Journal of Antimicrobial Chemotherapy Advance Access published August 10, 2006
Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkl338
In vitro activity of linezolid against clinical isolates of Fusobacterium spp. G. Daeschlein1, C. Hoehne1, O. Assadian2*, F. Daxboeck2, C. Meinl2, A. Kramer3 and A. S. Kekule´1 1
Institute for Medical Microbiology, Martin-Luther-University Halle-Wittenberg, Halle/Saale, Germany; 2 Department for Hygiene and Medical Microbiology, Medical University of Vienna, Vienna, Austria; 3 Institute for Hygiene and Environmental Medicine, Ernst-Moritz-Arndt University, Greifswald, Germany; Received 26 May 2006; returned 6 July 2006; revised 8 July 2006; accepted 26 July 2006;
Objectives: Although most susceptibility studies for linezolid have investigated aerobic bacteria, only a few have investigated anaerobe isolates. The aim of the present study was to determine the antibacterial activity of linezolid against a larger sample of clinical isolates of Fusobacterium spp. and to report on the detailed susceptibility, stratified by species. Methods: The in vitro susceptibility of 80 clinical isolates of Fusobacterium (Fusobacterium necrophorum, n = 34; Fusobacterium nucleatum, n = 20; Fusobacterium varium, n = 18; Fusobacterium mortiferum; n = 8) was tested and compared with the activity of the older compounds amoxicillin and amoxicillin/clavulanic acid. Results: The MIC of linezolid ranged from 0.016 to 1.0 mg/L, with the MIC90 being 0.5 mg/L. The highest MIC obtained for linezolid (1.0 mg/L) was measured for an F. varium isolate. The MIC90 for both, amoxicillin (range: 0.016–0.75 mg/L) and amoxicillin/clavulanic acid (range: 0.047–0.75 mg/L), was 0.5 mg/L. Overall, no resistant strains were found in the study. Conclusions: Compared with amoxicillin and amoxicillin/clavulanic acid, linezolid was less active against F. necrophorum (MIC90 0.25 mg/L) and F. nucleatum (MIC90 0.25 mg/L), equally active against F. varium (MIC90 0.75 mg/L) and slightly more active against F. mortiferum (MIC90 0.19 mg/L). Keywords: anaerobic bacteria, susceptibility, MIC, resistance, amoxicillin, amoxicillin/clavulanic acid
Introduction Linezolid is an oxazolidinone-class antibacterial agent which inhibits bacterial protein synthesis by specifically binding to the 50S ribosomal subunit. Although other antibacterial agents including chloramphenicol, fusidic acid, lincosamides, macrolides, streptogramins and tetracyclines act by inhibiting protein synthesis, linezolid’s specific binding to the 50S ribosomal subunit makes quick cross-resistance with existing antibacterial compounds unlikely.1–4 Linezolid is not active (MIC > 64.0 mg/L) against Pseudomonas spp., Acinetobacter spp. or Enterobacteriaceae, including Escherichia coli, Klebsiella pneumoniae and Proteus penneri. However, linezolid has good in vitro activity against a wide variety of Gram-positive aerobic bacteria, in particular against
methicillin-resistant staphylococci, including methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae and vancomycin-resistant enterococci (VRE), including Enterococcus faecalis and Enterococcus faecium. In addition, it shows activity against Nocardia spp. and good activity against Streptococcus pyogenes, Bacillus spp., Corynebacterium spp., Listeria monocytogenes, Mycobacterium tuberculosis and Rhodococcus spp.5,6 Linezolid is less active against Gram-negative aerobic bacteria and lacks significant in vitro effects against most Gramnegative pathogens but shows moderate in vitro activity (MIC 4.0–8.0 mg/L) against Moraxella catarrhalis, Haemophilus influenzae, Legionella spp. and Bordetella pertussis or Bordetella parapertussis. Linezolid also exhibits MICs of 2.0 and 4.0 mg/L against Flavobacterium meningosepticum and Pasteurella multocida, respectively.1,2
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Daeschlein et al. Similar to its activity against aerobic bacteria, linezolid has also shown greater in vitro activity against Gram-positive than Gram-negative anaerobes. Linezolid shows activity (MIC 1.0–2.0 mg/L) against Clostridium difficile and Clostridium perfringens. It also has good activity against Gram-negative anaerobes, including Bacteroides spp. (MIC 2.0–4.0 mg/L) and Prevotella spp. (MIC 1.0–2.0 mg/L).1,2 Although most susceptibility studies for linezolid have investigated aerobic bacteria, only a few have investigated anaerobe isolates. For fusobacteria, there are even fewer published reports.7–14 So far, the susceptibility of only a total of 129 fusobacteria has been published in eight papers, with the most frequent species being Fusobacterium nucleatum (33%, n = 42). With one exception, linezolid was effective against all strains of fusobacteria with MIC90 values ranging from 1.0 to 2.0 mg/L. The aim of the present study was to determine the antibacterial activity of linezolid against a larger sample of clinical isolates of Fusobacterium spp. and to report on the detailed susceptibility, stratified by species.
