Telithromycin resistance in Streptococcus pneumoniae is conferred by ...

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Dec 3, 2007 - Douthwaite, S. and W. S. Champney. 2001. Structures of ketolides and macrolides. 368 determine their mode of interaction with the ribosomal ...
AAC Accepts, published online ahead of print on 3 December 2007 Antimicrob. Agents Chemother. doi:10.1128/AAC.01074-07 Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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Telithromycin resistance in Streptococcus pneumoniae is

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conferred by a deletion in the leader sequence of erm(B) that

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increases rRNA methylation

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Running title: Telithromycin resistance due to increased methylation

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Nicole Wolter1*, Anthony M. Smith1, David J. Farrell2, John Blackman Northwood2,

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Stephen Douthwaite3 and Keith P. Klugman1, 4

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1. Respiratory and Meningeal Pathogens Research Unit, National Institute for

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Communicable Diseases, Medical Research Council and University of the

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Witwatersrand, Johannesburg, South Africa.

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2. GR Micro Ltd., London, United Kingdom.

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3. Department of Biochemistry and Molecular Biology, University of Southern

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Denmark, Odense, Denmark.

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4. Hubert Department of Global Health, Rollins School of Public Health, and Division

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of Infectious Diseases, School of Medicine, Emory University, Atlanta, GA, USA.

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*Corresponding author: Nicole Wolter, Respiratory and Meningeal Pathogens Research

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Unit, National Institute for Communicable Diseases, Private Bag X4, Sandringham,

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2131, South Africa. Phone: 27 11 555 0352. Fax: 27 11 555 0437. E-mail:

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[email protected]

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ABSTRACT

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A telithromycin-resistant, clinical isolate of Streptococcus pneumoniae (strain

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P1501016) has been found to contain a version of erm(B) that is altered by a 136 bp

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deletion in the leader sequence. By allele replacement mutagenesis, a second strain of S.

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pneumoniae (PC13) with a wild-type erm(B) gene was transformed to the

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telithromycin-resistant phenotype by introduction of the mutant erm(B) gene. Whereas

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the wild-type PC13 strain showed slight telithromycin resistance only after induction by

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erythromycin (telithromycin MIC increasing from 0.06 to 0.5 µg/ml), the transformed

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PC13 strain is constitutively resistant (MIC 16 µg/ml). Expression of erm(B) was

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quantified by real-time reverse transcription PCR in the presence of erythromycin or

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telithromycin; erm(B) expression was significantly higher in the transformed PC13

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strain than the wild-type strain. Furthermore, the transformed strain had significantly

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higher levels of ribosomal methylation in the absence as well as in the presence of the

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antibiotics. Growth studies showed that the transformed PC13 strain had a shorter lag

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phase than the wild-type strain in the presence of erythromycin. Telithromycin

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resistance is conclusively shown to be conferred by the mutant erm(B) gene that is

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expressed at a constitutively higher level than the inducible wild-type gene. Elevated

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erm(B) expression results in a higher level of rRNA methylation that presumably

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hinders telithromycin binding to the ribosome.

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INTRODUCTION

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Macrolide resistance in Streptococcus pneumoniae is conferred by two predominant

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mechanisms: target-site modification and active drug efflux due to the acquisition of

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erm(B) and mef(A) genes, respectively, or a combination of these mechanisms (16). The

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erm(B) gene encodes an rRNA methylase that methylates nucleotide A2058

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(Escherichia coli numbering) in 23S rRNA resulting in the macrolide-lincosamide-

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streptogramin B (MLSB) resistance phenotype (36). The mef(A) gene encodes an efflux

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pump and confers the M phenotype with resistance to 14- and 15-membered ring

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macrolides (29). In a small proportion of isolates, mutations in 23S rRNA and/or

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ribosomal proteins L4 and L22 were found to confer macrolide resistance (11, 30, 31).

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Telithromycin is a ketolide antibiotic, a semi-synthetic derivative of the 14-membered

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macrolide erythromycin A. Macrolides and ketolides bind close to the peptidyl

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transferase region of 23S rRNA and inhibit bacterial protein synthesis by blocking

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elongation of the peptide chain through the tunnel of the large ribosomal subunit (8, 41).

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Macrolides and ketolides also interfere with 50S ribosomal assembly (4). However

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despite common mechanisms of action, ketolides remain active against most macrolide-

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resistant strains expressing erm(B) and mef(A) (3, 14, 18).

