aac accepts, published online ahead of print on 2 may...

Induction of efflux-mediated macrolide resistance in Streptococcus pneumoniae 1

2

Scott Chancey1,3

, Xiaoliu Zhou1,3§

, Dorothea Zähner1,3

, David S. Stephens1,2,3

* 3

4

5

1 Division of Infectious Diseases, Department of Medicine 6

2 Department of Microbiology and Immunology, Emory University School of Medicine, 7

Atlanta, GA 30322 8

3 Department of Veterans Affairs Medical Center Atlanta, GA 30033 9

10

11

12

13

Running title: Inducers of macrolide resistance in S. pneumoniae 14

15

16

§ Present address: Centers for Disease Control and Prevention, 4770 Buford Highway, Chamblee, GA 17

30341, USA 18

19

20

21

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00060-11 AAC Accepts, published online ahead of print on 2 May 2011

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Abstract 22

The antimicrobial efflux system encoded by the operon mefE/mel on the mobile genetic element 23

MEGA in Streptococcus pneumoniae and other Gram-positive bacteria is inducible by macrolide 24

antibiotics and antimicrobial peptides. Induction may affect the clinical response to the use of 25

macrolides. We developed mefE reporter constructs and a disk diffusion induction and resistance assay 26

to determine the kinetics and basis of mefE/mel induction. Induction occurred rapidly, >15-fold increase 27

in transcription within one hour of exposure to sub-inhibitory concentrations of erythromycin. A 28

spectrum of environmental conditions including competence and non-macrolide antibiotics with distinct 29

cellular targets did not induce mefE. Using sixteen different structurally-defined macrolides, induction 30

was correlated with the amino sugar attached to C5 of the macrolide lactone ring, not with the size (e.g., 31

14-, 15- or 16-member) of the ring or with the presence of the neutral sugar cladinose at C3. Macrolides 32

with a monosaccharide attached to C5, known to block exit of the nascent peptide from the ribosome 33

after the incorporation of up to eight amino acids, induced mefE expression. Macrolides with a C5 34

disaccharide, which extends the macrolide into the ribosomal exit tunnel disrupting peptidyl transferase 35

activity, did not induce. The induction of mefE did not require macrolide efflux, but the affinity of 36

macrolides for the ribosome determined availability for efflux and pneumococcal susceptibility. The 37

induction of mefE/mel expression by inducing macrolides appears to be based on specific interactions of 38

the macrolide C5 saccharide with the ribosome alleviating transcriptional attenuation of mefE/mel. 39

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Introduction 40

Macrolides are broad spectrum antibiotics with complex macrocyclic 14-, 15- or 16-member 41

lactone rings that bind in the peptide exit tunnel of bacterial ribosomes and inhibit protein synthesis. 42

Macrolides are often recommended as the empirical first-line treatment of upper respiratory bacterial 43

infections including pneumococcal infections and community acquired pneumonia. However, bacterial 44

resistance to macrolides is expanding worldwide in Gram-positive bacteria and is now present in almost 45

a third of all invasive Streptococcus pneumoniae isolates (14). The two most common mechanisms of 46

macrolide resistance in bacterial pathogens are modification of the bacterial ribosome, either by 47

methylation or mutation, and extrusion of the drugs from the bacterial cell by an efflux pump. Genes of 48

the erm (erythromycin ribosomal methylases) family of rRNA methylases confer high-level resistance to 49

lincosamides and streptogramins as well as macrolides (the MLSB phenotype) and can be constitutive or 50

inducible. In the inducible form resistance develops only after exposure of the bacterium to the 51

macrolide. 52

The best-studied mechanism of inducible MLSB resistance involves the ermC gene found in S. 53

aureus and other Gram-positive pathogens (4, 32, 33). Translation of ermC is attenuated in the absence 54

of inducers due to secondary structures that render the ribosomal binding site inaccessible. Inducer-55

bound ribosomes stall during translation of a 19-amino acid leader peptide upstream of ermC resulting 56

in the refolding of the transcript to make available the RBS thus promoting translation (32). A later 57

comparison of inducers for ermC and ermSV in Streptomyces viridochromogenes showed that the 58

specific sequence of leader peptide determines the range of inducers (24). Macrolides with 14-59

membered rings and C3 cladinose induced ermC, and 16-membered macrolides induced ermSV (24). 60

The lincosamide celesticetin induced both ermC and ermSV demonstrating a common inducer between 61

the otherwise disparately induced genes (24). Attenuation of erm genes can also occur at the level of 62

transcription such as with ermK of Bacillus licheniformis (28, 29). Transcriptional attenuation occurs 63

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when secondary structures in the transcript function like rho-independent terminators to terminate 64

transcription short of the erm gene coding region. 65

Efflux-mediated macrolide-resistance was first reported in Staphylococcus epidermidis and later 66

in Streptococcus pyogenes and S. pneumoniae (30, 40, 45). In S. epidermidis macrolide efflux is 67

conferred by msrA and is induced by the 14- and 15-membered macrolides clarithromycin and 68

azithromycin and the ketolide telithromycin, but not by streptogramin B, even though the latter is a 69

substrate for MsrA-mediated efflux. In streptococci, macrolide efflux is conferred by proteins encoded 70

by mefE, mefA or less commonly mefI, that share >90% identity. The mef genes are transcribed as an 71

operon with the msrA homolog mel, comprising a dual efflux pump (3, 12). In S. pneumoniae, mefE/mel 72

is carried on the small mobile element MEGA, the predominant macrolide efflux determinant in the 73

pneumococcus (15, 43). MefE/Mel confers in vitro moderate-level resistance (1-32 µg/ml) to 14- and 74

15-membered macrolides, but reportedly not 16-membered macrolides, lincosamides, or streptogramins 75

