Susceptibility of Gram-positive cocci from 25 UK hospitals to antimicrobial agents including linezolid

C. J. Henwooda,*, D. M. Livermorea, A. P. Johnsona, D. Jamesa, M. Warnera, A. Gardinerb and The Linezolid Study Group

a Antibiotic Resistance Monitoring and Reference Laboratory, Central Public Health Laboratory, Colindale Avenue, London NW9 5HT; b Pharmacia Corporation, Davy Avenue, Knowlhill, Milton Keynes MK5 8PH, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The prevalence of antibiotic resistance amongst Gram-positive cocci from 25 UK hospitals was studied over an 8 month period in 1999. A total of 3770 isolates were tested by the sentinel laboratories using the Etest; these bacteria comprised 1000 pneumococci, 1005 Staphylococcus aureus, 769 coagulase-negative staphylococci (CNS) and 996 enterococci. To ensure quality, 10% of the isolates were retested centrally, as were any found to express unusual resistance patterns. The prevalence of penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant enterococci and methicillin-resistant S. aureus (MRSA) varied widely amongst the sentinel laboratories. The resistance rates to methicillin among S. aureus and CNS were 19.2 and 38.9%, respectively, with MRSA rates in individual sentinel sites ranging from 0 to 43%. No glycopeptide resistance was seen in S. aureus, but 6.5% of CNS isolates were teicoplanin resistant and 0.5% were vancomycin resistant. Vancomycin resistance was much more frequent among Enterococcus faecium (24.1%) than E. faecalis (0.5%) (P < 0.05), with most resistant isolates carrying vanA. The rate of penicillin resistance in pneumococci was 8.9%, and this resistance was predominantly intermediate (7.9%), with only six hospitals reporting isolates with high level resistance. The prevalence of erythromycin resistance among pneumococci was 12.3%, with the majority of resistant isolates having the macrolide efflux mechanism mediated by mefE. All the organisms tested were susceptible to linezolid with MICs in the range 0.12–4 mg/L. The modal MICs of linezolid were 1 mg/L for CNS and pneumococci, and 2 mg/L for S. aureus and enterococci. Linezolid was the most potent agent tested against Gram-positive cocci, including multiresistant strains, and as such may prove a valuable therapeutic option for the management of Gram-positive infections in hospitals.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gram-positive bacteria show increasing resistance to many antibiotics, complicating antimicrobial therapy. Particular problems include methicillin resistance in staphylococci, glycopeptide resistance in enterococci, penicillin and macrolide resistance in pneumococci and, to a much more limited extent, the emergence of glycopeptide-intermediate Staphylococcus aureus. Against this background there is an agreed need for more effective surveillance of resistance. This can be undertaken by gathering routine susceptibility test results generated by hospital laboratories,1,2 by central testing of collected isolates,35 or by ‘sentinel’ laboratories, which test isolates according to a strictly controlled protocol.68 Controlled sentinel surveys have the advantage, compared with the routine collection of data, that the isolates are tested against a standard set of antibiotics and to a defined protocol. Compared with central testing, they minimize transport costs and central workload.

In this study, 25 sentinel laboratories across the UK were selected to determine the prevalence of antibiotic resistance among Gram-positive pathogens. The organisms surveyed comprised S. aureus, coagulase-negative staphylococci (CNS), enterococci and pneumococci. The isolates were tested by each sentinel laboratory against a standard set of reference antibiotics and also linezolid, which is the first oxazolidinone agent to be licensed. Oxazolidinones inhibit the initiation of protein synthesis9 and linezolid shows considerable promise in treating severe infections caused by Gram-positive pathogens.10,11


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study design

Twenty-five sentinel laboratories with a wide geographical distribution in the UK were asked to collect and test consecutive clinically significant isolates (defined by an intention to treat), comprising: 40 pneumococci, 40 enterococci, 30 CNS and 30 S. aureus. Each laboratory was also requested to collect and test an additional 10 methicillin-resistant S. aureus (MRSA) isolates. Duplicate isolates from the same infection episode in the same patient were excluded. The laboratory completed a case record form for each isolate, giving the patient's age, gender, clinical diagnosis and the site of isolation.

Identification of bacteria by sentinel laboratories

Staphylococci were identified by Gram's stain and coagulase reaction; pneumococci on the basis of optochin susceptibility as tested on horse blood agar and enterococci with the API20-STREP or ATB32-STREP system (bioMérieux, La Balme les Grottes, France) or, in the case of one laboratory, using the BBL Crystal GP system (Becton Dickinson, Oxford, UK). CNS were not identified to species level.