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ribosome with greater affinity than macrolides such as erythromycin (3, 7). The primary

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contact site of erythromycin and telithromycin is at nucleotide A2058 of domain V of

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23S rRNA, and telithromycin possibly makes additional contacts within domain II of

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the 23S rRNA (1, 7, 13, 39), although the exact site of the other contacts remains

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controversial (34).

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Ketolides bind to the

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Telithromycin resistance in S. pneumoniae remains rare. In laboratory-generated strains

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with reduced susceptibility to telithromycin, mutations have been observed in domains

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II and V of 23S rRNA, ribosomal protein L22 and erm(B) (2, 35). Mutations in these

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genes, as well as ribosomal protein L4, have also been found in clinical telithromycin-

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resistant isolates (12, 20, 21, 22, 31), and a combination of mutated genes can result in a

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higher telithromycin resistance than mutation of only one gene (10, 20). Although

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deletions in the erm(B) leader sequence are a common molecular change associated

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with telithromycin resistance in pneumococci (32, 35), the exact effects of these

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mutations on the expression of erm(B) and rRNA methylation have not previously been

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quantified using isogenic pneumococcal strains. In this study, a clinical isolate of S.

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pneumoniae identified as resistant to telithromycin and containing a large deletion

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mutation in the leader sequence of erm(B) was investigated to characterize the

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resistance mechanism.

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MATERIALS AND METHODS

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Bacterial strains

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A clinical isolate of S. pneumoniae (P1501016), identified as resistant to telithromycin

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(MIC 8 µg/ml), was obtained from the PROTEKT surveillance study. Clinical patient

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information was not available and therefore previous treatment with telithromycin could

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not be determined. A strain (PC13) representative of pneumococcal clone 13 (South

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Africa19A) (17) was used as a recipient in allele replacement mutagenesis. PC13 was

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resistant to macrolides (MICs (µg/ml): erythromycin >256, clarithromycin >256) and

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clindamycin (>256 µg/ml), but susceptible to telithromycin (MIC 0.06 µg/ml).

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Pneumococci were routinely cultured at 37˚C in 5% CO2 on Mueller-Hinton agar

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supplemented with 5 % horse blood.

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Phenotypic and genotypic characterisation

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MICs were determined by the agar dilution method and the Etest (AB Biodisk, Solna,

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Sweden). Agar dilution plates were prepared according to the CLSI guidelines

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pertaining to the general preparation of agar dilution plates (5). They contained 5%

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horse blood and were incubated in ambient air for 20-24 hrs. MICs were interpreted as

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susceptible, intermediately resistant or resistant using CLSI breakpoints for broth

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microdilution (6). For telithromycin breakpoints were: ≤ 1 µg/ml for susceptible, 2

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µg/ml for intermediate and ≥ 4 µg/ml for resistant. S. pneumoniae ATCC49619 was

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used as a reference strain. P1501016 was serotyped by the quellung reaction with

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antisera from the Statens Serum Institut (Copenhagen, Denmark).

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Chromosomal DNA was extracted from overnight pneumococcal cultures as previously

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described (26). A duplex PCR was used to identify the presence of the erm(B) and

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mef(A) genes (28). The transposon (Tn1545 or Tn917) carrying erm(B) was identified

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as previously described (19) using transposon specific forward primers (Tn1545 primer

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ermBF:

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TGACGGTGACATCTCTC-3’) and a common reverse primer (ermBM-R2: 5’-

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CTGTCTAATTCAATAGACGT-3’). Genes encoding ribosomal proteins L4 and L22

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and all four alleles encoding 23S rRNA were amplified and sequenced according to

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previously described methods (11, 30).

5’-CTTAGAAGCAAACTTAAGAG-3’;

Tn917

amplified

using

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The

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CTTAGAAGCAAACTTAAGAG-3’)

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ATCGATACAAATTCCCCGTAG-3’). For each 50 µl reaction, 3 µl of chromosomal

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DNA was added to a mix containing 2.5 U of Taq DNA polymerase, 1x reaction buffer,

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1.5 mM MgCl2, 200 µM each of dATP, dCTP, dGTP and dTTP and 800 nM each of

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forward and reverse primer. Cycling parameters were: 94˚C for 2 min; 94 ˚C for 1 min,

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52˚C for 1 min and 72˚C for 3 min for 30 cycles; and 72˚C for 5 min. Amplified

