The D test is a simple laboratory method that reveals whether a bacterium carrying erythromycin resistance also harbors a hidden ability to resist clindamycin, a resistance that standard susceptibility testing can miss entirely. It works by placing an erythromycin disk near a clindamycin disk on a culture plate and watching whether the clindamycin zone of inhibition flattens on the side facing erythromycin, forming a characteristic D shape. That flattened zone signals that erythromycin is switching on a resistance gene that would otherwise stay silent, and it warns clinicians that prescribing clindamycin could lead to treatment failure.
Why Standard Testing Misses This Resistance
Bacteria such as Staphylococcus aureus can resist macrolides like erythromycin and lincosamides like clindamycin through two main routes. One is an efflux pump, encoded by the msrA gene, that actively pumps macrolides out of the bacterial cell. That pump does not work against clindamycin, so isolates with only an efflux mechanism test resistant to erythromycin and genuinely susceptible to clindamycin. The second route is ribosomal methylation, controlled by erm genes (primarily ermA, ermB, and ermC in staphylococci). When an erm gene is active, it chemically modifies the ribosome at a site shared by macrolides, lincosamides, and type B streptogramins, creating cross-resistance to all three drug classes at once.1Microbiology and Biotechnology Letters. Detection of Inducible Clindamycin Resistance Genes (ermA, ermB, and ermC) in Staphylococcus aureus and Staphylococcus epidermidis
The catch is that erm genes can sit in either of two regulatory states. In constitutive expression, the gene runs all the time, and the bacterium tests resistant to both erythromycin and clindamycin on a standard susceptibility panel. In inducible expression, the gene stays quiet until a macrolide like erythromycin shows up and flips the switch. Without that trigger, the bacterium looks susceptible to clindamycin in a routine test. A clinician who trusts that result and prescribes clindamycin may unknowingly select for mutants where the gene has become permanently turned on, converting inducible resistance to constitutive resistance in the middle of treatment.
How the D Test Actually Works
The mechanics are straightforward. A standard disk diffusion susceptibility plate is set up with the bacterial isolate, and two antibiotic disks are placed nearby on the agar: one containing erythromycin and the other clindamycin. As the antibiotics diffuse outward, erythromycin acts as the inducer. If the isolate carries an inducible erm gene, the erythromycin diffusing toward the clindamycin disk triggers expression of the methylase. The result is that bacteria between the two disks gain enough resistance to grow closer to the clindamycin disk than they otherwise would, flattening the clindamycin zone on the erythromycin-facing side into a D shape.
Disk spacing matters. The original validation work compared standard dispenser spacing (about 26 to 28 mm between disk centers) with closer placements at 15 or 20 mm. Even at the standard spacing, detection rates were high: the method caught 97% of S. aureus strains and all coagulase-negative staphylococcal strains carrying inducible resistance.2PubMed Central. Practical disk diffusion method for detection of inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci That finding made the test easy to adopt: labs could use the same disk dispensers they already had without custom positioning.
Reading the Results and What Each Phenotype Means
Not every erythromycin-resistant, clindamycin-susceptible isolate produces a D zone. The results typically sort into a few distinct patterns, and each one tells a different story about the underlying resistance mechanism.
- D phenotype: A clearly flattened, D-shaped clindamycin zone on the side nearest the erythromycin disk. This indicates inducible erm-mediated resistance. Clindamycin may fail during therapy because the resistance gene can switch to constitutive expression.
- D-plus phenotype: A D-shaped zone that also contains visible colonies growing within the flattened area, approaching the clindamycin disk. This variant often correlates with ermC rather than ermA and suggests a stronger or more readily induced resistance response.
- MS phenotype: The isolate is resistant to erythromycin and susceptible to clindamycin with a completely round, uninhibited clindamycin zone. No D shape appears. This pattern indicates the efflux pump (msrA) rather than erm-mediated methylation. Clindamycin can be prescribed safely.
- Constitutive resistance: The isolate is resistant to both erythromycin and clindamycin outright. No D test is needed because the lab already knows clindamycin will not work.
