Abstract
Studies were conducted to compare and evaluate antimicrobial susceptibility test results for enterococci obtained by six national committee disk diffusion procedures. Variations in the incidence of isolates in resistance categories and errors were associated with the use of ciprofloxacin, gentamicin, nitrofurantoin, rifampin, and teicoplanin in a number of committee procedures. Results indicate that laboratories performing disk diffusion antimicrobial susceptibility testing may have problems correctly identifying resistance in enterococci with agents used to combat infections and that it may be difficult to compare resistance data for surveillance purposes.
Enterococci have emerged as a leading cause of nosocomial infection worldwide (17). Many clinical isolates recovered today are resistant to conventional treatment regimens, and antimicrobial susceptibility testing through detection of phenotypic resistance plays a pivotal role in the management of infectious diseases (2).
National committee susceptibility testing recommendations have been outlined by the National Committee for Clinical Laboratory Standards (NCCLS) in the United States (19, 20). European committees include the British Society for Antimicrobial Chemotherapy (BSAC) (4, 27), the Comité de l′ Antibiogramme de la Société Française de Microbiologie (CA-SFM) (6, 7), the Swedish Reference Group for Antibiotics (SRGA) (22; http://www.ltkronoberg.se/ext/raf/raf.htm), and the Deutsches Institut für Normung (DIN) (8–11), among others. A standardized disk diffusion procedure, the calibrated dichotomous sensitivity (CDS) test, has also been developed in Australia (3).
Protocols differ in their choices of test medium, inoculum density, antibiotic disk content, and interpretative breakpoint criteria. Consequently, the primary objective of the present study was to compare and evaluate susceptibility test results following the performance of disk diffusion procedures in accordance with six national committee criteria.
A total of 61 clinical isolates consisting of 54 Enterococcus faecalis and 7 Enterococcus faecium (urinary tract infection) isolates were obtained from the Microbiology Laboratory at the Regional Hospital, Limerick, Ireland. Disk diffusion susceptibility testing was performed in accordance with NCCLS (19), BSAC (4), CA-SFM (6, 7), SRGA (22; http://www.ltkronoberg.se/ext/raf/raf.htm), DIN (8, 11), and CDS (3) literature.
Nine antibiotics were employed in the study, including ampicillin, chloramphenicol, ciprofloxacin, gentamicin, nitrofurantoin, rifampin, teicoplanin, tetracycline, and vancomycin. Certain antibiotic disk contents were not available commercially and were prepared in our laboratory (1).
To allow determination of errors, breakpoint broth macrodilution MIC tests were performed according to NCCLS (20), BSAC (27), CA-SFM (6), SRGA (22; http://www.ltkronoberg.se/ext/raf/raf.htm), and DIN (9) guidelines. Errors were determined following a comparison of the results of disk diffusion, which placed isolates in resistance categories, with those obtained from the corresponding breakpoint MIC method (i.e., NCCLS disk diffusion with NCCLS broth dilution, etc.). Very major, major, and minor errors were defined as outlined by Dubreuil et al. (12).
The six national committee procedures incorporating E. faecalis indicated all isolates as being susceptible to vancomycin, teicoplanin, and nitrofurantoin, with 1.85% (1 of 54), 20.4% (11 of 54), and 68.5% (37 of 54) of the test isolates being resistant to ampicillin, chloramphenicol, and tetracycline, respectively. However, the percentages of test isolates in every resistance category varied for ciprofloxacin (BSAC), rifampin (SRGA), and gentamicin (Table 1). Determination of errors revealed a total of 6 very major and 7 minor errors with detection of high-level resistance to gentamicin (Table 2), 24 major and 15 (or 23) minor errors with ciprofloxacin, and 5 very major, 41 major, and 16 minor errors with rifampin (Table 3).
TABLE 1.