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products were purified with the QIAquick gel extraction kit (Qiagen, Surrey, UK). DNA

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sequencing was performed using the BigDye Terminator Cycle Sequencing kit (Applied

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Biosystems, Foster City, CA) and an Applied Biosystems Model 310 automated DNA

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sequencer.

and

forward

reverse

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primer

5’-

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erm(B)

was

Tn917F:

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111 gene

primer

primer

ermBF

ermBR

(5’-

(5’-

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Allele replacement mutagenesis

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PC13 was chosen as a recipient strain for allele replacement mutagenesis as it contains a

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wild-type copy of erm(B), which can be replaced by an exogenous copy by homologous

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recombination. The laboratory strain R6 does not contain an erm(B) gene, and attempts

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to introduce an erm(B) gene into R6 by means of electroporation and conjugation were

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unsuccessful. The wild-type erm(B) gene in PC13 is encoded on transposon Tn1545

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and, when uninduced, the strain is susceptible to telithromycin (MIC 0.06 µg/ml); the

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genes in PC13 encoding 23S rRNA and ribosomal proteins L4 and L22 were confirmed

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to be wild-type. PC13 was made competent by culture in C-medium (33) and allele

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replacement mutagenesis performed as previously described (27). The erm(B) gene of

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P1501016 was used as donor DNA and transformants were selected on Mueller-Hinton

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agar supplemented with 5% horse blood and containing telithromycin (0.5 µg/ml).

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MICs of the transformants were determined, and the presence of the mutant erm(B)

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gene was confirmed by sequencing. In addition, the genes encoding 23S rRNA, L4 and

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L22 were resequenced.

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Induction assays

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Telithromycin MICs of P1501016, PC13 and the PC13-transformant were determined

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by the agar dilution method as described above, in the absence and presence of

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erythromycin (0.25 µg/ml).

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Relative quantification of erm(B) mRNA expression

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P1501016, PC13 and the PC13-transformant were grown in Tryptone Soya Broth (TSB)

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at 37°C, 5% CO2 to an OD600nm of 0.2. Three cultures were grown for each strain in the

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absence of antibiotic, the presence of telithromycin (at 0.008 µg/ml) or the presence of

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erythromycin (at 0.25 µg/ml). At the required OD, cultures were aliquoted and

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incubated with RNAProtect™ Bacteria Reagent (Qiagen), centrifuged and the pellet

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was stored at -70°C. Total RNA was extracted using the RNeasy Mini Kit for bacteria

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(Qiagen). RNA was reverse transcribed using the High-Capacity cDNA Archive Kit

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(Applied Biosystems), according to the manufacturer’s instructions. The target gene

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(erm(B)) and endogenous reference gene, glucose-6-phosphate dehydrogenase (gdh),

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were amplified separately from the same total RNA for each sample. For each PCR

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reaction, 5 µl of cDNA was added to a 45 µl mix containing 1x SYBR Green PCR

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Mastermix (Applied Biosystems) and 12.5 pmol each of forward primer and reverse

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primer (Applied Biosystems). The erm(B) cDNA was amplified using primers

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ermB747F

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CATTCCGCTGGCAGCTTAAG-3’). The cDNA of gdh, a housekeeping gene, was

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amplified using primers SPgdhF1 (5’-ATTCCGTGGTGTTCCTTTCTTTT-3’) and

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SPgdhR1

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performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems).

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Relative standard curves were constructed for each gene, and the ratio of erm(B) to gdh

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was calculated for each sample.

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(5’-AGGGTTGCTCTTGCACACTCA-3’)

and

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ermB806R

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(5’-TTCCTTTTTCAGTCAGTCGTTTAC-3’).

Real-time

PCR

(5’-

was

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Quantification of ribosomal dimethylation

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P1501016, PC13 and the PC13-transformant were grown in 20 ml TSB without

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antibiotic or with telithromycin or erythromycin, as described above. Cells were

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harvested by centrifugation at 1,500xg for 15 min; pellets were re-suspended in 1 ml of

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RNAProtect™ Bacteria Reagent (Qiagen) and incubated at room temperature for 5 min.

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Samples were centrifuged at 3,500xg for 10 min at 4oC and total RNA was extracted

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using the FastRNA® Pro Blue Kit (QBiogene, Inc., CA) and FastPrep® Instrument

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(QBiogene), for 40 sec at a speed setting of 6.0.

