Proposal to create subspecies of Rickettsia conorii based on multi-locus sequence typing and an emended description of Rickettsia conorii

Abstract

Background: Rickettsiae closely related to the Malish strain, the reference Rickettsia conorii strain, include Indian tick typhus rickettsia (ITTR), Israeli spotted fever rickettsia (ISFR), and Astrakhan fever rickettsia (AFR). Although closely related genotypically, they are distinct serotypically. Using multilocus sequence typing (MLST), we have recently found that distinct serotypes may not always represent distinct species within the Rickettsia genus. We investigated the possibility of classifying rickettsiae closely related to R. conorii as R. conorii subspecies as proposed by the ad hoc committee on reconciliation of approaches to bacterial systematics. For this, we first estimated their genotypic variability by using MLST including the sequencing of 5 genes, of 31 rickettsial isolates closely related to R. conorii strain Malish, 1 ITTR isolate, 2 isolates and 3 tick amplicons of AFR, and 2 ISFR isolates. Then, we selected a representative of each MLST genotype and used multi-spacer typing (MST) and mouse serotyping to estimate their degree of taxonomic relatedness.

Results: Among the 39 isolates or tick amplicons studied, four MLST genotypes were identified: i) the Malish type; ii) the ITTR type; iii) the AFR type; and iv) the ISFR type. Among these four MLST genotypes, the pairwise similarity in nucleotide sequence varied from 99.8 to 100%, 99.4 to 100%, 98.2 to 99.8%, 98.4 to 99.8%, and 99.2 to 99.9% for 16S rDNA, gltA, ompA, ompB, and sca4 genes, respectively. Representatives of the 4 MLST types were also classified within four types using MST genotyping as well as mouse serotyping.

Conclusion: Although homogeneous genotypically, strains within the R. conorii species show MST genotypic, serotypic, and epidemio-clinical dissimilarities. We, therefore, propose to modify the nomenclature of the R. conorii species through the creation of subspecies. We propose the names R. conorii subsp. conorii subsp. nov. (type strain = Malish, ATCC VR-613), R. conorii subspecies indica subsp. nov. (type strain = ATCC VR-597), R. conorii subspecies caspia subsp. nov. (type strain = A-167), and R. conorii subspecies israelensis subsp. nov. (type strain = ISTT CDC1). The description of R. conorii is emended to accomodate the four subspecies.

Figure 1

Unrooted phylogenetic tree derived from the comparison of sequences of the complete gltA gene from rickettsiae closely related to R. conorii and other validated Rickettsia species. The analysis used the Neighbor Joining method and the Kimura 2 parameter as described in the Methods section. Bootstrap values are indicated at the nodes of the phylogenetic tree. GenBank accession numbers are indicated in parentheses for each taxon.

Figure 2

A = Unrooted phylogenetic trees derived from the comparison of sequences of the 16S rDNA, gltA, ompA, ompB and sca4 genes using the Neighbor Joining method (A). Bootstraps values are indicated at the nodes of the phylogenetic tree. The scale bars represent the percentage of nucleotide differences. RC = R. conorii; ITTR = Indian tick typhus rickettsia; AFRA = AFR isolate A-167; AFRC = AFR isolate Chad; ISFR = Israeli spotted fever rickettsia. B = Unrooted phylogenetic trees derived from the comparison of concatenated sequences of the dksA / xerC, mppA / purC, and rpmE / tRNA-fMet intergenic spacers using the Neighbor Joining method (B). Bootstraps values are indicated at the nodes of the phylogenetic tree. The scale bars represent the percentage of nucleotide differences. RC = R. conorii; ITTR = Indian tick typhus rickettsia; AFRA = AFR isolate A-167; AFRC = AFR isolate Chad; ISFR = Israeli spotted fever rickettsia. C = Taxonomic dendrogram of rickettsiae closely related to Rickettsia conorii obtained by using the unweighted pair group method based on reactivity of mouse antisera to all tested rickettsiae. RC = R. conorii; ITTR = Indian tick typhus rickettsia; AFRA = AFR isolate A-167; AFRC = AFR isolate Chad; ISFR = Israeli spotted fever rickettsia.