Phenotypic and genetic characterization of a novel Borrelia burgdorferi sensu lato isolate from a patient with lyme borreliosis

Abstract

Borrelia burgdorferi sensu lato A14S was cultured from a skin biopsy specimen of a patient with erythema migrans in The Netherlands. This isolate had a unique DNA fingerprint pattern compared to 135 other B. burgdorferi sensu lato isolates. In this study, the isolate A14S was further characterized by protein analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reactivity with various monoclonal antibodies. In addition, the 16S rRNA, ospA, and ospC genes, as well as the 5S-23S rRNA intergenic spacer DNA, were amplified by PCR, cloned, and sequenced. SDS-PAGE protein profiles and phylogenetic analysis based on all of the analyzed genes confirmed that B. burgdorferi sensu lato A14S was phenotypically and genetically different from the three human pathogenic species B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, as well as from other B. burgdorferi sensu lato species. Our findings indicate that Borrelia genomic groups or isolates other than the three well-known human pathogenic species may also cause human Lyme borreliosis.

FIG. 1

Silver-stained SDS-PAGE gel of whole-cell lysates of B. burgdorferi sensu lato strains. Lanes 1 to 5, B. burgdorferi sensu stricto B31, B. garinii 20047, B. afzelii VS461, B. valaisiana AR-2, and Borrelia isolate A14S, respectively. The positions of flagellin (Fla), OspA, OspB, and OspC are indicated by arrows. The unique protein bands of isolate A14S are marked with asterisks. Molecular mass standards (Pharmacia) are shown on the right.

FIG. 2

Sequences of the 5S-23S rRNA intergenic spacers of Borrelia sp. strain A14S and the type strains of B. burgdorferi species known to be pathogenic to humans. Gaps were introduced to obtain maximum levels of homology. The nucleotides identical among all isolates are indicated with asterisks under the sequences, and the unique nucleotide positions of isolate A14S are shaded in black. The accession numbers of the 5S-23S rRNA intergenic spacer sequences of Borrelia strains used for comparison are L30127 (B. burgdorferi sensu stricto B31), L30119 (B. garinii 20047), L30135 (B. afzelii VS461), and L30126 (B. bissettii DN127).

FIG. 3

Phylogenetic tree of B. burgdorferi sensu lato strains based on 1,330 bp of 16S rRNA gene sequences (positions 34 to 1364). B. hermsii HS1 was used as an out-of-group control. Numbers at each of the branch nodes indicate results from bootstrap analysis. The accession numbers (in parentheses) of the 16S rRNA gene sequences of Borrelia strains used for comparison are as follows: B31 (U03396), DK7 (X85195), 20047 (D67018), PBi (X85199), DK1 (X85190), IP3 (M84815), HO14 (L40597), VS116 (X98232), UK (X98233), PotiB2 (X98228), 21038 (L46701), DN127 (L40596), Hk501 (D67023), Ya501 (D67022), and HS1(U42292).