JAC Advance Access published online on July 7, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn276
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Original research |
In vitro susceptibility of genotypically distinct and clonal Clostridium difficile strains to oritavancin
1 Department of Microbiology, Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK 2 Department of Microbiology, The General Infirmary, Old Medical School, Leeds LS1 3EX, UK
* Corresponding author. Tel: +44-113-3926818; Fax: +44-113-3435649; E-mail: mark.wilcox{at}leedsth.nhs.uk
Received 16 April 2008; returned 5 June 2008; revised 10 June 2008; accepted 11 June 2008
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Abstract |
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Objectives: Clostridium difficile infection is a nosocomial disease of increasing importance. First-line treatment is limited to metronidazole or vancomycin. Oritavancin is a lipoglycopeptide with activity against Gram-positive bacteria, including drug-resistant pathogens. MICs of oritavancin, metronidazole and vancomycin for genotypically distinct C. difficile strains, including epidemic C. difficile PCR ribotypes 001 and 027, were determined by agar incorporation and broth macrodilution methods. In agar incorporation methods, the impact of supplements on oritavancin MICs was tested to address oritavancin binding to surfaces.
Methods: Thirty-three genotypically distinct C. difficile strains were identified by PCR ribotyping. Wilkins Chalgren agar incorporation plates containing oritavancin, metronidazole and vancomycin were prepared with and without 0.002% polysorbate-80 (P80) and lysed horse blood (2%). Broth macrodilution MICs of oritavancin, metronidazole and vancomycin were determined in Brucella broth. Inoculated agar incorporation plates and broth macrodilution tubes were cultured anaerobically at 37°C for 48 h.
Results: Broth macrodilution MICs were lower than agar incorporation MICs for oritavancin, but not for metronidazole and vancomycin. Oritavancin broth macrodilution MIC90s were 2- to 4-fold lower than the corresponding agar incorporation MIC90s, while geometric mean MICs were >5-fold lower. Oritavancin broth macrodilution MIC90s were 
2- and 5-fold lower than those for metronidazole and vancomycin. Metronidazole was the most active antimicrobial agent against C. difficile using agar incorporation methods. Oritavancin agar incorporation MIC90s were unaffected by 0.002% P80 and/or 2% lysed horse blood.
Conclusions: Oritavancin was at least 4-fold more potent than vancomycin against the majority (25/33) of C. difficile strains tested by broth macrodilution. Oritavancin activity may be underestimated by agar incorporation methods, regardless of the use of P80 or lysed horse blood.
Key Words: C. difficile , susceptibility , oritavancin , metronidazole , vancomycin , ribotyping
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Introduction |
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Clostridium difficile is a major cause of morbidity in the hospitalized elderly and is almost exclusively associated with antimicrobial therapy. C. difficile infection (CDI) may range in severity from mild antibiotic-associated diarrhoea/colitis to life-threatening pseudomembranous colitis.1 Treatment strategies for CDI have changed little over the past two decades: oral metronidazole (400–500 mg three times a day) or vancomycin (125 mg four times a day) is most commonly used to treat CDI.2 Early studies demonstrated little difference between metronidazole and vancomycin in terms of response or recurrence rates,3,4 although response time was faster with the latter.5 More recent reports have questioned the efficacy of metronidazole therapy for CDI,6,7 particularly disease attributable to an apparently hypervirulent C. difficile PCR ribotype 027 (NAP1/BI). These studies have reinforced the need for the evaluation of the activity of new antimicrobial agents against C. difficile.
Oritavancin is a new lipoglycopeptide antimicrobial agent active against Gram-positive bacteria. In this study, we evaluated the activity of oritavancin against 33 genotypically distinct C. difficile using two methods: agar incorporation and broth macrodilution. Previous studies have demonstrated that incorporation of polysorbate-80 (P80) or lysed horse blood significantly reduced oritavancin surface binding and broth microdilution MICs in vitro.8,9 We therefore examined the effects of these factors on the measured MICs for C. difficile.
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Materials and methods |
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Bacterial strains
Thirty-three genotypically distinct (by PCR ribotyping) C. difficile isolates were selected from a library of strains at the Leeds General Infirmary (Leeds, UK). Representative isolates of epidemic C. difficile PCR ribotypes 001, 106 and 027 were included in the panel. Control isolates of Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and Bacteroides fragilis ATCC 25285 were included in all MIC determinations to ensure procedure accuracy.