Materials and methods A total of 80 clinical isolates of Fusobacterium spp. were investigated (Fusobacterium necrophorum, n = 34; F. nucleatum, n = 20; Fusobacterium varium, n = 18; Fusobacterium mortiferum, n = 8). All isolates were obtained from patients with clinical infections caused by Fusobacterium and cultured at the Institute for Medical Microbiology, Martin-Luther-University Halle-Wittenberg, Halle/ Saale. All strains were identified by standard criteria.15–18 Three American Type Culture Collection (ATCC) strains (Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741 and C. perfringens ATCC 13124) were included for quality control and assessment of reproducibility to each batch of test. Pre-produced supplemented brucella-agar (Oxoid, Hampshire, UK) containing haemin, vitamin K1 and 5% laked sheep blood was used as test medium. An inoculum was stirred into 4 mL of sterile NaCl until an optical density equivalent to that of a 0.5 McFarland standard was reached. From this suspension, 50 mL was plated on brucella-agar. As controls, the same procedure was performed using reference strains Bacillus fragilis (ATCC 25285) and Bacteroides thetaiotaomicron (ATCC 29741). MIC values were determined by Etest (AB Biodisk, Stockholm) according to the manufacturer’s recommendations. All MIC results were recorded after 48 h of incubation at 35–36 C in an anaerobic environment. The MIC of a test antibiotic for an organism was defined as the lowest concentration of an antimicrobial agent yielding no growth. Interpretation of linezolid susceptibility testing was performed by use of European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations for non-species-related breakpoints (susceptible: 4.0 mg/L; resistant: >4.0 mg/L).19 Susceptibility breakpoints for amoxicillin and amoxicillin/clavulanic acid were interpreted according to Deutsche Industrie Norm (DIN 58940-4) recommendations (susceptible 2.0 mg/L; intermediate >2.0 – 8.0 mg/L; resistant > 8.0 mg/L).20
Results Distribution of the MIC, MIC50 and MIC90 values of each antibiotic for all Fusobacterium spp. isolates (n = 80) are listed in Table 1. The MIC of linezolid ranged from 0.016 to 1.0 mg/L,
Table 1. Cumulative susceptibility of 80 Fusobacterium spp. strains to linezolid, amoxicillin and amoxicillin/clavulanic acid MIC (mg/L) Antibiotics Linezolid Amoxicillin Amoxicillin/clavulanic acid
range
MIC50
MIC90
0.016–1.0 0.016–0.75 0.047–0.75
0.125 0.032 0.032
0.5 0.5 0.5
with the MIC90 being 0.5 mg/L. The highest MIC obtained for linezolid (1.0 mg/L) was measured for an F. varium isolate. The MIC90 for both amoxicillin (range: 0.016 to 0.75 mg/L) and amoxicillin/clavulanic acid (range: 0.047 to 0.75 mg/L), was 0.5 mg/L. Overall, no resistant strains were found in the study. For F. necrophorum, F. nucleatum and F. mortiferum, the MIC50s for amoxicillin and amoxicillin/clavulanic acid were lower than the MIC50s obtained for linezolid. However, for F. varium, the MIC50s for all three antibiotics were identical (0.5 mg/L). For F. necrophorum and F. nucleatum, the MIC90s of linezolid were higher than the MIC90s of the two other antibiotics tested. For F. varium, the MIC90s of all three antibiotics were identical (0.75 mg/L), and again, the highest of all Fusobacterium species. The lowest MIC90 against F. mortiferum was found for linezolid. Compared with amoxicillin and amoxicillin/clavulanic acid, linezolid was less active against F. necrophorum and F. nucleatum, equally active against F .varium and slightly more active against F. mortiferum (Table 2).
