Use of the E test to predict high-level resistance to aminoglycosides among enterococci.

The E test and the reference agar dilution methods were compared for detecting high-level aminoglycoside resistance (HLAR) among 71 selected clinical isolates, including 62 Enterococcus faecalis and 9 Enterococcus faecium isolates. High-level gentamicin resistance alone was found in 11% (5 E. faecalis and 3 E. faecium strains) and high-level streptomycin resistance was found in 42% (28 E. faecalis, 2 E. faecium strains) of the strains tested, and 31% of the strains demonstrated high-level resistance to both antimicrobial agents (21 E. faecalis and 1 E. faecium strains). The E test detected all HLAR populations, but the streptomycin strip may require recalibration to achieve absolute MIC comparisons with the reference value (twofold less) or the use of an alternative interpretive resistance breakpoint, e.g., > 1,000 micrograms/ml. By the E test, MIC results indicate that ampicillin, imipenem, penicillin, piperacillin, and vancomycin remain active against the HLAR E. faecalis isolates; however, these tested drugs were less effective on the HLAR E. faecium isolates (< 50%).     

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

Since the early 1970s, the synergistic activity of an aminoglycoside with a cell wall-active agent has been predicted by determining the ability of an enterococcus to grow in the presence of high levels of the aminoglycoside (usually > or = 2,000 micrograms/ml). However, a variety of media and concentrations of aminoglycosides has been used for this screening procedure. In the present study, we sought to optimize the agar dilution, broth microdilution, and disk diffusion tests used to detect high-level gentamicin and streptomycin resistance in enterococci. For dilution tests, brain heart infusion agar or broth gave the best growth and performance. For agar dilution, 500 micrograms of gentamicin per ml, 2,000 micrograms of streptomycin per ml, and an inoculum of 1 x 10(6) CFU/ml were optimal, while for broth microdilution, 500 micrograms of gentamicin per ml, 1,000 micrograms of streptomycin per ml, and an inoculum of 5 x 10(5) CFU/ml were best. Growth of more than one colony in the agar dilution test was determined to be the best indicator of high-level resistance. For disk diffusion, Mueller-Hinton agar, 120-micrograms gentamicin disks, and 300-micrograms streptomycin disks with breakpoints of no zone for resistance and > or = 10 mm for susceptibility gave the best sensitivity and specificity if results for strains with zones of 7 to 9 mm are considered inconclusive, indicating that a broth or agar test should be performed to determine susceptibility or resistance.     

Molecular characterization and multilaboratory evaluation of Enterococcus faecalis ATCC 51299 for quality control of screening tests for vancomycin and high-level aminoglycoside resistance in enterococci.

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 kill 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 micrograms) and gentamicin (120 micrograms) 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 for inhibition were seen when E. faecalis ATCC 51299 was tested with these high-content disks.     

In vitro antibacterial activities of eleven antibiotics against S. faecalis

The in vitro antibacterial activities of penicillin, ampicillin, piperacillin, azlocillin, vancomycin, erythromycin, clindamycin, gentamicin, streptomycin, cefotaxime, and ceftriaxone against 198 S. faecalis strains were investigated, employing an agar dilution technique. Results were generally in accord with those of other studies. Ampicillin and penicillin showed equal activities. Twenty percent of the strains were resistant to erythromycin, which correlated with resistance to clindamycin. The activity of cefotaxime was fairly good, but the clinical importance of this observation warrants further investigation. Twenty percent showed high-level resistance to streptomycin. High-grade resistance to gentamicin was noted for one isolate only, making gentamicin the first-choice aminoglycoside for combination treatment at present.     

Comparison of microscan broth microdilution,synergy quad plate agar dilution,and disk diffusion screening methods for detection of high-level aminoglycoside resistance in enterococcus species

We compared the dried MicroScan microdilution panel, Synergy Quad plate agar dilution, and high-potency disk diffusion screening methods for the detection of high-level aminoglycoside resistance in 815 enterococcal bloodstream isolates. Agreement between the three methods was 99% when testing for high-level gentamicin resistance and 96% when testing for high-level streptomycin resistance.     

Investigation of the reformulated Remel Synergy Quad plate for detection of high-level aminoglycoside and vancomycin resistance among enterococci.