Susceptibility testing by sentinel laboratories

Isolates were grown overnight on a non-selective medium and resuspended in 0.9% Mueller–Hinton broth (Oxoid, Basingstoke, UK) to the density of a 0.5 McFarland standard, except for mucoid pneumococci, which were resuspended to the density of a 1.0 McFarland standard. The suspensions were inoculated on to 15 cm diameter plates of Mueller–Hinton agar (Oxoid). This medium was supplemented with 5% lysed horse blood for pneumococci. Oxacillin was tested separately against staphylococci on 9 cm plates with 2% NaCl added to the agar. Etest strips (Cambridge Diagnostic Services, Cambridge, UK) were applied to the plates and the results were read according to the manufacturer's directions. Staphylococci and enterococci were incubated in ambient air at 37°C, whereas pneumococci were incubated in 5% CO2 at 35–37°C. MIC results were read after 24 h incubation. Oxacillin, vancomycin and teicoplanin results for staphylococci were re-read after 48 h, with these latter results taken as definitive.

Quality assurance

Proficiency in testing was established by asking centres to test six reference strains from the Antibiotic Resistance Monitoring and Reference Laboratory (ARMRL) at the start of the study and to include these with each batch of isolates tested. These comprised S. aureus ATCC 29213, Streptococcus pneumoniae ATCC 49619 and Enterococcus faecalis ATCC 29212 together with one multi-resistant isolate of each of these species. The sentinel laboratories were asked to test these strains according to the study protocol and to return the results. The identity of the control strains was not known by the sentinel laboratories. Testing of clinical isolates was commenced only after a centre was obtaining MICs within one dilution of the MIC range designated for the control strains by the ARMRL.

Further quality control (QC) was achieved by the sentinel laboratories' sending every 10th isolate collected to the ARMRL for retesting. Isolates with unusual resistances were also collected for retesting. These comprised any isolates with vancomycin or linezolid MICs > 4 mg/L, any S. aureus with teicoplanin MICs > 4 mg/L, any enterococci identified as a species other than E. faecalis, and any staphylococci where routine and Etest results for oxacillin disagreed.

Re-identification at the ARMRL

Identification of enterococcal species other than E. faecalis was undertaken using published methods.12,13

Susceptibility testing at the ARMRL

MIC determinations at the ARMRL were undertaken using an agar incorporation method, except for trovafloxacin, where the Etest was used. Mueller–Hinton agar (Oxoid) was employed, supplemented with 5% lysed horse blood (Tissue Culture Services, Buckingham, UK) for pneumococci. Inocula of 104 cfu/spot were delivered with a multipoint inoculator. Incubation was as described above for the Etest. MICs were defined as the lowest drug concentrations to prevent growth completely and isolates were defined as sensitive or resistant based on NCCLS criteria.14

All antimicrobial powders for determining MICs were obtained from Sigma (Poole, UK), with the exception of ciprofloxacin (Bayer, Newbury, UK) and linezolid (Pharmacia Corporation, Milton Keynes, UK).

Molecular studies

All enterococci with vancomycin MICs > 4 mg/L were collected by the ARMRL and the presence of vanA and vanB genes was determined by PCR.15 Sentinel laboratories were also asked to send in erythromycin-resistant pneumococci for the detection of the mefE16,17 and ermB18 genes by PCR. An Escherichia coli control strain with the cloned ermB gene was kindly supplied by Dr Marilyn Roberts from the University of Washington (Seattle, WA, USA) and an S. pneumoniae control strain with mefE by Dr Virginia Shortridge from Abbott Laboratories (Abbott Park, IL, USA).

Statistical analyses

Statistical analysis was performed using the chi-squared test with Yates' correction, and with a P value <0.05 indicating significance.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria isolated

The sentinel laboratories collected and tested 3770 isolates. These comprised 1000 S. pneumoniae; 1005 S. aureus; 769 CNS and 996 enterococci. The enterococci comprised 875 E. faecalis, 108 E. faecium and 13 isolates belonging to other species including Enterococcus gallinarum, Enterococcus avium and Enterococcus raffinosus. Among the S. aureus strains, 755 were consecutive isolates and 250 were collected specifically as MRSA. A total of 940 isolates were retested at the ARMRL.

