Sequential Linezolid-Resistant Staphylococcus epidermidis Isolates with G2576T Mutation

We report on an analysis of sequential isolates of Staphylococcusepidermidis from cultures of blood obtained from a patient withacute myeloid leukemia while the patient was receiving linezolidtreatment. All 12 isolates had a linezolid MIC of 32 µg/ml.A 420-bp domain V region of the 23S rRNA gene from all isolateswas amplified, and their nucleotide sequences were determined.A G2576T mutation was identified in all isolates. It was estimatedthat 67% of the 23S rRNA genes carried this mutation. This isthe first report of the failure of linezolid treatment for Staphylococcusepidermidis bacteremia associated with a G2576T mutation inan immunocompromised patient.

INTRODUCTION

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INTRODUCTION

Linezolid is an important antibacterial agent with extendedactivity against gram-positive organisms, including resistantorganisms, such as methicillin-resistant Staphylococcus aureus,methicillin-resistant coagulase-negative staphylococci (CoNS),vancomycin-resistant enterococci, and multidrug-resistant Streptococcuspneumoniae. The mechanism of action is the inhibition of proteinsynthesis through binding to the domain V region of the 23SrRNA. Since its introduction in 2000, resistance to linezolidhas been reported in isolates of vancomycin-resistant enterococci,methicillin-resistant staphylococci, and CoNS. Linezolid resistancehas been associated with mutations in the central loop of thedomain V region of the 23S rRNA gene, with the G2576T mutation(the substitution of thymine for guanine at position 2576) mostfrequently reported in Enterococcus faecium, Enterococcus faecalis,and Staphylococcus aureus isolates (4, 6, 8).

Linezolid resistance has been reported to occur in Staphylococcusepidermidis isolates, and recently, the specific gene mutationG2576T was also reported to be associated with linezolid resistancein S. epidermidis (14). Here we report on an analysis of sequentiallinezolid-resistant Staphylococcus epidermidis isolates fromblood cultures in a case of apparent linezolid treatment failure,with identification of the G2576T mutation in 67% of the domainV regions of the 23S rRNA genes.

CASE REPORT

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CASE REPORT

A 66-year-old man with acute myeloid leukemia in second relapsewas admitted with neutropenic fever. He became afebrile whilehe was receiving empirical piperacillin-tazobactam and underwentplacement of a subclavian Hickman catheter for salvage chemotherapy.Six days later he developed a Hickman catheter-associated tunnelinfection, with cultures of blood being positive for viridansgroup streptococcus and Staphylococcus epidermidis. Becauseof a history of intolerance to vancomycin, he was started onlinezolid at 600 mg intravenously every 12 h, and the catheterwas removed. Blood cultures remained positive for Staphylococcusepidermidis for another 7 days. All isolates were found to beresistant to linezolid, with MICs of 32 µg/ml for allisolates by E-test. The patient became afebrile with negativeblood cultures 5 days after the treatment was changed to daptomycin.The possibility of endocarditis was raised. A transthoracicechocardiogram was negative. The patient had never had a documentedinfection with any CoNS isolate, nor had he ever received linezolid.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Bacterial isolates. Twelve sequential Staphylococcus epidermidis isolates were obtainedfrom blood cultures (standard aerobic and anaerobic media; BacT/ALERT3D; bioMerieux, Inc., Durham, NC) from the source patient. Clonalrelationships were determined by pulsed-field gel electrophoresiswith SmaI-macrorestricted genomic DNA.

Antimicrobial susceptibility testing. Susceptibility testing was performed by using a Vitek GNS cardand E-test (AB Biodisk, Solna, Sweden). Broth macrodilutionwas performed as recommended by the CLSI (1a).

16S rRNA gene sequencing. Primers 0008F (5'-AGAGTTTGATCCTGGCTCAG-3') and 0532R (5'-TACCGCGGCTGCTGGCAC-3')(4) were used to amplify the first 500 bp of the 16S rRNA gene.The PCR mixture, which was made up to 50 µl with sterilewater (Sigma), contained 1x GeneAmp PCR buffer, MgCl₂ (2.5 mM),200 µM each deoxynucleoside triphosphate, 500 nM eachprimer, 5 U of AmpliTaq Gold DNA polymerase (Applied Biosystems,Foster City, CA), and 10 µl purified sample DNA (range,50 to 250 ng). PCR conditions were 94°C 12 min and 40 cyclesof 94°C for 60 s, 59°C for 20 s, and 72°C for 20s, with a final extension at 72°C for 5 min. The mixturewas then held at 4°C. The success of amplification was determinedby electrophoresis of the PCR products in a 1% agarose gel andstaining with ethidium bromide. Amplicons of between 500 and550 bp were excised from the agarose gels (1% agarose gel inTris-borate EDTA) and purified with a QIAquick gel extractionkit (QIAGEN Inc., Valencia, CA).

