induction of efflux-mediated macrolide resistance in

Induction of efflux-mediated macrolide resistancein Streptococcus pneumoniaeScott Chancey, Emory UniversityXiaoliu Zhou, Emory UniversityDorothea Zahner, Emory UniversityDavid S Stephens, Emory University

Journal Title: Antimicrobial Agents and ChemotherapyVolume: Volume 55, Number 7Publisher: American Society for Microbiology | 2011-07, Pages 3413-3422Type of Work: Article | Final Publisher PDFPublisher DOI: 10.1128/AAC.00060-11Permanent URL: http://pid.emory.edu/ark:/25593/f24gx

Final published version: http://aac.asm.org/content/55/7/3413

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2011, p. 3413–3422 Vol. 55, No. 70066-4804/11/$12.00 doi:10.1128/AAC.00060-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Induction of Efflux-Mediated Macrolide Resistance inStreptococcus pneumoniae�

Scott T. Chancey,1,3 Xiaoliu Zhou,1,3§ Dorothea Zahner,1,3 and David S. Stephens1,2,3*Division of Infectious Diseases, Department of Medicine,1 and Department of Microbiology and Immunology,2

Emory University School of Medicine, Atlanta, Georgia 30322, and Department ofVeterans Affairs Medical Center, Atlanta, Georgia 300333

Received 15 January 2011/Returned for modification 7 March 2011/Accepted 25 April 2011

The antimicrobial efflux system encoded by the operon mef(E)-mel on the mobile genetic element MEGA inStreptococcus pneumoniae and other Gram-positive bacteria is inducible by macrolide antibiotics and antimi-crobial peptides. Induction may affect the clinical response to the use of macrolides. We developed mef(E)reporter constructs and a disk diffusion induction and resistance assay to determine the kinetics and basis ofmef(E)-mel induction. Induction occurred rapidly, with a >15-fold increase in transcription within 1 h ofexposure to subinhibitory concentrations of erythromycin. A spectrum of environmental conditions, includingcompetence and nonmacrolide antibiotics with distinct cellular targets, did not induce mef(E). Using 16different structurally defined macrolides, induction was correlated with the amino sugar attached to C-5 of themacrolide lactone ring, not with the size (e.g., 14-, 15- or 16-member) of the ring or with the presence of theneutral sugar cladinose at C-3. Macrolides with a monosaccharide attached to C-5, known to block exit of thenascent peptide from the ribosome after the incorporation of up to eight amino acids, induced mef(E)expression. Macrolides with a C-5 disaccharide, which extends the macrolide into the ribosomal exit tunnel,disrupting peptidyl transferase activity, did not induce it. The induction of mef(E) did not require macrolideefflux, but the affinity of macrolides for the ribosome determined the availability for efflux and pneumococcalsusceptibility. The induction of mef(E)-mel expression by inducing macrolides appears to be based on specificinteractions of the macrolide C-5 saccharide with the ribosome that alleviate transcriptional attenuation ofmef(E)-mel.

Macrolides are broad-spectrum antibiotics with complexmacrocyclic 14-, 15-, or 16-member lactone rings that bind inthe peptide exit tunnel of bacterial ribosomes and inhibit pro-tein synthesis. Macrolides are often recommended as the em-pirical first-line treatment for upper respiratory bacterialinfections, including pneumococcal infections and community-acquired pneumonia. However, bacterial resistance to macro-lides is expanding worldwide in Gram-positive bacteria and isnow present in almost a third of all invasive Streptococcuspneumoniae isolates (14). The two most common mechanismsof macrolide resistance in bacterial pathogens are modificationof the bacterial ribosome, either by methylation or mutation,and extrusion of the drugs from the bacterial cell by an effluxpump. Genes of the erm (erythromycin ribosomal methylases)family of rRNA methylases confer high-level resistance to lin-cosamides and streptogramins, as well as macrolides (theMLSB phenotype), and can be constitutive or inducible. In theinducible form, resistance develops only after exposure ofthe bacterium to the macrolide.

The best-studied mechanism of inducible MLSB resistanceinvolves the erm(C) gene found in S. aureus and other Gram-positive pathogens (4, 32, 33). Translation of erm(C) is atten-uated in the absence of inducers due to secondary structures

that render the ribosomal binding site inaccessible. Inducer-bound ribosomes stall during the translation of a 19-amino acidleader peptide upstream of erm(C), resulting in the refoldingof the transcript to make the ribosome binding site available,thus promoting translation (32). A later comparison of induc-ers for erm(C) and erm(SV) in Streptomyces viridochromogenesshowed that the specific sequence of the leader peptide deter-mines the range of inducers (24). Macrolides with 14-mem-bered rings and C-3 cladinose induced erm(C), and 16-mem-bered macrolides induced erm(SV) (24). The lincosamidecelesticetin induced both erm(C) and erm(SV), demonstratinga common inducer between the otherwise disparately inducedgenes (24). Attenuation of erm genes can also occur at the levelof transcription, as with erm(K) of Bacillus licheniformis (28,29). Transcriptional attenuation occurs when secondary struc-tures in the transcript function like rho-independent termina-tors to terminate transcription short of the erm gene codingregion.

Efflux-mediated macrolide resistance was first reported inStaphylococcus epidermidis and later in Streptococcus pyogenesand S. pneumoniae (30, 40, 45). In S. epidermidis, macrolideefflux is conferred by msr(A) and is induced by the 14- and15-membered macrolides clarithromycin and azithromycin andthe ketolide telithromycin but not by streptogramin B, eventhough the latter is a substrate for Msr(A)-mediated efflux. Instreptococci, macrolide efflux is conferred by proteins encodedby mef(E), mef(A), or less commonly, mef(I), which share�90% identity. The mef genes are transcribed as an operonwith the msr(A) homolog mel [also called msr(D)], comprising

* Corresponding author. Mailing address: Emory University, 1440Clifton Rd. NE, Suite 420, Atlanta, GA 30322. Phone: (404) 727-8357.Fax: (404) 778-5434. E-mail: [email protected].