A study that characterized these patterns by gene content confirmed the association: all D-phenotype isolates carried ermA, D-plus isolates overwhelmingly carried ermC, and every MS-phenotype isolate carried msrA alone.3PubMed Central. Testing for induction of clindamycin resistance in erythromycin-resistant isolates of Staphylococcus aureus Those clean genotype-phenotype links make the D test a surprisingly reliable proxy for the molecular basis of resistance without requiring genetic testing.
What Happens When Inducible Resistance Is Missed
The clinical stakes are not hypothetical. A published case report describes a surgical site infection caused by MRSA that tested susceptible to clindamycin. The patient was treated with clindamycin and did not improve. A post-treatment isolate was now overtly resistant to clindamycin, yet molecular typing proved it was the same strain as the original. When researchers went back and performed a D test on the pre-treatment isolate, it was positive: the inducible resistance had been present all along but was invisible to the initial susceptibility panel.4PubMed. Failure of clindamycin treatment of methicillin-resistant Staphylococcus aureus expressing inducible clindamycin resistance in vitro
That case is a textbook illustration of why the D test exists. Under clindamycin pressure, the bacterium’s erm gene transitioned from inducible to constitutive expression, likely through a small mutation or deletion in the regulatory region upstream of the gene. Similar regulatory-region changes, including deletions, duplications, and point mutations, have been documented as the molecular basis for that switch in both staphylococci and other bacteria.5PubMed Central. The diversity of inducible and constitutively expressed erm(C) genes and association to different replicon types in staphylococci plasmids6PubMed Central. Constitutive and Inducible Expression of the rRNA Methylase Gene erm(B) in Campylobacter The conversion is not a rare laboratory curiosity; it is a recognized route to clinical failure.
How Common Is Inducible Clindamycin Resistance
Rates vary enormously depending on geography, patient population, and whether the staphylococci in question are methicillin-resistant or methicillin-sensitive. One consistent finding across studies is that MRSA isolates carry inducible clindamycin resistance at far higher rates than MSSA. A study from the sub-Himalayan region of India found the inducible phenotype in about 28% of MRSA isolates versus roughly 9% of MSSA, with both inducible and constitutive resistance significantly more common in the methicillin-resistant group.7PubMed Central. Inducible Clindamycin Resistance among Clinical Isolates of Staphylococcus aureus from Sub Himalayan Region of India In Nepal, D-test screening of S. aureus from school-aged children found inducible resistance in about 23% of tested isolates, concentrated heavily among MRSA strains (over 76% of MRSA versus roughly 4% of MSSA).8PubMed Central. Inducible clindamycin resistance and erm genes in Staphylococcus aureus in school children in Kathmandu, Nepal
Other studies echo that MRSA-MSSA gap. Data from a northeastern Indian hospital similarly found higher rates of both inducible and constitutive phenotypes in MRSA compared to MSSA.9Journal of Infection in Developing Countries. Inducible and constitutive clindamycin resistance in Staphylococcus aureus in a northeastern Indian tertiary care hospital A study from another part of India reported inducible resistance in 10% of all S. aureus clinical isolates and constitutive resistance in 9%, with MRSA carrying about 20% inducible resistance compared to 6% in MSSA.10PubMed Central. Inducible Clindamycin Resistance in Staphylococcus aureus Isolated from Clinical Samples
These numbers underscore a practical point: in settings where MRSA is common, a significant fraction of isolates that appear clindamycin-susceptible on routine testing actually carry the inducible phenotype. Skipping the D test in those environments means accepting a meaningful blind spot.
Shifts Over Time in a Single Population
Rates of inducible resistance are not static. A longitudinal study of pediatric community-acquired MRSA in Dallas, Texas tracked erythromycin-resistant, clindamycin-susceptible isolates from 1999 through 2002. Inducible resistance was present in about 93% of tested isolates in 1999 but dropped to just 7% by 2002. All the non-inducible strains carried msrA (the efflux gene), while the inducible strains carried various erm genes, with ermB, ermC, and ermA all represented.11Antimicrobial Agents and Chemotherapy. Inducible clindamycin resistance and molecular epidemiologic trends of pediatric community-acquired methicillin-resistant Staphylococcus aureus in Dallas, Texas That dramatic shift probably reflected changes in the circulating clonal types of MRSA in the community rather than a change in testing. It is a reminder that local epidemiology drives the clinical relevance of D testing: in some times and places, nearly every erythromycin-resistant isolate will be inducible; in others, most will carry only the efflux mechanism and be genuinely susceptible to clindamycin.