Percentages of E. faecalis isolates in qualitative resistance categories following disk diffusion antimicrobial susceptibility testing with ciprofloxacin, gentamicin, and rifampin
| Antibiotic | Resistance category | % of E. faecalis isolates (n = 54) according to indicated methoda
|
|||||
|---|---|---|---|---|---|---|---|
| NCCLS | BSAC | CA-SFM | SRGA | DIN | CDS | ||
| Ciprofloxacin | Resistant | 42.6 | 100 | 44.5 | 44.5 | 42.6 | ND |
| Intermediate | 11.1 | 0 | 0 | 55.5 | 16.7 | ND | |
| Susceptible | 46.3 | 0 | 55.5 | 0 | 40.7 | ND | |
| Gentamicin | Resistant | 24.1 | 27.8 | 33.3 | 29.6 | ND | 35.2 |
| Intermediate | 11.1 | 0 | 1.9 | 0 | ND | 0 | |
| Susceptible | 64.8 | 72.2 | 64.8 | 70.4 | ND | 64.8 | |
| Rifampin | Resistant | 22.2 | ND | 0 | 100 | ND | ND |
| Intermediate | 0 | ND | 0 | 0 | ND | ND | |
| Susceptible | 77.8 | ND | 100 | 0 | ND | ND | |
ND, not determined.
TABLE 2.
Summary of qualitative resistance categories and assessment of high-level resistance to gentamicin from results of disk diffusion procedures and quantitative MICs from reference broth dilution procedures performed on seven E. faecalis isolates and one E. faecium clinical isolate
| Organism | Isolate | Test result by method of indicated national committeea
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| NCCLS
|
BSAC
|
CA-SFM
|
SRGA
|
||||||||||
| RC | MIC | Error | RC | MIC | Error | RC | MIC | Error | RC | MIC | Error | ||
| E. faecalis | 146 | I | >500 | Minor | R | >1,024 | None | R | >500 | None | R | >500 | None |
| 236 | I | >500 | Minor | S | >1,024 | Very major | R | >500 | None | S | >500 | Very major | |
| 648 | R | >500 | None | R | >1,024 | None | R | >500 | None | S | >500 | Very major | |
| 031 | I | >500 | Minor | S | >1,024 | Very major | R | >500 | None | R | >500 | None | |
| 151 | I | >500 | Minor | R | >1,024 | None | R | >500 | None | R | >500 | None | |
| 198 | I | >500 | Minor | S | >1,024 | Very major | I | >500 | Minor | R | >500 | None | |
| 836 | I | >500 | Minor | S | >1,024 | Very major | R | >500 | None | R | >500 | None | |
| E. faecium | 224 | R | >500 | None | R | >1,024 | None | R | >500 | None | S | >500 | Very major |
RC, resistance category; R, resistant; S, susceptible; I, intermediate resistance. “Very major error” (false susceptibility) indicates that the isolates were susceptible by disk diffusion and resistant by the reference method; “minor error” indicates that the isolates were intermediate by one method and resistant or susceptible by the other (14).
TABLE 3.
Percentages of E. faecalis isolates in qualitative resistance categories according to disk diffusion and reference broth dilution testing and errors associated with disk diffusion procedures for ciprofloxacin and rifampina
| Antibiotic | Method of testing | Incidence (%) of isolates in disk diffusion qualitative category:
|
Incidence (%) of isolates in MIC qualitative category:
|
Disk diffusion error classification(s) (no. of errors) | ||||
|---|---|---|---|---|---|---|---|---|
| R | I | S | R | I | S | |||
| Ciprofloxacin | NCCLS | 42.6 | 11.1 | 46.3 | 55.5 | 0 | 44.5 | Minor (6) |
| BSAC | 100 | 0 | 0 | 55.5 | 0 | 44.5 | Major (24) | |
| CA-SFM | 44.5 | 0 | 55.5 | 44.5 | 0 | 55.5 | None | |
| SRGA | 44.5 | 55.5 | 0 | 44.5 | 55.5 | 0 | None | |
| DINb | 42.6 | 16.7 | 40.7 | ND | ND | ND | Minor (9), minor (23)c | |
| Rifampin | NCCLS | 22.2 | 0 | 77.8 | 14.8 | 18.5 | 66.7 | Very major (5), major (5), minor (10) |
| CA-SFM | 0 | 0 | 100 | 0 | 11.1 | 88.9 | Minor (6) | |
| SRGA | 100 | 0 | 0 | 33.3 | 0 | 66.7 | Major (36) | |
ND, not determined; R, resistant; S, susceptible, I, intermediate resistance. “Very major error” (false susceptibility) indicates that the isolates were susceptible by disk diffusion and resistant by the reference method; “major error” (false resistance) indicates that the isolates were resistant by disk diffusion and susceptible by the reference method; “minor error” indicates that the isolates were intermediate by one method and resistant or susceptible by the other (14).
