Expanded Diversity among Californian Borrelia Isolates and Description of Borrelia bissettii sp. nov. (Formerly Borrelia Group DN127)

Up to now, the only species in the complex Borrelia burgdorferi sensu lato known to cause Lyme borreliosis in the United States has been B. burgdorferi sensu stricto. However, some atypical strains closely related to the previously designated genomic group DN127 have been isolated in the United States, mostly in California. To explore the diversity of B. burgdorferi sensu lato group DN127, we analyzed the nucleotide sequences of the rrf-rrl intergenic spacer regions from 19 atypical strains (18 from California and one from New York) and 13 North American B. burgdorferi sensu stricto strains (6 from California). The spacer region sequences from the entire B. burgdorferi sensu lato complex available in data banks were used for comparison. Phylogenetic analysis of sequences shows that the main species of the B. burgdorferi sensu lato complex (B. afzelii, B. garinii, B. andersonii, B. japonica, B. burgdorferi sensu stricto, B. valaisiana, and B. lusitaniae) each form a coherent cluster. A heterogeneous group comprising strains belonging to the previously designated group DN127 clustered separately from B. burgdorferi sensu stricto. Within this cluster, the deep branches expressing the distances between the rrf-rrl sequences reflect a high level of divergence. This unexpected diversity contrasts with the monomorphism exhibited by B. burgdorferi sensu stricto. To clarify the taxonomic status of this highly heterogeneous group, analysis of the rrs sequences of selected strains chosen from deeply separated branches was performed. The results show that these strains significantly diverge at a level that is compatible with several distinct genomic groups. We conclude that the taxonomy and phylogeny of North American B. burgdorferi sensu lato should be reevaluated. For now, we propose that the genomic group DN127 should be referred to as a new species, B. bissettii sp. nov., and that other related but distinct strains, which require further characterization, be referred to as Borrelia spp.In Eurasia, seven species of the complex Borrelia burgdorferi sensu lato have been reported. Only three of these species are associated with Lyme borreliosis. It has also been shown that each pathogenic species is associated predominantly with a given clinical presentation; Borrelia burgdorferi sensu stricto is associated with arthritis, B. garinii is associated with neuroborreliosis, and B. afzelii is associated with late cutaneous symptoms (2, 39). Up to now, B. burgdorferi sensu stricto is the only species associated with Lyme borreliosis in North America. However, two other B. burgdorferi sensu lato genospecies coexist in the United States, B. andersonii (22) and the genomic group DN127 (3, 32). B. andersonii seems to be restricted to a limited ecosystem involving cottontail rabbits and Ixodes dentatus ticks. In contrast, the genomic group DN127 appears to be involved in several enzootic transmission cycles (6, 29). A recent study demonstrated substantial genetic heterogeneity among Californian and other American strains (24). We took advantage of the unique structure of ribosomal genes in B. burgdorferi sensu lato to analyze the polymorphism of some strains isolated in California. A single copy of the rrs gene is separated by a large spacer (rrs-rrl; 3,000 to 5,000 bp) from two tandemly duplicated copies of rrl and rrf genes (13, 36). These two copies are separated by a small spacer, rrf-rrl, which is approximately 250 bp long. The genetic heterogeneity of the group DN127 was first evidenced by analysis of the restriction patterns of the rrf-rrl spacer (32). However, the results of DNA-DNA hybridization on a limited number of strains (32) allowed us to place them in a single genomic group. To clarify the genetic relationships between diverse North American strains, 20 atypical strains were compared with 13 B. burgdorferi sensu stricto strains. Identification procedures involved restriction polymorphism and sequencing studies of both the variable rrf-rrl spacer and the conserved rrs gene. Sequences of the rrf-rrl spacer and the rrs gene were used in a phylogenetic analysis. Some Californian strains are closely related to the genomic group DN127, for which we propose the name of B. bissettii sp. nov. Other atypical strains which do not fall into this group are designated merely as Borrelia spp. in this study. The latter strains cannot be assigned to specific genomic groups until more isolates representative of each group are available for further characterization.