Effects of medium and inoculum variations on screening for high-level aminoglycoside resistance in Enterococcus faecalis

Abstract

Enterococcus faecalis isolates that are refractory to aminoglycoside-penicillin synergy can be detected by their ability to grow in the presence of high concentrations of aminoglycoside (2,000 ,g/ml). In past studies investigators have used a variety of media and inoculum sizes to perform high-level aminoglycoside resistance screens, but little is known about how these variations affect test accuracy. We screened 63 E. faecalis strains on different media by using various inoculum sizes and correlated the results with synergy test results obtained by time-kill studies. Screens were done with dextrose-phosphate agar, brain heart infusion agar, Trypticase soy agar with 5% sheep blood, Mueller-Hinton agar with 5% sheep blood, dextrose-phosphate broth, and Mueller-Hinton broth. Agar screens were inoculated with 102, 104, and 106 CFU; and broth screens contained a final inoculum of 105 CFU/ml. The E. faecalis isolates were tested for high-level resistance to streptomycin, kanamycin, amikacin, gentamicin, and tobramycin. Of the 63 isolates tested, 21 did not show high-level resistance to any of the aminoglycosides tested, and 42 demonstrated high-level resistance to one or more drugs. The sensitivity of most screens was-90%. Regardless of the inoculum size or medium used, false-resistance results were seldom encountered. Screen specificity, which was used as the indicator of false susceptibility, was markedly influenced by both the inoculum size and the drug being tested. Specificity was low whenever a 102-CFU inoculum was used, when amikacin was tested with any inoculum, and when tobramycin was tested in broth media. Data for kanamycin could be used to predict amikacin-penicillin synergy, and the highly accurate gentamicin screen obviated the need for the testing of tobramycin. We recommend a 106-CFU inoculum for agar screens and a 105-CFU/ml inoculum for broth screens. The type of medium used did not substantially influence screen accuracy. Among the aminoglycosides, only streptomycin, gentamicin, and, occasionally, kanamycin need to be used to screen E. faecalis isolates for aminoglycoside-penicillin synergy.

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

“…Results of this investigation and those of previous studies have shown that false aminoglycoside susceptibility, not false resistance, is the problem most frequently encountered with available screening methods (2,4,12,15,17,18,21). In this investigation prolonging of the incubation time to 48 h substantially increased the percentage of resistant strains detected.…”

“…These findings are consistent with those of several previous studies which showed that false susceptibility occurs more frequently with streptomycin testing (2,4,14,15,(17)(18)(19). In an earlier study, false streptomycin susceptibility occurred most frequently with the use of a low inoculum (102 CFU/ml) on either Mueller-Hinton or Trypticase soy agar (12). However, it is not yet known whether discrepancies in the results for streptomycin are related to media, inoculum size, or strain characteristics.…”

“…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.…”

Influence of growth media on vancomycin resistance of Enterococcus isolates and correlation with resistance gene determinants

“…Several studies have been conducted to validate the accuracy of antimicrobial susceptibility testing of VRE [4,5]. The composition of plating/broth medium and the growth conditions of enterococcal isolates may in£uence the antibiotic susceptibility pro¢les and the minimum inhibitory concentration (MIC) values [6,7]. The effect of plating medium, however, was not correlated with the presence of vancomycin resistance genes in these studies.…”

The life and times of the Enterococcus.

“…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). Sahm and Torres evaluated dextrose phosphate agar, brain heart infusion agar, Mueller-Hinton agar with 5% sheep blood, and Trypticase soy agar with 5% sheep blood with different inocula of E. faecalis to screen for HLR (181). Excellent results were obtained with inocula of 104 and 106 CFU per spot with both gentamicin and streptomycin regardless of the medium used.…”