Agar incorporation MICs were determined based on the method of Freeman and Wilcox.10 Briefly, bacteria were cultured in Schaedler's anaerobic broth (Oxoid, Basingstoke, UK) at 37°C for 24 h in an anaerobic cabinet (Don Whitley Scientific, Shipley). Stock solutions of metronidazole and vancomycin (Sigma-Aldrich Co., Poole, UK) were prepared in de-ionized water. Oritavancin (Targanta Therapeutics, Cambridge, MA, USA) was prepared in 0.002% P80. All antimicrobial solutions were sterilized by filtration through 0.22 µm syringe filters. Oritavancin was filtered at 1280 mg/L as filtration at lower concentrations leads to significant proportional losses of drug owing to saturable binding (G. Moeck and F. Arhin, Targanta Therapeutics, Quebec, Canada—personal communication). Wilkins Chalgren agar (Oxoid) incorporating doubling dilutions of antimicrobial agents (0.03–16 mg/L) was prepared with and without 0.002% P80 and with and without 2% lysed horse blood (E&O Labs, Bonneybridge, UK). All media and diluents were pre-reduced overnight in the anaerobic cabinet. Bacterial cultures were diluted in sterile saline and inoculated onto the surface of agar incorporation agar plates using a multipoint inoculator (104 cfu). Agar incorporation plates were incubated anaerobically for 48 h. MIC endpoints were read as the lowest concentration of antimicrobial agent where there is no apparent growth (disregarding a visible haze of growth or a single colony).
Broth macrodilution MICs were determined following the method of Jousimies-Somer et al.11 Briefly, double-strength antimicrobial stock solutions were prepared in Brucella broth (Sigma) supplemented with haemin (5 mg/L, Sigma), NaHCO3 (1 mg/L, Sigma) and vitamin K1 (10 µL/L, Sigma). All broths for oritavancin MICs contained 0.002% P80.9 Bacterial strains were initially cultured overnight in Schaedler's anaerobic broth and subsequently diluted 1:200 (3x105 cfu/mL) in sterile pre-reduced supplemented Brucella broth. Antimicrobial stock solutions were diluted 1:2, following the addition of each strain inoculum. Broths were incubated anaerobically at 37°C for 48 h. MIC endpoints were read as the concentration of antimicrobial agent where no growth was observed compared with the control. MICs were determined in duplicate for all antimicrobial agents.
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Results and discussion |
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We previously demonstrated marked variations in the activity of antimicrobial agents against genotypically distinct C. difficile PCR ribotypes,10 yet many susceptibility studies fail to genetically distinguish between C. difficile isolates. We therefore evaluated the activity of the new lipoglycopeptide antimicrobial agent oritavancin against 33 genotypically distinct C. difficile using both agar incorporation and broth macrodilution methods. The panel of C. difficile strains used in this study included a representative isolate of PCR ribotype 027; the strain linked to recent epidemics of CDI in Europe, Canada and the USA.12 MICs for genotypically distinct C. difficile are shown in Table 1. Metronidazole agar incorporation geometric mean MICs were 2- to 5-fold lower than oritavancin MICs and 2- to 3-fold lower than vancomycin MICs. Oritavancin and vancomycin were similarly active against C. difficile for all agar conditions. Supplementation of Wilkins Chalgren agar with 0.002% P80 and 2% lysed horse blood did not greatly influence MICs of oritavancin (or vancomycin and metronidazole) for C. difficile or control organisms. Furthermore, incorporation of P80 with or without lysed horse blood did not affect the growth of C. difficile or control organisms on non-antimicrobial-containing (control) agar. Broth macrodilution MICs were 2- to 4-fold lower than agar incorporation MICs for oritavancin but not for metronidazole or vancomycin. Oritavancin broth macrodilution geometric mean MICs were
2- and 5-fold lower than those for metronidazole and vancomycin, respectively. When comparing individual C. difficile strain broth macrodilution MICs, 76% (25/33) of tested isolates were more susceptible to oritavancin (
2 doubling dilutions) than to vancomycin.
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Arhin et al.8 recently reported that oritavancin was rapidly lost from the solution in broth microdilution susceptibility assay test plates in the absence of P80 or 2% lysed horse blood. Prior studies have suggested incorporation of P80 or lysed horse blood may significantly reduce oritavancin MICs for staphylococci and enterococci (but not clinical streptococcal isolates) using broth microdilution methods.8,9 The authors postulated that the presence of lysed blood in culture media used to evaluate clinical streptococcal isolates may have abrogated oritavancin binding to surfaces; hence, the incorporation of P80 did not facilitate any further reduction in uuuuoritavancin MICs. Additionally, inclusion of P80 in agar incorporation methods does not reduce oritavancin MICs for S. aureus, coagulase-negative staphylococci, E. faecalis or Enterococcus faecium (D. Sahm, Eurofins Medinet Inc., VA, USA—personal communication). The present study evaluated the effect of P80 and lysed horse blood on oritavancin, vancomycin and metronidazole MICs using agar incorporation. The presence of P80±lysed horse blood in agar incorporation methods did not substantially reduce oritavancin MICs, thus reflecting previous agar-based MIC studies with facultative anaerobes (D. Sahm, Eurofins Medinet Inc., VA, USA—personal communication) and highlighting MIC variability between methods. Additionally, incorporation of P80±lysed horse blood in the present study did not affect vancomycin (or metronidazole) MICs, which also reflects prior in vitro susceptibility studies that did not demonstrate a shift in MICs of these antimicrobial agents in the presence of P80 or lysed horse blood.13