Discussion In humans, predominant pathogenic anaerobes include Bacteroides spp., Peptostreptococcus spp., Gram-positive nonspore-forming bacilli, Clostridium spp. and Fusobacterium spp.21 Members of the Gram-negative anaerobic genus Fusobacterium are important in both the normal flora and infection. They are common inhabitants of the oral cavity, gastrointestinal tract and female genital tract. In dental plaque they may represent as much as 4% of all anaerobic isolates.22 The most frequent isolates in human clinical infection are F. necrophorum, in liver abscesses, and F. nucleatum, in anaerobic pleuropulmonary infections including aspiration pneumonia, necrotizing pneumonia and lung abscess.23 Both can cause chronic sinusitis, brain abscess, osteomyelitis, septic arthritis and intra-abdominal infections. However, neutropenic haematological patients exhibit a higher risk for F. nucleatum infections.24 F. necrophorum is frequently found in patients with necrobacillosis (Lemierre’s disease).25,26 F. mortiferum and F. varium are mainly isolated in patients with intra-abdominal infections.25 Depending on the location of infection, the treatment of Fusobacterium infection entails surgical intervention and the use of appropriate antibiotics. Selection of appropriate antibiotics, however, is not a simple matter, since these infections are often polymicrobial. For many decades, penicillin G and cephalosporins were considered the drug of choice, with clindamycin and chloramphenicol as secondary alternatives. Later, however, a
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In vitro activity of linezolid against Fusobacterium spp. Table 2. Stratified susceptibility of four Fusobacterium species tested against linezolid, amoxicillin and amoxicillin/clavulanic acid MIC (mg/L) Fusobacterium spp.
No. of isolates
F. necrophorum
34
F. nucleatum
20
F. varium
18
F. mortiferum
Antibiotics linezolid amoxicillin amoxicillin/clavulanic linezolid amoxicillin amoxicillin/clavulanic linezolid amoxicillin amoxicillin/clavulanic linezolid amoxicillin amoxicillin/clavulanic
8
acid
acid
acid
acid
range
MIC50
MIC90
0.047–0.25 0.016–0.125 0.016–0.047 0.064–0.38 0.016–0.047 0.016–0.032 0.125–1.0 0.125–0.75 0.125–0.75 0.016–0.25 0.016–0.38 0.016–0.38
0.125 0.032 0.023 0.19 0.023 0.023 0.5 0.5 0.5 0.094 0.064 0.064
0.25 0.047 0.047 0.25 0.047 0.032 0.75 0.75 0.75 0.19 0.38 0.38
Table 3. Studies publishing linezolid susceptibility results against Fusobacterium spp. Linezolid MIC (mg/L) Fusobacterium spp. F. nucleatum Fusobacterium spp. (F. necrophorum) (F. nucleatum) (F. varium) Fusobacterium spp. F. nucleatum Fusobacterium spp. (F. gonidiaformans) (F. necrophorum) (F. russii) (others)a Fusobacterium spp. (F. nucleatum) (F. necrophorum) (F. mortiferum) (F. varium) Fusobacterium spp. (F. nucleatum) (F. mortiferum) (F. necrophorum) (F. varium) (others)b Fusobacterium spp. (F. mortiferum) (F. varium) (others)c Fusobacterium spp.