(M phenotype) (45, 47). MefE/Mel-mediated resistance has been shown to be induced by the 14- and 76

15-membered macrolides erythromycin, clarithromycin and azithromycin, but not by 16-membered 77

macrolides (3, 50). Ketolides, such as telithromycin, are considered poor inducers of mefE/mel and 78

retain antimicrobial activity against pneumococcal strains carrying these genes (34, 50). 79

In vivo exposure to macrolides or other potential inducers may result in resistance higher than 80

predicted by in vitro determined MIC’s. We recently reported that certain antimicrobial peptides 81

(AMP), including the human AMP LL-37, activated transcription of mefE/mel and resulted in induced 82

resistance to the macrolide erythromycin (51). This suggests that the MefE/Mel efflux pump can be 83

induced by human host defenses and therefore be primed prior to clinically administered macrolides. 84

The goals of this study were to define the range of compounds, macrolides as well as other antibiotics 85

and other conditions, that induce MefE/Mel-mediated resistance; to elucidate the kinetics of induction; 86

to determine the structural features of macrolides required for induction and to develop a model for the 87

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mechanism of induction. To accomplish these goals, mefE-lacZ reporter fusion constructs and a disk 88

diffusion-based assay were developed that allowed assessment of both the induction of mefE/mel and of 89

efflux-mediated macrolide resistance. 90

Methods 91

Bacterial strains. Construction of the mefE-lacZ reporter strain XZ7042 and the MEGA element 92

deletion derivative XZ8004 from the erythromycin resistant, MEGA-containing S. pneumoniae clinical 93

isolate GA17457 was as previously described (51). The negative control strains XZ7049 was generated 94

by insertion of the promoterless lacZ of pPP2 (18) into bgaA of GA17457. Likewise, XZ7067 was 95

generated by insertion of the comC-lacZ transcriptional fusion on plasmid pPC2 (18) into GA17457. 96

Quantitative β-galactosidase assays. The rate of induction of mefE by erythromycin (ERY) was 97

determined by adding 0.1 µg/ml erythromycin to mid-log phase cultures (OD600~0.3-0.4). Test strains 98

were incubated at 37°C in parallel with uninduced cultures. At 30 minute intervals, cultures were 99

sampled for assessment of β-galactosidase activity as described previously (35). To determine a 100

transcriptional dose response of mefE-lacZ, parallel cultures of XZ7042 and XZ8004 were grown to 101

mid-log phase (OD600 ~0.3-0.4) and exposed to concentrations of erythromycin varying by four orders 102

of magnitude or more. Each subculture was harvested one hour after induction for assessment of β-103

galactosidase activity. All experiments were performed in duplicate and assay readings were taken in 104

triplicate. 105

Antibiotics. Antibiotic susceptibility disks were either obtained commercially or prepared by 106

application of stock solutions to sterile blank disks, followed by drying for fifteen minutes in a laminar 107

flow hood. Susceptibility disks containing the macrolides azithromycin, clarithromycin, erythromycin, 108

telithromycin and tilmicosin were purchased from Becton, Dickinson and Company (Franklin Lakes, 109

NJ). Disks were prepared for the macrolides dirithromycin, josamycin, midecamycin, roxithromycin, 110

and spiramycin (Sigma-Aldrich, St. Louis, MO); and kitasamycin (MP Biomedicals, Solon, OH), 111

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oleandomycin (Crescent Chemical Co., Inc., Islandia, NY), troleandomycin (Enzo Life Sciences, 112

Plymouth Meeting, PA) and tylosin (Wako Pure Chemical Industries, Ltd, Osaka, Japan). The 113

macrolide tulathromycin was obtained as the animal health product Draxxin® from Pfizer Animal 114

Health (Kalamazoo, MI). Commercially prepared antibiotic disks were obtained containing bacitracin, 115

clindamycin, colistin, levofloxacin, lincomycin, polymyxin B, and trimethoprim/sulfmethoxazole 116

(Becton, Dickinson and Company, Franklin Lakes, NJ) and quinupristin/dalfopristin (Remel, Lenexa, 117

KS). Disks were prepared as described above for the non-macrolide antibiotics amoxicillin, ampicillin, 118

penicillin, cefotaxime, ceftriaxone, cefuroxime, ciprofloxacin, chloramphenicol, tetracycline, 119

gentamycin, kanamycin, spectinomycin, rifampin and vancomycin (Sigma-Aldrich, St. Louis, MO), 120

mupirocin (AppliChem GmbH, Darmstadt, Germany). Cyclopentadecanolide was obtained from Acros 121

Organics (Geel, Belgium). 122

Susceptibility assays. The minimum inhibitory concentration (MIC) of erythromycin was determined 123

by Etest as per manufacturer recommendations (AB bioMerieux, Solna, Sweden). Briefly, strains 124

incubated overnight at 37ºC in 5% CO2 on trypticase soy agar with 5% sheep blood (TSA-SB) (Becton, 125

Dickinson and Company, Franklin Lakes, NJ) were suspended in Mueller-Hinton broth to a 0.5 126

McFarland Standard and spread onto Mueller-Hinton agar with 5% sheep blood (MH-SB) (Becton, 127

Dickinson and Company, Franklin Lakes, NJ). E-test strips were placed on the plate surface and the 128

plates were incubated overnight at 37ºC in 5% CO2. Susceptibility to other macrolides was determined 129

by disk diffusion in accordance with the standards described by the Clinical and Laboratory Standards 130

Institute (11). To determine the effects of erythromycin induction on resistance, MICs were determined 131

by E-test as described above except strains were incubated on TSA-SB supplemented with 1/10 MIC 132

erythromycin prior to suspension and plating on MH-SB. Susceptibility to other macrolides was 133

determined by disk diffusion in accordance with the standards described by the Clinical and Laboratory 134