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Primer extension analysis was performed on the RNA as previously described (11, 25).

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Briefly, a 5´-32P-labelled deoxynucleotide primer (5´-AGTAAAGCTCCATGGGGTC)

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complementary to the G2061-U2079 region of the streptococcal 23S rRNA was

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extended with 1 U reverse transcriptase (Life Sciences) using a mixture of either 1 mM

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dTTP, dCTP, 5 mM ddGTP in the test samples or 1 mM dTTP, dCTP, dATP, dGTP in

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control samples; in both cases, 1.5 pmol of rRNA was used as the template. Extension

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reactions were performed in triplicate, and were run on denaturing polyacrylamide gels

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alongside dideoxy sequencing reactions performed on an unmodified rRNA template

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from S. pneumoniae strain R6. Gels were autoradiographed and quantified by

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phosphorimager scanning (Typhoon model, Amersham).

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Growth studies

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Pneumococci were inoculated from glycerol stocks into TSB (1:100 dilution), and TSB

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containing erythromycin (1 µg/ml), and incubated at 37˚C in 5% CO2. Growth was

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monitored by optical density measurements at 600 nm at intervals of 30 min, and

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doubling times during the exponential phase of growth were calculated.

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Statistical analysis

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Statistical differences between means were calculated using the paired t test with P

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values interpreted at the 95% confidence level.

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RESULTS

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The S. pneumoniae isolate P1501016 is serotype 23F, highly resistant to macrolides

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(MICs (µg/ml): erythromycin >256, clarithromycin >256) and clindamycin (>256

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µg/ml) and resistant to telithromycin (8 µg/ml). P1501016 is mef(A) negative and

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erm(B) positive, with the latter gene located in the transposon Tn1545. The four alleles

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encoding 23S rRNA, and ribosomal proteins L4 and L22 have unchanged wild-type

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sequences. However, the erm(B) operon has a 136 bp deletion that removed the last

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three codons of the leader and the entire Shine-Dalgarno (SD2) sequence immediately

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proximal to the erm(B) cistron (Fig. 1A). The putative mutant Erm(B) protein therefore

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consists of the remaining portion of the leader peptide fused in-frame with the full-

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length Erm(B) sequence. The mutant Erm(B) is translated from SD1 and is extended by

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24 amino acids at its N-terminal compared to the wild-type protein (Fig. 1B).

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Allele replacement mutagenesis was performed to determine whether the deletion in

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erm(B) was responsible for the observed telithromycin resistance. After transformation

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of PC13 with the mutant erm(B) from P1501016, the minimal concentration of

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telithromycin required to inhibit growth is 16 µg/ml (Table 1). The erm(B) gene of the

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transformants was confirmed to contain the deletion mutation identical to that of

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P1501016; genes encoding 23S rRNA, L4 and L22 were shown to have remained

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unchanged. The MIC of telithromycin for PC13 increased from 0.06 µg/ml to 0.5 µg/ml

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in the presence of erythromycin, whereas the MICs of telithromycin for P1501016 and

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the PC13-transformant remained unaltered by the presence of erythromycin (Table 1).

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The relative expression of erm(B) compared to the control gene gdh is shown in Fig. 2.

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In the absence of antibiotic, baseline relative expression of erm(B) in the clinical

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telithromycin–resistant isolate P1501016 (mean, ± standard error of the mean; 2.54 ±

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0.36) was significantly higher than PC13 (0.96 ± 0.08) (P=0.02). When the mutant

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erm(B) was introduced into PC13, in the absence of antibiotic there was a small but

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insignificant increase in the relative expression of erm(B) between PC13 (0.96 ± 0.08)

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and the PC13-transformant (1.52 ± 0.30) (P=0.12). Telithromycin exposure did not

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significantly increase the expression of erm(B) relative to baseline without antibiotic,

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but the expression was significantly higher in the PC13-transformant (1.31 ± 0.22) than

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in PC13 (0.66 ± 0.09) in the presence of telithromycin (P=0.02). In contrast, in the

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presence of erythromycin significant increases in erm(B) expression were found

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(P256

PC13

Wild-type

>256

>256

PC13-transformant

136 bp deletion in leader sequence

>256

>256

a

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tetracycline; TEL, telithromycin

TET TEL

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CLI >256

4

8

>256

16

0.06

>256

16

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Abbreviations: ERY, erythromycin; CLR, clarithromycin; CLI, clindamycin; TET,

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FIGURE LEGENDS

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FIG. 1A Diagram representing a wild-type erm(B) gene and the mutant erm(B) gene of

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isolate P1501016 (SD; Shine-Dalgarno sequence).

