rplC T460C Identified as a Dominant Mutation in Linezolid-Resistant
Mycobacterium tuberculosis Strains
Patrick Beckert,aDoris Hillemann,bThomas A. Kohl,cJörn Kalinowski,cElvira Richter,bStefan Niemann,aand Silke Feuerriegela
Molecular Mycobacteriology, Research Center Borstel, Borstel, Germanya; National Reference Center for Mycobacteria, Research Center Borstel, Borstel, Germanyb; and
Institute for Genome Research and Systems Biology, Center for Biotechnology, Bielefeld University, Bielefeld, Germanyc
TheribosomalL3proteinwasidentifiedasanoveltargetinlinezolid(LZD)-resistant Mycobacteriumtuberculosis strains.Next-
generationsequencingconfirmed rplCT460CasthesolemutationinanLZD-resistant M.tuberculosis H37Rvstrainselected in
vitro.Sequencinganalysisrevealedthe rplCT460CmutationineightfurtherLZD-resistantisolates(three invitro-selectedmu-
control worldwide. Since the appearance of multidrug-resistant
(MDR; resistant to at least isoniazid and rifampin) (8) and exten-
sively drug-resistant (XDR; additionally resistant to one fluoro-
application of reserve antibiotics has become more important.
Linezolid (LZD), which belongs to the oxazolidinone group (1),
has been used primarily for the treatment of methicillin-resistant
Staphylococcus aureus (MRSA) and vancomycin-resistant entero-
coccus infections (10) and has been used off-label to treat MDR
and XDR TB patients (9, 12). LZD acts on the 50S ribosomal
subunit, specifically, the peptidyl-transferase center (PTC), by
blocking the binding of tRNA and thus inhibiting bacterial cell
The first occurrence of LZD-resistant M. tuberculosis clinical
isolates was described by Richter et al. (11) As potential target
proteins L4 and L22), erm-37 (which encodes methyltransferase),
and whiB7 (which encodes a putative regulator) were sequenced.
However, since no mutations were detected in these genes, the
mechanism of LZD resistance in clinical M. tuberculosis isolates
has remained unclear. To gain further insight into the mecha-
nisms involved, LZD-resistant M. tuberculosis strains were se-
lected in vitro (2). Sequence analysis of the 23S rRNA gene re-
vealed mutations in 50% of the selected mutants, primarily at
nucleotide position 2061. However, no further polymorphisms
were found in the remaining selected clones.
Recently, mutations in rplC were described as a novel LZD
involved in LZD resistance in M. tuberculosis, we sequenced
rplC in (i) LZD-resistant, in vitro-selected mutants; (ii) LZD-
resistant clinical isolates; (iii) follow-up isolates from three
of the major phylogenetic lineages. The primers used for am-
plification were rplC_5=(?249) (5=-GCTGCGGCTGGACGAC
TC-3=) and rplC_3=(?668) (5=-CTCTTGCGCAGCCATCACTT
C-3=). The conditions used were (i) initial denaturation at 95°C
for 15 min, (ii) denaturation at 94°C for 30 s, (iii) annealing at
65°C for 30 s, (iv) elongation for 30 s at 72°C, and (v) terminal
elongation for 10 min at 72°C. Steps ii to iv were performed 35
times. The PCR products thus obtained were sequenced using an
(TB), still represents a huge challenge for infectious disease
Dye Terminator cycle sequencing kit (version 3.1) according to
the manufacturer’s instructions. Analysis of sequence data was
performed using the DNAStar Lasergene (version 8.0) software
First, the rplC genes of strains from the aforementioned selec-
tion experiments were sequenced (2) (selected on 4 ?g/ml LZD;
MICs for LZD of 4 to 16 ?g/ml). As references, the genes of the
parental strains used for in vitro selection were sequenced. All but
one of the five strains sequenced carried a mutation at nucleotide
an amino acid exchange from cysteine to arginine at codon 154
To verify the identified rplC T460C mutation as the only
polymorphism in the clones selected, and thus responsible for
the resistance phenotype, whole-genome sequencing of one of
carried out. Isolated genomic DNA was sequenced by GATC
Biotech in a paired end run on the Illumina platform. Derived
reads had a length of 46 bp for strain 9679/00 1.1.2. Reads were
sion no. NC_000962.2) using the CLC Genomics Workbench
software (CLC bio) with default settings. For strain 9679/00
1.1.2, 11,431,954 of the 12,400,138 reads were matched to the
reference genome, resulting in an average coverage of 119
reads. CLC Genomics Workbench was also employed for single
nucleotide polymorphism (SNP) detection using default set-
tings with a minimum coverage of four reads and a minimum
variant frequency of 75%. A total of 30 SNPs were detected
compared to the H37Rv reference sequence (see Table S1 in the
supplemental material). Of these SNPs, the only ones consid-
Received 23 November 2011 Returned for modification 15 December 2011
Accepted 17 February 2012
Published ahead of print 27 February 2012
Address correspondence to Silke Feuerriegel, email@example.com.