§ Present address: Centers for Disease Control and Prevention, 4770Buford Highway, Chamblee, GA 30341.

� Published ahead of print on 2 May 2011.

3413

a dual efflux pump (3, 12). In S. pneumoniae, mef(E)-mel iscarried on the small mobile element MEGA, the predominantmacrolide efflux determinant in the pneumococcus (15, 43). Invitro, Mef(E)/Mel confers moderate resistance (1 to 32 �g/ml)to 14- and 15-membered macrolides but not, reportedly, to16-membered macrolides, lincosamides, or streptogramins (Mphenotype) (45, 47). Mef(E)/Mel-mediated resistance hasbeen shown to be induced by the 14- and 15-membered mac-rolides erythromycin, clarithromycin, and azithromycin but notby 16-membered macrolides (3, 49). Ketolides, such as teli-thromycin, are considered poor inducers of mef(E)-mel andretain antimicrobial activity against pneumococcal strains car-rying these genes (34, 49).

In vivo exposure to macrolides or other potential inducersmay result in resistance higher than the levels predicted byMICs determined in vitro. We recently reported that certainantimicrobial peptides (AMP), including the human AMP LL-37, activated transcription of mef(E)-mel and resulted in in-duced resistance to the macrolide erythromycin (50). This sug-gests that the Mef(E)/Mel efflux pump can be induced byhuman host defenses and, therefore, can be primed prior toclinical administration of macrolides. The goals of this studywere to define the range of compounds, macrolides as well asother antibiotics and other conditions, that induce Mef(E)/Mel-mediated resistance, to elucidate the kinetics of induction,to determine the structural features of macrolides required forinduction, and to develop a model for the mechanism of in-duction. To accomplish these goals, mef(E)-lacZ reporter fu-sion constructs and a disk diffusion-based assay were devel-oped that allowed the assessment of both the induction ofmef(E)-mel and efflux-mediated macrolide resistance.

MATERIALS AND METHODS

Bacterial strains. Construction of the mef(E)-lacZ reporter strain XZ7042 andthe MEGA element deletion derivative XZ8004 from the erythromycin-resistant,MEGA-containing S. pneumoniae clinical isolate GA17457 was as previouslydescribed (50). The negative-control strain XZ7049 was generated by insertion ofthe promoterless lacZ of pPP2 (18) into bgaA of GA17457. Likewise, XZ7067was generated by insertion of the comC-lacZ transcriptional fusion on plasmidpPC2 (18) into GA17457.

Quantitative �-galactosidase assays. The rate of induction of mef(E) by eryth-romycin was determined by adding 0.1 �g/ml erythromycin to mid-log-phasecultures (optical density at 600 nm [OD600], �0.3 to 0.4). Test strains wereincubated at 37°C in parallel with uninduced cultures. At 30-min intervals,cultures were sampled for assessment of �-galactosidase (�-Gal) activity asdescribed previously (35). To determine a transcriptional dose response ofmef(E)-lacZ, parallel cultures of XZ7042 and XZ8004 were grown to mid-logphase (OD600, �0.3 to 0.4) and exposed to concentrations of erythromycinvarying by 4 orders of magnitude or more. Each subculture was harvested 1 hafter induction for assessment of �-galactosidase activity. All experiments wereperformed in duplicate, and assay readings were taken in triplicate.

Antibiotics. Antibiotic susceptibility disks were either obtained commerciallyor prepared by application of stock solutions to sterile blank disks, followed bydrying for 15 min in a laminar flow hood. Susceptibility disks containing themacrolides azithromycin, clarithromycin, erythromycin, telithromycin, and tilmi-cosin were purchased from Becton, Dickinson and Company (Franklin Lakes,NJ). Disks were prepared for the macrolides dirithromycin, josamycin, mideca-mycin, roxithromycin, and spiramycin (Sigma-Aldrich, St. Louis, MO) and forkitasamycin (MP Biomedicals, Solon, OH), oleandomycin (Crescent ChemicalCo., Inc., Islandia, NY), troleandomycin (Enzo Life Sciences, Plymouth Meeting,PA), and tylosin (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Themacrolide tulathromycin was obtained as the animal health product Draxxinfrom Pfizer Animal Health (Kalamazoo, MI). Commercially prepared antibioticdisks containing bacitracin, clindamycin, colistin, levofloxacin, lincomycin, poly-myxin B, and trimethoprim-sulfamethoxazole (Becton, Dickinson and Company,

Franklin Lakes, NJ) and quinupristin-dalfopristin (Remel, Lenexa, KS) wereobtained. Disks were prepared as described above for the nonmacrolide antibi-otics amoxicillin, ampicillin, penicillin, cefotaxime, ceftriaxone, cefuroxime, cip-rofloxacin, chloramphenicol, tetracycline, gentamicin, kanamycin, spectinomy-cin, rifampin, and vancomycin (Sigma-Aldrich, St. Louis, MO) and mupirocin(AppliChem GmbH, Darmstadt, Germany). Cyclopentadecanolide was obtainedfrom Acros Organics (Geel, Belgium).

Susceptibility assays. The MIC of erythromycin was determined by Etestaccording to the manufacturer’s recommendations (AB bioMerieux, Solna, Swe-den). Briefly, strains incubated overnight at 37°C in 5% CO2 on Trypticase soyagar with 5% sheep blood (TSA-SB) (Becton, Dickinson and Company, FranklinLakes, NJ) were suspended in Mueller-Hinton broth to a 0.5 McFarland stan-dard and spread onto Mueller-Hinton agar with 5% sheep blood (MH-SB)(Becton, Dickinson and Company, Franklin Lakes, NJ). Etest strips were placedon the plate surface, and the plates were incubated overnight at 37°C in 5% CO2.Susceptibility to other macrolides was determined by disk diffusion in accordancewith the standards described by the Clinical and Laboratory Standards Institute(11). To determine the effects of erythromycin induction on resistance, MICswere determined by Etest as described above except that strains were incubatedon TSA-SB supplemented with 1/10 MIC erythromycin prior to suspension andplating on MH-SB. Susceptibility to other macrolides was determined by diskdiffusion in accordance with the standards described by the Clinical and Labo-ratory Standards Institute (11). Susceptibility disks were purchased or preparedas described above. All susceptibility data represent the means � standarddeviations of at least five independent replications and were analyzed by two-tailed, paired t tests. P values of �0.05 were considered significant.