PCR amplification of the domain V region and DNA sequence. The domain V region spanning 2,280 to 2,699 bp (Escherichiacoli numbering) was amplified. The primers used were 5'-GCGGTCGCCTCCTAAAAG-3'(upper primer, corresponding to bases 2280 to 2297 of the S.aureus 23S rRNA gene; GenBank accession no. X68425) and 5'-ATCCCGGTCCTCTCGTACTA-3'(lower primer, consisting of the strand complementary to theupper primer and corresponding to bases 2680 to 2699 of theS. aureus 23S rRNA gene; GenBank accession no. X68425) (6).PCR conditions were 5 min of lysis and denaturation at 94°C;32 cycles of denaturation, annealing, and extension at 94°C(30 s), 55°C (30 s), and 72°C (1 min), respectively;and a final 10-min extension at 72°C. The products were420 bp and were separated by agarose (1%) gel electrophoresis.The PCR products were gel extracted and purified (QIAGEN). Theywere then sequenced by use of the BigDye (version 3.1) sequencingkit and an Applied Biosystems 310 genetic analyzer.

Restriction enzyme digestion. The 420-bp domain V fragment was digested with the NheI restrictionenzyme (Invitrogen) by using the conditions recommended by themanufacturer. After digestion, the DNA fragments were separatedon a 3% agarose gel and stained with ethidium bromide.

Cloning. The PCR-amplified 420-bp domain V fragments from the S. epidermidisstrain (including the wild type and the G2576T mutant) werecloned into the pGEM-T Easy vector, according to the manufacturer'sinstructions (Promega Corp., Madison, WI). The 420-bp domainV fragment was then reisolated from a single transformed bacterialcolony. The fragment was analyzed by NheI restriction enzymedigestion and nucleotide sequencing.

RESULTS

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RESULTS

Twelve CoNS blood culture isolates from the patient were identifiedas Staphylococcus epidermidis with the Vitek GPI card, and theiridentities were confirmed by 16S rRNA gene sequencing. The MICsof linezolid for all isolates were 32 µg/ml (resistant)by E-test, and these were confirmed by the CLSI macrodilutionmethod. Pulsed-field gel electrophoresis analysis indicatedthat all isolates belonged to a single strain (data not shown).

The nucleotide sequence available from GenBank indicates thatthe domain V region of the 23S rRNA gene nucleotide sequencefrom both Staphylococcus aureus (GenBank accession no. NC_007795)and Staphylococcus epidermidis (GenBank accession no. NC_004461)are identical. A 420-bp fragment (Fig. 1A) was amplified fromthe domain V region of the 23S rRNA genes of all isolates byusing the primer set originally designed for Staphylococcusaureus (6). The mutations associated with linezolid resistancereported from most other organisms were associated with thisdomain. Nucleotide sequence analysis of the amplified domainV region identified a G-to-T point mutation at position 2576(E. coli numbering) from all 12 isolates.

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FIG. 1. NheI restriction enzyme digestion of domain V of 23S rRNA gene. (A) Lanes 1 to 12, serial isolates from the patient subjected to PCR amplification of a 420-bp fragment of domain V region of 23S rRNA gene; lane Mw, molecular size marker. The gel was stained with ethidium bromide. (B) Same as panel A, plus NheI restriction enzyme digestion. 420 bp, NheI-resistant fragment; 322 bp and 98 bp, NheI-digested fragments.

The G-to-T point mutation introduces an NheI site into the mutantdomain V region. NheI restriction enzyme digestion of the PCR-amplifieddomain V region generates a 322-bp fragment and a 98-bp fragment.The result shown in Fig. 1 indicated incomplete digestion, evenafter prolonged incubation (overnight) with NheI. This resultsuggested that the PCR-amplified fragments were a mixture offragments with both G2576T mutant and wild-type sequences. Onthe basis of visual observation on an ethidium bromide-stainedagarose gel, we estimated that approximately 50% or a littlemore of the fragment was digested by the NheI restriction enzyme(Fig. 1B).

To confirm our estimation that 50% or a little more of the domainV fragment carries the G2576T mutation, we cloned the PCR-amplifiedfragments (a mixture of G2576T mutant and wild-type sequences)into the pGEM-T Easy Vector and transformed E. coli cells. Thirty-sixtransformed bacterial colonies were analyzed by purifying theplasmid, reisolating the 420-bp fragment, and identifying theG2576T mutation by NheI restriction enzyme digestion and nucleotidesequencing. The result indicated that 67% (24/36) of the clonescarried the G2576T mutation. This result correlates well withour estimation of partial restriction enzyme digestion.