Quality assurance

Two types of QC were built into the study. In the first instance, centres were asked to test six reference strains before commencing the study. These organisms comprised three susceptible ATCC strains and three resistant ARMRL strains. The sentinel laboratories' results fell within the ARMRL QC ranges for 90.5% and 86.7% of tests with the ATCC and ARMRL strains, respectively (Table IGo). Even when a sentinel laboratory's MIC result fell outside the ARMRL MIC ranges, the deviation was commonly only one dilution. There was 98% agreement between the ARMRL and the sentinel laboratories with regard to the susceptibility categorization. Categorization agreement was maintained for S. aureus strain ARU 30300 with gentamicin, penicillin and oxacillin and for S. aureus ATCC 29213 tested with erythromycin, although many sentinel laboratories obtained MIC values outside the ARMRL's control range in these cases. These differences were not confined to a single laboratory (Table IGo). In the case of S. pneumoniae ARU 3489, where the QC ranges spanned the intermediate/resistant border, both intermediate and resistant categories were accepted as correct.


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Table I. Susceptibility testing of control strains by 25 sentinel laboratories
 
As a second validation, 10% of all isolates collected were retested centrally. For pneumococci (n = 101), no difference in susceptibility categories was seen for any retested bacterium, except for one isolate found resistant to trovafloxacin by the source laboratory but deemed susceptible on retesting. Testing for S. aureus likewise was generally accurate, with only two S. aureus isolates identified incorrectly as MRSA by the sentinel laboratories. For CNS, however, MICs of oxacillin determined at the ARMRL were often two or more dilutions higher than the Etest results obtained by the sentinel laboratories. This did not change resistance categorization relative to the NCCLS breakpoint14 (MIC > 0.25 mg/L) but would have resulted in under-reporting of oxacillin resistance with the BSAC breakpoint (>2 mg/L). Most (95%) isolates referred as E. faecalis were found to have been identified correctly when retested by PCR at the ARMRL. All non-E. faecalis enterococci were sought by the ARMRL, since reports19 led us to anticipate problems with their identification using API20-STREP strips. Of the 124 isolates received as ‘non-E. faecalis', 28 (22.7%) were found to have been identified incorrectly. Among 106 isolates received as E. faecium, six were re-identified as E. faecalis, E. gallinarum or E. raffinosus whereas 100 were confirmed as E. faecium. Of 15 isolates received as E. gallinarum, 10 were E. faecium.

Isolates received by the ARMRL as unusually resistant

Sentinel laboratories were also asked to refer in all isolates with atypical resistances. Two MRSA isolates were reported initially as resistant to vancomycin with MICs of 8 mg/L, but were found susceptible on retesting (MIC 4 mg/L). Two centres initially reported linezolid resistance in enterococci (a total of eight isolates) and S. aureus (a total of 12 isolates). On retesting at the ARMRL, the linezolid MIC for all these 20 isolates was 4 mg/L. All enterococci (n = 38) received as vancomycin resistant were confirmed as such.

Prevalence of resistance

S. aureus.
A total of 755 S. aureus isolates were tested by the sentinel laboratories as consecutive isolates, and a further 250 as confirmed MRSA. Skin and wound infections were the most frequent sources (63.2%), followed by respiratory tract infections (10.2%) and blood (7.6%). The prevalence of MRSA amongst the consecutive isolates was 19.2%, but this rose to 29% (114/392) if out-patient isolates were excluded. MRSA rates for individual hospitals varied from 0 to 43%. Most methicillin-susceptible S. aureus (MSSA) isolates were resistant to penicillin (84.2%), but only 8.2% were resistant to erythromycin and 3.4% to ciprofloxacin. In contrast, 87.9% of MRSA isolates were resistant to erythromycin and 94.7% were resistant to ciprofloxacin. Resistance to gentamicin was also more prevalent among MRSA than MSSA but remained infrequent (8.8%).

No glycopeptide-intermediate S. aureus isolates were reported, but vancomycin MICs for 5% of MRSA isolates were on the breakpoint at 4 mg/L. Linezolid MIC ranges were similar for MSSA and MRSA, with most MIC values between 0.5 and 4 mg/L, although one MSSA (not retested at the ARMRL) was reported with a linezolid MIC of 0.064 mg/L. Modal MICs of linezolid for MRSA and MSSA were both 2 mg/L. The MIC90 for MRSA was 4 mg/L, compared with 2 mg/L for MSSA. This difference, however, reflected only a slightly higher proportion of MRSA for which the MIC was 4 mg/L and the MIC95 values for both MRSA and MSSA were 4 mg/L.

