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Journal of Clinical Microbiology, December 2007, p. 3954-3957, Vol. 45, No. 12
0095-1137/07/$08.00+0     doi:10.1128/JCM.01501-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Detection of Inducible Clindamycin Resistance in Staphylococci by Broth Microdilution Using Erythromycin-Clindamycin Combination Wells{triangledown}

Jana M. Swenson,1* William B. Brasso,2 Mary Jane Ferraro,3 Dwight J. Hardy,4 Cynthia C. Knapp,5 Linda K. McDougal,1 L. Barth Reller,6 Helio S. Sader,7 Dee Shortridge,8 Robert Skov,9 Melvin P. Weinstein,10 Barbara L. Zimmer,11 and Jean B. Patel1

Antimicrobial Resistance Team, Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,1 BD Diagnostic Systems, Sparks, Maryland 21152,2 Massachusetts General Hospital, Boston, Massachusetts 02114,3 University of Rochester Medical Center Hospital, Rochester, New York 14642,4 Trek Diagnostic Systems, Cleveland, Ohio 44131,5 Duke University Hospital, Durham, North Carolina 27710,6 JMI Laboratories, North Liberty, Iowa 52317,7 BioMérieux, Inc., Hazelwood, Missouri 63042,8 Statens Serum Institut, Copenhagen, Denmark 2300,9 Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903,10 Dade Behring MicroScan, Inc., West Sacramento, California 95691,11

Received 26 July 2007/ Returned for modification 11 September 2007/ Accepted 8 October 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
A study conducted by 11 laboratories investigated the ability of four combinations of erythromycin (ERY) and clindamycin (CC) (ERY and CC at 4 and 0.5, 6 and 1, 8 and 1.5, and 0.5 and 2 µg/ml) in a single well of a broth microdilution panel to predict the presence of inducible CC resistance. Each laboratory tested approximately 30 Staphylococcus aureus isolates and 20 coagulase-negative staphylococcus (CoNS) isolates in a panel using cation-adjusted Mueller-Hinton broth from three different manufacturers. Only the strains resistant to ERY and those susceptible or intermediate to CC were included in the analysis (S. aureus, n = 333; CoNS, n = 97). Results of the D-zone test were used as the gold standard. After an 18-h incubation, the combination of 4 µg/ml ERY and 0.5 µg/ml CC performed the best, with 98 to 100% sensitivity and 100% specificity for both organism groups. After a 24-h incubation, the ERY-CC combinations of 4 and 0.5, 6 and 1, and 8 and 1.5 µg/ml correlated well with the D-zone test.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The D-zone test, a disk approximation test using erythromycin (ERY) and clindamycin (CC) disks, which was proposed originally by Fiebelkorn et al. (5) and is currently described in the Clinical and Laboratory Standards Institute's (CLSI) antimicrobial susceptibility documents (1, 2, 4), has made the detection of inducible CC resistance in staphylococci an easy test for clinical microbiology laboratories to perform, especially if they are already performing disk diffusion tests. To further simplify the process for laboratories that routinely perform dilution susceptibility tests, a version using the broth microdilution test was investigated.

In a previous study at the Centers for Disease Control and Prevention (CDC), a test combining ERY and CC in a single well of a broth microdilution panel using results of growth or no growth had produced promising results, but further work was suggested by the authors to define the most accurate concentrations for the two drugs (7). Results of a follow-up study at CDC, using a combination of 4 µg/ml ERY and 0.5 µg/ml CC in a single well, showed excellent correlation with the D-zone test results for 349 clinical isolates of staphylococci (335 Staphylococcus aureus and 13 coagulase-negative staphylococcus [CoNS] isolates), with a sensitivity of 97.4% and a specificity of 99.1% (unpublished data).

