CONCISE REVIEWS OF PEDIATRIC INFECTIOUS DISEASES®

Macrolide-Inducible Resistance to Clindamycin and the D-TestCharles R. Woods, MD, MS

Key Words: D-test, macrolide resistance,clindamycin(Pediatr Infect Dis J 2009;28: 1115–1118)

Clindamycin has been used to treat seri-ous infections caused by susceptible

Staphylococcus aureus strains in children formore than 30 years.1 It remains effective formany infections caused by community-ac-quired methicillin-resistant S. aureus (CA-MRSA).2,3 Clindamycin also is useful forselected infections caused by pneumococci,group A streptococci, and a number of othermicrobes. Absorption after oral administra-tion is nearly complete, yielding serum con-centrations that approximate those from in-travenous (IV) administration.4 This permitsearly transition to outpatient management ofsusceptible infections without the complica-tions of continued IV access.5

Clindamycin resistance is commonamong health care-associated MRSA strains.Most CA-MRSA remain susceptible to date,but resistance rates vary by region.2 Pneu-mococcal resistance to clindamycin may ex-ceed 30% in some areas of the US,6 whileabout 4% of group A streptococcal isolatesare resistant.7 Macrolide-inducible resistanceto clindamycin was first recognized in thelaboratory in the early 1960s.8 Clinical iso-

lates resistant to clindamycin were first rec-ognized in 1968.9 Relapse of S. aureus in-fection in a rabbit model of endocarditisduring clindamycin therapy was observed inthe early 1970s.10 Clinical and bacteriologicrelapse in a patient with S. aureus endocar-ditis during the fourth week of clindamycintherapy after initial improvement was re-ported in 1976.11 The initial isolate wassusceptible to erythromycin and clindamycinwhile that from the relapse was resistant toboth. This led to abandonment of clindamy-cin for treatment of endocarditis.

The mechanism by which resistanceto clindamycin can emerge during therapy,the D-test used to detect it, and the clinicalimplications are discussed in this review.

INDUCIBLE MACROLIDERESISTANCE

The clinical implications of a posi-tive D-test begin with an understanding ofcross-resistance for 3 antibiotic familiesthat share a common binding site—macro-lides (eg, erythromycin, clarithromycin,azithromcyin), lincosamides (eg, clinda-mycin), and group B streptogrammins (eg,quinupristin). This cross-resistance, calledthe MLSB phenotype, results from enzymaticdimethylation of an adenine residue in theseantibiotics’ binding site in the 23S rRNAcomponent of the 50S ribosomal subunit.The methylase enzyme is encoded by a mul-tiallele plasmid-borne gene erm, which oc-curs predominantly as variants erm(C) orerm(A) in staphylococci (other variants oc-cur in pneumococci, hemolytic streptococci,and enterococci). When the MLSB pheno-type is the result of constitutive (“always

on”) erm expression, initial in vitro suscep-tibility tests show resistance to all 3 antibi-otic classes. When the inducible genotype(iMLSB) is present, in vitro tests show resis-tance to 14- and 15-membered ring macro-lides listed above, but susceptibility to clin-damycin and 16-membered ring macrolides(available in some countries other than theUS) is retained—these agents do not induceresistance to themselves.12–14

The inducible methylase system isregulated at the level of mRNA translation(translational attenuation) rather than genetranscription. The gene is transcribed tomRNA constitutively, but the mRNA cannotbe read into protein without the presence of atranslational inducer. In the inducible ermcassette, the open reading frame of the meth-ylase gene and its dedicated ribosomal bind-ing site is preceded by DNA sequences thatencode another ribosomal binding site, ashort 19 amino-acid peptide with a stopcodon, and an additional sequence that in-cludes 2 inverted repeats (IRs).15–17

In the attenuated state, the secondarystructure of the transcribed mRNA has 2-stemloops (Fig. 1 panel A). The first involves thelast 8 codons of the short peptide (functionally,IR1) and an adjacent IR2. The second consistsof an IR3 sequence (which follows IR2) thatbinds to the sequences encoding the ribosomalbinding site and start codon for the methylasegene (functionally, IR4). This effectively“hides” the ribosomal binding site and startcodon for the methylase in the second (IR3–IR4) stem-loop, such that the methylase en-zyme cannot be translated.15

When macrolide molecule capable ofinducing the methylase translation binds toits target site in a ribosome, the “inhibited”

From the Department of Pediatrics, University ofLouisville School of Medicine, Louisville, KY.