Automated Systems Versus the D Test
Many clinical laboratories use automated platforms such as the Vitek-2 system for susceptibility testing. These systems can flag isolates for possible inducible clindamycin resistance, but their accuracy compared to the D test is imperfect. One head-to-head comparison found that the Vitek-2 system had a sensitivity of about 91% and specificity of 100% for detecting the inducible phenotype when measured against D-test results. It missed three D-test-positive isolates entirely.12Journal of Medical Sciences and Health. Identification of Inducible Clindamycin Resistance in Staphylococcus aureus using Automated Vitek-2 Compact System and D test
A separate validation study reported slightly different numbers: the Vitek-2 achieved 95% sensitivity with 100% specificity against the D test, but its negative predictive value was only 72%, meaning that when the automated system said an isolate was not inducibly resistant, it was wrong about a quarter of the time.7PubMed Central. Inducible Clindamycin Resistance among Clinical Isolates of Staphylococcus aureus from Sub Himalayan Region of India In practical terms, labs relying solely on automated results without a confirmatory D test risk releasing false susceptibility reports. Many guidelines therefore recommend performing the D test on any erythromycin-resistant, clindamycin-susceptible isolate, regardless of what the automated system says.
Testing Beyond Staphylococci
The D test was developed primarily for staphylococci, but inducible clindamycin resistance driven by erm genes also occurs in beta-hemolytic streptococci (groups A, B, C, F, and G). The challenge is that the standard disk approximation test was originally validated only for staphylococci, and some laboratories were uncertain whether the same method worked reliably for streptococcal species.
Work on this question demonstrated that the erythromycin-clindamycin D-zone test accurately detects inducible resistance in streptococci: in one study, all 16 erythromycin-resistant, clindamycin-susceptible streptococcal isolates that were D-zone positive were confirmed to carry inducible clindamycin resistance by broth microdilution testing.13PubMed. Investigation of inducible clindamycin and telithromycin resistance in isolates of beta-hemolytic streptococci A collaborative evaluation later confirmed that a single-well broth microdilution test combining erythromycin and clindamycin at specific concentrations could serve as an alternative to the disk test and might be easier to incorporate into routine automated panels for streptococci.14PubMed Central. Collaborative evaluation of an erythromycin-clindamycin combination well for detection of inducible clindamycin resistance in beta-hemolytic streptococci by use of the CLSI broth microdilution method
The point for clinicians is that group A and group B streptococci are common causes of skin infections and neonatal sepsis, and clindamycin is a key drug in penicillin-allergic patients. Inducible resistance in these species is not merely a theoretical problem; it carries the same risk of in-vivo selection for constitutive resistance as in staphylococci.
The Molecular Underpinnings of Inducibility
Why does an erm gene sometimes stay silent until a macrolide arrives? The answer lies in a stretch of regulatory DNA upstream of the erm coding sequence. In the inducible state, this region forms a secondary structure in the messenger RNA that blocks translation of the erm protein. When erythromycin binds to a ribosome that is translating a short upstream peptide within that regulatory region, the ribosome stalls. That stalling rearranges the RNA structure and exposes the erm reading frame, allowing the methylase to be produced.
Any mutation, deletion, or duplication that disrupts that regulatory hairpin can lock the gene into permanent expression. Researchers have catalogued a wide range of such changes in ermC-carrying staphylococci from animal sources: deletions ranging from just 3 base pairs up to 118 base pairs, a 63-base-pair duplication, and various point mutations, all of which converted the gene from inducible to constitutive expression.5PubMed Central. The diversity of inducible and constitutively expressed erm(C) genes and association to different replicon types in staphylococci plasmids The same phenomenon has been documented for ermB in Campylobacter, where insertions and deletions in the regulatory region flipped expression from inducible to constitutive.6PubMed Central. Constitutive and Inducible Expression of the rRNA Methylase Gene erm(B) in Campylobacter The diversity of mutations that achieve this switch explains why conversion during therapy is not a freak event: there are many ways to break the regulatory hairpin, and antibiotic pressure provides strong selection for any of them.
