Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS

Abstract

Background: The genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays.

Results: In this study, the accurate identification of Brucella species using MALDI-TOF-MS was achieved by constructing a Brucella reference library based on multilocus variable-number tandem repeat analysis (MLVA) data. By comparing MS-spectra from Brucella species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for Brucella species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and B. suis biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for Brucella, even minimal genomic differences between these serovars translate to specific proteomic differences.

Conclusions: MALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library.

Figure 1

Partial dendrogram MLVA-16 clustering analysis of 170 Brucella isolates, with all 93 of the B. melitensis and 29 B. abortus isolates included in this study. The columns present the following data: original strain number [Strain id.], MLVA cluster number reference [Ref. cluster], epidemiologic relatedness (a-d indicate isolates from the same patient, 1-3 indicate isolates that are epidemiologically linked to each other)[Linked], highest logarithmic value of the four generated MS spectra [High LogValue], number of the 4 generated MS spectra corresponding with species identification using MLVA [N identified], genus [Genus], species [Species], and biovar [Biovar] identification based on the MLVA database. The similarity axis is presented in the top left corner. Each color reflects a different cluster with > 52.5% similarity. The group of 'melitensis-abortus' isolates clustered as follows: B. melitensis isolates grouped in Clusters 1, 2, and 3. B. abortus isolates grouped in Clusters 4, 6, and 7. Outlier B. abortus/melitensis W99 is a singleton (Cluster 5).

Figure 2

Partial dendrogram MLVA-16 clustering analysis of 170 Brucella isolates, including the 48 isolates from Brucella species that were not B. melitensis or B. abortus included in this study. The columns present data as described in Figure 1. The similarity axis is presented in the top left corner. Each color reflects a different cluster with > 52.5% similarity. The group of 'non-melitensis/abortus' isolates clustered as follows: Cluster 8 with B. suis biovar 3 and B. canis; Cluster 9 with B. suis biovar 1; Cluster 10 with B. suis biovar 2; and Cluster 11 with B. ovis isolates. The 'B. ceti/pinnipedialis/neotomae' subgroup clustered as follows: Clusters 12 and 13 with B. pinnipedialis isolates and Cluster 14 and 16 with B. ceti isolates. Furthermore, this subgroup also contained two clusters with only one isolate (singletons): Cluster 15 with a B. suis biovar 5 and Cluster 16 with a B. neotomae isolate.

Figure 3

Representative MALDI-TOF-MS spectra of the Brucella strains used as references in the generated Brucella reference library in the range of 1, 000 to 12, 000 Da. The relative intensity (R.i) is shown as a percentage of the total intensity on the y-axis, and the mass to charge ratio (M/Z) is shown on the x-axis. A) B. melitensis Ether. B) B. melitensis 16 M. C) B. melitensis 63/9. D) B. abortus 98/3033. E) B. abortus/melitensis W99. F) B. abortus B19. G) B. abortus Tulya. H) B. canis RM6/66. I) B. suis biovar 3 686. J) B. suis biovar 1 S2 Chine. K) B. suis Thomsen biovar 2. L) B. ovis Réo. M) B. pinnipedialis 09-00388. N) B. pinnipedialis 17 g-1. O) B. ceti M78/05/02. P) B. suis biovar 5 513. Q) B. ceti M 644/93/1. R) B. neotomae 5 K33.