Address for correspondence: Charles R. Woods, MD,MS, Pediatric Infectious Diseases, 571 S. FloydSt., Suite 321, Louisville, KY 40202. E-mail:charles.woods@louisville.edu.

Copyright © 2009 by Lippincott Williams & WilkinsISSN: 0891-3668/09/2812-1115DOI: 10.1097/INF.0b013e3181c35cc5

CONTENTSMacrolide-Inducible Resistance to Clindamycin and the D-Test

EDITORIAL BOARDCo-Editors: Margaret C. Fisher, MD, and Gary D. Overturf, MDEditors for this Issue: Michael Capello, MD, and Leonard Krilov, MDBoard MembersMichael Cappello, MDEllen G. Chadwick, MDJanet A. Englund, MDLeonard R. Krilov, MD

Charles T. Leach, MDKathleen McGann, MDJennifer Read, MDJeffrey R. Starke, MD

Geoffrey A. Weinberg, MDLeonard Weiner, MDCharles R. Woods, MD

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ribosome is still able to bind to and starttranslation of the mRNA sequence of theshort peptide. The ribosome stalls after read-ing several codons but remains complexedwith the mRNA strand (macrolides work byinhibiting elongation of peptide chains be-yond 3 or 4 residues). This attached stallingleads to a conformational change in the sec-ondary structure of the mRNA: the induc-ible-state stem loops are opened up, a newsingle stem loop with IR2 binding to IR3 isformed, and the ribosomal binding site andstart codon of the methylase gene are ex-posed (Fig. 1 panel B).15,17

These initials steps occur at very lowmacrolide concentrations (10–100 ng/mL oferythromycin, below the MIC for many sus-ceptible microbes), such as may occurshortly after a macrolide dose is adminis-tered to a patient but before accumulation ofMIC-equivalent concentrations at the site ofinfection. At this early stage, macrolide mol-ecules are bound to only a small fraction of

the mature ribosomes extant in the microbialcytoplasm. Most ribosomes remain macroli-de-free (uninhibited) and readily able to as-sociate with the newly exposed erm ribo-somal binding site. The mRNA then istranslated, producing sufficient methylase tomodify the MLSB binding sites of the ribo-somes that remain uninhibited. The microbethus is protected against further ribosomalinactivation despite increasing intracellularmacrolide concentrations. Phenotypic mac-rolide resistance is demonstrable in vitrowithin 20 to 40 minutes after initial macro-lide exposure. Of note, induction is lost after2 generations of growth in vitro in the ab-sence of an inducer.15–17

RELEVANCE TO CLINDAMYCINHow then is inducible macrolide re-

sistance relevant to clindamcyin? Clindamy-cin binding to the same 23S rRNA subunitdoes not lead to induction of methylase

translation—clindamycin does not induceresistance to itself, and in vitro testing ofisolates with the iMLSB genotype demon-strates clindamycin susceptibility. However,the macrolide-inducible DNA sequences thatprecede the erm(C) methlyase open readingframe undergo mutations, substitutions or de-letions that generate readily translatable (nowconstitutive MLSB) secondary mRNA struc-tures in about 1 in 2 million replications13,14

(Fig. 1 panel C). Many infections, especiallywhen purulent collections are present, havemicrobial burdens that are exceed the denom-inator of this mutation rate by 10-fold or more,such that small numbers of clindamycin-resis-tant microbes are likely common in infectionscaused by such iMLSB� strains.