No. of isolates
range
MIC50
MIC90
1 4 (2) (1) (1) 30 18 10 (1) (1) (6) (2) 24 (8) (4) (6) (6) 21 (13) (2) (4) (1) (1) 15 (6) (5) (4) 6
0.5 0.25–4.0
ND 0.5
ND 0.5
100 100
Zurenko et al. (1996)7 Yagi et al. (1997)8
0.25–8.0 0.03–1.0 0.125–1.0
ND ND ND
8.0 1.0 0.5
ND 100 100
Edlund et al. (1999)9 Goldstein et al. (1999)10
0.25–2.0
ND
2.0
100
Ednie et al. (2002)11
0.06–2.0
0.5
1.0
100
Behra-Miellet et al. (2003)12
0.25–2.0
ND
1.0
100
Citron et al. (2003)13
2.0
ND
2.0
100
Phillips et al. (2003)14
ND, not defined. a Including two isolates of Fusobacterium spp. b Includes one isolate of Fusobacterium spp. c Includes one isolate of Fusobacterium spp. and three isolates of Veillonella spp.
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susceptible (%)
Reference (year)
Daeschlein et al. possible rise in the incidence of b-lactam antibiotic resistant strains was anticipated, which emphasized the need for alternative treatment options in the future.27 Indeed, in 2001, Aldridge et al.28 reported on 22 clinical Fusobacterium spp. isolates of which 2 were resistant to penicillin G and clindamycin, and recently it was reported that significantly higher MIC values were noted in Spanish strains of F. nucleatum for penicillin G and ciprofloxacin.29 Although most antimicrobial agents are still active against Fusobacterium isolates, some authors express concern for what appears to be an increase in resistance to penicillin and clindamycin. Only a few studies have been published on testing of antibacterial activity of linezolid against Fusobacterium.7–14 Within these studies (Table 3), only a few isolates of Fusobacterium were tested, and, because of the low number of isolates, in most papers the Fusobacterium susceptibility was not stratified by species level. With one exception,9 all studies showed 100% susceptibility to linezolid. Edlund et al.9 found a linezolid MIC of 8.0 mg/L testing 30 undefined Fusobacterium species. Four studies tested fusobacteria without stratifying their results to species level. Ednie et al.11 investigated 24 isolates, BehraMiellit et al.12 investigated 21 isolates, Citron et al.13 investigated 15 isolates and Phillips et al.14 investigated 6 Fusobacterium isolates. All reported 100% susceptibility to linezolid, although MIC ranges and MIC90s were consistently similar or higher than in our study (MIC90 1.0–2.0 mg/L as compared with an MIC90 of 0.75 mg/L in our study). A study investigating susceptibility of F. necrophorum, F. nucleatum and F. varium was conducted by Yagi et al.8 The susceptibility of all isolates to linezolid was 100%. However, four strains (F. necrophorum n = 2, F. nucleatum n = 1, F. varium, n = 1) were tested without indicating results by species level; overall, the MIC ranged higher (maximum MIC 4.0 mg/L) compared with the MIC ranges observed among 80 strains in our study (highest MIC: 1.0 mg/L). Yet, in both studies, the MIC90s were identical (0.5 mg/L). In addition to 18 strains of F. nucleatum, Goldstein et al.10 tested 10 strains of different Fusobacterium species (F. necrophorum n = 1, Fusobacterium russii n = 6, Fusobacterium gonidiaformans n = 1, and Fusobacterium spp. n = 2) and found a higher MIC range (0.125–1.0 mg/L) but an identical MIC90 (0.5 mg/L) of linezolid as in the present study. In conclusion, with the exception of one study reporting on a linezolid-resistant organism of a non-defined species,9 our study is in agreement with previously published susceptibility studies indicating 100% susceptibility of fusobacteria to linezolid. However, the present study shows lower MIC ranges and MIC90. In vitro, linezolid was effective against 80 clinical isolates of Fusobacterium: F. necrophorum (n = 34), F. nucleatum (n = 20), F. varium (n = 18) and F. mortiferum (n = 8). The MIC of linezolid against all isolates was below the susceptibility limit of 4.0 mg/L as was proposed by EUCAST.19 These in vitro data, however, do not necessarily predict in vivo efficacy of linezolid. Moreover, according to EUCAST, nonspecies-related breakpoints have been determined mainly on the basis of PK/PD data and are independent of MIC distributions of specific species. They are to be used only for species that have not been given a species-specific breakpoint. Regrettably, the EUCAST document does not explicitly state whether these breakpoints also apply to Fusobacterium spp. or not, although for
