Antibiotic susceptibility of 65 isolates of Burkholderia pseudomallei and Burkholderia mallei to 35 antimicrobial agents

F. M. Thibault1,*, E. Hernandez2, D. R. Vidal1, M. Girardet2 and J.-D. Cavallo2

1 Centre de Recherches du Service de Santé des Armées Emile Pardé, Département de Biologie des Agents Transmissibles, 24 Avenue des Maquis du Grésivaudan, B.P. 87, F-38702 La Tronche; 2 Service de Biologie Médicale, Hôpital d'Instruction des Armées Begin, 69 Avenue de Paris, F-94160 Saint-Mandé, France

Received 30 April 2004; returned 24 July 2004; revised 9 September 2004; accepted 20 September 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Objectives: Fifty isolates of Burkholderia pseudomallei and 15 isolates of Burkholderia mallei were tested for their susceptibilities to 35 antimicrobial agents, including agents not previously tested against these bacteria.

Methods: MICs were determined by agar dilution in Mueller–Hinton medium.

Results: Among the antibiotics tested, lower MICs were obtained with imipenem, ceftazidime, piperacillin, piperacillin/tazobactam, doxycycline and minocycline. Fluoroquinolones and aminoglycosides had poor activities. A single clinical isolate of B. pseudomallei was resistant to ceftazidime, co-amoxiclav and doxycycline but remained susceptible to imipenem.

Conclusions: Although B. mallei MICs are often lower, the overall results underline the importance of resistance in both species. The susceptibilities measured are consistent with the current recommendations for the treatment of B. pseudomallei and B. mallei infections.

Keywords: melioidosis , glanders , MICs , resistance , biowarfare


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Burkholderia pseudomallei is responsible for melioidosis, which is considered a major community-acquired infection in endemic areas, leading the disease to be recognized as a medical problem in the armies involved in conflicts in Asia.1 Because relapses of melioidosis are common, clinical management requires long courses of chemotherapy.1 Burkholderia mallei is the causative agent of glanders and primarily infects horses, mules and donkeys. It is also able to infect humans; intentional releases of this bacterium have been documented.2

B. pseudomallei and B. mallei are considered as potential biological warfare or bioterrorism agents and have been included in the B list of the CDC.3 B. mallei and B. pseudomallei are intrinsically resistant to a wide range of antimicrobial agents including ß-lactam antibiotics, aminoglycosides and macrolides.4,5 However, few antibiotic susceptibility studies of B. mallei have been performed.6,7 As there is a need for effective treatments and post-exposure prophylaxis, the objective of this study was to assess the in vitro susceptibilities of a large panel of strains of B. mallei and B. pseudomallei to a wide variety of antibiotics.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Fifty isolates of B. pseudomallei and 15 isolates of B. mallei belonging to the collection of the Centre de Recherches du Service de Santé des Armées Emile Pardé (La Tronche, France) were included in this protocol. Strains were stored at –80°C in 20% glycerol. Strain origin is reported in Table 1. Species identification was confirmed by routine phenotypic characterization including Gram staining, motility tests and biochemical profiles on API 20 NE tests (bioMérieux, Marcy l'Étoile, France).8 All experiments were conducted in a BSL 3 laboratory.


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Table 1. Strains used in this study