We investigated the accuracy of the recently released Remel Synergy Quad plate, a commercially available agar screening method for detecting high-level aminoglycoside and vancomycin resistance among enterococci that is based on the National Committee for Clinical Laboratory Standards recommended guidelines (National Committee for Clinical Laboratory Standards, M7-A3, 1993). The Synergy Quad correctly determined the gentamicin and streptomycin resistance status for > or = 97% of 147 Enterococcus faecalis and Enterococcus faecium isolates tested. Detection of vancomycin resistance also was reliable, as no false susceptibility occurred with 36 vancomycin-resistant E. faecalis and E. faecium strains and false resistance occurred only once with the 47 susceptible strains tested. One strain each of Enterococcus gallinarum and Enterococcus casseliflavus failed to grow on the screen, but because the true nature and significance of resistance in such isolates is unknown the implication of their screen negativity is uncertain. In summary, the Remel Synergy Quad provides a highly accurate and convenient method for susceptibility testing of enterococci against gentamicin, streptomycin, and vancomycin.     

Evaluation of a new system, VITEK 2, for identification and antimicrobial susceptibility testing of enterococci

We evaluated the new automated VITEK 2 system (bioMérieux) for the identification and antimicrobial susceptibility testing of enterococci. The results obtained with the VITEK 2 system were compared to those obtained by reference methods: standard identification by the scheme of Facklam and Sahm [R. R. Facklam and D. F. Sahm, p. 308-314, in P. R. Murray et al., ed., Manual of Clinical Microbiology, 6th ed., 1995] and with the API 20 STREP system and, for antimicrobial susceptibility testing, broth microdilution and agar dilution methods by the procedures of the National Committee for Clinical Laboratory Standards. The presence of vanA and vanB genes was determined by PCR. A total of 150 clinical isolates were studied, corresponding to 60 Enterococcus faecalis, 55 Enterococcus faecium, 26 Enterococcus gallinarum, 5 Enterococcus avium, 2 Enterococcus durans, and 2 Enterococcus raffinosus isolates. Among those isolates, 131 (87%) were correctly identified to the species level with the VITEK 2 system. Approximately half of the misidentifications were for E. faecium with low-level resistance to vancomycin, identified as E. gallinarum or E. casseliflavus; however, a motility test solved the discrepancies and increased the agreement to 94%. Among the strains studied, 66% were vancomycin resistant (57 VanA, 16 VanB, and 26 VanC strains), 23% were ampicillin resistant (MICs, >/=16 microgram/ml), 31% were high-level gentamicin resistant, and 45% were high-level streptomycin resistant. Percentages of agreement for susceptibility and resistance to ampicillin, vancomycin, and teicoplanin and for high-level gentamicin resistance and high-level streptomycin resistance were 93, 95, 97, 97, and 96%, respectively. The accuracy of identification and antimicrobial susceptibility testing of enterococci with the VITEK 2 system, together with the significant reduction in handling time, will have a positive impact on the work flow of the clinical microbiology laboratory.     

Comparison of the new MicroScan Pos MIC Type 6 panel and AMS-Vitek Gram Positive Susceptibility Card (GPS-TA) for detection of high-level aminoglycoside resistance in Enterococcus species.

We compared the MicroScan Pos MIC Type 6 panel and AMS-Vitek Gram Positive Susceptibility Card (GPS-TA) to agar dilution screen plates for the detection of high-level aminoglycoside resistance in 182 enterococcal isolates. The specificity of the two commercial systems was 100%, with the exception of one susceptible isolate found to be streptomycin resistant by the Vitek system. The MicroScan and Vitek systems had comparable sensitivities for the detection of gentamicin resistance (90 and 95% respectively) and streptomycin resistance (85 and 78%, respectively). These results suggest that screening tests such as agar dilution screen plates, broth dilution, or disk diffusion should continue to be used to detect high-level gentamicin and streptomycin resistance.     