Gram-negative anaerobes it is stated that susceptibility testing is not recommended as Gram-negative anaerobes are a poor target for therapy with linezolid. Yet, previous data together with our recent results indicate that this statement may not be fully correct. In view of European breakpoint harmonization efforts this issue should be further discussed. Furthermore, clinical trials on the impact of linezolid on severe anaerobic infections (e.g. soft tissue infections, peritonsillar abscess, pleuropulmonary infections) caused by Fusobacterium as well as possible side effects, i.e. on normal enteric or vaginal flora composition, are needed.
Transparency declarations We have no conflicts of interest to declare in relation to this manuscript. We also declare that no financial or business interests were involved while conducting the study or during preparation of the manuscript.
References 1. Diekema D, Jones R. Oxazolidinones: a review. Drugs 2000; 59: 7–16. 2. Clemett D, Markham A. Linezolid. Drugs 2000; 59: 815–27. 3. Fines M, Leclercq R. Activity of linezolid against Gram-positive cocci possessing genes conferring resistance to protein synthesis inhibitors. J Antimicrob Chemother 2000; 45: 797–802. 4. Fines M, Leclercq R. Influence of mechanisms of resistance to antibiotics that bind to the 50S ribosomal subunit on the activity of linezolid against Gram-positive organisms. In: Programs and Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. C183, Paper 847. 118–8. 5. Perry CM, Jarvis B. Linezolid. A Review of its use in the management of serious Gram-positive infections. Drugs 2001; 61: 525–51. 6. Brauers J, Kresken M, Hafner D et al. Surveillance of linezolid resistance in Germany, 2001–2002. Clin Microbiol Infect 2005; 11: 39–46. 7. Zurenko GE, Yagi BH, Schaadt RD et al. In vitro activities of U-100592 and U-100766, novel oxazolidinone antibacterial agents. Antimicrob Agents Chemother 1996; 40: 839–45. 8. Yagi B, Zurenko G. In vitro activity of Linezolid and Eperezolid, two novel oxazolidinone antimicrobial agents, against anaerobic bacteria. Anaerobe 1997; 3: 301–6. 9. Edlund C, Oh H, Nord CE. In vitro activity of linezolid and eperezolid against anaerobic bacteria. Clin Microbiol Infect 1999; 5: 51–3. 10. Goldstein EJ, Citron DM, Merriam CV. Linezolid activity compared to those of selected macrolides and other agents against aerobic and anaerobic pathogens isolated from soft tissue bite infections in humans. Antimicrob Agents Chemother 1999; 43: 1469–74. 11. Ednie LM, Jacobs MR, Appelbaum PC. Anti-anaerobic activity of AZD2563, a new oxazolidinone, compared with eight other agents. J Antimicrob Chemother 2002; 50: 101–5. 12. Behra-Miellet J, Calvet L, Dubreuil L. Activity of linezolid against anaerobic bacteria. Int J Antimicrob Agents 2003; 22: 28–34. 13. Citron DM, Merriam CV, Tyrrell KL et al. In vitro activities of ramoplanin, teicoplanin, vancomycin, linezolid, bacitracin and four other antimicrobials against intestinal anaerobic bacteria. Antimicrob Agents Chemother 2003; 47: 2334–8. 14. Phillips OA, Rotimi VO, Jamal WY et al. Comparative in vitro activity of PH-027 versus linezolid and other anti-anaerobic antimicrobials against clinical isolates of Clostridium difficile and other anaerobic bacteria. J Chemother 2003; 15: 113–7.
