JAC Advance Access originally published online on April 8, 2008
Journal of Antimicrobial Chemotherapy 2008 62(1):208-210; doi:10.1093/jac/dkn146
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Research letters |
Susceptibility of 71 French isolates of Francisella tularensis subsp. holarctica to eight antibiotics and accuracy of the Etest® method
1 Centre de Recherches du Service de Santé des Armées, Laboratoire de Bactériologie, BP 87, 38702 La Tronche, France 2 Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d'Etudes et de Recherches en Pathologie Animale et Zoonoses, 23 avenue du général De Gaulle, 94700 Maison-Alfort, France 3 Hôpital d'Instruction des Armées Bégin, Service de Biologie Médicale, 69 avenue de Paris, 94163 Saint Mandé, France
* Corresponding author. Tel: +33-476636840; Fax: +33-476636917; E-mail: evalade{at}crssa.net
Keywords: method comparison , minimum inhibition concentration , tularaemia
Francisella tularensis subsp. holarctica causes tularaemia, which affects a large range of vertebrates, including humans and other mammals.1 This facultative intracellular bacterium is highly infectious and causes life-threatening infections, especially when transmitted via the respiratory route. In France, 20 to 25 cases in humans are reported to the national health authorities each year.2 This bacterium is naturally resistant to penicillins and cephalosporins, but is susceptible to many other antibiotics including aminoglycosides, chloramphenicol, tetracyclines, rifampicin and fluoroquinolones.3–6
In this study, we aimed to determine the in vitro antibiotic susceptibility of a representative panel of 71 French F. tularensis subsp. holarctica strains by the agar dilution method and to evaluate the accuracy of the Etest® method (AB Biodisk, Sweden) using supplemented Mueller–Hinton II reference medium (Becton Dickinson, France) or supplemented chocolate medium (Becton Dickinson).
Seventy-one isolates of F. tularensis subsp. holarctica were collected from human or animal samples in France between 1996 and 2005 by the French National Reference Centre for Tularaemia (AFSSA/LERPAZ). The isolates were selected to include those obtained from diverse geographical areas of France [Table S1, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. MICs were determined for eight antibiotics by the broth microdilution and the agar dilution methods using Mueller–Hinton II medium supplemented with 2% IsoVitaleXTM to satisfy the in vitro growth requirements of Francisella. MICs were read after 48 h of incubation in ambient air to comply with the recommendations of the CLSI. For antibiotics for which specific breakpoints have been established, strains were classified as susceptible or non-susceptible. In the absence of specific breakpoint data, CLSI general interpretative standards were used.
We first determined MICs by the broth microdilution method, but this method did not give reproducible results, except for gentamicin, ciprofloxacin and doxycycline. It was difficult to determine the MIC due to a regrowth phenomenon, which does not depend on the bactericidal or bacteriostatic activity of the antibiotic. Due to these technical difficulties, we therefore used the agar dilution method.
Consistent with the results of previous studies,3,5,6 100% of our isolates were susceptible to gentamicin (MIC 0.03–0.5 mg/L) and streptomycin (MIC < 0.5–1 mg/L). As streptomycin is rarely used in France, gentamicin remains the first-line antibiotic for the treatment of tularaemia. All isolates were also susceptible to doxycycline (MIC 0.125–1 mg/L), ciprofloxacin (MIC 0.015–0.03 mg/L), nalidixic acid (MIC 0.06–2 mg/L) and chloramphenicol (MIC 0.25–2 mg/L). Fluoroquinolones and doxycycline are now considered as treatment options and are the recommended antibiotics for post-exposure prophylaxis in cases of suspected or confirmed exposure to the pathogen.2 Fluoroquinolones seem to be effective for treatment, with a low rate of therapeutic failure. Doxycycline has been used to treat patients, but this antibiotic must be administered for long periods of time to avoid high relapse rates.1 Rifampicin was also found to be active in vitro against all the strains studied (MIC 0.015–0.5 mg/L), and the use of this compound in combination with quinolones or aminoglycosides has been proposed for the treatment of severe tularaemia. However, rifampicin should not be used in monotherapy because of the potential for rapid emergence of resistance. All isolates were also susceptible to telithromycin (MIC 0.125–0.25 mg/L), a recently developed macrolide that Maurin et al.4 considered effective for treatment, due to its bactericidal activity and high intracellular concentration.
The second aim of our study was to validate convenient methods for determining the antibiotic susceptibility of this fastidious bacterium, which could be used by routine laboratories, especially in case of deliberate release. Thus, the antimicrobial resistance of all the strains was assessed in parallel by the Etest® method on supplemented Mueller–Hinton II agar, except for telithromycin, for which no Etest® is yet available. The agreement between the two methods is shown in Table 1. The agreement was superior to 80% for ciprofloxacin, doxycycline and streptomycin. For all other antibiotics, strains were classified similarly in terms of their susceptibility, but agreement between the MICs determined by the Etest® method and the agar reference method was poor.
|
As supplemented Mueller–Hinton II agar must be custom-made and is not readily available for most laboratories, we tested supplemented chocolate agar medium, which can be purchased in a ready-to-use form. We therefore determined MICs by Etest® on this medium for three major antibiotics: doxycycline, ciprofloxacin and gentamicin. All the Francisella strains tested were classified as susceptible, and the results were highly concordant with those obtained on supplemented Mueller–Hinton II medium with the agar method for doxycycline and ciprofloxacin (Table 1).
These results indicate that the Etest® appears to be a practical and reliable method to assess the antibiotic susceptibility of F. tularensis. This method can be applied not only to the supplemented Mueller–Hinton II reference medium but also to supplemented chocolate agar medium.
 |
Funding |
|---|
 Top Funding Transparency declarationsSupplementary dataReferences |
|---|
This work was supported by grant nos 07co301 and 23e from the French Ministry of Defence.
|
Transparency declarations |
|---|
|
TopFundingTransparency declarationsSupplementary dataReferences |
|---|
None to declare.
|
Supplementary data |
|---|
|
TopFundingTransparency declarationsSupplementary dataReferences |
|---|
Table S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
|
Acknowledgements |
|---|
We thank R. Fabre, F. Desor, D. Riou and D. Cariou for excellent technical assistance and M. Pépin, Head AFSSA-LERPAZ, for authorizing this work. We thank the French Ministry of Health and Solidarity, and French departmental veterinary laboratories for providing F. tularensis isolates.
|
References |
|---|
|
TopFundingTransparency declarationsSupplementary dataReferences |
|---|
1 Ellis JP, Oyston C, Green M, et al. Tularaemia. Clin Microbiol Rev (2002) 15:631–46.
2 Institut National de Veille Sanitaire. Tularémie. http://www.invs.sante.fr/surveillance/tularemie/donnees.htm (10 March 2008, date last accessed).
3
Baker CN, Hollis DG, Thornsberry C. Antimicrobial susceptibility testing of Francisella tularensis with a modified Mueller–Hinton broth. J Clin Microbiol (1985) 22:212–5.
4
Maurin M, Mersali NF, Raoult D. Bactericidal activities of antibiotics against intracellular Francisella tularensis. Antimicrob Agents Chemother (2000) 44:3428–31.
5
Ikaheimo I, Syrjala H, Karhukorpi J, et al. In vitro antibiotic susceptibility of Francisella tularensis isolated from humans and animals. J Antimicrob Chemother (2000) 46:287–90.
6 Tomaso H, Al Dahouk S, Hofer E, et al. Antimicrobial susceptibilities of Austrian Francisella tularensis holarctica biovar II strains. Int J Antimicrob Agents (2005) 26:279–84.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||