Assays of oritavancin concentrations in samples collected from an in vitro triple-stage chemostat human gut model using large-plate bioassay indicate that diffusion of the drug in agar is slow; a pre-incubation diffusion step is required to enhance zone diameters (S. D. Baines, unpublished data). Glycopeptides are known to bind to polymer surfaces,14 a property that is influenced by surface charge of both the antimicrobial and polymer. For example, binding of teicoplanin to the surfaces of specimen vessels was on average four times greater than that of vancomycin. Pre-exposure of a polymer surface to human body fluids markedly reduces the binding of teicoplanin. Oritavancin is a semi-synthetic lipoglycopeptide that possesses either a single or double positive charge at neutral pH (Dr A. Rafai Far, Targanta Therapeutics, Quebec, Canada—personal communication). Therefore, complexing of oritavancin with components of agar, due to physicochemical properties of the antibiotic, may explain the elevated MICs (compared with those measured by broth macrodilution) for C. difficile and other bacteria. Indeed, adsorption of antimicrobial peptides to agar surfaces has been reported previously.15 The present study included all permutations of P80 and lysed horse blood in agar incorporation MICs and showed that neither of the two supplements significantly impacted the assessment of oritavancin susceptibility. Elevated oritavancin agar incorporation MICs compared with both broth micro- and macrodilution MICs have been observed in other institutions, but the definitive mechanism behind this phenomenon remains unexplained (Dr G. Moeck, Targanta Therapeutics, Quebec, Canada—personal communication).13
Agar incorporation MICs in the present study were similar for vancomycin and oritavancin, with metronidazole demonstrating the lowest geometric mean MICs. A trend to oritavancin/vancomycin MIC equivalence using agar incorporation was reproduced in a recent study, which evaluated the activity of oritavancin against a wide range of aerobic, facultatively anaerobic and obligately anaerobic bacteria, including 32 isolates of C. difficile.16 However, oritavancin MIC90s in the present study were 2- to 4-fold higher than those previously reported, which may be a consequence of differing methodologies between experiments or alternatively a failure to genetically discriminate between C. difficile isolates evaluated by Moeck et al.16 Indeed, some C. difficile strains in the present study demonstrated oritavancin agar incorporation MICs of 0.5 mg/L, which were equal to the MIC50 reported by Moeck et al.; thus, if only exquisitely susceptible clonal C. difficile strains were examined in prior published studies, this would inevitably skew MIC data. We found that expanded in vitro susceptibility testing of 30 randomly selected C. difficile PCR ribotype 001 isolates showed little intra-PCR ribotype variation in oritavancin MICs (MICs 2–4 mg/L; data not shown). We have recently reported the emergence of reduced susceptibility to metronidazole in 25% of C. difficile PCR ribotype 001 isolated from our institution in 2005–06 (geometric mean MIC = 5.94 mg/L) in comparison with historic (1995–2001) PCR ribotype 001 isolates (n = 72, geometric mean MIC = 1.03 mg/L) (P < 0.001).17,18 Within the extended panel of C. difficile PCR ribotype 001 isolates evaluated in this study, three strains demonstrated reduced susceptibility to metronidazole (geometric mean MIC for all agar combinations = 6.94 mg/L) (data not shown). Oritavancin and vancomycin were similarly active against these C. difficile strains; geometric mean MICs were 2.82 and 1.69 mg/L, respectively (data not shown).
In summary, oritavancin demonstrated similar activity against genotypically distinct C. difficile strains as vancomycin, as measured by agar incorporation, and greater activity according to broth macrodilution. Furthermore, both oritavancin and metronidazole were active against C. difficile PCR ribotype 001 isolates with reduced susceptibility to metronidazole. Assuming similar pharmacokinetics to vancomycin with respect to faecal levels following an oral dose, these results demonstrate that oritavancin has the potential to be used for the treatment for CDI.
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This work was funded by a research grant from Targanta Therapeutics.
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M. H. W. has received honoraria for consultancy work, financial support to attend meetings and research funding from Astra-Zeneca, Bayer, Genzyme, Nabriva, Novacta, Pfizer and Wyeth. S. D. B. has received financial support to attend meetings from Bayer and Targanta Therapeutics. J. F. has received financial support to attend meetings from Bayer and Wyeth. R. O.: none to declare.
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Acknowledgements |
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Parts of this work were recently communicated at the Forty-seventh Interscience Conference on Antimicrobial Agents and Chemotherapy.19
We thank Dr Warren Nigel Fawley for performing PCR ribotyping. We also thank Dr Greg Moeck, Dr Francis Arhin and Dr Adel Rafai Far for their helpful discussions during the preparation of this manuscript.
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19 . O'Connor R, Freeman J, Baines SD, et al. In vitro susceptibility of genotypically distinct Clostridium difficile strains to oritavancin. In: Abstracts of the Forty-seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007. Washington, DC, USA: American Society for Microbiology. Abstract E-1618, p. 206.
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