DIN procedures indicate zone diameter breakpoints and disk contents for disk diffusion susceptibility testing of ciprofloxacin; however, corresponding MICs are not provided.
Error classification was based on a comparison of DIN disk diffusion results, with NCCLS, BSAC, and CA-SFM broth dilution qualitative categories yielding 9 minor errors and with SRGA broth dilution qualitative categories yielding 23 minor errors.
Results from the six disk diffusion procedures performed on E. faecium identified all seven isolates as being resistant to ampicillin and chloramphenicol, with 14.3% (one of seven) and 42.9% (three of seven) of the isolates being resistant to vancomycin and tetracycline respectively. The numbers of isolates placed into each resistance category varied in testing with ciprofloxacin (SRGA), nitrofurantoin (NCCLS), rifampin (CA-SFM), teicoplanin, and gentamicin (Table 4). Errors associated with disk diffusion procedures included a single very major error with gentamicin (Table 2), a total of 2 minor errors with teicoplanin, and 11 very major and 9 minor errors with nitrofurantoin (Table 4).
TABLE 4.
Qualitative resistance categories and errors associated with E. faecium isolates (n = 7) tested by the disk diffusion procedures with ciprofloxacin, gentamicin, nitrofurantoin, rifampin, and teicoplanin
| Antibiotic | Method of testing | Incidence (%) of isolates in disk diffusion qualitative categorya:
|
Disk diffusion error classification (no. of errors)b | ||
|---|---|---|---|---|---|
| R | I | S | |||
| Ciprofloxacin | NCCLS | 85.7 | 0 | 14.3 | Minor (1) |
| BSAC | 100 | 0 | 0 | Major (1) | |
| CA-SFM | 85.7 | 0 | 14.3 | Minor (1) | |
| SRGA | 57.1 | 42.9 | 0 | Minor (2) | |
| DINc | 85.7 | 0 | 14.3 | Minor (1) | |
| CDSd | ND | ND | ND | ND | |
| Gentamicin | NCCLS | 71.4 | 0 | 28.6 | None |
| BSAC | 71.4 | 0 | 28.6 | None | |
| CA-SFM | 71.4 | 0 | 28.6 | None | |
| SRGA | 57.1 | 0 | 42.9 | Very major (1) | |
| DIN | ND | ND | ND | ND | |
| CDS | 71.4 | 0 | 28.6 | None | |
| Nitrofurantoin | NCCLS | 42.9 | 14.3 | 42.9 | Minor (2) |
| BSAC | 0 | 0 | 100 | Very major (4) | |
| CA-SFM | 0 | 0 | 100 | Very major (1), minor (3) | |
| SRGA | 0 | 0 | 100 | Very major (3) | |
| DIN | 0 | 14.3 | 85.7 | Minor (4) | |
| CDS | 0 | 0 | 100 | Very major (3) | |
| Rifampin | NCCLS | 42.9 | 0 | 57.1 | Minor (1) |
| BSAC | ND | ND | ND | ND | |
| CA-SFM | 0 | 0 | 100 | Minor (1) | |
| SRGA | 57.1 | 0 | 42.9 | Major (1), minor (1) | |
| DIN | ND | ND | ND | ND | |
| CDS | ND | ND | ND | ND | |
| Teicoplanin | NCCLS | 0 | 14.3 | 85.7 | Minor (1) |
| BSAC | 14.3 | 0 | 85.7 | None | |
| CA-SFM | 14.3 | 0 | 85.7 | None | |
| SRGA | 0 | 14.3 | 85.7 | Minor (1) | |
| DIN | ND | ND | ND | ND | |
| CDS | 14.3 | 0 | 85.7 | None | |
R, Resistant; S, susceptible; I, intermediate resistance; ND, not determined.