Standards Institute (11). Susceptibility disks were purchased or prepared as described above. All 135

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susceptibility data represents the mean ± standard deviation of at least five independent replications and 136

were analyzed by two-tailed, paired T-tests. p values > 0.05 were considered significant. 137

Disk diffusion assay for mefE-lacZ induction. The mefE-lacZ reporter strain XZ7042 was grown to 138

mid-log phase in Todd-Hewitt broth supplemented with 0.5% yeast extract. The liquid culture was 139

swabbed onto trypticase soy agar (TSA) supplemented with 300 U/ml catalase (Sigma-Aldridge, St. 140

Louis, MO) and 0.032 % X-gal. Susceptibility disks infused with test compounds were placed 141

immediately on the plates and incubated at 37ºC, 5 % CO2 for 24 h. Induction by synthetic competence-142

stimulating peptide-1 (CSP-1) (20) was tested by spotting 15 µl of a 100 ng/ml solution directly to the 143

center of indicator plate swabbed with the indicated strain. CSP-1 was synthesized by the Emory 144

University Microchemical Facility. 145

Results 146

Characterization of erythromycin-induced expression of mefE/mel. The kinetics and dose-147

dependence of mefE/mel induction were determined using the prototype macrolide erythromycin and a 148

series of isogenic reporter strains that contained a mefE-lacZ transcriptional fusion in the S. pneumoniae 149

strain GA17457 [Table 1, (51)]. The “wild type” reporter strain XZ7042 contained a functional MEGA 150

element encoding the MefE/Mel efflux pump that confers resistance to erythromycin (MIC 12 µg/ml), 151

while the MEGA-deletion derivative, XZ8004, was erythromycin-susceptible (MIC 0.1 µg/ml) (51). A 152

promoterless lacZ-reporter strain, XZ7049, which was otherwise identical to XZ7042, served as a 153

negative control. The induction of mefE transcription in XZ7042, expressed as β-galactosidase (β-gal) 154

activity, was measured over time following the addition of subinhibitory concentrations of erythromycin 155

(1/10 of the MIC). β-gal activity increased rapidly upon addition of erythromycin to more than 7-fold 156

within the first 30 minutes post-induction (Fig. 1a). Maximal induction (>15-fold increase) was 157

obtained at 1 hour (Fig. 1a). β-gal activities of XZ7042 cultures without induction were equivalent to 158

those of the induced and uninduced promoterless negative control, indicating a very low level of 159

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constitutive expression from the mefE-promoter in the absence of an inducer (Fig.1a). 160

The role of a functional MEGA element in mefE induction was assessed by measuring the 161

erythromycin dose-response in XZ7042 and the MEGA deletion derivative XZ8004. The strains were 162

incubated with erythromycin concentrations beginning with 0.0012 µg/ml and increasing by one-half or 163

one-quarter log intervals (Figure 1b). In XZ8004 mefE was induced at lower erythromycin 164

concentrations than in XZ7042 (0.012 and 0.04 µg/ml, respectively) and with higher maximal 165

expression (205.3 MU and 106.9 MU, respectively, Fig. 1b). However, peak expression of mefE in 166

XZ8004 occurred at a concentration equivalent to the erythromycin MIC (0.12 µg/ml) of the strain, 167

while in XZ7042 peak expression was observed at a concentration (0.4 µg/ml) 30-fold less than the 168

strains erythromycin MIC (12 µg/ml) (Fig. 1b). Expression in XZ8004 decreased rapidly with 169

increasing erythromycin concentrations above the MIC (Fig. 1b), consistent with an inhibitory effect of 170

erythromycin. The mefE-lacZ induction in XZ8004 indicated that a functional MEGA element was not 171

required for the induction of mefE/mel; however, strain susceptibility to the inducer and the ability to 172

modify intracellular concentrations of the inducer contributed to the magnitude and duration of 173

mefE/mel induction. 174

To evaluate mefE/mel expression over a range of inducers and inducer concentrations, a disk 175

diffusion assay using the XZ7042 reporter strain was developed (42). In contrast to the quantitative β-176

gal assays, this approach provided a concentration gradient, allowing the detection of mefE-lacZ 177

induction without a priori knowledge of the MIC of the test compound or the time-dependent induction 178

kinetics and was suitable for study of a broad range of potential inducers. Fig. 1c shows the results of 179

the disk diffusion assays using erythromycin as the test compound with the “wild type” reporter 180

XZ7042, the MEGA-deletion strain XZ8004, and the promoter-less control strain XZ7049. Strain 181

XZ7042 showed a characteristic large blue halo, indicative of the induction of mefE-lacZ, surrounding a 182

narrow zone of inhibited growth (Fig. 1c). As anticipated, the MEGA deletion strain XZ8004 showed a 183

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narrow range of induction, surrounding a large zone of inhibition (Fig. 1c). This result was consistent 184

with the quantitative assay (Fig. 1b), showing a wide range of inducing concentrations for XZ7042 and a 185

strong but narrow range of futile induction of mefE in strain XZ8004 in the absence of the resistance 186

determinant MEGA and the efflux pump (Fig. 1b). The promoterless-lacZ strain XZ7049 showed the 187

same zone of inhibition as XZ7042, as expected due to a functional MEGA element; however, no β-gal 188

activity (i.e., blue halo) was detected (Fig. 1c), confirming that the mefE-lacZ-expression in the reporter 189

strain XZ7042 exclusively depended on mefE. 190

Environmental or antibiotic (non-macrolide) stress and competence did not induce mefE-191

lacZ. To assess the spectrum of inducers of mefE/mel expression, different environmental conditions 192

and antibiotic compounds were analyzed in the disk diffusion assay using XZ7042. Non-macrolide 193

antibiotics were chosen to include representatives of major antimicrobial classes with diverse cellular 194

targets, thus providing a wide range of antibiotic stresses. Also tested were environmental conditions 195

involved in regulation of other genetic systems, as well as known substrates for multi-drug efflux 196

systems. None of the tested compounds and conditions inducted mefE-lacZ expression, with the 197

exception of the previously described antimicrobial peptides LL-37, CRAMP38, CRAMP39 (51). These 198

results demonstrated that mefE/mel induction is restricted to a narrow range of compounds, suggesting 199

one or more well-defined mechanism(s) of induction. 200

In addition, competence was tested as potentially inducing condition of mefE induction. 201