Coagulase-negative staphylococci.
MIC distributions and resistance rates for CNS are shown in Table IIGo. Isolates from blood or line infections accounted for 52.7% of the total infections. Based on the NCCLS breakpoint of 0.25 mg/L, 68.9% of the CNS were oxacillin resistant, whereas only 39% were resistant with respect to the BSAC breakpoint of 2 mg/L.


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Table II. MIC distributions for Staphylococcus aureus and coagulase-negative staphylococci
 
Teicoplanin-resistant CNS were collected at 10 hospitals, with local prevalence rates ranging up to 26%. These isolates were not examined to determine whether the phenomenon was present in a single species. Four isolates from different hospitals also showed reduced vancomycin susceptibility, with MICs of 8 mg/L. Resistance to ciprofloxacin, erythromycin and gentamicin was frequent among CNS, with prevalence rates exceeding 25%. All isolates were susceptible to linezolid, with MICs from 0.5 to 4 mg/L and a modal value of 1 mg/L.

Enterococci.
Resistance rates and MIC distributions for the enterococci are shown in Table IIIGo. E. faecalis and E. faecium accounted for 87.9 and 10.8% of the enterococcal isolates, respectively, after re-identification at the ARMRL. E. faecalis isolates were mostly from urine samples (70.5%), and were divided equally between those from hospital patients and those from community patients. By contrast, virtually all the E. faecium isolates (99/108) were from hospitalized patients and many (36%) were from blood. About 91% of the confirmed E. faecium isolates were resistant to ampicillin (MIC > 8 mg/L), as were single isolates of E. raffinosus, E. avium and E. gallinarum. Eight E. faecium isolates were sensitive to ampicillin, with MICs <= 8 mg/L as confirmed at the ARMRL. One of these was vancomycin resistant.


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Table III. MIC distribution for Enterococcus faecalis and Enterococcus faecium
 
Thirty-eight vancomycin-resistant enterococci (VRE) were received from 15 hospitals. Many (31%) were E. faecium from blood and only four were E. faecalis, three of them from urine samples from hospitalized patients and one from a burn patient in an intensive care unit. High-level gentamicin resistance (MIC > 500 mg/L) was also more prevalent in E. faecium (38.9%) than in E. faecalis (24.2%; P < 0.05, {chi}2 test). No resistance to linezolid was found in any of the enterococci, and MICs ranged from 1 to 4 mg/L for most isolates.

PCR-based identification of Van determinants was undertaken on all 38 VRE. Twenty-six, including the four resistant E. faecalis isolates, carried the vanA gene. These characteristically had high-level resistance to vancomycin (MIC >= 256 mg/L). The vanB gene was present in five isolates and was characteristically associated with lower-level vancomycin resistance (MIC <= 64 mg/L) and susceptibility to teicoplanin (MIC <= 4 mg/L). The seven remaining VRE were E. gallinarum isolates with low-level resistance to vancomycin (MICs 8–16 mg/L) but were susceptible to teicoplanin. This behaviour corresponds to the VanC phenotype, which is characteristic of this species. These isolates did not give PCR products with primers for vanA or vanB.

Pneumococci.
A total of 1000 S. pneumoniae isolates were tested by the sentinel laboratories, but two duplicates were identified from the case record forms received and were excluded. Pneumococci were most often isolated from the respiratory tract (53%), eyes (16.5%), blood (14.7%) and ears (10.8%). Overall, 8.9% of the pneumococcal isolates were resistant to penicillin. Most resistance was intermediate (MIC 0.12–1 mg/L), but 1% of the isolates had high-level resistance (MIC >= 2 mg/L). Resistance rates to cefotaxime (MIC > 0.5 mg/L) and erythromycin (MIC > 0.5 mg/L) were 2.0 and 12.3%, respectively. No resistance to linezolid or vancomycin was found and linezolid was active over a narrow range of MICs (0.125–4 mg/L), with a mode of 1 mg/L.

To analyse the susceptibility profiles of pneumococci more fully, the isolates were categorized according to their resistance to penicillin (Table IVGo). Of the 911 isolates susceptible to penicillin, 10.5% were resistant to erythromycin and none was resistant to any other drug tested. Of 78 isolates with intermediate resistance to penicillin, 15.4% had intermediate resistance to cefotaxime (MIC > 0.5 mg/L) and 29.8% were resistant to erythromycin. One penicillin-intermediate isolate from a respiratory tract infection in a community patient was resistant to trovafloxacin, with an MIC of 4 mg/L. The nine isolates resistant to penicillin were all resistant or intermediately resistant to cefotaxime (MIC > 0.5 mg/L), and five were resistant to erythromycin. These nine isolates were received from six hospitals and were isolated from sputa (six), eyes (two) and an ear (one). No highly resistant pneumococci came from blood, but 8.2% (12/147) of the bacteraemia isolates had intermediate resistance to penicillin and one had intermediate resistance to cefotaxime.