During the past 10 years, treatmentfailure has reported in handfuls of adult andpediatric cases when clindamycin was usedfor MRSA infections caused by strains thatinitially appeared susceptible to clindamycinbut resistant to macrolides. Clindamycin re-sistance was evident upon retesting of therecurring isolates.3,12,18 The common themeof these cases has been initial improvementon clindamycin with subsequent recrudes-cence days to weeks into or after completionof therapy. This is consistent with the abovelow frequency mutation rate from iMLSB

genotype to constitutive MLSB erm expres-sion, where a resistant subpopulation sur-vives the innate and early adaptive host im-mune responses and emerges to cause new orworsening of existing signs and symptoms.

THE D-TESTThe combination of resistance to

erythromycin with susceptibility to clinda-mycin in S. aureus (and other gram-positivemicrobes) can be due to the iMLSB genotypeor efflux pumps that remove macrolides butnot clindamycin from the microbe. The D-test, based on disk diffusion susceptibilitytesting, is recommended to determine if theiMLSB genotype is present.19 In the D-test,disks containing erythromycin (15 �g) andclindamcyin (2 �g) are placed 15 to 20 mmapart on an agar plate that has been inocu-lated with the clinical isolate (Fig. 2). Aclindamycin-susceptible, erythromycin-re-sistant isolate should have a zone of inhibi-tion �21 mm in diameter, with minimal ifany inhibition of growth around the erythro-mycin disk.

The round zones of erythromycin andclindamycin that diffuse outward from thedisks partially overlap in this configuration.Erythomycin molecules reach the outer re-gion of the clindamycin zone prior to clin-damycin molecules. When the iMLSB geno-type is present, this leads to methylasetranslation, permitting microbial growth inthis region despite subsequent diffusion of

FIGURE 1. A, Shows the schematic representation of the secondary structure of theinducible methylase cassette in the absence of macrolides. Two stem loops areformed by the interactions of inverted repeat 1 (IR1) with IR2 and IR3 with IR4. IR4consists of the ribosomal binding site and initial codons of the methylase mRNA.Ribsomes are unable to complex with the methylase binding site in this conforma-tion. B, Shows the secondary structure after a macrolide-bound ribosome com-plexes with the first few codons of the short peptide and stalls. Other ribosomesnot yet inhibited by macrolides then bind to and translate the methylase, whichprotects remaining ribosomes from further macrolide binding. Panel C shows anexample of mutation of the “inducible” mRNA sequences that can lead to consti-tutive methylase translation. The peptide and IR2 are deleted, and all mature ribo-somes are methylated. Adapted from Mol Cell. 2008;30:190–202.

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inhibitory concentrations of clindamcyin.This growth blunts the expected round zoneof growth-inhibition around the clindamycindisk into a D-shape facing the erythromycindisk (Fig. 2), which indicates a positive D-test. A negative D-test (round zone) indicatesefflux-mediated macrolide resistance withretained clindamycin susceptibility.

CLINICAL IMPLICATIONS OF APOSITIVE D-TEST

A positive D-test indicates the pres-ence of iMLSB genotype. This means that itis possible, but far from certain, that a sub-population of microbes resistant to clinda-mycin may emerge and lead to clinical fail-ure or recrudescence. Infections caused by S.aureus strains that carry the iMLSB genotypeoften respond to clindamycin therapy with-out relapse3—resistant subpopulations either

do not develop or are eliminated by hostresponses before progression of infection canoccur. However, because of the clinical re-ports of clindamycin failure associated withD-test� strains, the Clinical and LaboratoryStandards Institute recommends that labora-tories report D-test� isolates as resistant toclindamycin.20

Clinical and Laboratory Standards In-stitute recommendations also suggest inclu-sion of a comment that “this isolate is pre-sumed to be resistant based on detection ofinducible clindamycin resistance. Clindamy-cin may still be effective in some patients.”This is an important allowance, as relativelyminor infections such as cellulitis caused byD-test� S. aureus strains (which can beMRSA or MSSA) will respond adequately.These minor infections are far more commonthan serious invasive infections, and change

to other antimicrobial agents based simplyon the D-test� result in these cases mayserve only to increase sensitization risks tothe new agent or increase cost of therapy.Consequences of recrudescence in most ini-tially minor episodes also are unlikely to besevere, especially if recognized early on.