Disk diffusion assay for mef(E)-lacZ induction. The mef(E)-lacZ reporterstrain XZ7042 was grown to mid-log phase in Todd-Hewitt broth supplementedwith 0.5% yeast extract (THY). The liquid culture was swabbed onto TSAsupplemented with 300 U/ml catalase (Sigma-Aldridge, St. Louis, MO) and0.032% X-Gal (5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside). Susceptibil-ity disks infused with test compounds were immediately placed on the plates, andthe plates were incubated at 37°C, 5% CO2 for 24 h. Induction by syntheticcompetence-stimulating peptide-1 (CSP-1) (20) was tested by spotting 15 �l of a100 ng/ml solution directly to the center of an indicator plate swabbed with theindicated strain. CSP-1 was synthesized by the Emory University MicrochemicalFacility.

RESULTS

Characterization of erythromycin-induced expression ofmef(E)-mel. The kinetics and dose dependence of mef(E)-melinduction were determined using the prototype macrolideerythromycin and a series of isogenic reporter strains thatcontained a mef(E)-lacZ transcriptional fusion in the S. pneu-moniae strain GA17457 (Table 1) (50). The wild-type reporterstrain XZ7042 contained a functional MEGA element encod-ing the Mef(E)/Mel efflux pump that confers resistance toerythromycin (MIC 12 �g/ml), while the MEGA deletion de-rivative, XZ8004, was erythromycin susceptible (MIC of 0.1�g/ml) (50). A promoterless lacZ reporter strain, XZ7049,which was otherwise identical to XZ7042, served as a negativecontrol. The induction of mef(E) transcription in XZ7042, ex-pressed as �-Gal activity, was measured over time followingthe addition of subinhibitory concentrations of erythromycin(1/10 of the MIC). �-Gal activity increased rapidly upon theaddition of erythromycin, by more than 7-fold within the first30 min postinduction (Fig. 1a). Maximal induction (�15-foldincrease) was obtained at 1 h (Fig. 1a). The �-Gal activities ofXZ7042 cultures without induction were equivalent to those ofthe induced and uninduced promoterless negative control, in-dicating a very low level of constitutive expression from themef(E) promoter in the absence of an inducer (Fig. 1a).

The role of a functional MEGA element in mef(E) inductionwas assessed by measuring the erythromycin dose response inXZ7042 and the MEGA deletion derivative XZ8004. The

3414 CHANCEY ET AL. ANTIMICROB. AGENTS CHEMOTHER.

strains were incubated with erythromycin concentrations be-ginning with 0.0012 �g/ml and increasing by one-half- or one-quarter-log intervals (Fig. 1b). In XZ8004, mef(E) was inducedat lower erythromycin concentrations than in XZ7042 (0.012and 0.04 �g/ml, respectively) and with higher maximal expres-sion (205.3 Miller units [MU] and 106.9 MU, respectively)(Fig. 1b). However, the peak expression of mef(E) in XZ8004occurred at a concentration equivalent to the erythromycinMIC (0.12 �g/ml) of the strain, while in XZ7042, peak expres-sion was observed at a concentration (0.4 �g/ml) 30-fold lessthan the strain’s erythromycin MIC (12 �g/ml) (Fig. 1b). Ex-pression in XZ8004 decreased rapidly with increasing erythro-mycin concentrations above the MIC (Fig. 1b), consistent withan inhibitory effect of erythromycin. The mef(E)-lacZ induc-tion in XZ8004 indicated that a functional MEGA element wasnot required for the induction of mef(E)-mel; however, thestrain’s susceptibility to the inducer and its ability to modifyintracellular concentrations of the inducer contributed to themagnitude and duration of mef(E)-mel induction.

To evaluate mef(E)-mel expression over a range of inducersand inducer concentrations, a disk diffusion assay using theXZ7042 reporter strain was developed (42). In contrast to thequantitative �-Gal assays, this approach provided a concentra-tion gradient, allowing the detection of mef(E)-lacZ inductionwithout a priori knowledge of the MIC of the test compound orthe time-dependent induction kinetics and was suitable forstudy of a broad range of potential inducers. Figure 1c showsthe results of the disk diffusion assays, using erythromycin asthe test compound, with the wild-type reporter XZ7042, theMEGA deletion strain XZ8004, and the promoterless controlstrain XZ7049. Strain XZ7042 showed a characteristic largeblue halo, indicative of the induction of mef(E)-lacZ, sur-rounding a narrow zone of inhibited growth (Fig. 1c). As an-ticipated, the MEGA deletion strain XZ8004 showed a narrowrange of induction surrounding a large zone of inhibition (Fig.1c). This result was consistent with the results of the quanti-tative assay (Fig. 1b), showing a wide range of inducing con-centrations for XZ7042 and a strong but narrow range of futileinduction of mef(E) in strain XZ8004 in the absence of theresistance determinant MEGA and the efflux pump (Fig. 1b).The promoterless-lacZ strain XZ7049 showed the same zoneof inhibition as XZ7042, as expected due to a functionalMEGA element; however, no �-Gal activity (i.e., blue halo)was detected (Fig. 1c), confirming that the mef(E)-lacZ expres-sion in the reporter strain XZ7042 depended exclusively onmef(E).

