Genotyping of Brucella species using clade specific SNPs.
BACKGROUND
Brucellosis is a worldwide disease of mammals caused by Alphaproteobacteria in the genus Brucella. The genus is genetically monomorphic, requiring extensive genotyping to differentiate isolates. We utilized two different genotyping strategies to characterize isolates. First, we developed a microarray-based assay based on 1000 single nucleotide polymorphisms (SNPs) that were identified from whole genome comparisons of two B. abortus isolates , one B. melitensis, and one B. suis. We then genotyped a diverse collection of 85 Brucella strains at these SNP loci and generated a phylogenetic tree of relationships. Second, we developed a selective primer-extension assay system using capillary electrophoresis that targeted 17 high value SNPs across 8 major branches of the phylogeny and determined their genotypes in a large collection ( n = 340) of diverse isolates.


RESULTS
Our 1000 SNP microarray readily distinguished B. abortus, B. melitensis, and B. suis, differentiating B. melitensis and B. suis into two clades each. Brucella abortus was divided into four major clades. Our capillary-based SNP genotyping confirmed all major branches from the microarray assay and assigned all samples to defined lineages. Isolates from these lineages and closely related isolates, among the most commonly encountered lineages worldwide, can now be quickly and easily identified and genetically characterized.


CONCLUSIONS
We have identified clade-specific SNPs in Brucella that can be used for rapid assignment into major groups below the species level in the three main Brucella species. Our assays represent SNP genotyping approaches that can reliably determine the evolutionary relationships of bacterial isolates without the need for whole genome sequencing of all isolates.
Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 86011-4073, USA. jeff.foster@nau.edu
Jeffrey T
JT
Foster
Lance B
LB
Price
Stephen M
SM
Beckstrom-Sternberg
Talima
T
Pearson
William D
WD
Brown
Danika M
DM
Kiesling
Christina A
CA
Allen
Cindy M
CM
Liu
James
J
Beckstrom-Sternberg
Frank F
FF
Roberto
Paul
P
Keim
D000818:Animals; D002003:Brucella abortus / Q000145:classification* / Q000235:genetics; D017347:Brucella melitensis / Q000145:classification* / Q000235:genetics; D041021:Brucella suis / Q000145:classification* / Q000235:genetics; D002006:Brucellosis / Q000382:microbiology / Q000662:veterinary; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods; D005838:Genotype; D006801:Humans; D008322:Mammals; D046228:Microarray Analysis / Q000379:methods; D058889:Molecular Typing; D010802:Phylogeny; D020641:Polymorphism, Single Nucleotide
D004269:DNA, Bacterial