Linezolid-resistant ST36 methicillin-resistant ...

J Antimicrob Chemother 2010; 65: 442 – 445 doi:10.1093/jac/dkp494 Advance publication 20 January 2010

Linezolid-resistant ST36 methicillin-resistant Staphylococcus aureus associated with prolonged linezolid treatment in two paediatric cystic fibrosis patients Robert L. R. Hill1*, Angela M. Kearns1, James Nash2, Sarah E. North1, Rachel Pike1, Timothy Newson2, Neil Woodford1, Richard Calver2 and David M. Livermore1 1

Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK; 2William Harvey Hospital, Ashford, Kent, UK

*Corresponding author. Antibiotic Resistance Monitoring Reference Laboratory, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK. Tel: þ44-20-8327-7237; Fax: þ44-20-8327-6264; E-mail: [email protected]

Objectives: To describe the emergence of linezolid-resistant methicillin-resistant Staphylococcus aureus (MRSA) of sequence type (ST)36 lineage in two paediatric patients with cystic fibrosis, after long-term low-dose linezolid treatment. Methods: Two paediatric males with cystic fibrosis had sputum samples quantitatively cultured during hospitalization. After the isolation of MRSA from both patients, oral treatment with 300 mg linezolid twice daily was initiated for periods of 1– 2 months separated by up to 6 months. Isolates cultured 9 months after the start of treatment were tested for resistance to linezolid by agar dilution (BSAC). Resistant isolates were examined for 23S rDNA mutations, and typed by phage and macrorestriction with SmaI. Isolates from follow-up sputum samples were obtained until 44 –51 months after treatment with linezolid. Results: Colonization with MRSA was at a density of 106 cfu/mL sputum for both subjects. Initial isolates were susceptible to linezolid, but, 9 months later, isolates from both patients were resistant (MICs .16 mg/L). Both isolates were epidemic MRSA-16 variant A1 (ST36-MRSA-II), which is widespread in UK hospitals. Both isolates were heterozygous for a G2576T mutation in their 23S rDNA genes, but one was resistant to fusidic acid and tetracycline. In follow-up sampling, the younger patient yielded linezolid-resistant EMRSA-16 for a further 42 months, whilst the other lost the linezolid-resistant MRSA and had alternately Pseudomonas aeruginosa or linezolid-susceptible EMRSA-16 variant A1 isolated over 35 further months. Conclusions: Linezolid resistance emerged in two isolates of ST36 MRSA colonizing the lungs of two paediatric cystic fibrosis patients. Subtherapeutic levels of linezolid may have facilitated the selection of resistance. Keywords: subtherapeutic, colonization, follow-up

Introduction Although a methicillin-resistant Staphylococcus aureus (MRSA) epidemic type 16 (EMRSA-16) was successfully eradicated from an adult cystic fibrosis (CF) patient using 600 mg linezolid twice daily,1 it is uncertain as to whether this treatment could be successfully applied to paediatric cases. It is not just that there are insufficient data on the safety and efficacy of linezolid in patients ,18 years old, but there is also controversy as to the correct dosage.2 Indeed, whilst one MRSA pulmonary infection in a child was successfully treated with linezolid,3 another resulted in the emergence of linezolid-resistant MRSA.4 Nevertheless, linezolid has been put forward as a last line for treating paediatric CF patients with lung colonization by MRSA,5

as such colonization may be a contraindication to future lung transplantation. Downward adjustment of the adult dose of linezolid for paediatric use, although seemingly logical, may carry risks in trying to eradicate colonization. In this light, we describe here the emergence of linezolid-resistant EMRSA-16 variant A1 from two paediatric CF patients during long-term treatment with half the adult dose.

Methods Sputum samples were quantitatively cultured from two paediatric patients, one aged ,12 years and the other .12 years, who were hospitalized for reasons unconnected with infection. Cultures revealed MRSA in

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Received 16 July 2009; returned 1 September 2009; revised 13 November 2009; accepted 13 December 2009

JAC

Subtherapeutic linezolid and resistance

Results Before treatment with linezolid was initiated, both subjects were found to be colonized with linezolid-susceptible, oxacillinresistant S. aureus at a density of 106 cfu/mL sputum. Nine months later, isolates of MRSA from both patients were resistant to linezolid, with MICs of 16 mg/L (Table 1). These isolates were also resistant to erythromycin, clindamycin, moxifloxacin, gentamicin and rifampicin, but susceptible to quinupristin/dalfopristin, vancomycin and teicoplanin. One isolate was resistant to fusidic acid and tetracycline, whereas the other was susceptible (Table 1). The two linezolid-resistant isolates had differing phage patterns of 29inh/80inh/75/96 for isolate 367 from the patient aged .12 years and 29inh/52inh/80inh/83C/83A/77/96 for the patient aged ,12 years. However, these phage patterns both conformed to EMRSA-16, which was confirmed by macrorestriction. The PFGE profiles for both linezolid-resistant isolates were identical and were matched to the reference strain for EMRSA-16 variant A17 (Figure 1). Additional analyses showed that both isolates were ST36-MRSA-SCCmecII. Both isolates were heterozygous for the G2576T 23S rDNA mutation, which indicates the presence of G2576U on the corresponding 23S rRNA. Pyrosequencing determined copy numbers of mutated gene copies as 2/4 in the isolate from the older patient and 3/5 in that from the younger one (Table 1). Table 2 details the isolates cultured from sputum immediately before the initiation of linezolid treatment (time 0), at the end of treatment (month 9) and during subsequent follow-up. For the older patient, Pseudomonas aeruginosa was the only sputum isolate 1 month after the treatment period (month 10), but, at 43 months, a linezolid-susceptible EMRSA-16 type A1 was recovered; this, nevertheless, had a G2576T mutation in one 23S rRNA gene copy. Linezolid-resistant EMRSA-16 A1 continued to be isolated from the younger subject through months 9 –51. Except