Competence induction in S. pneumoniae has been known to affect expression of a broad range of genes 202

and is mediated by the com regulon (1, 2, 21, 31). Prudhomme et al. (39) showed that the com regulon 203

was induced in response to antibiotic stress. Since it was unknown whether the conditions of the disk 204

diffusion assay supported the development of natural competence, synthetic competence-stimulating 205

peptide (CSP-1) was used to induce competence and monitor the effect on mefE-lacZ induction in the 206

lacZ-disk diffusion assay. Figure 2 shows that erythromycin, but not CSP-1, induced mefE-lacZ in 207

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XZ7042. As a control for competence development, a reporter strain, XZ7067, which carried the lacZ 208

gene under the control of the promoter for the early competence gene comC, was constructed (Table 1). 209

XZ7067 was induced by CSP-1 confirming competence induction under the assay conditions. 210

Erythromycin did not induce comC (Fig. 2), which is consistent with the previous observation (36) that 211

erythromycin does not induce competence. Thus, general environmental and antibiotic stresses, as well 212

as the development of competence did not induce mefE/mel expression. 213

Induction of mefE by macrolides. To understand the relationship between macrolide structure 214

and mefE/mel induction, a panel of sixteen macrolides composed of 14-, 15- and 16-membered 215

compounds were studied using the disk diffusion assay. The 14-membered cladinosolides, compounds 216

with the lactone ring substituted with the sugar cladinose at C3 and the monosaccharide amino sugar 217

desosamine at C5 (Fig. 3a) (e.g., erythromycin, clarithromycin, dirithromycin and roxithromycin), each 218

strongly induced β-gal activity over a wide diffusion zone (i.e. concentration gradient) (Fig. 3a). 219

Induction was correlated with decreased susceptibility to these compounds in the MEGA-containing 220

reporter strain, but not in the susceptible MEGA-deletion strain (Fig. 3a and Table 2, p <0.001). In 221

addition, preincubation of the reporter strains with a subinhibitory concentration of erythromycin (1/10 222

MIC, 1.2 µg/ml) further increased the resistance to these compounds for the wild type MEGA 223

containing reporter strain (p <0.05, Table 2) background, but not for the MEGA-deletion strain. Thus, 224

resistance in the wild type strain required the efflux-encoding MEGA element and suggested that 225

erythromycin, clarithromycin, clarithromycin, and roxithromycin are substrates for MefE/Mel-mediated 226

efflux. These findings were consistent with earlier reports for 14- and 15-membered macrolides (3, 34, 227

50). 228

Two 14-membered macrolides oleandomycin and troleandomycin with substitution of oleandrose 229

for the cladinose in the lactone ring were tested (Fig. 3c). Induction of mefE-lacZ by oleandomycin 230

resembled the strong induction observed with the cladinosolides; however, troleandomycin was a poor 231

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inducer of mefE (Fig. 3c). Troleandomycin has three acetate substitutions of the hydroxyl groups of 232

oleandomycin (Fig. 3c), suggesting that one or more of these acetate groups interfered with mefE 233

induction. The wild type reporter strain XZ7042 was significantly more susceptible to troleandomycin 234

than to oleandomycin (p <0.001, Table 2). To determine whether the increased susceptibility of XZ7042 235

for troleandomycin compared to oleandomycin was due to the weak mefE induction by troleandomycin, 236

the reporter strain XZ7042 was preincubated with a subinhibitory concentration of erythromycin (0.12 237

µg/ml) to achieve high induction of mefE/mel prior to susceptibility testing with troleandomycin. Under 238

these conditions, the resistance to troleandomycin increased to the levels of oleandomycin indicating 239

that troleandomycin was a substrate for efflux, despite being a poor mefE/mel inducer. 240

The ketolide telithromycin is a 14-membered macrolide that belongs to the ketolide subclass due 241

to a C3 ketone substitution instead of cladinose (Fig. 3b). Telithromycin retains excellent antimicrobial 242

activity of against MefE/Mel-containing pneumococci (12, 50). However, more detailed reports showed 243

that the efflux pump conferred small increases in pneumococcal MIC’s of telithromycin after 244

preincubation with subinhibitory concentrations of telithromycin (12, 50) or in comparison of clinical 245

isolates with and without mefE/mel (46). To determine whether the lack of TEL resistance was due to 246

an inefficient induction of mefE/mel expression or poor efflux of telithromycin by MefE/Mel, the 247

inducing ability of telithromycin was tested in the disk diffusion assay. Telithromycin strongly induced 248

mefE and the observed induction pattern was quite similar to that observed for the MEGA deletion 249

mutant XZ8004 with erythromycin (Fig. 1c), i.e., a narrow zone of strong mefE-induction surrounding a 250

large zone of inhibition of growth, which indicated that the wild type reporter strain remained 251

susceptible to telithromycin despite the strong induction of mefE-lacZ. This was confirmed by 252

telithromycin resistance (Table 2). As expected, preincubation with erythromycin had no significant 253

impact on telithromycin resistance although, the MEGA mutant was more susceptible to telithromycin 254

than the MEGA-containing strain (Table 2, p <0.001). These data suggest that telithromycin, while a 255

