Genotyping Rickettsia conorii detected in patients with Mediterranean spotted fever in Algeria using multispacer typing (MST)

Abstract

The aetiologic agent of Mediterranean spotted fever (MSF), Rickettsia conorii, is an obligate intracellular bacterium transmitted by the dog tick, Rhipicephalus sanguineus, to humans. Because of the fastidious nature of R. conorii, amplification and sequencing of partial gltA and ompA genes were frequently used for detection of this bacterium in ticks and molecular diagnosis of R. conorii infection in humans [1]. However, gltA and ompA sequences are too conserved to explore the intraspecies diversity of R. conorii. Multispacer typing (MST) incorporating three spacer sequences, dksA-xerC, mppA-purC and rpmE-tRNA, proved to be a reproducible and high-resolution genotyping method allowing not only clear identification of rickettsial strains at the species level but also strain-typing within some species including R. conorii [2,3]. In this study, we used MST to directly subtype R. conorii strains detected in skin biopsies from Algerian patients with MSF. Skin biopsies with tick bite were removed from Algerian patients with clinical diagnosis of MSF in Oran, Algeria, during January 2004 to December 2005. Total genomic DNAs were extracted from the skin biopsies using the QIAamp Tissue kit (QIAGEN, Hilden, Germany) as described by the manufacturer. Amplification of partial gltA and rompA genes was performed to detect R. conorii in the skin biopsies using the primer pairs 409 and 1258 and the primer pairs 190–70 and 190–701, respectively, as described [1]. Amplification and sequencing of the three spacers, dksA-xerC, mppA-purC and rpmE-tRNA, were performed as described [2]. To avoid contamination, no positive control was used. Sterile water was used as a negative control in each PCR assay. Amplicons were sequenced in both directions using the BigDye 1.1 chemistry (Applied Biosystems) on an ABI 3130XL automated sequencer (Applied Biosystems) as described by the manufacturer. Nucleotide sequences were edited using the Autoassembler package (PerkinElmer). For each intergenic spacer, a genotype was defined as a sequence exhibiting unique polymorphism that was defined by sequencing the corresponding spacers using forward and reverse primers, respectively. MST genotypes were defined as unique combinations of the three spacer genotypes. The sequences of three studied spacers were concatenated for phylogenetic analysis. We incorporated 27 MST genotypes previously identified among 39 R. conorii strains [2] and those identified in this study for phylogenetic analysis. Multiple alignment of sequences was carried out using the CLUSTALW software (Courtaboeuf, France). Phylogenetic relationships were obtained using the neighbour-joining and maximum parsimony methods within the MEGA 2.1 software (Courtaboeuf, France) [2]. R. conorii was detected in 22 skin biopsies from 22 patients with MSF using PCR assays targeting the partial gltA and ompA genes. For spacers mppA-purC and rpmE-tRNA, the 22 R. conorii human strains hold identical sequences and genotypes, types B and B, respectively, which have been previously identified [2]. Seven genotypes of spacer dksA-xerC were identified among 22 studied strains. Five of them, genotypes AS, AT, AU, AV and AW, were new and the sequences of them were submitted to GenBank with accession number: EU665226–EU665230. In total, seven MST genotypes based on the combination of genotypes of three spacers were identified among the 22 R. conorii human strains (Fig. 1). Of these, genotype 3 including 15 strains (68%) was previously identified. The Corresponding author and reprint requests: D. Raoult, CNRSIRD UMR 6236, Faculte de Medecine, Universite de la Mediterranee, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France E-mail: didier.raoult@univmed.fr