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. MALDI-TOF-MS The 608 MS spectra derived from 152, mostly clinical, isolates were compared against the reference library generated for Brucella species. Representative MS spectra from the 18 isolates selected

for the Brucella reference library are shown (Figure 3). Minor visual differences (peaks and intensities) among the MS spectra are detectable. BIX 1294 A total of 25 MS spectra had a logarithmic score value from 2.000 to 2.299, indicating ‘secure genus identification, probable species identification’. The highest logarithmic score values of the remaining 583 MS spectra were between 2.300 and 3.000, which indicate ‘highly AC220 purchase probable species identification’. 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 Tubastatin A price 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 3-mercaptopyruvate sulfurtransferase 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.

Because Brucella abortus W99, a singleton strain, is equally similar to B. abortus as to B. melitensis, we interpreted this strain as a potential B. melitensis strain. When identification at the species level is based on a ‘majority rule’ (i.e., identification is based on the species indicated by at least three out of four MS spectra), 149 (98%) isolates were correctly identified at the species level. Further, when instead of the majority rule, the identification at the species level was based on the highest of the four logarithmic values, which was always > 2.299, 151 (99.3%) of the isolates were correctly identified at the species level, while only 1 (0.7%) isolate was mistakenly identified as B. canis instead of B. suis. The isolates 03-3081-2, 04-2987, and 02-00117, which were identified as B. suis biovar 3, 1 or 3 and 1 or 3, respectively, based on their MLVA profile similarity, were all grouped into cluster 9, which only contained B. suis biovar 1 isolates. Therefore, these three isolates are most likely B. suis biovar 1. The MLVA data further demonstrated that the B. suis biovars 1 (MLVA cluster 9) and 2 (MLVA cluster 10) are genetically distinct clusters, whereas B. suis biovar 3 grouped together with B.