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Journal of Clinical Microbiology, August 2004, p. 3747-3751, Vol. 42, No. 8
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.8.3747-3751.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Multicenter Evaluation of Use of Penicillin and Ampicillin as Surrogates for In Vitro Testing of Susceptibility of Enterococci to Imipenem

Melvin P. Weinstein,^1,2,3* Stanley Mirrett,^4,5 Saman Kannangara,¹ Jan Monahan,⁶ Lizzie J. Harrell,^4,5 Alan C. Wilson,¹ and L. Barth Reller^4,5,7

Departments of Medicine,¹ Pathology, Robert Wood Johnson Medical School,² Microbiology Laboratory, Robert Wood Johnson University Hospital, New Brunswick, New Jersey 08901-0019,³ Departments of Pathology,⁴ Medicine,⁵ Clinical Microbiology Laboratory, Duke University Medical Center, Durham, North Carolina 27710,⁶ Microbiology Laboratory, University of Colorado Hospital, Denver, Colorado 80220⁷

Received 10 February 2004/ Returned for modification 17 March 2004/ Accepted 20 April 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Imipenem is approved by the U.S. Food and Drug Administration (FDA) for treatment of infections caused by Enterococcus faecalis. However, there are no NCCLS guidelines for testing susceptibility of enterococci against imipenem. To assess whether or not ampicillin or penicillin could be used as a surrogate for broth microdilution (BMD) testing of imipenem versus Enterococcus species, 633 strains of E. faecalis, E. faecium, and other enterococci isolated from blood cultures of patients at three geographically distinct university hospitals were tested by the NCCLS BMD and disk diffusion (DD) methods. Using FDA susceptibility breakpoints for imipenem and NCCLS breakpoints for penicillin and ampicillin, categorical agreement (CA) for penicillin-imipenem and ampicillin-imipenem tested with E. faecalis and E. faecium by BMD was 94% but was 90% for other enterococci. Using the DD method, CA for ampicillin-imipenem tested with E. faecalis and E. faecium was 98% and was 92% for other enterococci; CA for penicillin-imipenem was 91% for E. faecalis, 98% for E. faecium, and 87% for other enterococci. Further analysis showed that testing E. faecalis with ampicillin resulted in no false-susceptible (FS) or false-resistant (FR) results by BMD, no FS results by DD, and a single FR result by DD (0.2%), whereas testing with penicillin resulted in no FS results by BMD or DD and two FR results by BMD (0.4%). For E. faecium and other enterococci, the combination of FS and FR results was such that surrogate testing with penicillin or ampicillin appears not to be sufficiently reliable to be used clinically. We conclude that ampicillin is an accurate predictor of the in vitro activity of imipenem against E. faecalis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although early studies examining the in vitro activity of imipenem suggested good activity against Enterococcus faecalis (1, 2, 4, 5, 9, 10) and the drug is approved by the U.S. Food and Drug Administration (FDA) for clinical use for infections caused by this microorganism, NCCLS consensus guidelines for susceptibility testing have been lacking. A recent pilot study (11) suggested that in vitro susceptibility results obtained by testing penicillin or ampicillin could accurately predict the susceptibility of these organisms to imipenem. However, this report had several limitations. First, all isolates came from a single institution (i.e., geographic region). Second, although 201 strains of E. faecalis were tested, there were insufficient numbers of E. faecium strains tested and no other enterococcal species were assessed. Third, only broth microdilution (BMD) MICs were determined, and no data or correlations were available to assess disk diffusion (DD) susceptibility methods. Subsequently, Jones (R. N. Jones, Letter, J. Clin. Microbiol. 39:3810-3811, 2001) reported additional data on 6,538 enterococcal isolates collected from 1997 to 2000 from multiple centers throughout the United States and abroad as part of the SENTRY Antimicrobial Surveillance Program. These results supported the initial observations (11). However, the data were not categorized according to species, and only BMD testing was done. Therefore, to further address the limitations of these reports, we studied 633 contemporary strains of enterococci isolated from the bloodstreams of patients hospitalized at three geographically distinct university medical centers in the United States (in New Jersey, North Carolina, and Colorado), including E. faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus raffinosus, Enterococcus avium, Enterococcus durans, and Enterococcus casseliflavus. All isolates were tested by the NCCLS BMD method and the agar DD method (6, 7).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Enterococcal isolates causing bacteremia in patients hospitalized at Robert Wood Johnson University Hospital (New Brunswick, N.J.), Duke University Medical Center (Durham, N.C.), and the University of Colorado Hospital (Denver, Colo.) were tested. Isolates were identified by standard microbiologic methods (3) in the clinical microbiology laboratories of each institution. All isolates were frozen at –70°C until testing was done. Susceptibility testing was done at Robert Wood Johnson Medical School (New Brunswick, N.J.). Prior to testing, isolates were thawed and subcultured twice to ensure purity and viability. E. faecium isolates were selected such that at least 50% of strains were vancomycin susceptible on the premise that such strains were less likely to show clonality. The first three and last three (chronologically) vancomycin-resistant E. faecium isolates from each center were tested for clonality by pulsed-field gel electrophoresis; this testing was performed at Duke University Medical Center.