The prevalence of fecal colonization of enterococci, the resistance of the isolates to ampicillin, vancomycin, and high-level aminoglycosides, and the clonal relationship among isolates

The gastrointestinal tract carriage of enterococci was searched in 150 hospitalized patients and 100 outpatients, and clonal relatedness of the isolates and their resistance to ampicillin, vancomycin, and high-level streptomycin and gentamicin were investigated. A stool sample or rectal swab collected from each patient was inoculated into appropriate media within an hour. Enterococcus species were identified by using conventional biochemical tests, API-20 Strep assay, and BBL crystal kit. Antibiotic susceptibility tests were performed using Kirby-Bauer disk diffusion method. A polymerase chain reaction (PCR) was used to detect vanA and vanB genes. Pulsed-field gel electrophoresis (PFGE) and arbitrarily primed-polymerase chain reaction (AP-PCR) methods were used for molecular typing of the strains. Enterococci were isolated from 90 (60%) of the specimens collected from 150 inpatients. Of these 90 isolates, 37 (41%) had high-level gentamicin resistance, 36 (40%) had high-level streptomycin resistance, and 50 (55.6%) had ampicillin resistance. Fecal colonization was found in 30% of the outpatients. Resistances to ampicillin, high-level streptomycin, and gentamicin were 13%, 10%, and 3%, in these patients' isolates, respectively. No vancomycin-resistant enterococci were detected by both agar diffusion and PCR assays in our study. Both typing procedures were applied on 78 Enterococcus strains isolated from inpatients. AP-PCR typing showed that 30 (50.8%) of the 59 E. faecium and 5 (50%) of the 10 E. faecalis strains were clonally related. These values were found to be 12 (20.3%) and two (20%) by PFGE, respectively. The typing procedures did not find any clustered strains in the six E. durans and three E. avium isolates. Neither PFGE nor AP-PCR result was significantly different among the sensitive and resistant strains. Our results indicate that the high prevalence of colonization with ampicillin and highlevel aminoglycoside-resistant enterococci is an important problem in our medical center. The high clonal diversity among the isolates indicates limited spread of antibiotic-resistant strains between patients.     

Synergy characterization for Enterococcus faecalis strains displaying moderately high-level gentamicin and streptomycin resistance.

Synergy of 14 Enterococcus faecalis strains displaying moderately high-level aminoglycoside resistance (MICs, 500 and 256 to 1,000 micrograms/ml for gentamicin and streptomycin, respectively) was characterized by time-kill studies. All strains proved resistant to penicillin plus the respective aminoglycoside. Strains with moderately high-level aminoglycoside resistance should be considered to exhibit high-level resistance in severe infections.     

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

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 micrograms of streptomycin and either 120 or 2,000 micrograms 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 micrograms 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 micrograms 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 isolates that are resistant to aminoglycoside-penicillin synergy.     

Vancomycin-resistant enterococci colonizing the intestinal tracts of hospitalized patients.

A point prevalence culture survey was carried out to investigate the prevalence of fecal carriage of vancomycin-resistant enterococci (VRE) among patients admitted to an 800-bed general hospital where no VRE had been isolated previously. Twenty-two of 636 patients (3.5%) were found to be VRE carriers. Eighteen strains were identified as Enterococcus faecium, three were identified as Enterococcus gallinarum, and one was identified as Enterococcus faecalis. The susceptibilities of the enterococci to ampicillin, vancomycin, and teicoplanin were determined by the disk diffusion and the agar dilution methods. High-level resistance (HLR) to gentamicin and streptomycin was determined by the agar screening method. Eighteen strains (82%) were highly resistant to vancomycin, and four strains (18%) were moderately resistant to vancomycin. Five strains were susceptible to teicoplanin (23%; MICs, < or = 8 micrograms/ml). Only one strain (4.5%, E. faecium) showed HLR to gentamicin, and six strains (27%) showed HLR to streptomycin (one E. faecalis and five E. faecium strains). All 18 E. faecium and 1 E. faecalis strain carried the vanA gene, and 3 E. gallinarum strains carried the vanC gene. An epidemiological investigation revealed several risk factors for VRE colonization: hospitalization and duration of stay in the hematology department and prior vancomycin treatment. The study demonstrates that the patient's gastrointestinal tract is a possible reservoir for VRE, even in hospitals where VRE infections have not yet been observed. Therefore, we conclude that infection control precautions and restriction of glycopeptide usage may be key issues in limiting the emergence and spread of nosocomial VRE infections.     