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Table IV. Activity of antimicrobials against S. pneumoniae
 
Of the 124 pneumococci with erythromycin MICs >= 0.5 mg/L, only 64 isolates were available for testing for the presence of ermB and mefE. Of these, 42 (66%) had mefE, 21 (33%) had the ermB gene and one had both. All the isolates with ermB were resistant to clindamycin (MICs 4–>8 mg/L, results not shown), whereas those with mefE only were susceptible (MICs <= 0.25 mg/L).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Studies of consecutive isolates are a valuable tool for monitoring antibiotic resistance. In this study, sentinel laboratories tested specific antibiotics against specific bacteria to an agreed protocol and the results were pooled. This strategy requires a tight quality assurance system and to achieve this, resistant and susceptible QC strains were sent to each sentinel laboratory. Only 90.5% of the sentinel laboratories' MICs for the ATCC strains, and 86.7% of those for the multi-resistant strains, fell within the ARMRL's MIC ranges. Nevertheless, deviations were mostly small and there was excellent (>98%) agreement with respect to categorization of susceptibilities. For the isolates referred to the ARMRL as part of the second QC validation, there was generally good agreement between the ARMRL and sentinel laboratory categorization, but some problems were encountered in the identification of non-E. faecalis enterococci by the sentinel laboratories. Specifically, E. gallinarum was often falsely identified as E. faecium. Others19 have reported the same problem, which was overcome here by central re-identification of all non-faecalis enterococci.

The overall rate of resistance to methicillin among S. aureus isolates was 19.2%, but rose to 29% among isolates from hospitalized patients and 28% among isolates from bacteraemias. Routine laboratory reports for blood culture isolates in England and Wales, as sent to the Public Health Laboratory Service (PHLS),2 show the prevalence of methicillin resistance among S. aureus, increasing from 1.7% in 1990 to 34% in 1998. The latter figure is in good agreement with the present study, allowing that only 60 S. aureus bacteraemias were represented here. The MRSA prevalence rate in the UK remains lower than some southern European countries but is much higher than in Scandinavian countries.20

The prevalence of methicillin resistance amongst S. aureus varied between the sentinel hospitals: six hospitals had methicillin resistance rates <5%, whereas others had prevalence rates as high as 43%. Analogous surveys by Andrews et al.4,5 have revealed similar variation, and have also shown that rates in individual hospitals can vary widely between two consecutive years.

Higher resistance rates to other antibiotics were seen amongst MRSA than amongst MSSA. Specifically, most MRSA isolates were resistant to ciprofloxacin and erythromycin, though resistance to gentamicin remained rare (8.8% amongst MRSA). This profile is typical of the epidemic MRSA (EMRSA) 15 and 16 types currently prevalent in the UK.21 None of the S. aureus isolates was resistant to teicoplanin or vancomycin but MICs of these glycopeptides were on the breakpoint of 4 mg/L for c. 5% of the isolates. All the S. aureus isolates were susceptible to linezolid with MICs <= 4 mg/L.

The resistance rate to oxacillin among CNS was 68.9% based on the new NCCLS breakpoint14 (>0.25 mg/L) but this would decrease to 38.9% based upon the BSAC breakpoint (2 mg/L). PCR examination of CNS isolates with oxacillin MICs of 0.5–2 mg/L showed these isolates to lack mecA (results not shown), supporting the BSAC breakpoint.

E. faecalis was the predominant enterococcal species, with most isolates coming from urine specimens (70.5%), collected in almost equal proportions from hospital and community patients. On the other hand, the E. faecium isolates were predominantly from hospitalized patients, with 36% of the isolates from bacteraemias. Vancomycin resistance was seen mostly in E. faecium (24.1%) and was much rarer in E. faecalis (0.5%). VanA was the most prevalent phenotype. The prevalence rate for vancomycin resistance in E. faecium agreed well with data for bacteraemias in England and Wales in 1998 (24%), as reported to the PHLS,2 but the prevalence rate for this resistance in E. faecalis was lower. This discrepancy perhaps reflects the high proportion of isolates from urine samples from community patients in the present collection and the fact that 15–20% of ‘E. faecalis’ bacteraemias reported to the PHLS involve ampicillin-resistant organisms more likely to be E. faecium.2 The overall rates of VRE in this survey were similar to those reported by Andrews et al.5 in a 1997–1998 UK survey.