Each isolate was tested by the BMD and DD methods with penicillin, ampicillin, imipenem, and vancomycin. For BMD testing, solutions of all antimicrobials were prepared from standard powders of known potencies obtained either from the manufacturer of the compound or from a commercial source (Sigma, St. Louis, Mo.). MICs were determined in duplicate by the method of the NCCLS using cation-adjusted Mueller-Hinton broth (7). For DD testing, commercially manufactured disks (Becton Dickinson, Sparks, Md.) containing each of the antimicrobials were used. Testing was done in duplicate according to the NCCLS DD method (6).

Categorical agreements (CA) and interpretive errors were determined according to guidelines of the NCCLS (8). Regression statistics and correlation data for imipenem-penicillin and imipenem-ampicillin were determined by using SAS software (SAS/GRAPH, version 8.2 for Windows NT, 1999-2001).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Six hundred thirty-three isolates were tested, including 442 E. faecalis isolates, 151 E. faecium isolates, 11 E. avium isolates, 17 E. casseliflavus isolates, 3 E. durans isolates, 3 E. gallinarum isolates, and 6 E. raffinosus isolates. None of the E. faecium isolates tested from each institution demonstrated clonality.

Using the published NCCLS breakpoints for penicillin and ampicillin versus enterococci (6, 7) and the FDA-approved breakpoints for imipenem published in the manufacturer's package insert (MIC: susceptible, 4 µg/ml; intermediate, 8 µg/ml; resistant, 16 µg/ml) (DD: susceptible, 16 mm; intermediate, 14 to 15 mm; resistant, 13 mm), CA between penicillin and imipenem and ampicillin and imipenem were calculated, as shown in Tables 1 and 2. For BMD MICs, CA for E. faecalis and E. faecium was 94% for both penicillin-imipenem and ampicillin-imipenem but 90% or less for the other enterococci. In DD testing, CA was 98% for E. faecium and both penicillin-imipenem and ampicillin-imipenem and for E. faecalis and ampicillin-imipenem.

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TABLE 1. Correlation of penicillin and ampicillin NCCLS BMD susceptibility breakpoints for enterococci with imipenem FDA BMD susceptibility breakpointsa

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TABLE 2. Correlation of penicillin and ampicillin NCCLS DD susceptibility breakpoints for enterococci with imipenem FDA DD susceptibility breakpointsa

Correlation data for E. faecalis are shown in Fig. 1, and error rates are shown in Table 3. For BMD testing, with use of penicillin to predict results with imipenem, two imipenem-susceptible isolates would have been called resistant (0.4%) and no imipenem-resistant isolates would have been called susceptible. With use of ampicillin to predict results with imipenem, there would have been no false-susceptible (FS) or false-resistant (FR) results. For DD testing, with use of penicillin to predict results with imipenem, 38 FR results (8.6%) and no FS results would have occurred. With use of ampicillin to predict results with imipenem, there would have been one FR result (0.2%) and no FS results.

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FIG. 1. Correlation data for in vitro susceptibility testing of E. faecalis: imipenem versus penicillin by BMD (A), imipenem versus ampicillin by BMD (B), imipenem versus penicillin by DD (C), and imipenem versus ampicillin by DD (D).

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TABLE 3. Error rates for imipenem susceptibility results with enterococci if penicillin or ampicillin were used as a surrogate to predict results for imipenem

Correlation data for E. faecium are shown in Fig. 2, and error rates are shown in Table 3. For BMD testing, with use of penicillin to predict results with imipenem, there would have been three FS results (2.0%) and one FR result (0.7%). With use of ampicillin, there would have been six FS results (4.0%) and no FR results. For DD testing, with use of penicillin to predict results with imipenem, there would have been no FS results and three FR results (2.0%). If ampicillin were used, there would have been two FS results (1.3%) and no FR results.