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FIG. 1B Amino acid sequence alignment of the wild-type Erm(B) protein of

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telithromycin-susceptible strain PC13 with the putative mutant Erm(B) protein of

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isolate P1501016. The arrow indicates the break between the control (leader) peptide

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and structural protein in the wild-type protein.

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FIG. 2 Relative expression of erm(B), determined by quantitative real-time reverse

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transcription PCR, in isolate P1501016, PC13 and the PC13-transformant. Cultures

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were grown either in the absence of antibiotic, presence of telithromycin (0.008 µg/ml)

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or presence of erythromycin (0.25 µg/ml). Error bars represent the standard error of the

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mean (n=4).

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C A

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FIG. 3 Percentage dimethylation at position A2058 of 23S rRNA of P1501016, PC13

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and the PC13-transformant. Cultures were grown either in the absence of antibiotic,

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presence of telithromycin (0.008 µg/ml) or presence of erythromycin (0.25 µg/ml).

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Error bars represent the standard error of the mean (n=3).

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FIG. 4 Growth curves at 37˚C of isolate P1501016, PC13, and the PC13-transformant,

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in the absence (A) and presence (B) of erythromycin (1 µg/ml).

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FIG. 1A Wild-type erm(B) gene:

P1501016 erm(B) gene:

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136 bp deletion

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C A

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FIG. 1B Control peptide

PC13

1 MLVFQMRNVDKTSTVLKQTKNSDYADK MNKNIKYSQNFLTSEKVLNQIIKQLNLKETDTV

P1501016

1 MLVFQMRNVDKTSTVLKQTKNSDY----MNKNIKYSQNFLTSEKVLNQIIKQLNLKETDTV ************************

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T P

*********************************

PC13

34 YEIGTGKGHLTTKLAKISKQVTSIELDSHLFNLSSEKLKLNTRVTLIHQDILQFQFPNKQ

P1501016

58 YEIGTGKGHLTTKLAKISKQVTSIELDSHLFNLSSEKLKLNTRVTLIHQDILQFQFPNKQ

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************************************************************ PC13 P1501016

94 RYKIVGNIPYHLSTQIIKKVVFESRASDIYLIVEEGFYKRTLDIHRTLGLLLHTQVSIQQ

C A

118 RYKIVGSIPYHLSTQIIKKVVFESRASDIYLIVEEGFYKRTLDIHRTLGLLLHTQVSIKQ ****** *************************************************** *

PC13

154 LLKLPAECFHPKPKVNSVLIKLTRHTTDVPDKYWKLYTYFVSKWVNREYRQLFTKNQFHQ

P1501016

178 LLKLPAECFHPKPKVNSALIKLTRHTTDVPDKYWKLYTYFVSKWVNREYRQLFTKNQFHQ ***************** ******************************************

PC13

214 AMKHAKVNNLSTITYEQVLSIFNSYLLFNGRK (245 amino acids)

P1501016

238 AMKYAKVNDLSTVTYEQVLSIFNSYLLFNGRK (269 amino acids) *** **** *** *******************

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FIG. 2

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P1501016

Relative expression of erm(B)

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PC13 12

PC13-transformant 10

T P

8

6

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4

2

0

C A Untreated

Telithromycin

Erythromycin

Treatment of cultures

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FIG. 3

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Percentage dimethylation of A2058

P1501016 70

PC13 PC13-transformant

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60

50

T P

40

30

20

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10

Treatment of cultures

0

C A

Untreated

Telithromycin

Erythromycin

Treatment of cultures

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FIG. 4A

1.4

1.2

P1501016

D E

PC13

Absorbance (600 nm)

1.0

PC13-transformant 0.8

T P

0.6

E C

0.4

0.2

C A 0.0

0

1

2

3

4

5

6

7

8

Time (Hrs)

31

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FIG. 4B

1.2

P1501016

D E

1.0

Absorbance (600 nm)

PC13 0.8

PC13-transformant

T P

0.6

0.4

E C

0.2

0.0 0

1

2

C A

3

4

5

6

7

8

9

10

11

Time (Hrs)

32

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