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
26
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
34
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).
C A
Ketolides bind to the
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3
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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
5
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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:
T P
111 gene
primer
primer
ermBF
ermBR
(5’-
(5’-
C A
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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
6
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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
148
absence of antibiotic, the presence of telithromycin (at 0.008 µg/ml) or the presence of
149
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
7
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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
156
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
158
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
T P
(5’-TTCCTTTTTCAGTCAGTCGTTTAC-3’).
Real-time
PCR
(5’-
was
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C A
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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.
175 176
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)
178
complementary to the G2061-U2079 region of the streptococcal 23S rRNA was
8
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extended with 1 U reverse transcriptase (Life Sciences) using a mixture of either 1 mM
180
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
189
containing erythromycin (1 µg/ml), and incubated at 37˚C in 5% CO2. Growth was
190
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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196
RESULTS
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The S. pneumoniae isolate P1501016 is serotype 23F, highly resistant to macrolides
198
(MICs (µg/ml): erythromycin >256, clarithromycin >256) and clindamycin (>256
199
µg/ml) and resistant to telithromycin (8 µg/ml). P1501016 is mef(A) negative and
200
erm(B) positive, with the latter gene located in the transposon Tn1545. The four alleles
201
encoding 23S rRNA, and ribosomal proteins L4 and L22 have unchanged wild-type
202
sequences. However, the erm(B) operon has a 136 bp deletion that removed the last
203
three codons of the leader and the entire Shine-Dalgarno (SD2) sequence immediately
204
proximal to the erm(B) cistron (Fig. 1A). The putative mutant Erm(B) protein therefore
205
consists of the remaining portion of the leader peptide fused in-frame with the full-
206
length Erm(B) sequence. The mutant Erm(B) is translated from SD1 and is extended by
207
24 amino acids at its N-terminal compared to the wild-type protein (Fig. 1B).
208
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C A
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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
212
telithromycin required to inhibit growth is 16 µg/ml (Table 1). The erm(B) gene of the
213
transformants was confirmed to contain the deletion mutation identical to that of
214
P1501016; genes encoding 23S rRNA, L4 and L22 were shown to have remained
215
unchanged. The MIC of telithromycin for PC13 increased from 0.06 µg/ml to 0.5 µg/ml
216
in the presence of erythromycin, whereas the MICs of telithromycin for P1501016 and
217
the PC13-transformant remained unaltered by the presence of erythromycin (Table 1).
218 219
The relative expression of erm(B) compared to the control gene gdh is shown in Fig. 2.
220
In the absence of antibiotic, baseline relative expression of erm(B) in the clinical
10
221
telithromycin–resistant isolate P1501016 (mean, ± standard error of the mean; 2.54 ±
222
0.36) was significantly higher than PC13 (0.96 ± 0.08) (P=0.02). When the mutant
223
erm(B) was introduced into PC13, in the absence of antibiotic there was a small but
224
insignificant increase in the relative expression of erm(B) between PC13 (0.96 ± 0.08)
225
and the PC13-transformant (1.52 ± 0.30) (P=0.12). Telithromycin exposure did not
226
significantly increase the expression of erm(B) relative to baseline without antibiotic,
227
but the expression was significantly higher in the PC13-transformant (1.31 ± 0.22) than
228
in PC13 (0.66 ± 0.09) in the presence of telithromycin (P=0.02). In contrast, in the
229
presence of erythromycin significant increases in erm(B) expression were found
230
(P256
PC13
Wild-type
>256
>256
PC13-transformant
136 bp deletion in leader sequence
>256
>256
a
491
tetracycline; TEL, telithromycin
TET TEL
D E
T P
489 490
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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C A
25
492
FIGURE LEGENDS
493
FIG. 1A Diagram representing a wild-type erm(B) gene and the mutant erm(B) gene of
494
isolate P1501016 (SD; Shine-Dalgarno sequence).
495 496
FIG. 1B Amino acid sequence alignment of the wild-type Erm(B) protein of
497
telithromycin-susceptible strain PC13 with the putative mutant Erm(B) protein of
498
isolate P1501016. The arrow indicates the break between the control (leader) peptide
499
and structural protein in the wild-type protein.
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T P
500 501
FIG. 2 Relative expression of erm(B), determined by quantitative real-time reverse
502
transcription PCR, in isolate P1501016, PC13 and the PC13-transformant. Cultures
503
were grown either in the absence of antibiotic, presence of telithromycin (0.008 µg/ml)
504
or presence of erythromycin (0.25 µg/ml). Error bars represent the standard error of the
505
mean (n=4).
E C
C A
506 507
FIG. 3 Percentage dimethylation at position A2058 of 23S rRNA of P1501016, PC13
508
and the PC13-transformant. Cultures were grown either in the absence of antibiotic,
509
presence of telithromycin (0.008 µg/ml) or presence of erythromycin (0.25 µg/ml).
510
Error bars represent the standard error of the mean (n=3).
511 512
FIG. 4 Growth curves at 37˚C of isolate P1501016, PC13, and the PC13-transformant,
513
in the absence (A) and presence (B) of erythromycin (1 µg/ml).
514
26
FIG. 1A Wild-type erm(B) gene:
P1501016 erm(B) gene:
D E
136 bp deletion
T P
E C
C A
27
FIG. 1B Control peptide
PC13
1 MLVFQMRNVDKTSTVLKQTKNSDYADK MNKNIKYSQNFLTSEKVLNQIIKQLNLKETDTV
P1501016
1 MLVFQMRNVDKTSTVLKQTKNSDY----MNKNIKYSQNFLTSEKVLNQIIKQLNLKETDTV ************************
D E
T P
*********************************
PC13
34 YEIGTGKGHLTTKLAKISKQVTSIELDSHLFNLSSEKLKLNTRVTLIHQDILQFQFPNKQ
P1501016
58 YEIGTGKGHLTTKLAKISKQVTSIELDSHLFNLSSEKLKLNTRVTLIHQDILQFQFPNKQ
E C
************************************************************ 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) *** **** *** *******************
28
FIG. 2
16
P1501016
Relative expression of erm(B)
14
D E
PC13 12
PC13-transformant 10
T P
8
6
E C
4
2
0
C A Untreated
Telithromycin
Erythromycin
Treatment of cultures
29
FIG. 3
80
Percentage dimethylation of A2058
P1501016 70
PC13 PC13-transformant
D E
60
50
T P
40
30
20
E C
10
Treatment of cultures
0
C A
Untreated
Telithromycin
Erythromycin
Treatment of cultures
30
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
9
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
12
