Inducible clindamycin resistance in staphylococci can be detected by disk diffusion method using Clindamycin and Erythromycin disks. D test is performed by disk diffusion, placing a 15-μg Erythromycin disk in proximity to a 2-μg Clindamycin disk on an agar plate that has been inoculated with a staphylococcal isolate; the plate is then incubated overnight.
A flattening of the zone of inhibition around the Clindamycin disk proximal to the Erythromycin disk (producing a zone of inhibition shaped like the letter D) is considered a positive result and indicates that the Erythromycin has induced Clindamycin resistance (a positive “D-zone test”). For Erythromycin-resistant isolates, induction tests can help laboratories determine whether results for Clindamycin should be reported as susceptible (when the induction test is negative) or as resistant (when the induction test is positive).
Mechanism of Action of Erythromycin and Clindamycin and Development of Resistance
Erythromycin (a macrolide) and clindamycin (a lincosamide) represent two distinct classes of antimicrobial agents that inhibit protein synthesis by binding to the 50S ribosomal subunits of bacterial cells. In staphylococci, resistance to both of these antimicrobial agents can occur through methylation of their ribosomal target site. Such resistance is typically mediated by erm genes.
Clinical significance of Inducible Clindamycin resistance:
Macrolide-lincosamide-streptogramin B (MLSB) resistance, which is mediated by target side modification mechanism, results in resistance to Erythromycin, Clindamycin, and streptogramin B. This mechanism can be
- 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 (Note: Erythromycin is an effective inducer of Macrolide-lincosamide-streptogramin B (MLSB) resistance). Isolates with inducible resistance are resistant to Erythromycin but appear susceptible to Clindamycin in routine invitro testing.
Clinical significance of Inducible Clindamycin resistance testing (D Test)
Clindamycin is an attractive agent for empirical therapy for suspected S. aureus infections because of its excellent pharmacokinetic and pharmacodynamic properties. Clinical failures of clindamycin therapy for treatment of 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, In vivo, 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 (inducible clindamycin resistance) test is employed to detect inducible clindamycin resistance.
Procedure for Inducible Clindamycin Resistance (D) Test:
- Prepare 0.5 McFarland standard suspension of Erythromycin resistant Staphylococcus aureus isolates
- Make a lawn culture of bacteria in Muller Hinton Agar (MHA) plates.
- Put Clindamycin (2-μg ) and Erythromycin (15-μg ) disks approximately 15 mm apart (measured edge to edge).
- Incubate the plate for 16 to 18 hours 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 or D+. Four other non-induction phenotypes (designated as negative [Neg], hazy D zone [HD], resistant [R], and susceptible [S]) are also observed in disk diffusion results