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

Abstract

The ability of seven methods to detect high-level gentamicin (58 strains) and streptomycin resistance (56 strains) among 107 Enterococcusfaecalis isolates was investigated at the University of Chicago Medical Center and the University of Nebraska Medical Center. Methods included a standard agar screen plate, high-content disk diffusion, Remel (Lenexa, Kans.) EF Synergy Quad plates, standard microdilution panels prepared in house, Pasco MIC Gram-Positive panels (Difco Laboratories, Detroit, Mich.), MicroScan MIC Type 5 dry panels (Baxter Healthcare Corp., MicroScan Div., West Sacramento, Calif.), and Vitek GPS-TA cards (Vitek Systems Inc., Hazelwood, Mo.). Results indicating false resistance were not obtained by any method, and there was 100% agreement between the results of the disk diffusion and standard agar screen methods. Prolonging incubation from 24 to 48 h increased resistance detection for both agar and microdilution screens. EF Synergy Quad plates inoculated with micropipettes detected 100% of the streptomycinand gentamicin-resistant isolates. Resistance detection for streptomycin and gentamicin, respectively, was 93 and 96% by standard microdilution, 93 and 98% by Pasco panels, 88 and 89% by MicroScan panels, and 88 and 91% by Vitek GPS-TA cards. False susceptibility occurred more frequently with streptomycin-resistant isolates than it did with gentamicin-resistant strains and appeared to be strain related in some instances. The use of an increased inoculum size enhanced resistance detection with these strains, but it complicated interpretation of results and led to the selection of streptomycin-resistant mutants. Until results of further studies delineate optimum test conditions, a delay in the final interpretation of agar and microdilution screen results until 48 h for isolates showing no or light growth at 24 h may help to minimize the occurrence of false susceptibility reporting.

Detection of high-level aminoglycoside resistance in enterococci other than Enterococcus faecalis

“…Prolonging of incubation to 48 h had no notable effect on the inhibition zone diameters that were obtained. These findings are consistent with those of previous studies that have shown high-content disk diffusion to be a reliable method for detecting resistance in E. faecalis (9,11,13). False resistance did not occur by either the standard or Pasco microdilution methods ( Table 2).…”

“…Additionally, with swab inoculation, several resistant isolates showed weak growth in the presence of 2,000 ,tg of gentamicin per ml. As has been recommended for E. faecalis (9,14), prolonging of incubation to 48 h when weak or light growth, in comparison with the growth control, is observed at 24 h enhances resistance detection in some isolates. BHI results indicate that some of these problems might be related to the growth medium.…”

“…By using a micropipette, the BHI and EF Synergy Quad plates were spot inoculated with 106 CFU. As described previously for E. faecalis testing (9), each quadrant of the EF Synergy Quad plates was also inoculated with the tip of a swab that had been immersed in the organism suspension. After inoculation, standard and Pasco microdilution wells contained 104 CFU and Vitek GPS-TA cards had 105 CFU per well, as recommended by the manufacturers.…”

“…All screens were incubated at 35°C in an ambient atmosphere and were examined for growth after 24 and 48 h. The aminoglycoside concentrations tested are given in Tables 1 and 2. To establish consistency and minimize false susceptibility, weak growth (light haze or individual colonies) on the agar surface was interpreted as resistance (9,14).…”

Detection of vancomycin resistance in Enterococcus species

“…The results presented here also demonstrate the inadequa-cies inherent in the MicroScan and Vitek systems for the detection of enterococcal resistance to other antimicrobial agents commonly used for therapy. The problem of detecting high-level aminoglycoside resistance has been addressed previously, with the recommendation that a single-concentration agar screen plate or a high-potency disk be used in conjunction with the commercial systems (12,13,20,21,25). Similarly, the lack of sensitivity of the MicroScan panels and the lack of specificity of the Vitek cards to detect ampicillin resistance have been noted previously (12).…”