Use of Highly Variable Intergenic Spacer Sequences for Multispacer Typing ofRickettsia conoriiStrains

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Abstract
By use of the nearly perfectly colinear genomes ofRickettsia conoriiandRickettsia prowazekii, we compared the usefulness of three types of sequences for typing ofR. conoriiisolates: (i) 5 variable coding genes comprising the 16S ribosomal DNA,gltA,ompB, andsca4(gene D) genes, which are present in both genomes, and theompAgene, which is degraded inR. prowazekii; (ii) 28 genes degraded inR. conoriibut intact inR. prowazekii, including 23 split and 5 remnant genes; and (iii) 27 conserved and 25 variable intergenic spacers. The 4 conserved and 23 split genes as well as the 27 conserved intergenic spacers each had identical sequences in 34 human and 5 tick isolates ofR. conorii. Analysis of theompAsequences identified three genotypes ofR. conorii. The variable intergenic spacers were significantly more variable than conserved genes, split genes, remnant genes, and conserved spacers (P< 10−2in all cases). Four of the variable intergenic spacers (dksA-xerC,mppA-purC,rpmE-tRNAfMet, and tRNAGly-tRNATyr) had highly variable sequences; when they were combined for typing, multispacer typing (MST) identified 27 different genotypes in the 39R. conoriiisolates. Two batches from the sameR. conoriistrain, Malish (Seven), with different culture passage histories were found to exhibit the same MST type. MST was more discriminatory for strain genotyping than multiple gene sequencing (P< 10−2). Phylogenetic analysis based on MST sequences was concordant with the geographic origins ofR. conoriiisolates. Our study supports the usefulness of MST for strain genotyping. This tool may be useful for tracing a strain and identifying its source during outbreaks, including those resulting from bioterrorism.