Table 1. Characteristics of linezolid-resistant isolates Isolates 367a

368b

MIC (mg/L) linezolid oxacillin fusidic acid tetracycline erythromycin clindamycin quinupristin/dalfopristin moxifloxacin gentamicin chloramphenicol rifampicin vancomycin teicoplanin

16 (R) .16 (R) 0.25 (S) 1 (S) .8 (R) .16 (R) 0.5 (S) 8 (R) 256 (R) .8 (R) .2 (R) 2 (S) 2 (S)

16 (R) .16 (R) .8 (R) .8 (R) .8 (R) .16 (R) 0.5 (S) 8 (R) 256 (R) .8 (R) .2 (R) 2 (S) 2 (S)

Mutation of 23S rRNA

G2576U

G2576U

Copies mutated (%) measured rounded up

51 50

64 60

Estimated copies of 23S rRNA genes

4

5

MLST

ST36

ST36

SCCmec

SCCmecII

SCCmecII

Macrorestriction

EMRSA-16 A1 (UK)

EMRSA-16 A1 (UK)

a

Patient .12 years old. Patient ,12 years old. R, resistant; S, susceptible.

b

with regard to linezolid, the antibiograms of these subsequent isolates remained identical to the initial linezolid-resistant isolates shown in Table 1.

Discussion Linezolid resistance emerged in two ST36 MRSA colonizing the cystic lungs of two paediatric patients. Both isolates were EMRSA-16 A1, a common variant of the classical EMRSA-16 strain highly prevalent in the UK. Linezolid binds to domain V of the 23S rRNA9 and resistance has been associated with mutations in the central loop of this domain, usually G2576T, as found here. These mutations remain a rare occurrence in clinical isolates of S. aureus, where at least two of the 4 –6 gene copies normally present need to be altered to confer a resistant phenotype.10 Both patients studied here continued to have EMRSA-16 isolated from their sputum, but only the younger subject was persistently colonized with linezolid-resistant variants. G2576T-mediated linezolid resistance in S. aureus has been found to be unstable, with reversion to susceptibility occurring,

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both patients, who were then prescribed 300 mg of linezolid twice daily for periods of 1– 2 months duration separated by up to 6 months. Isolates from both subjects were cultured 9 months later and at irregular subsequent intervals for up to 44 months for the older patient and up to 51 months for the younger. All isolates of S. aureus were screened for resistance to linezolid by BSAC disc method. The MICs of linezolid, oxacillin, fusidic acid, tetracycline, erythromycin, clindamycin, quinupristin/ dalfopristin, moxifloxacin, gentamicin, chloramphenicol, rifampicin, teicoplanin and vancomycin were then determined for linezolid-resistant isolates by the BSAC agar dilution method. Isolates confirmed as resistant to linezolid were screened for T2504C and G2576T 23S rDNA mutations by sequencing of PCR amplicons,6 these being aligned with the corresponding nucleotide sequences from reference strains of Escherichia coli. Percentage copy numbers of detected mutations in each isolate were estimated by pyrosequencing.6 Phage-typing was performed with the 23 phages of the Basic International Set at 100 routine test dilution, plus the four UK phages 88A, 90, 83C and 932.7 PFGE profiles were obtained by macrorestriction of chromosomal DNA with SmaI, electrophoresed with a run time of 30 h at pulse switch times of 1 –80 s.7 Isolates were subjected to multilocus sequence typing (MLST), and allelic profiles and sequence type (ST) designations were assigned by comparison with characterized strains on the MLST database (www.mlst.net). The staphylococcal cassette chromosome mec (SCCmec) genomic island was also characterized.8

Hill et al.