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strong inducer of mefE/mel, was not sufficiently extruded by the MefE/Mel efflux pump to allow for 256

resistance to develop. 257

The two 15-membered macrolides, azithromycin and tulathromycin (Table 2, Fig. 3d), were 258

studied. Tulathromycin differs from azithromycin by a demethylation of N10 and by a propylamino side 259

chain substitution on the C3 cladinose (Fig. 3d). However, while mefE induction by azithromycin was 260

comparable to that of erythromycin, induction by tulathromycin was weaker. XZ7042 was slightly more 261

susceptible to tulathromycin than to azithromycin, suggesting that the increase in antimicrobial activity 262

of tulathromycin could be due to the weaker induction of mefE/mel. To test this, XZ7042 was 263

preincubated with erythromycin (1.2 µg/ml). Increased resistance to tulathromycin to the level of 264

azithromycin was noted. However, the weaker induction by TUL was still sufficient to render the 265

MEGA-containing strain XZ7042 less susceptible than the MEGA deletion strain XZ8004 (Table 2). 266

16-membered macrolides can induce mefE/mel expression. A current paradigm is that the 267

MefE/Mel efflux pump does not confer resistance to 16-membered macrolides (34). The inability of 16-268

membered macrolides to induce the efflux pump, structural feature(s) of the compounds that preclude 269

them as substrates for the efflux pump, or sequestration, e.g., at the ribosome, making these compounds 270

unavailable for efflux are possible explanations. Seven 16-membered macrolides, josamycin, 271

kitasamycin, midecamycin, rosamicin, spiramycin, tilmicosin and tylosin, were studied for induction of 272

mefE-lacZ (Figs. 4). Josamycin, kitasamycin, midecamycin, spiramycin and tylosin did not induce 273

mefE-lacZ expression (Fig. 4). Surprisingly, two 16-membered macrolides, tilmicosin and rosamicin 274

induced the mefE-lacZ reporter (Fig. 4b). All the16-membered macrolides lacked a cladinose at C3. 275

While the cladinose has been suggested as an important feature of inducing 14- and 15-membered 276

macrolides (8), the above noted 14- or 15- membered macrolides with modifications of the cladinose 277

(oleandomycin and troleandomycin) or with a ketone substitution (telithromycin) at C3 were also 278

inducers. In contrast, the common feature of the16-membered inducers (tilmicosin and rosamicin) and 279

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the inducing 14- and 15-membered macrolides including telithromycin was the presence of a 280

monosaccharide at C5 (Fig. 4b). All non-inducing macrolides possessed a disaccharide moiety at this 281

position. The requirement of a monosaccharide at C5 was supported by the data with tylosin, a non- 282

inducing 16-membered macrolide structurally closely related to tilmicosin and rosamicin, but 283

distinguished by the disaccharide in the C5 position (Fig. 4b). Thus, the ability to induce mefE/mel 284

transcription was not dependent upon the size (e.g., 14-, 15- or 16-membered) of the lactone ring or the 285

presence of a cladinose at C3. Rather, mefE/mel expression was linked to a monosaccharide amino 286

sugar attached at C5 of the macrolide lactone ring. 287

Differential MefE/Mel-mediated resistance to 16-membered macrolides: further evidence 288

of uncoupling of induction and resistance. As observed for troleandomycin, which did not induce 289

mefE-lacZ but was readily effluxed upon preincubation with erythromycin, the susceptibility of 290

pneumococci to the non-inducing 16-membered macrolides could result from a lack of induction of the 291

efflux pump. Therefore, the wild type reporter XZ7042 was preincubated with erythromycin and 292

susceptibility to the non-inducing 16-membered compounds were determined (Table 2). The results did 293

not show increased resistance towards these 16-membered macrolides suggesting that they were not 294

subjected to efflux in the presence of the MefE/Mel pump. This was confirmed by double disk diffusion 295

assays for induction in which no D-zone of clearing was produced by the non-inducing macrolides in the 296

presence of erythromycin (data not shown). 297

Still remaining was the question of whether the induction of mefE observed with the inducing 16-298

membered macrolides rosamicin and tilmicosin translated into increased resistance to these compounds. 299

The strong, narrow induction ring observed in the disk-diffusion induction assay with tilmicosin 300

indicated a narrow range of inducing concentrations; however, the small zone of inhibition indicated 301

pneumococcal resistance to tilmicosin (Fig. 4b). Preincubation of XZ7042 with erythromycin resulted 302

in further resistance of tilmicosin compared to the uninduced control (Table 2), indicating that tilmicosin 303

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was subjected to MefE/Mel-mediated efflux. Conversely, the induction of mefE by rosamicin (Fig. 4b), 304

resembled that observed with telithromycin (Fig. 3b) and the MEGA deletion strain XZ8004 (Fig. 1c), 305

and XZ7042 remained a susceptible phenotype despite induction (Table 2). Indeed, there was no change 306

in susceptibility to rosamicin between the MEGA-deletion strain XZ8004 and the wild type strain 307

XZ7042 despite the induction of mefE/mel (Table 2). 308

These data further demonstrate the uncoupling of macrolide induction of mefE/mel expression, and 309

macrolide efflux. The results with 16-membered macrolides further demonstrated that for induction of 310

mefE/mel a monosaccharide in position C5 of the macrolide was required but this was not sufficient to 311

confer MefE/Mel-mediated resistance. Thus, MEGA-containing isolates of S. pneumoniae remained 312

susceptible to most 16-membered macrolides, independent of the capacity to induce mefE/mel. 313

Discussion 314

Mechanisms of S. pneumoniae resistance to macrolides include modification of macrolide target 315

sites on the 23S ribosomal subunit, either by mutation or erm methylases, and macrolide efflux by the 316

