a Department of Bacteriology, Glasgow Royal Infirmary, 84 Castle Street, Glasgow G4 0SF, UK
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Abstract |
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Introduction |
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Since its discovery a number of studies have been directed towards measurement of the in vitro28 and in vivo9 activity of linezolid. The agent appears to be bacteriostatic toward staphylococci and bactericidal towards streptococci.2 However, linezolid does not show much activity against Gram-negative bacteria or anaerobes, but does show some potential against mycobacteria.2
The primary objective of this study was to generate in vitro susceptibility data for linezolid and comparator agents on a large number of recent bacterial clinical isolates from numerous sites within Europe. The use of common test methods allowed the pooling of data from the multiple sites, in addition to site-to-site comparisons. The secondary objective of this study was to compare linezolid susceptibility of some species obtained using NCCLS broth microdilution methodology and the epsilonometric assay (Etest).
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Materials and methods |
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Each laboratory collected recent isolates of the following species: Staphylococcus aureus, methicillin susceptible (MSSA) (20); S. aureus, methicillin resistant (MRSA) (30); Staphylococcus epidermidis, methicillin susceptible (MSSE) (20); S. epidermidis, methicillin resistant (MRSE) (30); Enterococcus species, vancomycin susceptible (20); Enterococcus species, vancomycin resistant (20); Streptococcus pyogenes (20); Streptococcus agalactiae (20); Streptococcus pneumoniae, penicillin susceptible (20); S. pneumoniae, penicillin intermediate (20); S. pneumoniae, penicillin resistant (20); Haemophilus influenzae (20); Moraxella catarrhalis (20). The enterococci were not speciated in this study.
The following quality control strains were available in each laboratory in order to ensure equivalent inter-laboratory standardization: S. aureus ATCC 29213, E. faecalis ATCC 29212 and S. pneumoniae ATCC 49619.
NCCLS broth microdilution method
MICs were determined using the NCCLS standard broth microdilution method10 using MuellerHinton broth (Oxoid, Basingstoke, UK). For testing of Streptococcus species and H. influenzae, the standard broth medium was supplemented with lysed horse blood, as described. Microplates were incubated without carbon dioxide. The inoculum suspension was standardized (0.5 McFarland reading to 1 x 108 cfu/mL) and diluted 1:10, and 5 µL were inoculated into each well giving a final concentration of 5 x 104 cfu/well.
Etest method
The Etest was conducted according to the manufacturer's instructions (AB Biodisk, Solna, Sweden). Testing of all organisms except streptococci and H. influenzae was conducted with MuellerHinton agar (Oxoid). Tests with streptococci were conducted using MuellerHinton agar supplemented with 5% defibrinated sheep blood. Tests with H. influenzae were conducted using MuellerHinton agar supplemented with 1% haemoglobin and 1% Isovitalex.
Antimicrobial agents
The concentration range of antimicrobial agents included in the NCCLS microdilution MIC tests were: for staphylococci and enterococci: linezolid (640.06 mg/L), vancomycin, teicoplanin, gentamicin, oxacillin, ciprofloxacin, ampicillin, erythromycin (1280.12 mg/L); for streptococci, H. influenzae and M. catarrhalis: linezolid (640.06 mg/L), vancomycin, penicillin, co-amoxiclav 160.06 mg/L, ciprofloxacin, clindamycin and clarithromycin (160.06 mg/L). Linezolid MIC ranges of 14 mg/L were acceptable for S. aureus ATCC 29213 and E. faecalis ATCC 29212 and 0.52 mg/L for S. pneumoniae ATCC 29619.
The antimicrobial agents included in the Etest were: for staphylococci and enterococci, linezolid and vancomycin; for streptococci, linezolid and penicillin G; for H. influenzae and M. catarrhalis, linezolid and co-amoxiclav.
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Results |
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For the enterococci, linezolid MIC50s and MIC90s of 12 mg/L and 24 mg/L, respectively, were obtained for both vancomycin-susceptible and -resistant isolates. Its activity was comparable to vancomycin for vancomycin-susceptible isolates and better activity was shown against vancomycin-resistant isolates as well as teicoplanin-resistant isolates.
For isolates of M. catarrhalis and H. influenzae, linezolid (MIC50 4 and 16 mg/L and MIC90 16 and 32 mg/L, respectively) compared unfavourably with its comparators. Most agents (ciprofloxacin, erythromycin, clindamycin, clarithromycin, penicillin and co-amoxiclav) had lower MIC50s and MIC90s.
