D-test detects inducible clindamycin resistance in Staphylococcus aureus by double disk diffusion using Clindamycin(2ug) and Erythromycin (15 ug) disks. Erythromycin is a macrolide and clindamycin is a lincosamide, two distinct classes of antimicrobial agents that inhibit protein synthesis by binding to the 50S ribosomal subunits of bacterial cells. Resistance in Staphylococcus can occur to both of these antimicrobial agents through methylation of their ribosomal target site. Such resistance is typically mediated by erm genes.
D test is performed by spreading the test organism on Muller Hinton agar (MHA) for Staphylococci and MHA with 5% Sheep blood (for Group B Sreptococci) and placing a 15-μg Erythromycin disk near to a 2-μg Clindamycin disk. After overnight incubation, a flattening of zone of inhibition around the Clindamycin disk near the Erythromycin disk (producing a zone of inhibition shaped like the alphabet D) is considered a positive result and indicates that the Erythromycin has induced Clindamycin resistance (a positive “D-zone test”). This test can help us to determine whether Clindamycin should be reported as susceptible (when D test is negative) or as resistant (when D test is positive) in erythromycin resistant strains.
Significance of Inducible Clindamycin resistance:
Macrolide-lincosamide-streptogramin B (MLSB) resistance, which is mediated by target side modification, resulting into resistance to Erythromycin, Clindamycin, and streptogramin B.
This mechanism can be either
- Constitutive, where the rRNA methylase is always produced,
If in vitro testing is done, Staphylococcus aureus isolates with constitutive resistance are resistant to Erythromycin and Clindamycin.
- Or can be inducible, where methylase is produced only in the presence of an inducing agent. Isolates with inducible resistance are resistant to Erythromycin but appear susceptible to Clindamycin in routine in vitro testing.
Note: Erythromycin is an effective inducer of Macrolide-lincosamide-streptogramin B (MLSB) resistance.
Significance of D Test
Clindamycin is used in empirical therapy for suspected Staphylococcal infections because of its excellent pharmacokinetic and pharmacodynamic properties. Clinical failures of clindamycin therapy for treatment of Methicilin Resistant Staphylococcus (MRSA) infections have been documented for strains that were clindamycin sensitive but erythromycin resistant. The failures were due to inducible resistance to clindamycin.
In such cases, therapy with Clindamycin may select for constitutive erm mutants, which may lead to clinical failure. Clindamycin resistance may be constitutive or inducible. Routine antibiotic susceptibility tests cannot identify these strains. The D test (inducible clindamycin resistance) can be used to detect inducible clindamycin resistance.
Procedure for detection of Inducible Clindamycin Resistance (D Test):
- Prepare a suspension of Staphylococcus aureus isolates. Equal to 0.5 McFarland turbidity standard
- Using a sterile cotton swab, inoculate the entire surface of Mueller-Hinton agar plate with the prepared suspension.
- Place antibiotic disks along with Clindamycin (2-μg ) and Erythromycin (15-μg ) disks approximately 15 -26 mm apart (measured edge to edge).
- Observe the plate after 16 to 18 hours of incubation at 37oC.
Interpretation of result of D Test
A clear, D-shaped zone of inhibition around the Clindamycin disk is designated as the D phenotype which is labeled as
D – When blunted, D shaped clear zone around Clindamycin disk near to the Erythromycin disk is observed.
D+– When blunted, D-shaped zone around Clindamycin disk near to the Erythromycin disk and small colonies growing to Clindamycin disk in otherwise clear zone is observed.
Four other non-induction phenotypes are designated as
Negative [Neg] When clear zone around Clindamycin disk is observed.
Hazy D zone [HD] When two zones of growth appear around the Clindamycin disk is observed. One zone is light, hazy growth extending from the Clindamycin disk to the second zone where the growth is much heavier. The inner, hazy zone is blunted proximal to the Erythromycin disk
Resistant [R] When growth up to Clindamycin and Erythromycin disks is observed.
Susceptible [S] When clear susceptible zone diameters around both the disks is observed.
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