LETTER
Burkholderia pseudomallei is the causative agent of melioidosis, a serious disease endemic in southeast Asia. Accurate identification of B. pseudomallei is important, since treatment of melioidosis requires prolonged antibiotics to prevent relapse (9). Although B. pseudomallei differs greatly from other Burkholderia species in pathogenicity and epidemiology, identification of B. pseudomallei is often difficult, as phenotypic tests and even 16S rRNA gene sequencing may not offer adequate discrimination from related species, such as B. thailandensis and B. cepacia complex (BCC) (3–6, 8).
Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has recently emerged as a revolutionary technique for rapid bacterial identification at a low cost. Although MALDI-TOF MS has been shown to be useful for the identification of various nonfermenting Gram-negative bacilli, including some Burkholderia species (1), its application in identifying B. pseudomallei has not been explored.
Using 76 Burkholderia strains, we evaluated the performance of MALDI-TOF MS for the identification of B. pseudomallei (Table 1). All isolates were phenotypically identified by using the API 20NE system (bioMérieux) supplemented with biochemical methods. The identities of B. pseudomallei and B. thailandensis isolates were confirmed by groEL gene sequencing, and that of BCC by recA gene sequencing (6). All isolates were grown on horse blood agar at 37°C for 18 to 24 h and analyzed by the direct transfer method (2) (except that two BCC strains were analyzed by ethanol-formic acid extraction due to suboptimal results) in biosafety level II cabinets. Samples were processed in a MALDI-TOF MS spectrometer (Bruker Daltonik) with 1 μl of matrix solution (Bruker α-cyano). Spectra were obtained with an accelerating voltage of 20 kV in linear mode and analyzed within an m/z range of 2,000 to 20,000 Da. Spectra were analyzed with MALDI Biotyper 3.0 and Reference Library version 3.1.2.0 (Bruker Daltonik), which contained 41 Burkholderia main spectra (MSPs) comprising 26 species. Since B. pseudomallei is not represented in the Bruker database, 21 B. pseudomallei strains and, later, one B. thailandensis strain were added as reference strains.
Table 1.
Identification of Burkholderia species by MALDI-TOF MS
| Species | No. of strains of species | Origin or reference; strain(s) (no. of strains from origin) | No. of isolates identified usinga: |
|
|---|---|---|---|---|
| B. pseudomallei reference strains | B. pseudomallei and B. thailandensis reference strains | |||
| B. cepacia complex | 20 | Clinical isolates (n = 17) | 20 | 20 |
| BCCM/LMG collection; LMG1222, LMG14191, LMG20980 (n = 3) | ||||
| B. gladioli | 1 | BCCM/LMG collection; LMG2216 | 1 | 1 |
| B. pseudomallei | 52 | Clinical isolates (n = 15) | 52 | 52 |
| Veterinary isolates (n = 25) | ||||
| Environmental isolates (n = 12) | ||||
| B. thailandensis | 3 | 7 | 0 (misidentified as B. pseudomallei) | 3 |
Isolates were identified by MALDI-TOF MS using extended database with B. pseudomallei reference strains or with B. pseudomallei and B. thailandensis reference strains in this study.
The spectra obtained with B. pseudomallei compared to those obtained with other species are shown in Fig. 1. Using the Bruker database extended with B. pseudomallei reference strains, all isolates except for B. thailandensis were correctly identified (the score of the top match was ≥2.0, and the score of the second match was lower by ≥10%) (Table 1). Notably, the three B. thailandensis isolates were misidentified as B. pseudomallei. Further extension of the database with one additional B. thailandensis reference strain enabled the correct identification of two other B. thailandensis isolates. The misidentification of B. thailandensis by using the Bruker database is probably due to the inclusion of only one MSP from the species, which fails to cover intraspecies variability.
Fig 1.
MALDI-TOF MS spectra of B. pseudomallei compared to those of other species. Intens. [a.u.], intensity in arbitrary units.
MALDI-TOF MS is potentially useful for accurate routine identification of B. pseudomallei and B. thailandensis. However, this requires optimization of the database by adding reference MSPs for B. pseudomallei and expanding the number of MSPs for B. thailandensis. While the present BCC isolates were correctly identified as BCC, species identification may require additional reference strains (1). Expansion of commercial databases with pathogens endemic in different localities is important to improve the usefulness of MALDI-TOF MS.
ACKNOWLEDGMENTS
We thank Wai-Sing Chan, Ting-Yin Wong, and Yu-Ting Tong for technical assistance.
This work is supported by the HKSAR Research Fund for the Control of Infectious Diseases (Commissioned Study) of the Health, Welfare and Food Bureau.
Footnotes
Published ahead of print 20 June 2012
Contributor Information
Susanna K. P. Lau, State Key Laboratory of Emerging Infectious Diseases The University of Hong Kong Hong Kong, China
Bone S. F. Tang, Department of Pathology Hong Kong Sanatorium Hospital Hong Kong, China
Shirly O. T. Curreem, Department of Microbiology The University of Hong Kong Hong Kong, China
Tsz-Ming Chan, Department of Pathology Hong Kong Sanatorium Hospital Hong Kong, China.
Paolo Martelli, Ocean Park Corporation Hong Kong, China.
Cindy W. S. Tse, Department of Pathology Kwong Wah Hospital Hong Kong, China
Alan K. L. Wu, Department of Pathology Pamela Youde Nethersole Eastern Hospital Hong Kong, China
Patrick C. Y. Woo, Research Centre of Infection and Immunology The University of Hong Kong Hong Kong, China
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