All the isolates were resistant to oxacillin and susceptibleto vancomycin, quinupristin-dalfopristin, and daptomycin.

DISCUSSION

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DISCUSSION

Staphylococcus epidermidis has been documented as a pathogenin many cases of bacteremia; native and prosthetic valve endocarditis;surgical wounds; ophthalmologic infections; and prosthetic joint-,ventricular shunt-, peritoneal dialysis-, and intravascularcatheter-related infections. Linezolid is an important alternativeagent for the treatment of severe infections caused by S. epidermidis.Our report describes the first case of clinical failure of linezolidtreatment due to linezolid-resistant Staphylococcus epidermidisin an immunocompromised patient.

Among a number of domain V mutations associated with linezolidresistance (9, 10, 12), the G2576T mutation is the only onereported from clinical isolates of Enterococcus faecium (9),Staphylococcus aureus (6, 7), and Staphylococcus epidermidis(14). Our report confirms that this mutation is associated withlinezolid resistance in Staphylococcus epidermidis. The domainV regions of the 23S rRNA genes of both S. aureus and S. epidermidisare identical, and we speculate that homologous recombinationbetween the wild-type and mutant genes could be the mechanismsby which this mutation is spreading among genetically similarorganisms. Alternatively, S. epidermidis may also become resistantto linezolid when it generates its own G2576T mutation in oneof the five to six alleles of the domain V region of the 23SrRNA gene and then homologous recombination occurs within thesame cell.

Most organisms carry multiple copies of rRNA genes. Accordingto GenBank data (accession nos. NC_002976 and NC_004461) (2,13), Staphylococcus epidermidis carries five or six copies ofthe 23S rRNA gene. Not all genes from our isolates carried theG2576T mutation. It was estimated, on the basis of the NheIdigestion and cloning data, that approximately 67% of the genescarried this mutation (Fig. 1B); therefore, three of the fivegene copies or four of the six gene copies may carry this mutation.This is different from the case for the linezolid-resistantStaphylococcus aureus isolates characterized by Pillai et al.(7), who reported that all five 23S rRNA genes carried the G2576Tmutation. The MICs for all of our isolates were 32 µg/ml,which is not as high as that reported by Potoski et al. (MIC> 256 µg/ml) (8). We believe that there might be acorrelation between the number of genes carrying the G2576Tmutation and the MIC of linezolid for S. epidermidis, as hasbeen described for enterococci (5) and S. aureus (11). It wouldbe interesting to determine whether most or all of the 23S rRNAgenes in isolates with much higher linezolid MICs (>256 µg/ml)carry the mutation. The important message is that our strainwith a linezolid MIC of 32 µg/ml was capable of causingthe clinical failure of linezolid therapy in an immunocompromisedpatient.

Linezolid was added to our hospital formulary in January 2001,with usage requiring prior approval by an infectious diseasephysician. Despite a significant increase in the rate of consumptionsince its introduction, with the numbers of defined daily dosesof linezolid rising from 0.31 per 100 patient-days in 2001 to0.82 per 100 patient-days in 2005, this was our first case ofa microbiologically documented treatment failure related tolinezolid resistance. We have been performing routine linezolidsusceptibility testing of all gram-positive isolates in ourmicrobiology laboratory since 2004. We detected linezolid resistancein only 1 other CoNS isolate of 1,771 isolates tested throughAugust 2006, resulting in a resistance rate of <0.1%. Thisis in keeping with the low rates from the 2002 and 2003 surveillanceprograms (1).

The previous administration of linezolid has been reported tobe an independent predictor of linezolid resistance in CoNS(8). This was not the case in our patient, who had never receivedlinezolid prior to becoming infected with the resistant strainof Staphylococcus epidermidis. Cases of patients developinginfections with linezolid-resistant CoNS in the absence of priorexposure to linezolid have also been reported (8). The sourceof the resistant strain remains undetermined, but the clonalspread of CoNS has been reported to occur within hospitals (8),and therefore, the possibility of nosocomial transmission frompatients colonized with linezolid-resistant CoNS following linezolidexposure needs to be entertained.

ACKNOWLEDGMENTS

We thank Edward J. Bottone for critical comments on the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pathology, Clinical Microbiology Laboratory, Hackensack University Medical Center, 30 Prospect Ave, Hackensack, NJ 07601. Phone: (201) 996-4854. Fax: (201) 996-2156. E-mail: thong{at}humed.com

Published ahead of print on 1 August 2007.

REFERENCES

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