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NS EMRSA-16a (linezolid resistant) P. aeruginosa EMRSA-16a (linezolid resistant) NS, no sample. Linezolid resistant due to G2576T mutation. b Linezolid susceptible, but G2576T detected. a

P. aeruginosa NS P. aeruginosa NS

EMRSA-16 EMRSA-16

EMRSA-16a (linezolid resistant) EMRSA-16a (linezolid resistant)

EMRSA-16b NS

51 33 10

Patient .12 years Patient ,12 years

Table 2. Time course of isolates from sputum

although often with the retention of one mutated copy of the 23S rRNA.11 This is consistent with the finding of a G2576T mutation in a follow-up linezolid-susceptible isolate of EMRSA-16 from the older subject. Although it was difficult to distinguish recolonization from relapse, as the colonization was with a widespread strain of MRSA, the retention of one copy of mutated 23S rRNA in the isolates from the older patient suggests that the original colonizing isolate persisted, losing resistance over time. It is also possible that, rather than the isolate ‘reverting’ to linezolid susceptibility, there may have been outgrowth of a minority population that had not gained full resistance. Sputum levels of linezolid in adult CF patients 12 h after a 600 mg dose are in the region of 3 mg/L, which is under the current clinical breakpoint of 4 mg/L. In addition, there is faster clearance of linezolid in children ,12 years of age and the dosing interval has to be considered to avoid a lower area under the drug concentration –time curve.2 The pharmacokinetics of linezolid in children aged 12 years is not significantly different from that in adults2 and it may be more appropriate to consider body mass when dose adjusting. The patient aged ,12 years that we studied here may have received a dose that was more subtherapeutic than that received by the older patient, despite having a lower body mass. The clearance of linezolid may also be affected by CF itself and, in any event, the doses used here (300 mg twice daily) are only half those


Figure 1. PFGE profiles of linezolid-resistant MRSA isolates. Lanes 1 and 5, l phage DNA; lane 2, isolate from patient .12 years old (isolate 367); lane 3, isolate from patient ,12 years old (isolate 368); lane 4, reference EMRSA-16 A1.

43

5

9

4

0.0

3

20.5

2

Sampling period (months)

1

Subtherapeutic linezolid and resistance

normally administered to adults (i.e. .18 years old). It should be noted that the doses of linezolid given to our patients was subtherapeutic and did not directly eliminate colonization. We therefore advocate caution in the intermittent use of linezolid in paediatric CF patients without taking into account the adverse pharmacokinetics for this patient group.

Funding

JAC 3 Ferrin M, Zuckerman JB, Meagher A et al. Successful treatment of methicillin-resistant Staphylococcus aureus pulmonary infection with linezolid in a patient with cystic fibrosis. Pediatr Pulmonol 2002; 33: 221–3. 4 Gales AC, Sader HS, Andrade SS et al. Emergence of linezolid-resistant Staphylococcus aureus during treatment of pulmonary infection in a patient with cystic fibrosis. Int J Antimicrob Agents 2006; 27: 300– 2. 5 Royal Brompton & Harefield NHS Trust, UK. Clinical Guidelines for the Care of Children with Cystic Fibrosis. http://www.rbht.nhs.uk/childrenCF/ (May 2009, date last accessed). 6 Woodford N, North SE, Ellington MJ. Detecting mutations that confer oxazolidinone resistance in gram-positive bacteria. Methods Mol Biol 2007; 373: 103–14.

Transparency declarations

7 Murchan S, Aucken HM, O’Neill GL et al. Emergence, spread, and characterisation of phage variants of epidemic methicillin-resistant Staphylococcus aureus 16 in England and Wales. J Clin Microbiol 2004; 42: 5154– 60.

N. W. has received research support and/or honoraria for lectures or conferences from GlaxoSmithKline, Janssen-Cilag, Johnson and Johnson, Pfizer, Sanofi-Aventis and Wyeth. D. M. L. has received research or conference support or lecture honoraria from several companies developing anti-Gram-positive agents (Pfizer, Wyeth, Chiron, Vicuron) and holds shares either directly, or as enduring attorney, in AstraZeneca, Dechra, Eco-Animal Health, GlaxoSmithKline, Pfizer and Schering-Plough. Other authors: none to declare.

8 Oliveira DC, de Lencastre H. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2002; 46: 2155 –61.

References

9 Xiong L, Kloss P, Douthwaite S et al. Oxazolidinone resistance mutations in 23S rRNA of Escherichia coli reveal the central region of domain V as the primary site of drug action. J Bacteriol 2000; 182: 5325– 31.

1 Serisier DJ, Jones G, Carroll M. Eradication of pulmonary methicillin-resistant Staphylococcus aureus (MRSA) in cystic fibrosis with linezolid. J Cystic Fibros 2004; 3: 61.

10 Wilson P, Andrews JA, Charlesworth R et al. Linezolid resistance in clinical isolates of Staphylococcus aureus. J Antimicrob Chemother 2003; 51: 186–8.

2 Jungbluth GL, Abdel-Rahman SM, Hopkins NK. Linezolid pharmacokinetics in pediatric patients: an overview. Pediatr Infect Dis J 2003; 22: S153–7.

11 Meka VG, Gold HS, Cooke A et al. Reversion to susceptibility in a linezolid-resistant clinical isolate of Staphylococcus aureus. J Antimicrob Chemother 2004; 54: 818– 20.

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The HPA Centre for Infections supported this work.