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J Clin Microbiol. Nov 2002; 40(11): 4298–4300.
PMCID: PMC139696

Detection of Oxazolidinone-Resistant Enterococcus faecalis and Enterococcus faecium Strains by Real-Time PCR and PCR-Restriction Fragment Length Polymorphism Analysis

Abstract

A real-time PCR assay identified linezolid-resistant Enterococcus faecalis and Enterococcus faecium isolates with a G2576U rRNA mutation. PCR-restriction fragment length polymorphism analysis of ribosomal DNA amplicons with NheI also detected this mutation. Both assays detected isolates heterozygous at this position. Recognition of isolates with what is presently the most frequent oxazolidinone resistance mutation may aid surveillance and individual case management.

The oxazolidinones are a new antibiotic class that displays excellent activity against gram-positive organisms; linezolid was the first member licensed for clinical use (6, 11), but other analogs, such as AZD2563 (3), are in development. It is difficult to select for resistance in vitro, but mutants may have any of several different point mutations in the gene encoding 23S rRNA, specifically in the central loop of domain V, responsible for peptidyl transferase activity (7). Resistance to linezolid has also been observed in a few clinical isolates of Enterococcus faecium (4; R. D. Gonzales, P. C. Schreckenberger, M. B. Graham, S. Kelkar, K. DenBesten, and J. P. Quinn, Letter, Lancet 357:1179, 2001; G. E. Zurenko, W. M. Todd, B. Hafkin, B. Myers, C. Kaufmann, J. Bock, J. Slightom, and D. Shinabarger, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 848, 1999) and methicillin-resistant Staphylococcus aureus (MRSA) (9), mostly selected during prolonged courses of therapy. These clinical strains consistently have a G→T change at the position equivalent to nucleotide 2576 of the Escherichia coli sequence (GenBank accession no. AF053964), giving a G→U change in the corresponding rRNA (5, 7). This mutation may take a lesser toll on the fitness of the bacterial cell than do other oxazolidinone resistance mutations selected in vitro. Rapid methods for detecting G2576T are therefore desirable to ascertain whether this mutation maintains its dominance; moreover, a facility for rapid confirmation of oxazolidinone resistance will influence the management of cases of serious gram-positive infection, where linezolid is likely to be used. The key involvement of a single mutation in resistance suggests the value of development of a real-time PCR assay (1). Such assays have proven useful for the rapid detection of genes and mutations conferring resistance to other drugs in diverse pathogens (2, 8, 10). The aim of this work was therefore to develop and evaluate such an assay for detecting the G2576T mutation in linezolid-resistant enterococci.

Twenty-seven enterococci were examined, consisting of 16 linezolid-resistant isolates (MIC, ≥8 μg/ml) and 11 linezolid-susceptible isolates (MIC, ≤4 μg/ml) recovered from six hospital patients in Europe, all of whom had received linezolid (Table (Table1)1) for periods ranging from 3 weeks to 3 months. Linezolid-resistant and -susceptible isolates were available from four patients and were shown to be highly related or indistinguishable by pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA (M.E. Kaufmann, personal communication), thereby indicating that linezolid resistance had arisen during therapy. Full details of these cases will be published elsewhere. The novel LightCycler (Roche, Lewes, United Kingdom) assay consisted of (i) primers 5′-GCA TCC TGG GGC TGT AGT C and 5′-GGA CCG AAC TGT CTC ACG AC, designed to amplify a 96-bp fragment of the relevant section of 23S rDNA, and (ii) a fluorescent dye-labeled detection probe, LC Red 640-GAA CCC AGC TCG CGT GCC-P (TIB Molbiol, Berlin, Germany), which spanned position 2576 (the base underlined above). The primers and probe matched conserved sequences of E. faecium and Enterococcus faecalis. The probe matched exactly the sequence present in oxazolidinone-susceptible enterococci but had a single mismatch compared with those possessing the G2576T mutation. Samples were processed using a LightCycler-FastStart DNA Master SYBR Green I kit (Roche), with the hot-start protocol recommended by the supplier. The cycling conditions were as follows: initial denaturation (hot-start) at 95°C (ramp rate, 20°C/s, held for 10 min); 50 cycles of amplification at 92°C (ramp rate, 20°C/s), 50°C (ramp rate, 20°C/s), 55°C (ramp rate, 3°C/s), and 74°C (ramp rate, 20°C/s, held for 10 s; acquisition mode, single). This was followed by a single probe melting cycle: 95°C (ramp rate, 20°C/s), 45°C (ramp rate, 20°C/s), and 95°C (ramp rate, 0.2°C/s) (acquisition mode, continuous).

TABLE 1.
Enterococci used in this study and susceptibilities to linezolid

In addition, a 633-bp fragment of the 23S rDNA was amplified with primers described by Tsiodras et al. (9) and then sequenced on both strands with a dye-labeled dideoxynucleoside triphosphate terminator cycle sequencing Quickstart kit (Beckman Coulter UK Ltd., High Wycombe, United Kingdom) to confirm the presence of the G2576T mutation. The samples were analyzed on a CEQ 2000 automated sequencer (Beckman Coulter UK Ltd.). The G2576T mutation creates a cutting site for the restriction endonuclease NheI, so PCR-RFLP analysis of the 633-bp amplicons was evaluated as an alternative method for detecting this mutation: small amounts (5 μl) of the PCR products were digested with 10 U of NheI (Invitrogen, Paisley, United Kingdom) for 4 h at 37°C, and the fragments were separated by electrophoresis through 2.5% agarose gels.