FIG. 1. Induction of mef(E)-lacZ in isogenic S. pneumoniae strains.All strains were derived from the MEGA-containing, invasive pneu-mococcal isolate GA17457; they include XZ7042, the MEGA deletionmutant XZ8004, and the promoterless lacZ negative control XZ7049.(a) �-Galactosidase expression by XZ7042 (solid lines, circles) andXZ7049 (dashed lines, squares). Strains were untreated (open sym-bols) or exposed to 0.1 �g/ml erythromycin (closed symbols) at 0 h, andcells were harvested and assayed at half-hour intervals. Error barsindicate standard deviations of the results of duplicate replications. (b)Erythromycin dose-response curves for XZ7042 (solid lines, opensquares) and XZ8004 (dashed lines, closed triangles). Arrows indicatethe erythromycin MIC of each strain (solid, XZ7042, and dashed,XZ8004). Error bars indicate standard deviations of the results ofduplicate replications. (c) Disk diffusion assay for induction of mef(E)-lacZ by erythromycin. Disks containing 15 �g/ml erythromycin wereplaced on plates swabbed with XZ7042 (first panel), XZ8004 (middlepanel), and XZ7049 (last panel). Expression of the reporter is indi-cated by a blue halo surrounding a zone of inhibition.

TABLE 1. Strains and plasmids used in this work

Strain or plasmid Description Reference or source

S. pneumoniae strainsGA17457 Wild type, parent; MEGA Eryr 50XZ7042 GA17457 derivative; bgaA::mef(E)-lacZ MEGA Eryr 50XZ8004 GA17457 derivative; bgaA::mef(E)-lacZ MEGA::aad9 Erys 50XZ7067 GA17457 derivative; bgaA::comC-lacZ MEGA Eryr This studyXZ7049 GA17457 derivative; bgaA::lacZ (promoterless) MEGA Eryr This study

PlasmidspPP2 pPP1 derivative, lacZ reporter gene with the htrA ribosomal binding site 18pPC2 pPP2 derivative, carries comC-lacZ fusion 18

VOL. 55, 2011 INDUCERS OF MACROLIDE RESISTANCE IN S. PNEUMONIAE 3415

Environmental or antibiotic (nonmacrolide) stress andcompetence did not induce mef(E)-lacZ. To assess the spec-trum of inducers of mef(E)-mel expression, different environ-mental conditions and antibiotic compounds were analyzed inthe disk diffusion assay using XZ7042. Nonmacrolide antibiot-ics were chosen to include representatives of major antimicro-bial classes with diverse cellular targets, thus providing a widerange of antibiotic stresses. Also tested were environmentalconditions involved in the regulation of other genetic systems,as well as known substrates for multidrug efflux systems. Noneof the tested compounds and conditions induced mef(E)-lacZexpression, with the exception of the previously described an-timicrobial peptides LL-37, CRAMP38, and CRAMP39 (50).These results demonstrated that mef(E)-mel induction is re-stricted to a narrow range of compounds, suggesting one ormore well-defined mechanism(s) of induction.

In addition, competence was tested as a potentially inducingcondition of mef(E) induction. Competence induction in S.pneumoniae has been known to affect the expression of a broadrange of genes and is mediated by the com regulon (1, 2, 21,31). Prudhomme et al. (39) showed that the com regulon wasinduced in response to antibiotic stress. Since it was unknownwhether the conditions of the disk diffusion assay supportedthe development of natural competence, synthetic compe-tence-stimulating peptide (CSP-1) was used to induce compe-tence and monitor the effect on mef(E)-lacZ induction in thelacZ disk diffusion assay. The results in Fig. 2 show that eryth-romycin but not CSP-1 induced mef(E)-lacZ in XZ7042. As acontrol for competence development, a reporter strain,XZ7067, which carried the lacZ gene under the control of thepromoter for the early competence gene comC, was con-structed (Table 1). XZ7067 was induced by CSP-1, confirmingcompetence induction under the assay conditions. Erythromy-cin did not induce comC (Fig. 2), which is consistent with theprevious observation (36) that erythromycin does not inducecompetence. Thus, general environmental and antibiotic

stresses, as well as the development of competence, did notinduce mef(E)-mel expression.

Induction of mef(E) by macrolides. To understand the rela-tionship between macrolide structure and mef(E)-mel induc-tion, a panel of 16 macrolides composed of 14-, 15-, and 16-membered compounds were studied using the disk diffusionassay. The 14-membered cladinosolides, compounds with thelactone ring replaced with the sugar cladinose at C-3 and themonosaccharide amino sugar desosamine at C-5 (e.g., eryth-romycin, clarithromycin, dirithromycin, and roxithromycin)(Fig. 3a), each strongly induced �-Gal activity over a widediffusion zone (i.e., concentration gradient) (Fig. 3a). Induc-tion was correlated with decreased susceptibility to these com-pounds in the MEGA-containing reporter strain but not in thesusceptible MEGA deletion strain (P � 0.001) (Fig. 3a andTable 2). In addition, preincubation of the reporter strains witha subinhibitory concentration of erythromycin (1/10 MIC, 1.2�g/ml) further increased the resistance to these compounds forthe wild-type MEGA-containing reporter strain (P � 0.05)(Table 2) but not for the MEGA deletion strain. Thus, resis-tance in the wild-type strain required the efflux-encodingMEGA element and suggested that erythromycin, clarithromy-cin, clarithromycin, and roxithromycin are substrates forMef(E)/Mel-mediated efflux. These findings were consistentwith those of earlier reports for 14- and 15-membered macro-lides (3, 34, 49).