Which erm Genes Show Up and Why It Matters
Laboratories that go beyond phenotypic detection and perform molecular testing on D-test-positive isolates find all three major erm genes in circulation. A study from Isfahan, Iran identified ermC in about half of inducible isolates, ermB in two, and ermA in one, with two isolates harboring no detectable erm gene at all, suggesting additional, less-characterized resistance determinants may be involved.15PubMed Central. Distribution of erm genes among Staphylococcus aureus isolates with inducible resistance to clindamycin in Isfahan, Iran The Dallas pediatric study found all three genes among inducible strains, with ermB most common, followed by ermC and then ermA.11Antimicrobial Agents and Chemotherapy. Inducible clindamycin resistance and molecular epidemiologic trends of pediatric community-acquired methicillin-resistant Staphylococcus aureus in Dallas, Texas
From a treatment standpoint, the specific gene matters less than the phenotype: if the D test is positive, the clinical implication is the same regardless of whether ermA, ermB, or ermC is responsible. But from a surveillance and infection-control perspective, knowing which gene predominates can help track the spread of particular clonal lineages. For example, ermA is classically associated with hospital-acquired MRSA lineages, while ermC is more often found on small plasmids circulating in community strains. Tracking gene prevalence over time helps infection-control teams understand whether community or hospital strains are driving local resistance trends.
Inducible Clindamycin Resistance in Veterinary Medicine
Dogs and other companion animals get staphylococcal infections too, and the species involved, Staphylococcus pseudintermedius, can carry the same erm-mediated resistance. Clindamycin is widely prescribed in veterinary dermatology, making the question of inducible resistance directly relevant. A study screening 608 S. pseudintermedius isolates from dogs found eight that were positive for inducible clindamycin resistance by D testing and PCR for ermB.16PubMed Central. Incidence of inducible clindamycin resistance in Staphylococcus pseudintermedius from dogs
A Thai study focused specifically on methicillin-resistant S. pseudintermedius and detected inducible clindamycin resistance in 5% of isolates. All the inducibly resistant strains were methicillin-resistant, multidrug-resistant, and carried ermB. Molecular typing revealed they belonged to a single clonal complex, suggesting that inducible resistance was spreading through clonal expansion rather than independent gene acquisition in separate lineages.17PubMed. Occurrence and characterization of inducible clindamycin resistance in canine methicillin-resistant Staphylococcus pseudintermedius The recommendation from both studies is the same as in human medicine: D-zone testing should be performed on erythromycin-resistant, clindamycin-susceptible veterinary isolates before prescribing clindamycin.
The veterinary parallel also raises a broader concern. Companion animals and their owners share skin bacteria, and methicillin-resistant staphylococci can move between species. Inducible resistance genes circulating in pet-associated strains are part of the same resistance ecology that affects human medicine, even if the staphylococcal species are different.
When Not to Worry About the D Test
If an isolate is susceptible to both erythromycin and clindamycin, no D test is needed. The test is only relevant when erythromycin resistance and clindamycin susceptibility coexist. Similarly, if the isolate is already resistant to both drugs, the answer is clear: clindamycin will not work, and there is nothing hidden to uncover.
The MS phenotype, where the D test is negative and erythromycin resistance comes from efflux only, is another situation where clindamycin can be used. The efflux pump encoded by msrA does not act on lincosamides, so there is no cross-resistance to worry about and no conversion risk during treatment. Labs that perform D testing routinely allow clinicians to distinguish these genuinely safe-to-treat cases from the inducible ones, preserving clindamycin as a treatment option when it is truly appropriate rather than abandoning it reflexively whenever erythromycin resistance appears.