For sepsis, pneumonia, osteomyelitis,and other serious invasive S. aureus infec-tions, even the small risk of emergence ofresistance indicated by a positive D-test re-sult generally should lead to avoidance ofclindamycin, or prompt change from clinda-mycin to another agent to which the isolate issusceptible.9 When starting or continuingclindamycin for less severe infections causedby D-test� strains, close follow-up for po-tential failure and late relapse is needed.Patients and/or their caregivers should becounseled regarding potential relapse and toseek care early if they have concerns ofrecurrence.

At the community level, relative fre-quency of constitutive MLSB-related versusiMLSB-related clindamycin resistance shouldbe monitored to provide appropriate guidancefor empiric therapy for clinical scenarios wherecommunity-acquired S. aureus infection is sus-pected. Reporting aggregate clindamycin resis-tance statistics without separating out the pro-portion deemed resistant on the basis of apositive D-test could lead to premature aban-donment of clindamycin as an empiric optionin clinical situations for which this may still bean appropriate agent. The risk of failure in thefirst hours to days of empiric clindamycin ther-apy prior to availability of D-test results seemsexceedingly small based on clinical reports todate—but ongoing vigilance is required.

Clindamycin suppresses production ofPanton-Valentine leukocidin, alpha-hemoly-sin, and toxic-shock syndrome toxin 1 by S.aureus in vitro at the translational (ribo-somal) level.21 Improved outcomes for se-vere group A streptococcal infections treatedwith clindamycin versus beta-lactam agentshave been demonstrated in a mouse myositismodel22 and in a clinical case series in chil-dren.23 This is the basis for use of clindamy-cin for toxin suppression in addition to a cellwall-active agent for treatment of life-threat-ening streptococcal and staphylococcal in-fections (eg, toxic shock syndrome, necrotiz-ing fasciitis).

Toxin production by isolates resistantto clindamcyin via constitutive methylaseexpression will not be impacted by “adjunc-tive” clindamycin. However, for infection bystrains with the iMLSB genotype, suppres-sion of toxin production by clindamycin canreasonably be expected, at least during thecritical first hours to days of therapy whenthis effect is most likely to be clinicallyimportant. A miniscule fraction at most

FIGURE 2. A, Is a photo of a positive D-test. The erythromycin and clindamycindisks are on the left and right, respectively. The expected O-shape of the clindamy-cin zone of growth inhibition, which would be seen in a negative D-test, is bluntedon the side facing the erythomycin disk, resulting in a D-shaped zone. B, Is a styl-ized cartoon of the D-test. Erythromycin molecules diffuse into the region of theclindamycin zone labeled “a” before clindamycin molecules, inducing the methyl-ase, conferring resistance and allowing microbial growth despite subsequent arrivalof otherwise inhibitory concentrations of clindamycin. Growth-inhibiting concen-trations of clindamycin reach the region labeled “b” before erythromycin mole-cules can arrive to induce resistance. Arrows denote approximate equidistant diffu-sion of each antibiotic to a line bisecting the distance between the 2 antibioticdisks. The actual “intersection” of erythromycin versus clindamycin effects is oftenslightly bowed as in panel A. Gray areas indicate microbial growth on the agar sur-face; white areas, growth inhibition; E, erythromycin disk; C, clindamycin disk.

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(�0.01%) of the total progeny of such astrain might be impervious to clindamycinearly on, due to the spontaneous mutationsthat can confer constitutive expression. Aswith empiric use of clindamycin for sus-pected S. aureus infections above, the deci-sion to switch from empiric use of clindamy-cin for toxin suppression to anotherribosomally active agent such as linezolidcan be based on local rates of constitutiveresistance among clinical isolates of S. au-reus and group A streptococci.

REFERENCES1. Feigin RD, Pickering LK, Anderson D, et al.

Clindamycin treatment of osteomyelitis and sep-tic arthritis in children. Pediatrics. 1975;55:213–223.

2. Martinez-Aguilar G, Hammerman W, Mason EJr, et al. Clindamycin treatment of invasive infec-tions caused by community-acquired, methicillin-resistant and methicillin-susceptible Staphylococ-cus aureus in children. Pediatr Infect Dis J.2003;22:593–598.