MefE/Mel efflux pump encoded on the MEGA element (16, 23, 44, 45, 47, 52). In North America and 317

many other parts of the world, MefE/Mel is the predominant mechanism of pneumococcal macrolide 318

resistance (16, 43). Approximately half of the macrolide resistant pneumococci isolated in the United 319

States in 2006 contained mefE/mel as the sole macrolide resistance determinant and an additional 25% 320

contained both mefE/mel and ermB (22). Resistance levels conferred by MefE/Mel in vitro 321

(erythromycin 1-32 µg/ml) are lower compared to levels conferred by ribosome modification; 322

nevertheless, these levels are correlated with macrolide treatment failures (25, 26). Exposure in vivo to 323

inducers of the efflux pump may lead to resistance levels at the site of infection higher than those 324

predicted by in vitro assays. MefE/Mel resistance can be induced by up to a 200-fold increase in MICs 325

(3, 49), and therefore could be of significant clinical impact. This background led us to further study 326

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induction of macrolide efflux, to identify structural feature that differentiate inducing and non-inducing 327

macrolides and to determine the relationship between induction and resistance. 328

A range of compounds and conditions were studied for the ability to induce expression of the 329

mefE-lacZ reporter using the disk diffusion assay. This assay allowed the efficient screening of a broad 330

range of inducer concentrations and conditions. Non-macrolide antibiotics or general environmental 331

stresses and conditions (e.g., competence induction), except for certain antimicrobial peptides (51) and 332

macrolides, did not induce expression of mefE/mel. The non-macrolide antibiotics studied represented 333

different classes with distinct cellular targets including ribosomal binding sites on 23S rRNA 334

overlapping the macrolide binding site (chloramphenicol, lincosamides, linezolid and streptogramins) 335

and 16S rRNA binding drugs (e.g., aminoglycosides and tetracycline), suggesting that disruption of 336

protein synthesis per se does not influence mefE/mel expression. Likewise, drugs that disrupt DNA 337

synthesis, cell wall synthesis, or membrane integrity, and various environmental conditions did not 338

induce expression, indicating that the range of inducers for mefE/mel expression is quite specific. 339

The current paradigm for the MefE/Mel efflux pump is that it confers resistance to 14- and 15-340

membered, but not to the 16-membered macrolides (5, 9, 10, 17, 34, 36); and 14- and 15-membered 341

induce MefE/Mel-mediated resistance (3, 36, 50). Our data confirm strong induction of mefE/mel 342

expression by erythromycin, clarithromycin and azithromycin, as previously reported (3, 50), and 343

showed that the most commonly studied 16-membered macrolides indeed did not induce mefE/mel 344

expression. However, the use a broader spectrum of structurally diverse macrolides revealed 14- 345

membered and 15-membered macrolides with reduced ability to induce mefE/mel expression and 16-346

membered macrolides that induced mefE/mel. In addition, the ketolide telithromycin, despite retaining 347

excellent activity against mefE-containing pneumococci, strongly induced mefE expression. 348

Telithromycin has not previously been demonstrated to induce mefE/mel. 349

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The induction of mefE/mel by 14- and 15-membered macrolides, but not common 16-membered 350

macrolides coincides with differences in macrolide binding to the ribosome. Most 14- and 15-351

membered macrolides contain the monosaccharide desosamine at position C5 of the lactone ring, 352

whereas 16-membered macrolide typically have a C5 disaccharide. C5 substituents form extensive and 353

distinct bonds with the ribosome (Figs 3-8). The disaccharides in the 16-membered macrolides extend 354

deep into the exit tunnel of the ribosome (19, 38), allowing the mycarose (Fig. 4) to interact with G2505 355

and U2506 of the 23S rRNA (E. coli numbering, Fig. 4), interfering with peptidyl transferase activity 356

(19, 37, 38) and resulting in truncated peptides (19). Macrolides with C5 monosaccharides do not 357

interfere with peptidyl transferase activity, instead block egress of the nascent peptide from the exit 358

tunnel leading to peptides up to eight amino acids long (13, 19, 27, 38, 42). 359

We found that the inducing 16-membered macrolides tilmicosin and rosamicin like inducing 14- 360

and 15-membered macrolides have a monosaccharide at C5 and therefore are expected to have a similar 361

affect on peptide synthesis. The weak inducing 14-membered macrolide troleandomycin has 362

acetylations of the C3 and C5 sugars that force a substantially altered conformation when bound to the 363

ribosome resulting in a location of the macrolide farther down the exit tunnel than inducing macrolides 364

(6). The pivotal role of the C5 substituent for induction over the lactone-ring size or the C3 substituent is 365

also supported by our first identification of inducing 16-membered macrolides, which in addition lack 366

the C3 cladinose that is typically found in the common 14- and 15-membered macrolides. Taken 367

together, the induction mechanism involves specific binding of the macrolide to the ribosome and in 368

particular the C5 substituent of the macrolide. 369

Specific macrolide binding to the ribosome is also required for the induction of macrolide 370

resistance by many erm methylases (24, 28, 33, 41, 48), in mechanisms that are controlled by 371

transcriptional or translational attenuation. Weisblum (48) found that translational attenuation of ermC 372

in Staphylococcus aureus is alleviated by 14- but not 16-membered macrolides (48) and proposed that 373

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when bound by a 14-membered macrolides, ribosomes stall due to constriction of the exit tunnel after a 374

short peptide is synthesized. Ribosome stalling at the end of the leader peptide synthesis promotes 375

refolding of the transcript into an anti-attenuator structure (48). Conversely, C5 disaccharides of many 376