“Very major error” (false susceptibility) indicates that the isolates were susceptible by disk diffusion and resistant by the reference method; “major error” (false resistance) indicates that the isolates were resistant by disk diffusion and susceptible by the reference method; “minor error” indicates that the isolates were intermediate by one method and resistant or susceptible by the other (14).
DIN procedures indicate zone diameter breakpoints and disk contents for disk diffusion susceptibility testing of ciprofloxacin; however, MICs corresponding to the qualitative resistance categories are not indicated. Consequently, a determination of MIC qualitative categories was not possible. Error classification was based on a comparison of DIN disk diffusion results with determinations of NCCLS, BSAC, and CA-SFM broth dilution qualitative categories.
The CDS literature has indicated both zone sizes and corresponding MICs of antibiotics. Only disk diffusion procedures are presented in the literature. Errors were determined following application of MIC breakpoints to MICs after NCCLS, BSAC, CA-SFM, and SRGA broth dilution procedures were carried out.
The incidence of resistant, susceptible, and intermediately resistant isolates differed from procedure to procedure following the performance of disk diffusion (and MIC) assays. Breakpoints recommended by various committees for the same agent may differ significantly. Establishment of breakpoints requires consideration of various factors (MIC distributions, pharmacology, and clinical and bacteriological response rates), and the importance accorded to each differs from committee to committee (5). Procedures also vary in their choices of test medium inoculum density, and antibiotic disk content (disk diffusion), factors that also affect test results and consequently necessitate differences in breakpoints (15). It is probably not surprising, therefore, that susceptibility test results generated with the six national committee procedures varied.
Determination of MICs is considered the reference method against which disk diffusion procedures are calibrated (13). Zones of inhibition delineating susceptible, resistant, and intermediately resistant isolates from disk diffusion procedures must correlate with corresponding MICs (24). Results presented in this study reveal poor correlation between disk diffusion zones of inhibition and their analogous MICs, most notably for E. faecalis by BSAC ciprofloxacin and SRGA rifampin procedures (Table 3). Such discrepancies between results from published standardized susceptibility testing methods developed by the same committee seem unacceptable. Multidrug resistance in enterococci has necessitated the use of alternative treatment regimens, including those antibiotics presented in this study (16), and errors associated with susceptibility testing may result in the unwarranted utilization or elimination of these antibiotics as part of possible treatment regimens.
No truly global antimicrobial resistance surveillance system has as yet been established, and the development of such a system could be complicated by the lack of a universal susceptibility testing method (26). There has been no agreement on an optimal procedure, and no one existing method is considered to be superior to be adopted as a worldwide standard (25).
Within Europe, other national committees have developed their own criteria including the Finnish Study Group for Antimicrobial Resistance, the National Reference Laboratory in the Czech Republic (28), the Werkgroep Richtlijnen Gevoeligheidsbepalingen in Holland (15), and the Norwegian Working Group for Antibiotics (21). In Denmark and Belgium many laboratories use a commercial test procedure referred to as the Neo-Sensitab method (15; Neo-Sensitab Susceptibility Testing, User's Guide, 11th ed., ROSCO, Tarstrup, Denmark), and recommendations in Spain have been developed by the Spanish Antibiogram Committee (the MENSURA group) (18). Many European clinical diagnostic laboratories, not including those from countries with well-established national committees, adhere to NCCLS criteria (14). It is not surprising that within Europe, at least, coordination of susceptibility testing methods to facilitate resistance surveillance has been suggested (23).
Given the disparity among susceptibility test results presented here, development of an international standardized antimicrobial susceptibility testing procedure could facilitate direct and meaningful comparison of resistance data generated within different national and international laboratories. Such a procedure could also provide a testing system that offers the most accurate guidance for the administration and outcome of antimicrobial therapy.
Acknowledgments
We acknowledge the assistance afforded by Denis Barron and Marie Lenihan and other personnel of the Microbiology Laboratory at the Regional Hospital, Limerick, Ireland, and Rachel Gorman in the Microbiology Laboratory at the University of Limerick. We also thank Kevin Kavanagh of the Medical Mycology Unit at the National University of Ireland, Maynooth, for his constructive criticism of the manuscript.
This work was funded by an Enterprise Ireland Strategic Research grant (no. ST99/043).
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