Two 14-membered macrolides, oleandomycin and trolean-domycin, with the substitution of oleandrose for the cladinosein the lactone ring, were tested (Fig. 3c). The induction ofmef(E)-lacZ by oleandomycin resembled the strong inductionobserved with the cladinosolides; however, troleandomycinwas a poor inducer of mef(E) (Fig. 3c). Troleandomycin hasthree acetate substitutions of the hydroxyl groups of oleando-mycin (Fig. 3c); the results suggest that one or more of theseacetate groups interfered with mef(E) induction. The wild-typereporter strain XZ7042 was significantly more susceptible totroleandomycin than to oleandomycin (P � 0.001) (Table 2).To determine whether the increased susceptibility of XZ7042to troleandomycin compared to its susceptibility to oleando-mycin was due to the weak mef(E) induction by troleandomy-cin, the reporter strain XZ7042 was preincubated with a sub-inhibitory concentration of erythromycin (0.12 �g/ml) toachieve high induction of mef(E)-mel prior to susceptibilitytesting with troleandomycin. Under these conditions, the re-sistance to troleandomycin increased to the levels of resistanceto oleandomycin, indicating that troleandomycin was a sub-strate for efflux, despite being a poor mef(E)-mel inducer.

The ketolide telithromycin is a 14-membered macrolide thatbelongs to the ketolide subclass due to a C-3 ketone substitu-tion instead of cladinose (Fig. 3b). Telithromycin retains ex-cellent antimicrobial activity against Mef(E)/Mel-containingpneumococci (12, 49). However, more detailed reports showedthat the efflux pump conferred small increases in pneumococ-cal MICs of telithromycin after preincubation with subinhibi-tory concentrations of telithromycin (12, 49) or in comparisonto the MICs of clinical isolates with and without mef(E)-mel(46). To determine whether the lack of telithromycin resis-tance was due to an inefficient induction of mef(E)-mel expres-sion or poor efflux of telithromycin by Mef(E)/Mel, the induc-ing ability of telithromycin was tested in the disk diffusion

FIG. 2. Competence-stimulating peptide 1 (CSP-1) did not inducemef(E)-lacZ. Disk diffusion induction assay of the mef(E)-lacZ(XZ7042) and comC-lacZ (XZ7067) reporter strains by erythromycin(ERY) and CSP-1. CSP-1 was spotted directly onto THY indicatorplates streaked with XZ7042 or XZ7067. As controls, diffusion diskscontaining erythromycin (15 �g) were placed onto TSA indicatorplates streaked with each strain. A blue spot or blue halo indicatescomC-lacZ or mef(E)-lacZ induction, respectively.


assay. Telithromycin strongly induced mef(E), and the ob-served induction pattern was quite similar to that observed forthe MEGA deletion mutant XZ8004 with erythromycin (Fig.1c), i.e., a narrow zone of strong mef(E) induction surroundinga large zone of inhibition of growth, which indicated that thewild-type reporter strain remained susceptible to telithromycin

despite the strong induction of mef(E)-lacZ. This was con-firmed by telithromycin resistance (Table 2). As expected, pre-incubation with erythromycin had no significant effect on teli-thromycin resistance, although the MEGA mutant was moresusceptible to telithromycin than the MEGA-containing strain(P � 0.001) (Table 2). These data suggest that telithromycin,

FIG. 3. Structure and disk diffusion induction assays for the 14- and 15-membered macrolides used in this study. Discs containing 15 �g/ml ofeach macrolide were placed onto TSA indicator plates spread with XZ7042. (a) Cladinosolides. Erythromycin and derivatives each contain a C-3cladinose. Previously described interactions between cladinosolides and the 23S rRNA (E. coli numbering) are indicated by dashed arrows. 1,erythromycin; 2, clarithromycin; 3, dirithromycin; 4, roxithromycin. (b) Telithromycin. The C-11/C-12 carbamate with a aryl-akyl side chain isresponsible for increased ribosomal affinity and compensates for the absence of cladinose at C-3 (19). 1, erythromycin; 2, telithromycin. (c)Oleandomycins. Oleandomycin and troleandomycin differ only by the acetylation of hydroxyl groups at three positions in troleandomycin (R1, R2,and R3). The dashed arrow indicates interaction of the acetyl group of the oleandrose of troleandomycin and the 23S rRNA nucleotide U790 (E.coli numbering). 1, erythromycin; 2, oleandomycin; 3, troleandomycin. (d) 15-Membered azalides. Previously described interaction betweenazithromycin cladinose hydroxyl group and the 23S rRNA nucleotide G2505 (E. coli numbering, dashed arrow) (19). C-5 desosamine is indicatedin red. 1, erythromycin; 2, azithromycin; 3, tulathromycin.


while a strong inducer of mef(E)-mel, was not sufficiently ex-truded by the Mef(E)/Mel efflux pump to allow resistance todevelop.

The two 15-membered macrolides, azithromycin and tulath-romycin (Table 2 and Fig. 3d), were studied. Tulathromycindiffers from azithromycin by a demethylation of N-10 and by apropylamino side chain substitution on the C-3 cladinose (Fig.3d). However, while mef(E) induction by azithromycin wascomparable to that of erythromycin, induction by tulathromy-cin was weaker. XZ7042 was slightly more susceptible to tu-lathromycin than to azithromycin, suggesting that the increasein antimicrobial activity of tulathromycin could be due to theweaker induction of mef(E)-mel. To test this, XZ7042 waspreincubated with erythromycin (1.2 �g/ml). Increased resis-tance to tulathromycin, to the level of resistance to azithromy-cin, was noted. However, the weaker induction by tulathromy-cin was still sufficient to render the MEGA-containing strainXZ7042 less susceptible than the MEGA deletion strainXZ8004 (Table 2).