16-membered macrolides disrupt ribosyltransferase activity preventing synthesis of the leader peptide so 377

that the erm transcript remains folded in the attenuator structure (19). 378

We propose a similar mechanism for induction of mefE/mel and efflux-mediated macrolide 379

resistance in pneumococci. Macrolides with C5 disaccharides may prevent synthesis of an unidentified 380

regulatory leader peptide and thus may not induce. Macrolides with a monosaccharide at C5 promote 381

stalling at the precise residue of the leader peptide due to the length of the nascent peptide that is 382

synthesized before being blocked in the ribosomal exit tunnel. This is independent of lactone ring size 383

or the C3 sugar substituent. 384

The MefE/Mel efflux pump does not confer resistance to common 16-membered macrolides. 385

Induction of the efflux pump with erythromycin prior to exposure to the 16-membered macrolides, 386

demonstrated that pneumococcal susceptibility to these macrolides was not due to a failure to induce 387

MefE/Mel. The lack of efflux of 16-membered macrolides could result either from an unavailability of 388

the compounds for export by the efflux pump due to high affinity ribosomal binding or that the 389

macrolides are not recognized as substrates. Most of 16-membered macrolides have a C6 390

acetylaldehyde that can form a covalent bond with the rRNA, resulting in high affinity binding to the 391

ribosome (19). In contrast, the 14- and 15-membered macrolides, which are known substrates for the 392

efflux pump, bind ribosomes through hydrophobic interactions and hydrogen bonding and are easily 393

dissociated from the ribosome (7, 19, 37, 42). Of the two16-membered macrolides, rosamicin and 394

tilmicosin that induced mefE/mel expression, only tilmicosin had reduced activity against MEGA-395

containing pneumococci, suggesting it was subject to efflux. The primary structural difference between 396

these two compounds is the C6 acetylaldehyde of rosamicin (but not tilmicosin), with the potential to 397

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form the covalent bond effecting efflux by MefE/Mel. Further, telithromycin with 700-fold higher 398

affinity for the ribosome than erythromycin is predicted to have limited availability for export by 399

MefE/Mel. Despite strong induction of the efflux pump by telithromycin, pneumococci remain 400

susceptible. Thus, because of high affinity ribosomal binding most 16-membered macrolides and the 14-401

membered ketolides even if mefE/mel is induced are not effectively available for efflux, and 402

pneumococcal isolates remain susceptible to these compounds. 403

To summarize, induction of the mefE/mel was shown to be limited to a narrow range of 404

macrolides and antimicrobial peptides. The model indicates induction is mediated by ribosomal binding 405

and that inducing 14-, 15- and 16-membered macrolides contain a monosaccharide in position C5 of the 406

lactone ring. The C5 monosaccharide forms a characteristic macrolide-ribosome-mRNA complex 407

consistent with the induction mechanism described for methylase-mediated macrolide resistance, an 408

anti-attenuation-based mechanism for mefE/mel. Most16-membered macrolides and the 14-membered 409

ketolide have high affinity binding to the ribosome and this strong ribosomal binding limits the 410

availability for efflux from the pneumococcus, even in the presence of MefE/Mel induction. The 411

antimicrobial peptides such LL-37 and CRAMP that induce are not expected to bind ribosomes, thus 412

induction by these agents is anticipated to occur by an alternate pathway. Investigations are currently 413

underway to confirm and define the proposed macrolide transcriptional attenuator mechanism and to 414

study the basis for induction by antimicrobial peptides. 415

Acknowledgments 416

This work was supported by grants from the National Institutes of Health (5R01AI070829) and from the 417

Medical Research Service of the Department of Veterans Affairs (to DSS). 418

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569

570

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Figure 1. Induction of mefE-lacZ in isogenic S. pneumoniae strains. All strains were derived from the 1

MEGA-containing, invasive pneumococcal isolate GA17457 and include XZ7042, the MEGA deletion 2

mutant XZ8004 and the promoterless lacZ negative control XZ7049. (a) β-galactosidase expression by 3

XZ7042 (solid lines, circles) and XZ7049 (dashed lines, squares). Strains were untreated (open symbols) or 4

exposed to 0.1 µg/ml erythromycin (closed symbols) at 0 hours and cells were harvested and assayed at 5

half-hour intervals. Error bars indicate standard deviation of duplicate replications. (b) Erythromycin dose 6

response curves of the XZ7042 (solid lines, open squares) and XZ8004 (dashed lines, closed triangles). 7

Arrows indicate the erythromycin MIC of each strain (solid, XZ7042; dashed, XZ8004). Error bars indicate 8

standard deviation of duplicate replications. (c) Disk diffusion assay for induction of mefE-lacZ by 9

erythromycin. Disks containing 15 µg/ml erythromycin were placed on plates swabbed with XZ7042 (first 10

panel), XZ8004 (middle panel) and XZ7049 (last panel). Expression of the reporter is indicated as a blue 11

halo surrounding a zone of inhibition. 12

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Figure 2. Competence stimulating peptide 1 (CSP-1) did not induced mefE-lacZ. Disk diffusion 1

induction assay of the mefE-lacZ (XZ7042) and comC-lacZ (XZ7067) reporter strains by 2

erythromycin (ERY) and CSP-1. CSP-1 was spotted directly onto THY indicator plates streaked 3

with XZ7042 or XZ7067. As controls, diffusion disks containing erythromycin (15 µg) were 4

placed onto TSA indicator plates streaked with each strain. A blue spot or blue halo indicated 5

comC-lacZ or mefE-lacZ induction, respectively. 6

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Figure 3. 14- and 15-membered macrolides. Structure and disk diffusion induction assays for the 14-

and 15-membered macrolides used in this study. Discs containing 15 µg/ml of each macrolide were

placed onto TSA indicator plates spread with XZ7042. (a) Cladinosolides. Erythromycin and

derivatives each contain a C3 cladinose. 1, erythromycin; 2, clarithromycin; 3, dirithromycin; 4,

roxithromycin. (b) Telithromycin. The C11/C12 carbamate with a aryl-akyl side chain is responsible for

increased ribosomal affinity and compensates for the absence of cladinose at C3 (19). 1, erythromycin;

2, telithromycin. (c) Oleandomycins. Oleandomycin and troleandomycin differ only by acetylation of

hydroxyl groups at three positions in troleandomycin (R1, R2, and R3). The dashed arrow indicates

interaction of acetyl group of the oleandrose of troleandomycin and the 23 rRNA nucleotide U790 (E.

coli numbering). 1, erythromycin; 2, troleandomycin; 3, oleandomycin. (d). 15-membered azalides.