There was little inter-laboratory variation with regard to the results obtained for most of the test bacteria except for strains of MSSA in one site in The Netherlands. At this site the linezolid MIC90 was 8 mg/L as compared with the other Dutch centre, which reported an MIC90 of 4 mg/L. Repeat tests in the coordinator's laboratory showed that the Dutch isolates were more susceptible to linezolid (MIC90 2 mg/L) than originally reported by either Dutch laboratory. However, since this study was designed to evaluate the results obtained in different laboratories using the NCCLS method, the original results remained in the database for analysis. The same centre also gave slightly higher linezolid MICs (MIC50 4 mg/L) for vancomycin-susceptible enterococci. It is likely that differences in interpretation of the end point were responsible for the higher MICs at this centre.
Overall the NCCLS broth microdilution assay10 gave MIC90s of linezolid of 24 mg/L for all groups of staphylococci (total of 1328 isolates), which was comparable to those of vancomycin and teicoplanin. Linezolid was equally active against methicillin-susceptible and -resistant strains. A significant level of resistance to gentamicin, ciprofloxacin, erythromycin and ampicillin was demonstrated (Table I). MIC90s of linezolid were 24 mg/L for the enterococci (total of 439 isolates), including both vancomycinsusceptible and -resistant isolates. The activity of linezolid was comparable to that of vancomycin for vancomycinsusceptible isolates (MIC90 2 mg/L) and it retained activity against the vancomycin-resistant strains. A significant level of enterococcal resistance was seen for all of the other agents tested. MIC90s of linezolid were 1.0 mg/L for all S. pneumoniae isolates tested (total of 551 isolates), including penicillin-susceptible, -intermediate and -resistant strains. The activity of linezolid was similar to that of vancomycin for all susceptibility groups (Table I
). With the exception of ciprofloxacin, the MIC90s for the comparator agents increased significantly for the penicillin-intermediate and -resistant strains. The MIC90 of linezolid for 281 isolates of S. agalactiae (2 mg/L) was comparable to that of vancomycin, and significantly less than that of some of the other comparator agents. The linezolid MIC90 for 268 isolates of S. pyogenes was 1 mg/L, which was slightly less active than vancomycin. Overall, linezolid demonstrated MIC90s of
2 mg/L for all of the streptococci tested. Against M. catarrhalis (258 isolates) and H. influenzae (257 isolates), linezolid was less active with MIC90s of 16 and 32 mg/L, respectively. Several of the comparator agents demonstrated excellent activity against these Gram-negative organisms.
Comparison of mean MIC determined by NCCLS microdilution method with mean MIC determined by investigational Etest
The mean MICs of linezolid and certain comparator agents for each organism group at each site determined by these different methods was calculated and is presented in Tables II and III. The vancomycin mean microdilution MIC agreed closely with the mean Etest MIC for staphylococci and enterococci, except for vancomycin-resistant enterococci; in this instance the values were so discrepant (59.69 mg/L versus 192.84 mg/L) that interpretation was difficult. Excellent agreement was also achieved for penicillin with the streptococci, and co-amoxiclav for H. influenzae and M. catarrhalis (Table III
). Mean linezolid Etest values were lower than the mean microdilution MICs for all organism groups. In general, the mean Etest MIC was approximately one two-fold dilution lower than the mean microdilution MIC.
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Discussion |
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The present multicentre study compares favourably with others in North America2,46,11,12 and elsewhere.7,8 Overall there are clear indications that linezolid displays good activity against a range of Gram-positive bacteria (MIC50 range 0.54 mg/L).
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Acknowledgments |
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The participating investigators were: A. Bouvet, Hôpital Hotel Dieu, Paris, France; I. Braveny, Technische Universitaet Muenchen, Munich, Germany; R. I. A. Diepersloot, Diakonessen Hospital, Utrecht, The Netherlands; R. Finch, Nottingham City Hospital, Nottingham, UK; G. French, St Thomas' Hospital, London, UK; B. Halle, Universittätsklinikum Charité Institut fur Medizinische Mikrobiologie, Berlin, Germany; J. Kluytmans, Ignatius Hospital Breda, Breda, The Netherlands; J. Linares, Hospital de Bellvitge, Barcelona, Spain; Q. Noury, Université Bordeaux, Bordeaux, France; M. Rylander, Karolinska Hospital, Stockholm, Sweden; G. Schito, Universita degli Studi di Genova, Genova, Italy; F. Soriano, Fundacíon Jimenez Diaz, Madrid, Spain; and G. Tempera, Universita di Catania, Catania, Italy.
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Notes |
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The participating investigators are listed in the Acknowledgements.
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References |
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Received 12 July 2000; returned 1 March 2001; accepted 19 March 2001