1 Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 2 Menzies School of Health Research, Charles Darwin University and Northern Territory Clinical School, Flinders University, Darwin, Australia; 3 Medical Department, Sappasithiprasong Hospital, Ubon Ratchathani, Thailand; 4 Biomedical Sciences, DSTL, Salisbury; 5 Department of Infection and Tropical Medicine, Heartlands Hospital, Birmingham; 6 Centre for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford OX3 7LJ, UK
Received 17 January 2005; returned 17 February 2005; revised 22 March 2005; accepted 8 April 2005
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Abstract |
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Patients and methods: We performed disc diffusion and Etest on isolates from the first positive culture for all patients presenting to Sappasithiprasong Hospital, Ubon Ratchathani, Thailand, with culture-confirmed melioidosis between 1992 and 2003.
Results: The estimated resistance rate for 1976 clinical Burkholderia pseudomallei isolates was 13% by Etest and 71% by disc diffusion. All isolates classed as either susceptible (n=358) or as having intermediate resistance (n=218) on disc diffusion were susceptible by Etest. Only 258 of the 1400 (18%) isolates classed as resistant on disc diffusion were resistant by Etest.
Conclusions: Disc diffusion testing of B. pseudomallei may be useful as a limited screening tool in resource poor settings. Isolates assigned as susceptible or intermediate by disc diffusion may be viewed as susceptible; those assigned as resistant require further evaluation by MIC methodology.
Keywords: Burkholderia pseudomallei , melioidosis , susceptibility , Etest
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Introduction |
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Patients and methods |
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Isolates were stored from time of isolation to testing in trypticase soy broth with 15% glycerol at 70°C. The freezer vial was scraped with a sterile loop and the organism streaked onto Columbia agar and incubated for 48 h at 37°C. A subculture was diluted in sterile normal saline to obtain a final concentration of between 1x108 and 5x108 cfu/mL using spectrophotometric methods and plated onto MuellerHinton agar (Oxoid).
Agar dilution and disc diffusion tests were performed as described previously.4
Co-trimoxazole discs (Oxoid) contained 1.25 µg of trimethoprim and 23.75 µg of sulfamethoxazole. Trimethoprim/sulfamethoxazole Etests (AB Biodisk, Solna, Sweden) were used according to the manufacturer's instructions, and the cultures read after incubation in air at 37°C for 1620 h. Bacteriostatic drugs give diffuse end-points and tests were read at the 80% inhibition point, taken as the first point of significant inhibition as judged by the naked eye. Interpretive standards for disc diffusion were based on NCCLS guidelines for Pseudomonas aeruginosa, listing resistant as 10 mm, intermediate 1115 mm and susceptible
16 mm.5
Interpretative standards for agar dilution and Etest were based on NCCLS guidelines for MIC testing of B. pseudomallei by broth microdilution, which lists susceptible organisms as
2/38 mg/L and resistant organisms as
4/76 mg/L.5
Escherichia coli ATCC 25922 was used as a susceptible control. Isolates with an estimated trimethoprim/sulfamethoxazole MIC by Etest of 3/57 mg/L were re-tested by agar dilution MIC. If the same result was obtained, these isolates were classed as resistant.
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Results and discussion |
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There was no relationship between clinical specimen type and Etest susceptibility results (P > 0.05). Resistance rates as based on Etest varied over time (P < 0.001), and ranged from a low of 0.6% in 2001 to a high of 24% in 2003 (Figure 1). Geographical localization suggestive of a disease outbreak with a resistant strain was not seen (data not shown). We are unaware of any agricultural practices that may have impacted on trimethoprim/sulfamethoxazole resistance, and cannot explain this variation in resistance over time.
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The correlation between disc diffusion and Etest results is depicted in Figure 2. All isolates classed as either susceptible (n=358) or as having intermediate resistance (n=218) on disc diffusion were susceptible by Etest. Based on these results, disc diffusion could be used as a limited first-line screen in the tropical setting, whereby isolates assessed as either susceptible or have intermediate resistance by disc diffusion could be classified as susceptible without further testing. However, this accounts for only one-third of isolates in this study. Of the 1400 isolates classed as resistant on disc diffusion, only 258 (18%) were resistant by Etest. All isolates that did not grow right up to the disc were susceptible by Etest; these could be classified as probably susceptible in a resource-poor setting, although this should be tentative and verified wherever possible. Only 20% of the 1280 isolates that grew up to the disc were resistant by Etest; this group requires further susceptibility testing.
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Acknowledgements |
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References |
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2 . Lumbiganon P, Tattawasatra U, Chetchotisakd P et al. Comparison between the antimicrobial susceptibility of Burkholderia pseudomallei to trimethoprim-sulfamethoxazole by standard disk diffusion method and by minimal inhibitory concentration determination. J Med Assoc Thai 2000; 83: 85660.[Medline]
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4 . National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk Susceptibility TestingSixth Edition: Approved Standard M2-A7. Wayne, PA, USA: NCCLS, 2000.
5 . National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing14th Informational Supplement M100-S14. Wayne, PA, USA: NCCLS, 2004.
6 . Jenney AW, Lum G, Fisher DA et al. Antimicrobial agent susceptibility of Burkholderia pseudomallei from tropical northern Australia and implications for therapy of melioidosis. Int J Antimicrob Agents 2001; 17: 10913.[CrossRef][ISI][Medline]