Previously described interaction between azithromycin cladinose hydroxyl group and the 23S rRNA

nucleotide G2505 (E. coli numbering, dashed arrow) (19). C5 desosamine is indicated in red. 1,

erythromycin; 2, tulathromycin; 3, azithromycin.

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Figure 4. Structures and disk diffusion induction assays for all 16-membered macrolides used in this study. 1

(a) Common non-inducing 16-membered macrolides. Positions of the carbon atoms in the lactone ring are 2

indicated and the C5 disaccharide composed of the amino sugar mycaminose and neutral sugar mycarose is 3

shown in red. Previously described points of interactions between 16-membered macrolides and 23S rRNA 4

nucleotides (E. coli numbering) are indicated by dashed arrows (43, 47). The C6 acetylaldehyde and the 5

reversible covalent bond (wavy line) formed with the nucleotide A2062 (solid arrow) are indicated in blue 6

(19). 1, josamycin; 2, kitasamycin; 3, midecamycin; 4, spiramycin; center disk, erythromycin control. Abbr. 7

foro, forosamide. (b) Tylosin and C5 monosaccharide-containing derivatives rosamicin and tilmicosin. The 8

C5 monosaccharides desosamine and mycaminose are indicated in red. 1, rosamicin; 2. erythromycin control; 9

3, tilmicosin; 4, tylosin. Discs containing 15 µg/ml of each macrolide were placed onto TSA indicator plates 10

spread with XZ7042. 11

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Table 1. Strains and plasmids used in this work

Strains or

plasmid Description

Reference

or Source

S. pneumoniae

GA17457 Wild type, parent; MEGA; ERYR (51)

XZ7042 GA17457-derivative; bgaA::mefE-lacZ; MEGA; ERYR (51)

XZ8004 GA17457-derivative; bgaA::PmefE-lacZ; MEGA::aad9; ERYS (51)

XZ7067 GA17457-derivative; bgaA::PcomC-lacZ; MEGA; ERYR This study

XZ7049 GA17457-derivative; bgaA::lacZ (promoterless); MEGA; ERYR This study

Plasmids

pPP2 pPP1 derivative, lacZ reporter gene with the htrA ribosomal binding site (18)

pPC2 pPP2 derivative; carries PcomC-lacZ fusion (18)

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Table 2. Induction of the MefE/Mel efflux pump and pneumococcal susceptibility. 1

Zone of Inhibition Diameter (mm)

XZ7042 XZ8004

Test drug Ring

membersa

C3b C5

b

C6c

ac-AH

-ERY +ERY -ERY +ERY

Erythromycin 14 cla des - 9.7±0.5 7.8±0.4 29.0±1.4 27.4±1.1

Clarithromycin 14 cla des - 9.4±0.5 8.2±0.4 28.0±2.0 28.2±1.3

Dirithromycin 14 cla des - 8.1±0.4 7.0±0.8 22.2±0.8 21.6±1.7

Roxithromycin 14 cla des - 8.4±0.5 6.4±0.5 24.4±1.1 25.6±0.9

Oleandomycin 14 ole des - 9.6±0.5 6.9±0.9 19.6±0.5 19.8±0.5

Troleandomycin 14 ole-ac des-ac - 12.5±1.0 7.3±0.8 16.5±0.5 17.4±1.7

Telithromycin 14 OH des - 20.6±1.1 19.0±1.4 31.2±0.4 31.0±0.7

Azithromycin 15 cla des - 7.6±0.5 6.4±0.5 22.4±1.1 20.2±0.8

Tulathromycin 15 cla* des - 8.7±0.5 6.6±0.5 17.4±2.7 16.6±0.5

Josamycin 16 OH myn-myr + 23.4±3.8 20.4±1.1 22.2±1.3 22.8±1.3

Kitasamycin 16 OH myn-myr + 23.2±1.3 22.2±1.3 23.2±1.3 23.2±0.8

Midecamycin 16 OH myn-myr + 20.2±1.3 19.8±0.8 21.2±0.8 21.8±0.8

Tylosin 16 OH myn-myr + 20.2±0.8 19.2±1.2 20.3±0.8 20.8±1.2

Rosamicin 16 OH des + 19.0±0.7 19.4±0.5 18.4±1.1 18.8±0.8

Spiramycin 16 OH myn-myr + 19.0±1.4 18.0±0.7 19.2±1.9 19.2±1.6

Tilmicosin 16 OH myn - 10.5±1.2 8.2±0.7 13.2±0.9 12.7±0.8 a Number atoms in the lactone ring;

b side group attached at the C3 or C5 position of the lactone ring;

c 2

presence of acetylaldehyde side group at C6 of the lactone ring. Complete resistance, defined by growth 3

the edge of the disk, was scored as 6 mm, i.e., equal to the diameter of the disk. All disks contained 15 4

µg of the macrolide. Values in bold vary significantly (p <0.05) from those of the uninduced control 5

(XZ7042 +ERY) to the same drug. Values within a box are significantly different (p <0.05). Abbr. ac-6

AH, acetylaldehyde group; cla, cladinose; cla*, modified cladinose; ole, oleandrose; ole-ac, acetylated 7

oleandrose; des, desosamine; des-ac, acetylated desosamine; myn, mycaminose; myr, mycarose. 8

9

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aac accepts, published online ahead of print on 2 may...

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