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FIG. 2. Correlation data for in vitro susceptibility testing of E. faecium: imipenem versus penicillin by BMD (A), imipenem versus ampicillin by BMD (B), imipenem versus penicillin by DD (C), and imipenem versus ampicillin by DD (D).

Correlation data for Enterococcus spp. other than E. faecalis and E. faecium are shown in Fig. 3, and error rates are shown in Table 3. For BMD testing, with use of penicillin to predict results with imipenem, there would have been no FS results and three FR results (7.5%). With use of ampicillin, there would have been no FS results and two FR results (5.0%). For DD testing, with use of penicillin to predict results with imipenem, there would have been no FS results and five FR results (12.8%). If ampicillin were used, there would have been one FS result (2.6%) and two FR results (5.1%).

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FIG. 3. Correlation data for in vitro susceptibility testing of enterococcal species other than E. faecalis and E. faecium: imipenem versus penicillin by BMD (A), imipenem versus ampicillin by BMD (B), imipenem versus penicillin by DD (C), and imipenem versus ampicillin by DD (C).

Top Abstract Introduction Materials and Methods Results Discussion References

 
This multicenter study has demonstrated the potential for penicillin or ampicillin to serve as a surrogate for in vitro testing of enterococcus susceptibility to imipenem and confirms the results of preliminary studies (11; Jones, letter). Our results suggest that ampicillin is the better agent to utilize and that results obtained for E. faecalis have greater reliability than those obtained for other enterococcal species. Since imipenem is not indicated clinically for species other than E. faecalis, the lesser predictive efficacy for these strains should not be of concern.

For E. faecalis testing using the BMD method, CA for both penicillin-imipenem and ampicillin-imipenem was excellent (>95% agreement), whereas for the DD method, CA for ampicillin-imipenem (99.8%) was superior to that for penicillin-imipenem (91.4%). The chance that a penicillin or ampicillin susceptible MIC result or zone of inhibition would be present for an imipenem-resistant isolate appears to be nil (Table 3). The risk of an FR result using ampicillin as a surrogate for imipenem similarly appears to be virtually nil. In contrast, whereas penicillin-susceptible MICs with BMD would yield an FR result only rarely (0.4%), DD test results using penicillin would yield an FR result more frequently (8.6%) and be less reliable.

For E. faecium, CA for both penicillin-imipenem and ampicillin-imipenem was excellent (94% for BMD, 98% for DD). However, some FS results would occur with surrogate testing (Table 3). FR results would not occur with use of ampicillin for surrogate testing and would be uncommon using penicillin (Table 3). For other enterococcal species, CA was excellent for both penicillin-imipenem and ampicillin-imipenem, using the BMD method (Table 1), and for ampicillin-imipenem with use of the DD method (Table 2). However, CA for penicillin-imipenem was lower with the DD method (Table 2). Surrogate testing using either method would result in no FS results using penicillin, and there would be no FS results using ampicillin with the DD method (Table 3). However, there would be FS results with ampicillin with the DD method, and the number of FR results with both drugs and use of either method would limit the potential value of surrogate testing (Table 3).

In summary, this multicenter study has documented the feasibility of using ampicillin and penicillin as surrogate agents for assessing the in vitro activity of imipenem against enterococci. Based on CA observations and the frequency of FS and FR results, ampicillin was superior to penicillin as a predictor of imipenem activity against E. faecalis, especially with use of the DD test method. Accordingly, we conclude that ampicillin can serve as an accurate predictor of the in vitro activity of imipenem against E. faecalis. Because of more-numerous FS and FR results, neither ampicillin nor penicillin is sufficiently reliable to be used as a surrogate for imipenem against other enterococcal species, including E. faecium.

 
* Corresponding author. Mailing address: Departments of Medicine and Pathology, UMDNJ-Robert Wood Johnson Medical School, 1 Robert Wood Johnson Place, New Brunswick, NJ 08901-0019. Phone: (732) 235-7713. Fax: (732) 235-7951. E-mail: weinstei{at}umdnj.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, August 2004, p. 3747-3751, Vol. 42, No. 8
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.8.3747-3751.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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