In this report, we present the results of a multicenter laboratory evaluation undertaken to validate the preliminary findings at CDC.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
The current study involved cation-adjusted Mueller-Hinton broth (CAMHB) from three manufacturers, the 4-µg/ml ERY-0.5-µg/ml CC combination described above, and three additional combinations of ERY and CC (6 and 1, 8 and 1.5, and 0.5 and 2 µg/ml, respectively). Frozen broth microdilution panels containing ERY (8 to 0.12 µg/ml) and CC (4 to 0.03 µg/ml) (BBL CAMHB; BD, Sparks, MD) and the four combinations of ERY and CC in Mueller-Hinton broth from three different manufacturers (BBL, Difco [BD], and Oxoid [Basingstoke, Hampshire, England]; with cation content adjusted if necessary) were prepared at CDC, according to the CLSI reference method (1) using U-bottom MIC trays. The frozen panels were shipped to all participants, along with 15-µg ERY disks and 2-µg CC disks (BBL). Using these panels and their current routine lot of Mueller-Hinton agar for disk diffusion testing, each laboratory tested 50 clinical isolates (30 S. aureus and 20 CoNS isolates) selected from their own culture collections (as either fresh or frozen cultures) and 5 quality control strains for each test day (S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212, S. aureus BAA-976, and S. aureus BAA-977 were all used for determining MIC only, and S. aureus ATCC 25923 was used for disk diffusion only) (8). Additional strains were tested at CDC.

For the evaluation of the ERY-CC broth microdilution combination wells, only isolates that tested resistant to erythromycin (MIC ≥ 8 µg/ml) and susceptible or intermediate to clindamycin (MIC ≤ 2 µg/ml) were included in the analysis (a total of 333 S. aureus and 97 CoNS isolates). The ERY-CC combination wells were read as either growth or no growth, with any growth recorded as positive. Results of the D-zone disk diffusion test with disks spaced exactly 15 mm apart were used as the gold standard for data analysis. Discrepancies between the D-zone test results and those of the combination wells were resolved at CDC, using molecular testing by PCR for the macrolide resistance genes ermA, ermB, ermC, and msrA, with previously described methods (7).

Additional testing of S. aureus strains ATCC 29213, BAA-976, and BAA-977 was done in all laboratories so that at least 8 test day results would be collected for each strain in each laboratory in order to establish the usefulness of the strains for the quality control of the ERY-CC combination wells. At least 80 test results were collected for each control strain.


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Quality control results. For the 11 participating laboratories, the percentages of MICs within the range for S. aureus ATCC 29213 with ERY and CC were 91.0% and 95.5%, respectively. For one laboratory, four of eight test results (50%) were out of range at the high end for ERY. With that laboratory excluded, 95.1% of the values were within range. Therefore, data from that laboratory for the ERY-CC combination wells were not included in data analysis for the study.

Two of the quality control strains (S. aureus ATCC 29213 and BAA-976) do not express inducible resistance to CC and thus would not be expected to grow in the combination wells. S. aureus strain BAA-977, however, is inducibly resistant to CC and would be expected to grow. Results for these strains, with the ERY and CC combination wells for the three media and the 18-h MICs obtained with one medium, are shown in Table 1. Two of the ERY-CC combination wells, the 4-µg/ml ERY-0.5-µg/ml CC and the 6-µg/ml ERY-1-µg/ml CC combination, provided the most consistent results compared to the expected outcomes. Both ATCC 29213 and ATCC BAA-976 did not grow in any of the combinations in any of the three media used. Strain BAA-977 failed to grow in BBL CAMHB with the 8-µg/ml ERY-1.5-µg/ml CC combination on one occasion after 18 h of incubation at five of eight laboratories (5 of 80 test results, or 6.3%) and after 24 h of incubation in two of eight laboratories (2 of 80 test results, or 2.5%). None of these three quality control strains grew in the final combination well, which had the 0.5-µg/ml ERY-2 µg/ml CC combination.