16-Membered macrolides can induce mef(E)-mel expression.A current paradigm is that the Mef(E)/Mel efflux pump doesnot confer resistance to 16-membered macrolides (34). Possi-ble explanations include the inability of 16-membered macro-lides to induce the efflux pump, structural feature(s) of thecompounds that preclude them as substrates for the effluxpump, or sequestration, e.g., at the ribosome, making thesecompounds unavailable for efflux. Seven 16-membered macro-lides, josamycin, kitasamycin, midecamycin, rosamicin, spira-mycin, tilmicosin, and tylosin, were studied for induction ofmef(E)-lacZ (Fig. 4). Josamycin, kitasamycin, midecamycin,spiramycin, and tylosin did not induce mef(E)-lacZ expression(Fig. 4). Surprisingly, two 16-membered macrolides, tilmicosinand rosamicin, induced the mef(E)-lacZ reporter (Fig. 4b). All

the 16-membered macrolides lacked a cladinose at C-3. Whilethe cladinose has been suggested as an important feature ofinducing 14- and 15-membered macrolides (8), the above-noted 14- or 15-membered macrolides with modifications ofthe cladinose (oleandomycin and troleandomycin) or with aketone substitution (telithromycin) at C-3 were also inducers.In contrast, the common feature of the 16-membered inducers(tilmicosin and rosamicin) and the inducing 14- and 15-mem-bered macrolides, including telithromycin, was the presence ofa monosaccharide at C-5 (Fig. 4b). All noninducing macrolidespossessed a disaccharide moiety at this position. The require-ment of a monosaccharide at C-5 was supported by the data fortylosin, a noninducing 16-membered macrolide that is struc-turally closely related to tilmicosin and rosamicin but is distin-guished by the disaccharide in the C-5 position (Fig. 4b). Thus,the ability to induce mef(E)-mel transcription was not depen-dent upon the size (e.g., 14-, 15-, or 16-membered) of thelactone ring or the presence of a cladinose at C-3. Rather,mef(E)-mel expression was linked to a monosaccharide aminosugar attached at C-5 of the macrolide lactone ring.

Differential Mef(E)/Mel-mediated resistance to 16-mem-bered macrolides: further evidence of uncoupling of inductionand resistance. As observed for troleandomycin, which did notinduce mef(E)-lacZ but was readily effluxed upon preincuba-tion with erythromycin, the susceptibility of pneumococci tothe noninducing 16-membered macrolides could result from alack of induction of the efflux pump. Therefore, the wild-typereporter XZ7042 was preincubated with erythromycin, and itslevels of susceptibility to the noninducing 16-membered com-pounds were determined (Table 2). The results did not showincreased resistance toward these 16-membered macrolides,suggesting that they were not subjected to efflux in the pres-ence of the Mef(E)/Mel pump. This was confirmed by double-

TABLE 2. Induction of the Mef(E)/Mel efflux pump and pneumococcal susceptibility

Test drug No. of ringmembersa

Side group atb:Presence of

ac-AH atC-6c

Inhibition zone diameter (mm) ford:

C-3 C-5XZ7042 XZ8004

�ERY �ERY �ERY �ERY

Erythromycin 14 Cla Des � 9.7 � 0.5 7.8 � 0.4 29.0 � 1.4 27.4 � 1.1Clarithromycin 14 Cla Des � 9.4 � 0.5 8.2 � 0.4 28.0 � 2.0 28.2 � 1.3Dirithromycin 14 Cla Des � 8.1 � 0.4 7.0 � 0.8 22.2 � 0.8 21.6 � 1.7Roxithromycin 14 Cla Des � 8.4 � 0.5 6.4 � 0.5 24.4 � 1.1 25.6 � 0.9Oleandomycin 14 Ole Des � 9.6 � 0.5 6.9 � 0.9 19.6 � 0.5 19.8 � 0.5Troleandomycin 14 Ac-ole Ac-des � 12.5 � 1.0 7.3 � 0.8 16.5 � 0.5 17.4 � 1.7Telithromycin 14 OH Des � 20.6 � 1.1 19.0 � 1.4 31.2 � 0.4 31.0 � 0.7Azithromycin 15 Cla Des � 7.6 � 0.5 6.4 � 0.5 22.4 � 1.1 20.2 � 0.8Tulathromycin 15 Cla* Des � 8.7 � 0.5 6.6 � 0.5 17.4 � 2.7 16.6 � 0.5Josamycin 16 OH Myn-myr � 23.4 � 3.8 20.4 � 1.1 22.2 � 1.3 22.8 � 1.3Kitasamycin 16 OH Myn-myr � 23.2 � 1.3 22.2 � 1.3 23.2 � 1.3 23.2 � 0.8Midecamycin 16 OH Myn-myr � 20.2 � 1.3 19.8 � 0.8 21.2 � 0.8 21.8 � 0.8Tylosin 16 OH Myn-myr � 20.2 � 0.8 19.2 � 1.2 20.3 � 0.8 20.8 � 1.2Rosamicin 16 OH Des � 19.0 � 0.7 19.4 � 0.5 18.4 � 1.1 18.8 � 0.8Spiramycin 16 OH Myn-myr � 19.0 � 1.4 18.0 � 0.7 19.2 � 1.9 19.2 � 1.6Tilmicosin 16 OH Myn � 10.5 � 1.2 8.2 � 0.7 13.2 � 0.9 12.7 � 0.8

a Number of atoms in the lactone ring.b Side group attached at the C-3 or C-5 position of the lactone ring. Cla, cladinose; Cla*, modified cladinose; Ole, oleandrose; Ac-ole, acetylated oleandrose; Des,

desosamine; Ac-des, acetylated desosamine; Myn, mycaminose; Myr, mycarose.c Presence of acetylaldehyde (ac-AH) side group at C-6 of the lactone ring.d Complete resistance, defined by growth at the edge of the disk, was scored as 6 mm, i.e., equal to the diameter of the disk. All disks contained 15 �g of the macrolide.

Values in bold vary significantly (P � 0.05) from that of the uninduced control (XZ7042 � ERY) for the same drug. Shaded pairs of values are significantly different(P � 0.05).


disk diffusion assays for induction, in which no D-zone ofclearing was produced by the noninducing macrolides in thepresence of erythromycin (data not shown).

