GPS-TA card detected 81 and 100% of the gentamicinand streptomycin-resistant isolates, respectively. In general, most methods used to detect high-level aminoglycoside resistance in E. faecalis appear to be reliable for the testing of the other enterococcal species. However, further investigations with a greater number of resistant E. raffinosus, E. avium, and E. mundtii isolates, when they are available, will be useful for establishing the full range of enterococci that can reliably be tested by the various methods.

Studies were conducted to validate the use of Enterococcus faecalis ATCC 51299 (which is vancomycin resistant and resistant to high levels of gentamicin and streptomycin) and E. faecalis ATCC 29212 (which is susceptible to vancomycin and against which gentamicin or streptomycin and cell wall-active agents have synergistic killing activity) as controls in an agar screening test for vancomycin resistance and high-level streptomycin and gentamicin resistance and a broth microdilution screening test for high-level streptomycin and gentamicin resistance. Both organisms performed as expected in these tests and will serve as appropriate controls. However, E. faecalis ATCC 29212 was occasionally noted to produce light growth on the vancomycin screening plate with certain lots of agar. Quality control ranges for disk diffusion tests with disks with large amounts of streptomycin (300 g) and gentamicin (120 g) were established for E. faecalis ATCC 29212; zone limits are 16 to 22 mm for gentamicin and 14 to 19 mm for streptomycin. No zones of inhibition were seen when E. faecalis ATCC 51299 was tested with these high-content disks.

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