Journal of Antimicrobial Chemotherapy (2009) 64, 990– 992 doi:10.1093/jac/dkp309 Advance Access publication 27 August 2009
Decreased linezolid uptake in an in vitro-selected linezolid-resistant Staphylococcus epidermidis mutant J. M. Sierra1*, M. Ortega1, C. Tarrago´1, C. Albet1, J. Vila2, J. Terencio1 and A. Guglietta1 1
Pharmaceutical Research and Development Center, Ferrer International S.A., Barcelona, Spain; 2 Department of Microbiology, CDB, Hospital Clı´nic de Barcelona, Spain Received 30 April 2009; returned 15 June 2009; revised 3 August 2009; accepted 3 August 2009
Objectives: The aim of this study was to characterize the mechanism of resistance to linezolid in an in vitro-selected linezolid-resistant Staphylococcus epidermidis mutant. Methods: A linezolid-resistant strain of S. epidermidis was selected by serial passages with increasing concentrations of linezolid. The MICs of linezolid, ciprofloxacin, tetracycline, rifampicin, vancomycin, gentamicin, tobramycin, chloramphenicol and oxacillin were determined. The 23S rRNA gene was amplified and sequenced, to search for mutations conferring linezolid resistance. The MIC of linezolid was also determined in the presence of reserpine. Finally, the accumulation of linezolid was measured and quantified by HPLC/UV. Results: The obtained resistant strain had an MIC of linezolid of 64 mg/L and was stable after several passages on blood agar. The MIC measured in the presence of 25 mg/L reserpine, an efflux pump inhibitor, was not altered (MIC of 64 mg/L). The sequence of the 23S rRNA gene showed that the mutation G2576T (Escherichia coli numbering) was not present and no other mutation was found. An analysis of the accumulation of linezolid was performed, comparing the uptake of the resistant strain with that of the susceptible one. This showed that the resistant strain had significantly lower levels of linezolid accumulation than its susceptible parental strain. Conclusions: The mechanism of resistance to linezolid, in this resistant strain, may be related to a decrease in the antimicrobial uptake. This new mechanism of resistance was also related to a little loss of fitness. Keywords: accumulation, antimicrobial resistance, staphylococci, oxazolidinones
Introduction Currently, linezolid is the only member of a new class of antimicrobials, the oxazolidinones. It has been used in the clinical setting for the last 10 years. Linezolid has been approved for the treatment of skin and respiratory infections caused by Gram-positive pathogens, including methicillin-resistant and -susceptible Staphylococcus aureus, penicillin-susceptible and -resistant Streptococcus pneumoniae, and vancomycinsusceptible and -resistant enterococci, and others, such as Moraxella spp. and Bacteroides spp. The activity of linezolid is being evaluated by two surveillance programmes, ZAAPS and LEADER. In general, the resistance to linezolid remains at a very low rate. However, a high rate of linezolid resistance (2%) has been identified in
Enterococcus faecium. Generally, the appearance of resistance has been associated with prolonged therapy. The most frequent mechanism of resistance to linezolid is related to its mechanism of action. Linezolid inhibits protein synthesis by binding to domain V of 23S rRNA, and there are variations in the number of 23S rRNA genes in different microorganisms, generally between 4 and 7. Modifications and mutations in the binding site of linezolid in the 23S rRNA are widely described.1 It is important to note that this resistance has been associated with a phenomenon called ‘gene dosage’, a relation between the number of 23S rRNA genes mutated and the level of resistance to linezolid.2 In addition, another mechanism of resistance to linezolid mediated by the cfr gene has been described. This gene encodes a methyltransferase that acts in the binding site of linezolid, at position A2503,3 and reduces the
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*Corresponding author. Present address: Infectious Diseases Dptm., Hospital Clı´nic of Barcelona, Villarroel 170, Ed Helios, 08036 Barcelona, Spain. Tel: þ34-93-227-5708; E-mail:
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Decreased linezolid uptake affinity of the antimicrobial agent for the altered target. This mechanism of resistance has been described for one clinical isolate of S. aureus from Colombia and for two clinical isolates (one S. aureus and one Staphylococcus epidermidis) from the USA.3,4 Another mechanism of resistance to linezolid has recently been described by Escribano et al.,5 who suggested the implication of an efflux system in the acquisition of linezolid resistance in Mycobacterium tuberculosis strains. It is known that efflux systems are responsible for intrinsic resistance to linezolid in Gram-negative bacteria.6 The main aim of this study was to characterize the mechanism of resistance to linezolid in an in vitro-selected linezolid-resistant S. epidermidis mutant.