Enterococcus faecium isolated from bone marrow transplant patients in Tunisia: high prevalence of antimicrobial resistance and low pathogenic power

Objectives

Investigation of the occurrence and antibiotic susceptibility of Enterococcus faecium isolates, collected during four years from neutropenic patients at the Tunisian bone marrow transplantation centre.

Materials and methods

E. faecium strains were identified by conventional methods and by the Api20 Strep (Bio-Mérieux, France). Antibiotic susceptibility was determined by the disk diffusion method on Mueller-Hinton agar and interpreted as recommended by CA-SFM. MICs of ampicillin, vancomycin, and teicoplanin were determined by E-test method.

Results

Two hundred and thirty five E. faecium isolates were recovered from stool cultures or rectal swabs (229), throat (three), urine (two), and pus of wound (one). None was responsible for bacteraemia. Ampicillin resistance, without production of β-lactamase, was observed in 43.8% of isolates. All the isolates were susceptible to glycopeptides. High rates of resistance were observed: high-level resistance (HLR) to gentamicin (33.6%), HLR to kanamycin (55.7%), HLR to streptomycin (47.6%), erythromycin (86.4%), ciprofloxacin (78.7%), rifampicin (85%), and tetracycline (43%). Strains with HLR to gentamicin were significantly more resistant to ampicillin and streptomycin. Multiple drug resistance was observed in most isolates.

Conclusion

These findings demonstrated the low pathogenic power of E. faecium in our patients, and the high frequencies of resistance to ampicillin and aminoglycosides. In the absence of glycopeptide-resistance, vancomycin remains an alternative treatment against multidrug resistant strains.     

Interlaboratory variation of antibiograms of methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains with conventional and commercial testing systems.

Laboratory-prepared (conventional) and commercial susceptibility testing systems were compared by using a group of methicillin-resistant (MR) and methicillin-susceptible (MS) strains of Staphylococcus aureus. A group of 25 MR and 15 MS S. aureus strains were coded and tested blindly by disk diffusion, agar dilution, broth microdilution, Sensititre, Micro-Media, Sceptor, API 3600S, MicroScan, Autobac I, and MS-2 systems. All systems were incubated at 35 degrees C and read with either a manual or automated reader at the recommended times. Where applicable, systems were also read at 48 h. Among the conventional assays, the broth and agar dilution methods were comparable, both detecting 88% of the MR strains at 24 h and detecting 92 and 96%, respectively, at 48 h. The disk diffusion method was less efficient, detecting only 36 and 72% at 24 and 48 h, respectively. Detection of cephalothin resistance was low for all systems at both time periods, with agar dilution and disk diffusion being the most and least efficient, respectively. Some variability was also seen with detection of resistance to clindamycin and gentamicin. Among the MS strains, variability among the conventional systems occurred with methicillin, gentamicin, ampicillin, and penicillin. Comparison of the commercial systems with manual readers with the broth microdilution method (reference method) showed that for MR strains, the Sceptor system gave identical results at 24 and 48 h. Sensititre detected 68 and 88% of the MR strains, whereas Micro-Media was least effective detecting 12 and 80% at 24 and 48 h, respectively. None of the commercial systems detected cephalothin resistance well, with only one strain being indicated by the Sceptor and Sensititre systems at 48 h. Slight differences were also seen among the systems with clindamycin and gentamicin. With regard to the MS strains, variability among the systems was seen with methicillin, penicillin, ampicillin, clindamycin, and gentamicin. Among commercial systems with automated readers, the API system detected a greater number of MR strains than did the reference method at 24 and 48 h, 96 and 100%, respectively. The MicroScan method was comparable to the reference method detecting 80 and 88% of the MR strains at both time periods, respectively. Both Autobac I and MS-2 were much less effective in detecting MR strains, noting only 32 and 16%, respectively, at the 3- to 6-h readings. Poor detection of cephalothin resistance among MR strains was evident in all systems. Variability also occurred among the systems with clindamycin, gentamicin, and ampicillin. A single strain of the MR group was reported to be vancomycin resistant by the API system. Among the MS group, the greatest variability was seen with methicillin. Less variability occurred with penicillin, ampicillin, gentamicin, and vancomycin.     