Still remaining was the question of whether the induction ofmef(E) observed with the inducing 16-membered macrolidesrosamicin and tilmicosin translated into increased resistance tothese compounds. The strong, narrow induction ring observedin the disk-diffusion induction assay with tilmicosin indicated anarrow range of inducing concentrations; however, the smallzone of inhibition indicated pneumococcal resistance to tilmi-cosin (Fig. 4b). Preincubation of XZ7042 with erythromycinresulted in further resistance to tilmicosin compared to theresistance of the uninduced control (Table 2), indicating thattilmicosin was subjected to Mef(E)/Mel-mediated efflux. Con-versely, the induction of mef(E) by rosamicin (Fig. 4b) resem-bled that observed with telithromycin (Fig. 3b) and the MEGAdeletion strain XZ8004 (Fig. 1c), and XZ7042 remained asusceptible phenotype despite induction (Table 2). Indeed,there was no change in susceptibility to rosamicin between the

MEGA deletion strain XZ8004 and the wild-type strainXZ7042 despite the induction of mef(E)-mel (Table 2).

These data further demonstrate the uncoupling of macrolideinduction of mef(E)-mel expression and macrolide efflux. Theresults with 16-membered macrolides further demonstrated thatfor induction of mef(E)-mel, a monosaccharide in position C-5 ofthe macrolide was required, but this was not sufficient to conferMef(E)/Mel-mediated resistance. Thus, MEGA-containing iso-lates of S. pneumoniae remained susceptible to most 16-mem-bered macrolides, independent of the capacity to induce mef(E)-mel.

DISCUSSION

Mechanisms of S. pneumoniae resistance to macrolidesinclude modification of macrolide target sites on the 23Sribosomal subunit, either by mutation or erm methylases,and macrolide efflux by the Mef(E)/Mel efflux pump en-coded on the MEGA element (16, 23, 44, 45, 47, 51). In

FIG. 4. Structures and disk diffusion induction assays for all 16-membered macrolides used in this study. (a) Common noninducing 16-membered macrolides. Positions of the carbon atoms in the lactone ring are indicated, and the C-5 disaccharide composed of the amino sugarmycaminose and neutral sugar mycarose is shown in red. Previously described points of interactions between 16-membered macrolides and 23SrRNA nucleotides (E. coli numbering) are indicated by dashed arrows (43, 47). The C-6 acetylaldehyde and the reversible covalent bond (wavyline) formed with the nucleotide A2062 (solid arrow) are indicated in blue (19). 1, josamycin; 2, kitasamycin; 3, midecamycin; 4, spiramycin; centraldisk, erythromycin control. foro, forosamide. (b) Tylosin and C-5 monosaccharide-containing derivatives rosamicin and tilmicosin. The C-5monosaccharides desosamine and mycaminose are indicated in red. 1, Rosamicin; 2, erythromycin control; 3, tilmicosin; 4, tylosin. Discs containing15 �g/ml of each macrolide were placed onto TSA indicator plates spread with XZ7042.


North America and many other parts of the world, Mef(E)/Mel is the predominant mechanism of pneumococcal mac-rolide resistance (16, 43). Approximately half of the mac-rolide-resistant pneumococci isolated in the United Statesin 2006 contained mef(E)-mel as the sole macrolide resis-tance determinant, and an additional 25% contained bothmef(E)-mel and erm(B) (22). The resistance levels conferredby Mef(E)/Mel in vitro (erythromycin 1 to 32 �g/ml) arelower than the levels conferred by ribosome modification;nevertheless, these levels are correlated with macrolidetreatment failures (25, 26). Exposure in vivo to inducers ofthe efflux pump may lead to levels of resistance at the site ofinfection higher than those predicted by in vitro assays.Mef(E)/Mel resistance can be induced by up to a 200-foldincrease in MICs (3, 49) and, therefore, could be of signif-icant clinical impact. This background led us to further studythe induction of macrolide efflux, in order to identify struc-tural features that differentiate inducing and noninducingmacrolides and to determine the relationship between in-duction and resistance.

A range of compounds and conditions were studied for theability to induce the expression of the mef(E)-lacZ reporterusing the disk diffusion assay. This assay allowed the efficientscreening of a broad range of inducer concentrations and con-ditions. Nonmacrolide antibiotics or general environmentalstresses and conditions (e.g., competence induction), exceptfor certain antimicrobial peptides (50) and macrolides, did notinduce the expression of mef(E)-mel. The nonmacrolide anti-biotics studied represented different classes with distinct cel-lular targets, including ribosomal binding sites on 23S rRNAoverlapping the macrolide binding site (chloramphenicol, lin-cosamides, linezolid, and streptogramins) and 16S rRNA bind-ing drugs (e.g., aminoglycosides and tetracycline), suggestingthat disruption of protein synthesis per se does not influencemef(E)-mel expression. Likewise, drugs that disrupt DNA syn-thesis, cell wall synthesis, or membrane integrity and variousenvironmental conditions did not induce expression, indicatingthat the range of inducers for mef(E)-mel expression is quitespecific.

The current paradigm for the Mef(E)/Mel efflux pump isthat it confers resistance to 14- and 15-membered but not16-membered macrolides (5, 9, 10, 17, 34, 36) and that 14- and15-membered macrolides induce Mef(E)/Mel-mediated resis-tance (3, 36, 49). Our data confirm strong induction of mef(E)-mel expression by erythromycin, clarithromycin, and azithro-mycin, as previously reported (3, 49), and show that the mostcommonly studied 16-membered macrolides indeed did notinduce mef(E)-mel expression. However, the use of a broaderspectrum of structurally diverse macrolides revealed 14-mem-bered and 15-membered macrolides with reduced ability toinduce mef(E)-mel expression and 16-membered macrolidesthat induced mef(E)-mel. In addition, the ketolide telithromy-cin, despite retaining excellent activity against mef(E)-contain-ing pneumococci, strongly induced mef(E) expression. Teli-thromycin has not previously been demonstrated to inducemef(E)-mel.