Materials and methods Selection of the resistant S. epidermidis mutant A linezolid-susceptible strain of S. epidermidis (strain 412, presenting an MIC of linezolid of 1 mg/L) was incubated daily in the presence of increasing concentrations of linezolid until the MIC was 64 mg/L. The starting concentration of linezolid was 0.5 MIC. After the resistant strain was obtained (strain 411), 20 passages on blood agar without antibiotic were performed in order to establish the stability of the resistance mechanism.
Susceptibility testing The MICs of ciprofloxacin, tetracycline, rifampicin, vancomycin, gentamicin, tobramycin, chloramphenicol and oxacillin were determined by the CLSI microdilution method. The MIC of linezolid was determined in the absence and presence of reserpine (25 mg/L) in Mueller –Hinton cation-adjusted broth.
The strains were cultured on blood agar plates for 24 h and then an inoculum with an OD600 of 0.10 was prepared in buffered saline. After that, a 1:100 dilution was made in 50 mL of LB medium. Samples were taken each hour for 8 h and were measured at 600 nm. Three independent experiments were performed.
Results and discussion The MIC of linezolid for the wild-type clinical isolate of S. epidermidis was 1 mg/L, whereas the obtained resistant strain had an MIC of linezolid of 64 mg/L and was stable after 20 passages on blood agar without antibiotic pressure, showing the same level of resistance. Furthermore, the MIC of linezolid was determined in the presence and absence of 25 mg/L reserpine, to evaluate the possibility that a reserpine-sensitive efflux pump was related to the acquisition of resistance. The two strains did not show any decrease in the MIC and they presented the same MIC either in the presence or absence of the inhibitor. It is known that linezolid is not a substrate for the efflux systems inhibited by reserpine in Gram-positive bacteria, but efflux systems are the intrinsic mechanism of resistance in Gram-negative bacteria.6 However, Escribano et al. 5 have recently described a reduction in the MIC of linezolid for M. tuberculosis when the MIC was determined in the presence of reserpine, suggesting that the resistance or the decreased susceptibility to linezolid was due to the presence of an efflux pump. Moreover, in the same study, some linezolid-resistant Table 1. MICs (mg/L) for the wild-type and resistant strains Strain Antimicrobial
412 (wild-type)
411 (linezolid resistant)
1 2 0.03 0.25 1 0.06 0.125 8 16
64 2 0.03 0.25 1 0.06 0.125 8 16
Detection of mutation in 23S rRNA The 23S rRNA gene was amplified and sequenced to search for mutations conferring linezolid resistance. Domain V of the rRNA gene was amplified as previously described.7 Restriction fragment length polymorphism (RFLP) was performed with NheI to detect mutations at nucleotide 2576. To find other mutations, the PCR products were sequenced (BigDye kit v3.1; Applied Biosystems, Foster City, CA, USA).
Accumulation of linezolid The accumulation of linezolid was measured and quantified by HPLC/UV as previously described.8 Calibration curves of linezolid were performed from 5 mg/L to 5 mg/L. Briefly, an overnight culture of S. epidermidis was centrifuged and resuspended in PBS plus 0.4% glucose (OD600 ¼ 1.5 and 1.9), and linezolid was added to a final concentration of 20 mg/L before incubating at 378C for 30 min. After that, cells were washed with fresh PBS and the pellet was lysed in glycine buffer ( pH 3), overnight. Finally, the samples were centrifuged and the supernatant was recovered and frozen until use. The experiment was performed twice on duplicate samples.