 
MICs were determined by the agar dilution method. Antibiotic powders were obtained from their respective manufacturers and dissolved according to their recommendations. The antibiotics tested were: amoxicillin (Inava, France), co-amoxiclav (clavulanic acid 2 mg/L) (Smith-Kline-Beecham, France), piperacillin (Dakota, France), piperacillin/tazobactam (tazobactam 4 mg/L) (Wyeth-Lederle, France), ticarcillin and ticarcillin/clavulanate (Smith-Kline-Beecham, France), cefoxitin (Merck-Sharp-Dohme-Chibret, France), cefoperazone (Pfizer, France), cefsulodin (Takeda, France), cefotaxime (Roussel-Diamant, France), clavulanate (Smith-Kline-Beecham, France), tazobactam (Wyeth-Lederle, France), aztreonam (Sanofi-Winthrop, France), ceftazidime (Glaxo-Wellcome, France), imipenem (Merck-Sharp-Dohme-Chibret, France), nalidixic acid (Sanofi-Winthrop, France), pefloxacin (Bellon, France), ciprofloxacin (Bayer, France), ofloxacin (Roussel-Diamant, France), norfloxacin (Glaxo-Wellcome, France), gatifloxacin (Grünenthal, France), levofloxacin (Aventis, France), trimethoprim/sulfamethoxazole (co-trimoxazole; Roche, France), gentamicin (Schering-Plough), tobramycin (Lilly, France), netilmicin (Schering-Plough, France), amikacin (Bristol-Myers Squibb, France), chloramphenicol (Chauvin, France), fosfomycin (Sanofi-Winthrop, France), doxycycline (Asta-medica, France), minocycline (G-Gam, France), rifampicin (Roussel-Diamant, France), erythromycin (Abbot, France), clindamycin (Pharmacia-Upjohn, France) and novobiocin (Sigma). Bacteria were recovered prior to the experiment by placing the glycerol stock in Mueller–Hinton broth incubated at 37°C for 48 h. Colonies were obtained by plating 10 µL of the initial broth on blood agar medium incubated for 48 h at 37°C. After growth, each isolate was suspended in PBS and 100 µL of the suspension was added to 3.9 mL of Mueller–Hinton broth and incubated for 48 h in a water bath at 37°C. Mueller–Hinton agar plates were inoculated using a multiple Steer inoculator (Dynatech, UK) with a final inoculum of 104 cfu/spot. The plates were incubated at 37°C to comply with the recommendations of the ‘Comité de l'Antibiogramme de la Société Française de Microbiologie’ (CASFM). These recommendations are freely available at http://www.sfm.asso.fr. Because B. mallei growth is usually poor after 24 h of incubation, the plates were incubated for 48 h and read at 24 and 48 h. Escherichia coli ATCC 25922 was used as a control strain. As there are no specific recommendations for the interpretation of susceptibility tests of B. mallei and B. pseudomallei, interpretative criteria were, when existing, those defined by the CASFM for B. cepacia (Table 2). When specific breakpoints were not available, general recommendations were applied.


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Table 2. MIC50s, MIC90s and ranges for 50 B. pseudomallei and 15 B. mallei strains

 

    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The results of susceptibility tests are shown in Table 2.

Whatever the antibiotic family, the present results demonstrated no differences between the strains isolated from humans and those isolated from animal or environmental sources.

Consistent with previous studies, the isolates tested in this protocol were highly resistant to amoxicillin, ticarcillin, cefoxitin, cefoperazone, cefsulodin and aztreonam. One strain, isolated from human infection, appeared resistant to ceftazidime with an MIC of 64 mg/L (MICs of ceftazidime for the other strains were between 1–4 mg/L). This strain also presented a cross-resistance to ticarcillin/clavulanate, doxycycline and minocycline and was categorized intermediate for co-amoxiclav with an MIC of 16 mg/L. All other strains of B. mallei and B. pseudomallei were susceptible to co-amoxiclav. Cefotaxime activity was low but was partially restored by clavulanate or tazobactam, demonstrating the effectiveness of ß-lactamase inhibitors against the two species. The resistance profiles of both species for ß-lactam antibiotics were similar; however, lower MICs were observed in B. mallei for ß-lactam inhibitor combinations. MICs of piperacillin were low (0.125–8 mg/L) and those of the combination of piperacillin and tazobactam were three- or four-fold lower, suggesting that this antibiotic was hydrolysed by the chromosomal ß-lactamases of both species.

All the isolates were susceptible to imipenem. This antibiotic was, with doxycycline and minocycline, one of the most active antibiotics tested. This has been observed previously in a study involving 211 clinical strains,5 and is of interest because this antibiotic is considered as a good alternative to ceftazidime in the treatment of disseminated disease. It has been recommended by the European Agency for the Evaluation of Medicinal Products (EMEA) for the treatment of suspected or confirmed melioidosis.9 The low MICs encountered with minocycline and doxycycline are also of interest because doxycycline has been used alone for the treatment of localized infection and has also been recommended by the EMEA in association with imipenem or meropenem for the treatment of severe cases of melioidosis.9 Despite the lack of recommendations, we assume that oral doxycycline could be useful for post-exposure prophylaxis.10

With 50% of isolates intermediate or resistant to ciprofloxacin (MIC breakpoint of 2 mg/L), this antibiotic cannot be recommended for treatment and/or prophylaxis. Clinical experience in maintenance therapy has demonstrated a poor efficacy for preventing relapses.11 Both species demonstrated resistance to pefloxacin, ofloxacin and norfloxacin. MICs of gatifloxacin and levofloxacin were equivalent to those of ciprofloxacin for B. pseudomallei. All the strains of this species were resistant to erythromycin and clindamycin, and nearly all were resistant to all the aminoglycosides tested (gentamicin, tobramycin, netilmicin, amikacin). This resistance is due to the presence of a unique multidrug efflux system (AmrAB-OprA) in B. pseudomallei, which is specific for both aminoglycosides and macrolide antibiotics.12 In contrast, MICs of aminoglycosides for B. mallei were lower and all strains appeared susceptible to netilmicin with MICs in the range 0.125–0.25 mg/L. All the MICs of clindamycin were high in both species, but only B. pseudomallei exhibited high MICs of erythromycin. This observation is consistent with those obtained with azithromycin.7 B. pseudomallei was categorized as moderately susceptible to chloramphenicol. The MIC50 and MIC90 of co-trimoxazole were 8 and 16 mg/L, respectively, and the majority of the strains were categorized as intermediate or resistant (breakpoints ≤2/38, >8/152) to this antibiotic. This relative in vitro resistance is not correlated with clinical experience as co-trimoxazole has been traditionally used for the therapy of melioidosis. Such discrepancies between results obtained with co-trimoxazole by different susceptibility testing methods and clinical data have already been documented.1