Page 4 of 5
In vitro activity of linezolid against Fusobacterium spp. 15. Holdeman LV, Moore WEC. Anaerobic Laboratory Manual, 4th edn. Blacksburg: Virginia Polytechnic Institute and State University, 1977. 16. Murray PR, Baron EJ, Pfaller MA et al. Manual of Clinical Microbiology, 6th edn. Washington DC: American Society for Microbiology, 1995. 17. Mutters R, Ihm P, Pohl S et al. Reclassification of the genus Pasteurella Trevisan 1887 on the basis of deoxyribonucleic acid homology, with proposals for the new species Pasteurella dagmatis, Pasteurella canis, Pasteurella stomatis, Pasteurella anatis, and Pasteurella langaa. Int J Syst Bacteriol 1985; 35: 309–22. 18. Jousimies-Somer HR, Summanen P, Citron DM et al. Wadsworth—KTL Anaerobic Bacteriology Manual, 6th edn.. Belmont, CA: Star Publishing Co., 2002. 19. European Committee on Antimicrobial Susceptibility Testing (EUCAST). Linezolid breakpoints. EUCAST definitive document E. Def. 4.1. Clin Microbiol Infect 2001; 7: 283–4. 20. Deutsche Industrie Norm (DIN). Empfindlichkeitspru¨fung von mikrobiellen Krankheitserregern gegen Chemotherapeutika—Teil 4: Bewertungsstufen fu¨r die minimale Hemmkonzentration; MHKGrenzwerte von antimikrobiellen Wirkstoffen. DIN 58940-4 Bbl 1; 2004-02. 21. Brook I. Recovery of anaerobic bacteria from clinical specimens in 12 years at two military hospitals. J Clin Microbiol 1988; 26: 1181–8.
22. Gajardo M, Silva N, Gomez L et al. Prevalence of periodontopathic bacteria in aggressive periodontitis patients in a Chilean population. J Periodontol 2005; 76: 289–94. 23. Marina M, Strong CA, Civen R et al. Bacteriology of pleuropulmonary infections: preliminary report. Clin Inf Dis 1993; 16 Suppl 4: 256–62. 24. Landsaat PM, van der Lelie H, Bongaerts G et al. Fusobacterium nucleatum, a new invasive pathogen in neutropenic patients? Scand J Infect Dis 1995; 27: 83–4. 25. Finegold SM, George WL. Anaerobic Infections in Humans. New York: Academic Press Inc., 1989; 793–818. 26. Epaulard O, Brion JP, Stahl JP et al. The changing pattern of fusobacterium infections in humans: recent experience with fusobacterium bacteraemia. Clin Microbiol Infect 2006; 12: 178–81. 27. George WL, Kirby BD, Sutter VL et al. Gram-negative anaerobic bacilli: Their role in infection and patterns of susceptibility to antimicrobial agents. II. Little-known Fusobacterium species and miscellaneous genera. Rev Infect Dis 1981; 3: 599–626. 28. Aldridge KE, Ashcraft D, Cambre K et al. Multicenter survey of the changing in vitro antimicrobial susceptibilities of clinical isolates of Bacteroides fragilis group, Prevotella, Fusobacterium, Porphyromonas, and Peptostreptococcus species. Antimicrob Agents Chemother 2001; 45: 1238–43. 29. van Winkelhoff AJ, Herrera D, Oteo A et al. Antimicrobial profiles of periodontal pathogens isolated from periodontitis patients in The Netherlands and Spain. J Clin Periodontol 2005; 32: 893–8.
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