Linear and circular plasmid content in Borrelia burgdorferi clinical isolates

Abstract

The genome of Borrelia burgdorferi, the etiologic agent of Lyme disease, is composed of a linear chromosome and more than 20 linear and circular plasmids. Typically, plasmid content analysis has been carried out by pulsed-field gel electrophoresis and confirmed by Southern hybridization. However, multiple plasmids of virtually identical sizes (e.g., lp28 and cp32) complicate the interpretation of such data. The present study was undertaken to investigate the complete plasmid complements of B. burgdorferi clinical isolates cultivated from patients from a single region where early Lyme disease is endemic. A total of 21 isolates obtained from the skin biopsy or blood samples of Lyme disease patients were examined for their complete plasmid complements by Southern hybridization and plasmid-specific PCR analysis. All clinical isolates harbored at least six of the nine previously characterized cp32s. Fourteen isolates harbored all B31-like linear plasmids, and seven isolates simultaneously lacked lp56, lp38, and some segments of lp28-1. The distinctive plasmid profile observed in these seven isolates was specific to organisms that had ribosomal spacer type 2 and pulsed-field gel type A, which implies a clonal origin for this genotype. The presence of nearly identical complements of multiple linear and circular plasmids in all of the human isolates suggests that these plasmids may be particularly necessary for infection, adaptation, and/or maintenance in the infected host.

FIG. 1.

Simultaneous detection of 10 plasmids of B. burgdorferi by multiplex PCR. PCR amplification by using genomic DNA from strain B372 or B297 as a template was performed as detailed in Materials and Methods with the primers listed in Table 1. Products obtained using primer sets A, B, and C are shown in lanes 3 and 4, 5 and 6, and 7 and 8, respectively. Lanes 1 and 2 contain DNA molecular size markers and a control lacking DNA, respectively.

FIG. 2.

Plasmid analysis for four representative isolates by Southern hybridization. (A) PFG electrophoresis blot hybridized with an lp38-specific probe (ospD); (B) blot hybridized with an lp28-1-specific probe (bbf01); (C) blot in panel B stripped and hybridized with a second lp28-1-specific probe (vlsE); (D) blot hybridized with an lp56-specific probe (bbq67). Lanes 1 through 4 contain undigested genomic DNA (A) or EcoRI-digested genomic DNA (B through D) from isolates BL219, B149, BL162, and B297, respectively.

FIG. 3.

Detection of multiple cp32 plasmids by PCR amplification of the orfC-orf3 locus. PCR analysis was performed for each cp32 as described in Materials and Methods by using the primers shown in Table 1. (A) Isolate B297; (B) isolate B372. In each panel, the lanes contain products obtained with primers specific for cp32-1 (lane 1), cp32-2/7 (lane 2), cp32-3 (lane 3), cp32-4 (lane 4), cp32-5 (lane 5), cp32-6 (lane 6), cp32-8 (lane 7), cp32-9 (lane 8), and lp56 (lane 9). Migration positions for DNA molecular size markers are indicated to the left of each panel.