The induction of mef(E)-mel by 14- and 15-membered mac-rolides but not by common 16-membered macrolides coincideswith differences in macrolide binding to the ribosome. Most14- and 15-membered macrolides contain the monosaccharide

desosamine at position C-5 of the lactone ring, whereas 16-membered macrolides typically have a C-5 disaccharide. C-5substituents form extensive and distinct bonds with the ribo-some (Fig. 3 and 4). The disaccharides in the 16-memberedmacrolides extend deep into the exit tunnel of the ribosome(19, 38), allowing the mycarose (Fig. 4) to interact with G2505and U2506 of the 23S rRNA (Escherichia coli numbering) (Fig.4), thus interfering with peptidyl transferase activity (19, 37,38) and resulting in truncated peptides (19). Macrolides withC-5 monosaccharides do not interfere with peptidyl transferaseactivity but instead block the egress of the nascent peptidefrom the exit tunnel, leading to peptides up to 8 amino acidslong (13, 19, 27, 38, 42).

We found that the inducing 16-membered macrolides tilmi-cosin and rosamicin, like inducing 14- and 15-membered mac-rolides, have a monosaccharide at C-5 and, therefore, are ex-pected to have a similar affect on peptide synthesis. The weaklyinducing 14-membered macrolide troleandomycin has acetyla-tions of the C-3 and C-5 sugars that force a substantially al-tered conformation when bound to the ribosome, resulting ina location of the macrolide farther down the exit tunnel thaninducing macrolides (6). The pivotal role of the C-5 substituentfor induction, over and above that of the lactone ring size orthe C-3 substituent, is also supported by our first identificationof inducing 16-membered macrolides, which additionally lackthe C-3 cladinose that is typically found in the common 14- and15-membered macrolides. Taken together, the inductionmechanism involves specific binding of the macrolide to theribosome and, in particular, the C-5 substituent of the macro-lide.

Specific macrolide binding to the ribosome is also requiredfor the induction of macrolide resistance by many erm meth-ylases (24, 28, 33, 41, 48) in mechanisms that are controlled bytranscriptional or translational attenuation. Weisblum (48)found that translational attenuation of erm(C) in Staphylococ-cus aureus is alleviated by 14- but not by 16-membered mac-rolides (48) and proposed that when bound by 14-memberedmacrolides, ribosomes stall due to constriction of the exit tun-nel after a short peptide is synthesized. Ribosome stalling atthe end of the leader peptide synthesis promotes refolding ofthe transcript into an antiattenuator structure (48). Con-versely, C-5 disaccharides of many 16-membered macrolidesdisrupt ribosyltransferase activity, preventing synthesis of theleader peptide such that the erm transcript remains folded inthe attenuator structure (19).

We propose a similar mechanism for the induction ofmef(E)-mel and efflux-mediated macrolide resistance in pneu-mococci. Macrolides with C-5 disaccharides may prevent thesynthesis of an unidentified regulatory leader peptide and,thus, may not induce. Macrolides with a monosaccharide atC-5 promote stalling at the precise residue of the leader pep-tide due to the length of the nascent peptide that is synthesizedbefore being blocked in the ribosomal exit tunnel. This isindependent of the lactone ring size or the C-3 sugar substit-uent.

The Mef(E)/Mel efflux pump does not confer resistance tocommon 16-membered macrolides. Induction of the effluxpump with erythromycin prior to exposure to the 16-mem-bered macrolides demonstrated that pneumococcal suscepti-bility to these macrolides was not due to a failure to induce


Mef(E)/Mel. The lack of efflux of 16-membered macrolidescould result either because of an unavailability of the com-pounds for export by the efflux pump due to high-affinity ribo-somal binding or because the macrolides are not recognized assubstrates. Most 16-membered macrolides have a C-6 acetylal-dehyde that can form a covalent bond with the rRNA, resultingin high-affinity binding to the ribosome (19). In contrast, the14- and 15-membered macrolides, which are known substratesfor the efflux pump, bind ribosomes through hydrophobic in-teractions and hydrogen bonding and are easily dissociatedfrom the ribosome (7, 19, 37, 42). Of the two 16-memberedmacrolides, rosamicin and tilmicosin, that induced mef(E)-melexpression, only tilmicosin had reduced activity againstMEGA-containing pneumococci, suggesting that it was subjectto efflux. The primary structural difference between these twocompounds is the C-6 acetylaldehyde of rosamicin (but nottilmicosin), which has the potential to form the covalent bondeffecting efflux by Mef(E)/Mel. Furthermore, telithromycin,with 700-fold higher affinity for the ribosome than erythromy-cin, is predicted to have limited availability for export byMef(E)/Mel. Despite strong induction of the efflux pump bytelithromycin, pneumococci remain susceptible. Thus, becauseof high-affinity ribosomal binding, most 16-membered macro-lides and the 14-membered ketolide are not effectively avail-able for efflux even if mef(E)-mel is induced, and pneumococ-cal isolates remain susceptible to these compounds.

To summarize, induction of the mef(E)-mel operon wasshown to be limited to a narrow range of macrolides andantimicrobial peptides. The model indicates that induction ismediated by ribosomal binding and that the inducing 14-, 15-,and 16-membered macrolides contain a monosaccharide inposition C-5 of the lactone ring. The C-5 monosaccharideforms a characteristic macrolide-ribosome-mRNA complexthat is consistent with an antiattenuation-based mechanism formef(E)-mel regulation. Most 16-membered macrolides and the14-membered ketolide have high-affinity binding to the ribo-some, and this strong ribosomal binding limits the availabilityfor efflux from the pneumococcus, even in the presence ofMef(E)/Mel induction. The antimicrobial peptides, such LL-37and CRAMP, that induce are not expected to bind ribosomes,and thus, induction by these agents is anticipated to occur byan alternate pathway. Investigations are under way to confirmand define the proposed macrolide transcriptional attenuatormechanism and to study the basis for induction by antimicro-bial peptides.

ACKNOWLEDGMENTS

This work was supported by grants from the National Institutes ofHealth (5R01AI070829) and from the Medical Research Service of theDepartment of Veterans Affairs (to D.S.S.).

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induction of efflux-mediated macrolide resistance in

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