In conclusion, this study confirms the high level of antibiotic resistance in B. mallei and B. pseudomallei, including towards agents not tested in previous studies. Nevertheless, the overall resistance is lower in B. mallei. The resistance profiles appear to be independent of the origin of isolates. Imipenem, ceftazidime, co-amoxiclav, piperacillin, piperacillin/tazobactam and doxycycline appear as the more effective drugs tested on this panel of isolates. These results remain consistent with the current recommendations for the treatment of melioidosis and glanders. However, the emergence of ceftazidime-resistant clinical isolates and the wide distribution of B. pseudomallei in Southeast Asia increase the risk of malicious use of those resistant strains. Piperacillin/tazobactam could be a useful alternative for treatment of both glanders and melioidosis. Nevertheless, the disparity between in vitro results and clinical response underlines the necessity to validate this association by time–kill studies, animal models and clinical experience.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We are indebted to Isabelle Perrichon for excellent technical work. This work was supported by the French Ministry of Defense, grant no. 3-e/LR/EMA.


    Footnotes
 
* Corresponding author. Tel: +33-4-76-63-69-20; Fax: +33-4-76-63-69-17; Email: fthibault{at}crssa.net


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . White, N. J. (2003). Melioidosis. Lancet 361, 1715–22.[CrossRef][ISI][Medline]

2 . Neubauer, H., Meyer, H. & Finke, E. J. (1997). Human glanders. International Review of Armed Forces Medical Services 70, 258–65.

3 . Centers for Disease Control and Prevention. (2003). Bioterrorism Agents/Diseases. [Online.] http://www.bt.cdc.gov/agent/agentlist-category.asp (6 September 2004, date last accessed).

4 . Heine, H. S., England, M. J., Waag, D. M. et al. (2001). In vitro antibiotic susceptibilities of Burkholderia mallei (causative agent of glanders) determined by broth microdilution and E-test. Antimicrobial Agents and Chemotherapy 45, 2119–21.[Abstract/Free Full Text]

5 . Dance, D., Wuthiekanun, V., Chaowagul, W. et al. (1989). The antimicrobial susceptibility of Pseudomonas pseudomallei. Emergence of resistance in vitro and during treatment. Journal of Antimicrobial Chemotherapy 24, 295–309.[Abstract]

6 . Batmanov, V. P. (1994). Sensitivity of Pseudomonas mallei to tetracyclines and their effectiveness in experimental glanders. Antibiotiki I Khimioterapiia 39, 33–7.

7 . Kenny, D. J., Russell, P., Rogers, D. et al. (1999). In vitro susceptibilities of Burkholderia mallei in comparison to those of other pathogenic Burkholderia spp. Antimicrobial Agents and Chemotherapy 43, 2773–5.[Abstract/Free Full Text]

8 . Ashdown, L. R. (1979). Identification of Pseudomonas pseudomallei in the clinical laboratory. Journal of Clinical Pathology 32, 500–4.[Abstract]

9 . European Agency for the Evaluation of Medicinal Products. (2002). Guidance document on use of medicinal products for treatment and prophylaxis of biological agents that might be used as weapons of bioterrorism. [Online.] http://www.emea.eu.int/pdfs/human/bioterror/404801.pdf (9 September 2004, date last accessed).

10 . Russell, P., Eley, S. M., Ellis, J. et al. (2000). Comparison of efficacy of ciprofloxacin and doxycycline against experimental melioidosis and glanders. Journal of Antimicrobial Chemotherapy 45, 813–8.[Abstract/Free Full Text]

11 . Chetchotisakd, P., Chaowagul, W., Mootsikapun, P. et al. (2001). Maintenance therapy of melioidosis with ciprofloxacin plus azithromycin compared with cotrimoxazole plus doxycycline. American Journal of Tropical Medicine and Hygiene 64, 24–7.[Abstract/Free Full Text]

12 . Moore, R. A., DeShazer, D., Reckseidler, S. et al. (1999). Efflux-mediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei. Antimicrobial Agents and Chemotherapy 43, 465–70.[Abstract/Free Full Text]