S.N. and S.F. contributed equally to this work.
Supplemental material for this article may be found at http://aac.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0066-4804/12/$12.00 Antimicrobial Agents and Chemotherapyp. 2743–2745 aac.asm.org
ered were those not located in repetitive elements (possible
mapping artifacts) and absent from the genomes of three fur-
ther H37Rv variants selected in vitro for resistance to other
antibiotics (possible failures in the H37Rv reference sequence).
The rplC T460C mutation was the only SNP detected in the in
vitro-selected LZD-resistant strain (9679/00 1.1.2) under these
To confirm the clinical significance of the rplC T460C muta-
the sequence analyzed.
To further investigate the significance of rplC T460C for LZD
three different patients were sequenced (Table 2). The isolates
infected with M. tuberculosis strains which acquired LZD resis-
tance during treatment. Samples were taken over a time span of
several years. IS6110 fingerprint genotyping was used to confirm
entire treatment period (no reinfection). Furthermore, IS6110
fingerprinting confirmed that the three patients do not belong to
one cluster, as the patterns of the strains showed marked differ-
ences (data not shown).
rplC sequence analysis of the follow-up isolates showed a clear
correlation between the occurrence of the LZD resistance pheno-
of the susceptible isolates obtained before the emergence of LZD
resistance had no mutation in rplC (n ? 13).
Besides mutations in rplC, alterations in the rplD gene,
which codes for ribosomal protein L4, have been described as
mediating LZD resistance in Staphylococcus aureus (7), Clos-
tridium perfringens (3), and pneumococci (13). To further elu-
cidate previously unknown resistance mechanisms in the
strains analyzed in this study, the rplD genes of all of the strains
were sequenced by using primers rplD_5=(?153) (5=-CCGGG
1), and the follow-up isolates collected from patients 1 and 2 (Table
of the follow-up isolates from patient 3 (Table 2). However, as this
polymorphism occurs in both susceptible and resistant isolates, it
obviously does not play a role in mediating LZD resistance. Whole-
genome sequencing might be interesting to determine putative pre-
of in vitro-selected, LZD-resistant mutant 9679/00 4.4.1 and the ele-
To investigate if mutations in rplC, especially T460C, occur in
genes of 71 LZD-susceptible strains of the M. tuberculosis com-
plex, comprising all of the major phylogenetic lineages (see Table
S2 in the supplemental material). The collection comprises 14 dif-
TABLE 1 LZD susceptibility test and rplC sequencingdresults of in
vitro-selected mutants and resistant clinical isolates
(?g/ml)rplC L3 protein
In vitro-selected mutants
9679/00 1.1. 2
Resistant clinical isolates
aBACTEC MGIT 960 (critical concentration, 1 ?g/ml.). S, susceptible; R, resistant.
bND, not determined.
cParental strain used for in vitro selection of LZD-resistant mutant strains. 9679/00, M.
tuberculosis H37Rv; 6446/02, M. tuberculosis red Euro-American; 4414/06, M.
dThe WT rplD sequence of each strain was determined.
TABLE 2 LZD resistance and rplC sequencing results for follow-up
isolates from three different patients
Patient no. and samplea
rplC gene Protein L3
aThe last two digits after the slash indicate the year of sample collection. The rplD
sequences in all of the strains were analyzed. All of the strains collected from patients 1
and 2 showed the WT rplD sequence. Follow-up isolates taken from patient 3 all
displayed a synonymous SNP (Asn214Asn [AAC/AAT]) in rplD, irrespective of
resistance or susceptibility to LZD.
bBACTEC MGIT 960 (critical concentration, 1 ?g/ml). ND, not determined; S,
susceptible; R, resistant.
Beckert et al.
aac.asm.orgAntimicrobial Agents and Chemotherapy
phylogenetic SNP was detected in all of the M. canettii strains Download full-text
the T460C SNP, it is highly unlikely to play a role in LZD resistance
development, as the A459G mutation is a synonymous SNP which
has not been found in any of the strains analyzed and can therefore
tance in M. tuberculosis complex strains. The same mutation in the
LZD-resistant Staphylococcus strains at positions Gly152Asp,
Gly155Arg, Ala157Arg, and Met169Leu, located in the PTC region
and S. aureus illustrates that the rplC T460C mutation (Cys154Arg)
located directly in the ribosome and drug binding region. However,
resistance, the generation of an isogenic mutant strain via homolo-
detected for strains displaying mutations in the 23S rRNA gene (2).
sible for lower-level LZD resistance, whereas mutations in the 23S
rRNA gene might, alone or in combination with rplC mutations,
This study highlights the clinical relevance of the rplC T460C
mutation for LZD resistance, which should be incorporated into
molecular assays for the detection of second-line drug resistance
in M. tuberculosis.
We thank I. Razio, Research Center Borstel, for excellent technical assis-
ics Workbench Software.
TB-PAN-NET (FP7-223681) project.
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rplC Mutation in Linezolid-Resistant M. tuberculosis
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