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  		TABLE 1. ERY and CC MICs and growth of quality control strains in the ERY-CC combinationa

 
Performance of the combination wells with the clinical strains. Results for the four different ERY and CC combinations with the three CAMHB preparations are shown in Table 2. Sensitivity error values (i.e., false negatives) are derived from those strains that showed a D zone but with no growth in the combination wells; specificity error values (i.e., false positives) are derived from those strains that were D-zone negative but with growth in the combination wells. Overall, the combination of 4 µg/ml ERY and 0.5 µg/ml CC performed best for both S. aureus and CoNS at both the 18-h and 24-h incubations, with ≥98% sensitivity and specificity. The ERY-CC combinations of 6 and 1 and 8 and 1.5 µg/ml, respectively, performed well at 24 h (sensitivity and specificity, ≥98%); but at 18 h, although specificity was high for both organism groups (>99%), sensitivity was acceptable only for S. aureus (95% to 98.9%). For CoNS, sensitivity at the 18-h incubation was 88.2% to 94.1% for these two combinations. The combination of 0.5 µg/ml ERY and 2 µg/ml CC performed poorly at both 18 and 24 h, with sensitivities of ≤30% for S. aureus and <75% for CoNS.


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  		TABLE 2. Results for ERY-resistant and CC-intermediate or -resistant strains compared to D-zone test resultsa

 
The discrepancies for the ERY-CC combination of 4 and 0.5 µg/ml at 18- and 24-h incubations are shown in Table 3. The only sensitivity errors that occurred with this combination were at the 18-h incubation for three S. aureus strains (all three grown in BBL CAMHB and two in both the Difco and the Oxoid CAMHB) and one CoNS strain (grown in both the Difco and the Oxoid CAMHB). There were three specificity errors for the 4-µg/ml ERY-0.5-µg/ml CC combination, two S. aureus and one CoNS strain. One D-zone-negative S. aureus strain (C-204) grew under all combinations of media and drugs at both the 18- and the 24-h incubations. When it was retested, this strain did show a slight D zone but only when the two disks were placed closer together than 15 mm. This isolate was not positive for ermA, ermB, ermC, or msrA by PCR, but multiple phenotypic tests suggest that this isolate may indeed express inducible clindamycin resistance, perhaps mediated by a different erm gene. One D-zone test-negative S. aureus strain that was positive for the msrA gene grew in Oxoid CAMHB at both times. One D-zone test-negative CoNS strain, positive for both the ermA and the msrA genes, also grew in the 4-µg/ml ERY-0.5-µg/ml CC combination in BBL medium at 18 h and in both BBL and Difco CAMHB at 24 h.


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  		TABLE 3. Discrepancies of the D-zone test versus the 4-µg/ml ERY-0.5-µg/ml CC combination wella

 
Although the clinical need to test for inducible CC resistance in staphylococci continues to be debated, laboratories should be capable of detecting it if asked to do so (6). Using one combination well in a microdilution format makes the detection of inducible resistance to CC easy to perform, especially if a combination well were to be included in a commercial susceptibility testing system. But as with any single-well test, quality control can be problematic. The difficulty is that in order to do adequate quality control, it is necessary to test both a CC inducibly resistant strain and a strain that is not inducibly resistant to CC. In order to confirm that both ERY and CC are in the well, it would be necessary to test both BAA-976 and BAA-977. With only ERY (at 4 µg/ml) in the well, both BAA-977 and 29213 would test the same as when both ERY and CC are together in the well. Therefore, testing 29213 and BAA-977 only would not detect the absence of clindamycin in the well. To avoid testing three quality control strains each time the test is performed, we recommend that both BAA-976 and BAA-977 be tested once for each new lot of MIC panels. Further testing can then be limited to either S. aureus ATCC BAA-976 or S. aureus ATCC 29213 as the susceptible strain (which should not grow in the test well) and the S. aureus inducible strain ATCC BAA-977 (which should grow in the test well).