The resistance rate to penicillin amongst pneumococci was 8.9%. This shows an increase in resistance from that found in previous multi-centre PHLS surveys,3 which indicated rates of 1.5% in 1990 and 3.9% in 1995. Penicillin resistance was mostly intermediate level, with considerable variation in prevalence between hospitals. Past surveys in the UK have shown similar variations in resistance rates between regions,22 but an upward trend in intermediate resistance is apparent at many hospitals.5 In contrast to the USA,23,24 differences in penicillin resistance rates were not observed in relation to age groups or isolation sites. Resistance to erythromycin appears to have stabilized at around 8–13%, with no significant differences to the rates found in the 1995 UK survey3 (P > 0.05). This conclusion is also supported by the routine data for pneumococci from bacteraemia, as reported to the PHLS.2 Investigation of mechanisms of macrolide resistance amongst the S. pneumoniae isolates by PCR suggested that mefE was more prevalent than ermB, but the sample size was small.

Linezolid is being developed to treat serious infections caused by Gram-positive bacteria, including those resistant to the antibiotics currently available. Previous studies have shown linezolid to have promising laboratory2526 and clinical1011 activity. Its in vitro activity was confirmed here for UK isolates. Linezolid was equally active against methicillin-susceptible and -resistant staphylococci, vancomycin-susceptible and -resistant enterococci and against all pneumococci, irrespective of their penicillin or macrolide resistance. For each group of bacteria, linezolid MICs spanned a very narrow range of dilutions, with MICs never exceeding 4 mg/L. Together with promising clinical results,27 the present observations suggest that linezolid will become a valuable agent for the treatment of serious infections caused by Gram-positive cocci.


    Acknowledgments
 
We would like to thank all the staff who helped at the sentinel laboratories, also Terri Parsons and Luke Tysall from the ARMRL for technical assistance, and Pharmacia Corporation for financial assistance. We would also like to thank colleagues in the Laboratory of Hospital Infection for identifying the enterococci, Marilyn Roberts from Washington University for the control strain for ermB and Virginia Shortridge from Abbott Laboratories for the mefE control strain. Linezolid Study Group: I. Gould, K. Milne (Aberdeen Royal Infirmary, Aberdeen), N. Kirk, S. Baillie (Ashford Hospital, Ashford), A. M. Walker, K. T. Dunkin (Bangor Hospital, Bangor), J. Watts, G. Wilson (Royal United Hospital, Bath), J. Paul, L. McCormick (Royal Sussex County Hospital, Brighton), R. C. Spencer, U. NiRiain (Bristol Royal Infirmary, Bristol), D. F. J. Brown, E. Keuleyan (Addenbrooke's Hospital, Cambridge), A. Paull, I. Hosein (University Hospital of Wales, Cardiff), P. T. Mannion, S. B. Fraser (Countess of Chester Hospital, Chester), I. Thangkhiew, G. Ackland (Coventry and Warwickshire Hospital, Coventry), D. Bullock, S. Brown (Derbyshire Royal Infirmary, Derby), P. Chadwick, J. Elliott (Hope Hospital, Salford), M. Wilcox, A. Secker (Leeds General Infirmary, Leeds), B. Oppenheim, D. Weston (Withington Hospital, Manchester), E. McKay-Ferguson{dagger} (South Cleveland Hospital, Middlesbrough), B. Das, C. Jones (Milton Keynes General Hospital, Milton Keynes), D. Crook, D. Griffiths (John Radcliffe Hospital, Oxford), D. A. B. Dance, S. Marshall (Derriford Hospital, Plymouth), M. Dryden (Royal Hampshire County Hospital), A. Sefton, M. Yuan (Royal London Hospital, London), A. Bint, C. Marshall (Royal Victoria Infirmary, Newcastle upon Tyne), R. Warren, K. Howells (Royal Shrewsbury Hospital, Shrewsbury), A. MacGowan, K. Bowker (Southmead Hospital, Bristol), A. Tuck (Southampton General Hospital, Southampton), A. Anderson, L. Smith (York District Hospital, York).


    Notes
 
* Corresponding author. Tel: +44-20-8200-4400, ext. 4282; Fax: +44-20-8358-3292; E-mail: chenwood{at}phls.nhs.uk Back

{dagger} Deceased; this paper is dedicated to his memory. Back


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 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 19 April 2000; returned 2 July 2000; revised 21 July 2000; accepted 15 August 2000