Evaluation of in vitro spectra of activity of azithromycin, clarithromycin, and erythromycin tested against strains of Neisseria gonorrhoeae by reference agar dilution, disk diffusion, and Etest methods.

The macrolide-azilide susceptibility testing (agar dilution, disk diffusion, Etest) criteria for 105 Neisseria gonorrhoeae strains were evaluated. In addition, the potencies of azithromycin, clarithromycin, and erythromycin were studied. The most active macrolide-azilide agent was azithromycin (MIC at which 90% of the isolates are inhibited [MIC90], 0.5 microgram/ml) compared with clarithromycin (MIC90, 1.5 to 2 micrograms/ml) and erythromycin (MIC90, 2 to 4 micrograms/ml). The Etest (AB Biodisk, Solna, Sweden) was observed to produce MIC results very similar to those of the reference agar dilution test (GC agar base), with 100% of the results within 1 log2 dilution step of the reference MICs. The disk diffusion test zone diameters for all three drugs correlated at an acceptable level (r = -0.81 to -0.92) with the reference agar dilution MICs. Interpretive criteria for susceptibility were proposed for azithromycin at a MIC of < or = 2 micrograms/ml and a disk diffusion test zone of > or = 25 mm. No category for resistance was proposed because of the paucity of strains for which MICs were > 2 micrograms/ml. These tentative criteria should be further validated by correlations with clinical trial data for gonococcal strains (as they emerge) that have azithromycin MICs above the proposed susceptible category range.     

Therapy of enterococcal infections

Because enterococci are typically tolerant of the bactericidal effects of cell wall-active antimicrobial agents, bactericidal therapy has required use of these agents in combination with aminoglycosides. For strains which do not demonstrate high-level aminoglycoside resistance, either streptomycin or gentamicin can be used in combination with penicillin, ampicillin or vancomycin. At some centers, as many as 50 % of isolates display high-level gentamicin resistance. A minority of such isolates will not be highly streptomycin-resistant, and the latter drug can be used in combination with a cell wall-active drug. Optimal treatment of serious infections due to strains highly resistant to both streptomycin and gentamicin is unknown. While no agent is predictably bactericidal against such isolates, ampicillin, penicillin or vancomycin alone would be expected to cure some patients. Other drugs or drug combinations do not offer any predictable therapeutic advantages.     

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

The ability of six screening methods to detect high-level aminoglycoside resistance in enterococcal species other than Enterococcus faecalis was investigated. The 85 Enterococcus isolates, which included 55 E. faecium, 11 E. gallinarum, 9 E. casseliflavus, 5 E. raffinosus, 4 E. 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 micrograms 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 streptomycin-resistant strains were not detected by standard microdilution. The Vitek GPS-TA card detected 81 and 100% of the gentamicin- and 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.     

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

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 micrograms/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 10(2), 10(4), and 10(6) CFU; and broth screens contained a final inoculum of 10(5) 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 greater than or equal to 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 10(2)-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 10(6) -CFU inoculum for agar screens and a 10(5) -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.     

Problems with the disk diffusion test for detection of vancomycin resistance in enterococci.

A total of 53 strains of enterococci, including recently isolated strains with high-level resistance to vancomycin, were tested for vancomycin susceptibility by broth microdilution and disk diffusion using Mueller-Hinton media with and without supplementation with 5% blood. By using currently published parameters of the National Committee for Clinical Laboratory Standards for the disk diffusion test, we found that strains for which MICs were 8 to 32 micrograms/ml were incorrectly placed in the susceptible or intermediate category, which caused both very major (1.9%) and minor (11.5%) errors. When we used newer, recently proposed breakpoints for vancomycin, we found 13.5% minor errors but no very major errors. Changing disk diffusion breakpoints to less than or equal to 14 mm for resistant [corrected] and greater than or equal to 15 mm for susceptible [corrected] would eliminate the problem for the strains with MICs of 32 micrograms/ml but not for those with MICs of 8 micrograms/ml. For those strains, it is necessary to perform an MIC test to differentiate them from strains with MICs of less than or equal to 4 micrograms/ml.