Fluorescence traces from the LightCycler assay divided isolates into three categories (Fig. (Fig.1):1): 10 of 11 linezolid-susceptible isolates (MIC range, 2 to 4 μg/ml) showed a single peak with a melting temperature (Tm) of ca. 70°C (standard deviation, ±0.5°C), one linezolid-resistant isolate (MIC, 64 μg/ml) showed a single peak with a lower Tm of ca. 61°C, and the remaining 15 linezolid-resistant isolates (MIC range, 8 to 64 μg/ml) and one linezolid-susceptible isolate (MIC, 4 μg/ml) showed both of these peaks. Sequencing indicated that all isolates with the fluorescence peak with a Tm of 61°C contained the G2576T rDNA mutation, thereby confirming that the single mismatch with the detection probe lowered the Tm of the probe-PCR product complex. The occurrence of twin fluorescence peaks for 15 resistant isolates and one susceptible isolate probably reflects the fact that enterococci have several 23S rDNA genes, giving a potential for heterozygosity at position 2576; some isolates have both wild-type (G2576) and mutant (T2576) copies in their genomes. For example, E. faecalis strain V583 has four copies (http://www.tigr.org), while E. faecium strains have five or six copies (http://rrndb.cme.msu.edu/rrndb/servlet/controller). The number of G2576 relative to the number of T2576 copies may influence levels of oxazolidinone resistance, but no simple relationship was apparent, for the highest linezolid MICs (64 μg/ml) were noted not only for the one homozygous (T2576) isolate but also for eight heterozygous isolates.

FIG. 1.
Typical results of a LightCycler assay to detect the G2576T mutation in the 23S rDNA of oxazolidinone-resistant enterococci. Traces shown represent a homozygote with the T2756 mutation (solid line), a homozygote with wild-type G2576 (dashed line), a heterozygote ...

Although the heterozygous genotype was consistently indicated clearly in the LightCycler assay by the twin fluorescence peaks, the T2576 mutation was missed on initial analysis of sequence data for some heterozygous isolates. In these cases, G was the predominant base called by the sequencer, and the T was only detected upon careful reexamination of the sequencing traces. Thus, in our experience, the LightCycler data were less prone to subjective errors of interpretation than were sequencing data.

The results of NheI PCR-RFLP analysis of 633-bp rDNA amplicons (Fig. (Fig.2)2) showed 100% concordance with LightCycler data: amplicons from isolates with only G2576 lacked an NheI cutting site and so remained undigested; amplicons from isolates with only T2576 were cleaved to give products of 591-bp and 42-bp, with the latter running off the gel; heterozygous strains showed both the 633-bp and the 591-bp bands. An amplicon from a homozygous T2576 strain was included in all experiments to ensure that complete digestion had occurred.

FIG. 2.
Results of PCR-RFLP analysis of rDNA amplicons with NheI. Lanes: 1, a homozygote with the T2576 mutation; 2, a 123-bp ladder (Invitrogen); 3 and 4, homozygotes with wild-type G2576; 5 to 9, heterozygotes with both mutant (T2576) and wild-type (G2576) ...

The real-time PCR assay described here identified the G2576T rDNA mutation in oxazolidinone-resistant isolates of E. faecalis and E. faecium in less than 2 h. This mutation is the only mechanism of resistance to oxazolidinones so far identified in clinical isolates of gram-positive bacteria, although other mutations have been identified in laboratory mutants. There was no need for prior identification of enterococci to the species level, which is an essential consideration if the method is to be used in busy clinical laboratories. The PCR primers would also amplify the relevant 23S rDNA sequence from MRSA, although the enterococcal detection probe has a single base mismatch with the sequence of linezolid-susceptible S. aureus (e.g., GenBank accession no. X68425). Hence, with a minor modification to the probe, this assay could be used to detect the G2576T mutation in oxazolidinone-resistant isolates of MRSA, such as the strain described by Tsiodras et al. (9). The NheI PCR-RFLP assay provided a slower means of detecting the G2576T mutation potentially suitable for laboratories without access to sequencing or real-time PCR facilities. Both assays detected qualitatively heterozygous strains with G2576 and T2576 in different rDNA copies, but quantitative analysis requires further development. Both methods will be useful for molecular characterization and surveillance of emergent resistance, and the speed of real-time PCR may also be an advantage in case management for individual patients. However, as with all molecular methods of detecting antibiotic resistance, microbiological confirmation of oxazolidinone susceptibility by disk diffusion testing or MIC determination remains essential for isolates not identified as resistant, so as to exclude the presence of other resistance mechanisms.

Acknowledgments

We are grateful to Pharmacia for providing financial support for this work.

We are grateful to colleagues in clinical laboratories who referred isolates included in this study.

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