Answering your questions; D test for MLSB resistance.

Q Could you please explain the "D" test and how to validate it?

A Resistance to the macrolides in staphylococci may be due to an active efflux mechanism (encoded by the mrsA gene) or can be due to modification of the ribosomal target (macrolide-lincosamide-streptogramin B [MLSB] resistance; usually encoded by ermA or ermC genes). MLSB resistance is either constitutive (always produced) or inducible following exposure to a macrolide. (1)

If an isolate of staphylococci tests resistant to erythromycin and susceptible to clindamycin (a lincosamide), it could still possess the inducible MLSB- (iMLSB-) resistance mechanism. Induction tests to determine clindamycin resistance utilize erythromycin and clindamycin disks located on an agar plate in close proximity (a 2-ug disk of clindamycin placed 15 millimeters away from the edge of a 15-ug disk of erythromycin). This can be performed by the standard disk-diffusion procedure or by using a standard blood-agar plate normally utilized for inoculum purity check. After overnight incubation, if there is a flattening of the clindamycin zone adjacent to the erythromycin disk (a "D-zone"), this indicates that the organism has iMLSB resistance. According to the 2006 Clinical and Laboratory Standards Institute (CLSI) susceptibility documents, these organisms should be reported as clindamycin resistant. (2)

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A study by Fiebelkorn and colleagues showed that the simple placement of erythromycin and clindamycin disks at a distance achieved with a standard disk dispenser allowed detection of 97% of Staphylococcus aureus strains and 100% of coagulase negative staphylococci strains with inducible MLSB resistance in this study. (1)

Siberry and colleagues reported a case of a surgical-site infection caused by clindamycin-susceptible, erythromycin-resistant, methicillin-resistant S aureus (MRSA) that did not respond to treatment with clindamycin. (3) The MRSA isolate obtained after treatment was resistant to clindamycin but was found to be identical by pulsed-field gel electrophoresis to the clindamycin-susceptible isolate obtained before treatment. A D-zone test confirmed the presence of iMLSB in the pre-treatment isolate. D-zone testing further confirmed the presence of iMLSB in 90 (56%) of 161 erythromycin-resistant, clindamycin-susceptible clinical isolates of S aureus overall and in a significantly higher proportion (78%) of methicillin-susceptible S aureus isolates from pediatric patients. The authors' clinical laboratory currently tests all S aureus isolates for iMLSB resistance prior to reporting clindamycin susceptibility.

Validation of the D-zone test starts with standard testing of both clindamycin and erythromycin disks by normal quality-control procedures to assure appropriate antibiotic concentration. After evaluating several laboratory isolates of staphylococci that are erythromycin-R and clindamycin-S with the D-zone test, you will find isolates with the iMLSB mechanism of resistance (a positive D-zone). This organism can then be used for future training for your staff in recognizing this phenomenon and for competency assessment purposes. The CLSI includes in its 2006 antimicrobial-susceptibility testing documents (page 73, footnote C) a type strain that can be used for these purposes; but if you test several in your own laboratory, you will find these strains.

References

(1.) Fiebelkorn KR, Crawford SA, McElmeel ML, Jorgensen JH. Practical Disk Diffusion Method for Detection of Inducible Clindamycin Resistance in Staphylococcus aureus and Coagulase-Negative staphylococci. J Clin Microbiol. 2003;41(10):4740-4744.

(2.) Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; M100-S16. 2006 Catalog Supplement. 2006;26(3).

(3.) Siberry GK, Tekle T, Carroll K, Dick J. Failure of clindamycin treatment of methicillin-resistant Staphylococcus aureus expressing inducible clindamycin resistance in vitro. Clin Infect Dis. 2003;37(9): 1257-1260.

--Susan E. Sharp, PhD (DABMM)

Director of Microbiology

Kaiser Permanente Pathology

Regional Laboratory;

Associate Professor

Oregon Health and Science University

Portland, OR

Edited by Daniel M. Baer, MD

Daniel M. Baer, MD, is professor emeritus of laboratory medicine at Oregon Health and Science University in Portland, OR, and a member of MLO's editorial advisory board.

MLO's "Tips from the Clinical Experts" provides practical, up-to-date solutions to readers' technical and clinical issues from a panel of experts in various fields. Readers may send questions to Dan Baer by e-mail at [e-mail].