In summary, ERY and CC tested together in a single well of a broth microdilution panel performed satisfactorily for the detection of inducible CC resistance in both S. aureus and CoNS. The use of the 4-µg/ml ERY-0.5-µg/ml CC combination for the detection of inducible clindamycin resistance in Staphylococcus spp., read at 18 to 24 h, was recently approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing and will be included in the M100-S18 tables to be published in January 2008 (3).


   ACKNOWLEDGMENTS
 
We thank all those who performed technical work for this study: Tracey Gill and John P. Douglass (BD Diagnostic Systems); Jean Spargo (Massachusetts General Hospital); David Vicino (University of Rochester Medical Center); Amy Miskov, Nikki Holliday, and Scott Killian (Trek Diagnostic Systems); Stanley Mirrett, Dolores H. Calley, and Hina S. Patel (Duke University Hospital); Doug Biedenbach and Ron N. Jones (JMI Laboratories); Rita Griffith (BioMérieux, Inc.); Frank Hansen (Statens Serum Institut); Judy Rothberg (Robert Wood Johnson Medical School); and Linda Van Pelt and Guadalupe Mendoza-Morales (Dade Behring MicroScan).

The findings and conclusions in this report are ours and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Use of trade names is for identification purposes only and does not constitute endorsement by the Public Health Service or the U.S. Department of Health and Human Services.


   FOOTNOTES
 
* Corresponding author. Mailing address: Antimicrobial Resistance Team, Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention, Mailstop G08, 1600 Clifton Rd., Atlanta, GA 30333. Phone: (404) 639-0196. Fax: (404) 639-1381. E-mail: jms1{at}cdc.gov Back

{triangledown} Published ahead of print on 17 October 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Clinical and Laboratory Standards Institute/NCCLS. 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-7th ed. CLSI/NCCLS M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA.
  2. Clinical and Laboratory Standards Institute/NCCLS. 2006. Performance standards for antimicrobial disk susceptibility tests; approved standard-9th ed. CLSI/NCCLS M2-A9. Clinical and Laboratory Standards Institute, Wayne, PA.
  3. Clinical and Laboratory Standards Institute/NCCLS. 2008. Performance standards for antimicrobial susceptibility testing: 18th ed. CLSI/NCCLS M100-S18. Clinical and Laboratory Standards Institute, Wayne, PA.
  4. Clinical and Laboratory Standards Institute/NCCLS. 2007. Performance standards for antimicrobial susceptibility testing: 17th ed. CLSI/NCCLS M100-S17. Clinical and Laboratory Standards Institute, Wayne, PA.
  5. Fiebelkorn, K. R., S. A. Crawford, M. L. McElmeel, and J. H. Jorgensen. 2003. Practical disk diffusion method for detection of inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci. J. Clin. Microbiol. 41:4740-4744.[Abstract/Free Full Text]
  6. Lewis, J. S., II, and J. H. Jorgensen. 2005. Inducible clindamycin resistance in staphylococci: should clinicians and microbiologists be concerned? Clin. Infect. Dis. 40:280-285.[CrossRef][Medline]
  7. Steward, C. D., P. M. Raney, A. K. Morrell, P. P. Williams, L. K. McDougal, L. Jevitt, J. E. McGowan, Jr., and F. C. Tenover. 2005. Testing for inducible clindamycin resistance in erythromycin-resistant isolates of Staphylococcus aureus. J. Clin. Microbiol. 43:1716-1721.[Abstract/Free Full Text]
  8. Zelazny, A. M., M. J. Ferraro, A. Glennen, J. F. Hindler, L. M. Mann, S. Munro, P. R. Murray, L. B. Reller, F. C. Tenover, and J. H. Jorgensen. 2005. Selection of strains for quality assessment of the disk induction method for detection of inducible clindamycin resistance in staphylococci: a CLSI collaborative study. J. Clin. Microbiol. 43:2613-2615.[Abstract/Free Full Text]

Journal of Clinical Microbiology, December 2007, p. 3954-3957, Vol. 45, No. 12
0095-1137/07/$08.00+0     doi:10.1128/JCM.01501-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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