3. Frank AL, Marcinak J, Mangat P, et al.Clindamycin treatment of methicillin-resistantStaphylococcus aureus infections in children. Pe-diatr Infect Dis J. 2002;21:530–534.

4. Chambers HF. Protein synthesis inhibitors. In:Brunton LL, ed. The Pharmacologic Basis ofTherapeutics. 11th ed. New York, NY: McGraw-Hill; 2006:1188–1190.

5. Ruebner R, Keren R, Coffin S, et al. Complicationsof central venous catheters used for the treatment of

acute hematogenous osteomyelitis. Pediatrics.2006;117:1210–1215.

6. Jacobs MR, Good CE, Beall B, et al. Changes inserotypes and antimicrobial susceptibility of in-vasive Streptococcus pneumoniae strains inCleveland: a quarter century of experience. J ClinMicrobiol. 2008;46:982–990.

7. Richter SS, Heilmann K, Beekman S, et al.Macrolide-resistant Streptococcus pyogenes inthe United States, 2002–2003. Clin Infect Dis.2005;41:599–608.

8. Barber M, Waterworth P. Antibacterial activity oflincomycin and pristinamycin: a comparison witherythromycin. Br Med J. 1964;2:603–606.

9. McGehee RF, Barrett F, Finland M. Resistance ofStaphylococcus aureus to lincomycin, clinimy-cin, and erythromycin. Antimicrob Agents Che-mother. 1968;8:392–397.

10. Sande M, Johnson M. Antimicrobial therapy ofexperimental endocarditis caused by Staphylo-coccus aureus. J Infect Dis. 1975;131:367–375.

11. Watanakunakorn C. Clindamycin therapy ofStaphylococcus aureus endocarditis. Am J Med.1976;60:419–425.

12. Lewis JS, Jorgensen J. Inducible clindamycinresistance in staphylococci. Clin Infect Dis. 2005;40:280–285.

13. Daurel C, Huet C, Dhalluin A, et al. Differencesin potential for selection of clindamycin-resistantmutants between inducible erm(A) and erm(C)Staphylococcus aureus genes. J Clin Microbiol.2008;46:546–550.

14. Leclercq R. Mechanisms of resistance to macro-lides and lincosamides. Clin Infect Dis. 2002;34:482–492.

15. Weisblum B. Insights into erythromycin action.Antimicrob Agents Chemother. 1995;39:797–805.

16. Dubnau D. Translational attenuation: the regula-tion of bacterial resistance. CRC Crit Rev Bio-chem. 1984;16:103–132.

17. Vazquez-Laslop N, Thum C, Mankin A.Molecular mechanism of drug-dependent ribo-some stalling. Mol Cell. 2008;30:190–202.

18. Siberry GK, Tekle T, Carroll K, et al. Failure ofclindamycin treatment of methicillin-resistantStaphylococcus aureus expressing inducible clin-damycin resistance in vitro. Clin Infect Dis. 2003;37:1257–1260.

19. Steward CD, Raney P, Morrell A, et al. Testingfor induction of clindamycin resistance in eryth-romycin-resistant isolates of Staphylococcus au-reus. J Clin Microbiol. 2005;43:1716–1721.

20. NCCLS (CLSI). Performance standards for anti-microbial susceptibility testing. NCCLS (CLSI).Wayne, PA, 2004. M100-S14.

21. Stevens DL, Ma Y, Salmi D, et al. Impact ofantibiotics on expression of virulence-associ-ated exotoxin genes. J Infect Dis. 2007;195:202–211.

22. Stevens DL, Gibbons A, Bergstrom R, et al. TheEagle effect revisited: efficacy of clindamycin,erythromycin, and penicillin in the treatment ofstreptococcal myositis. J Infect Dis. 1988;158:23–28.

23. Zimbelman J, Palmer A, Todd J, et al. Improvedoutcome of clindamycin compared with beta-lactam antibiotic treatment for invasive Strepto-coccus pyogenes infection. Pediatr Infect Dis J.1999;18:1096–1100.

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