Linezolid Ciprofloxacin Rifampicin Tetracycline Vancomycin Gentamicin Tobramycin Chloramphenicol Oxacillin
Table 2. Intracellular accumulation of linezolid in wild-type and linezolid-resistant S. epidermidis Intracellular accumulation of linezolid (ng/mL)+SD Strain Wild-type strain 412a Mutant strain 411b
OD600 ¼ 1.5
OD600 ¼ 1.9
632.5+120.9 102+19.8
1012+107.5 281.5+55.8
Growth curves Growth curves were performed in LB medium, with continuous shaking at 378C. Both strains presented the same initial inoculum.
Each value is the mean of at least two independent experiments. a MIC¼1 mg/L. b MIC ¼64 mg/L.
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Sierra et al. M. tuberculosis strains with an unknown mechanism of resistance were also detected. In the same sense, Livermore et al. 9 described a linezolid-resistant Enterococcus faecalis clinical isolate that was resistant to linezolid without a mutation in the 23S rRNA region, for which the mechanism of resistance remained unknown. In order to evaluate possible cross-resistance with other antimicrobials, the susceptibility to ciprofloxacin, tetracycline, rifampicin, vancomycin, gentamicin, tobramycin, chloramphenicol and oxacillin was determined by the microdilution method for both strains. As shown in Table 1, both strains presented the same susceptibility pattern, with the exception of linezolid, suggesting that the selection of linezolid resistance did not select for cross-resistance, at least with the tested antimicrobial agents. The central loop of the V domain of the 23S rRNA gene was amplified and a PCR product of 420 bp was obtained. To analyse the common G2576T mutation, RFLP with the restriction enzyme NheI was performed. In our strains the PCR product remained undigested, showing that the mutation was not present in any of the copies of the 23S rRNA gene. It has been described that mutations other than G2576T are related to the acquisition of linezolid resistance.1 The PCR products of the 23S rRNA of strains 411 (linezolid resistant) and 412 (wild-type) were sequenced and compared, and no other mutation was found in the region analysed. As mentioned above, linezolid-resistant strains without mutations in the 23S rRNA have been described.9 The results of the intracellular accumulation of linezolid are summarized in Table 2. The in vitro-selected linezolid-resistant strain accumulated lower levels of linezolid than the linezolidsusceptible strain. These results suggest that the linezolid-resistant strain had a decreased uptake of linezolid as a mechanism of resistance. This is the first publication describing a decreased uptake of linezolid in Gram-positive bacteria. Finally, growth curves of these strains were performed to establish if this new mechanism of resistance could have any fitness cost. It has been described that resistance to linezolid due to mutations in position G2576T of the 23S rRNA gene reduces the fitness of the bacteria. In addition, when more copies of the 23S rRNA gene are mutated, the bacteria grow slower, indicating a relationship between the fitness cost and the number of copies of the 23S rRNA gene that are altered.10 In our study, the linezolid-resistant S. epidermidis showed a fitness slightly lower than that of the wild-type strain. In summary, the mechanism of resistance to linezolid, in this resistant strain, seems to be related to a decrease in the antimicrobial uptake, due to alterations in the permeability of the bacterial membrane or due to an overexpression of an efflux system not inhibited by reserpine. Further studies are needed to elucidate the specific mechanism of resistance.
Funding J. M. S. was funded by the Torres-Quevedo programme PTQ-04-3-0479 from the Ministry of Education of Spain.
Transparency declarations J. M. S., M. O., C. T., C. A., J. T. and A. G. are (or were) employees of Pharmaceutical Research and Development Center, Ferrer International S.A., but no one owns any stocks or options. J. V.: none to declare.
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