Characterization of Borrelia burgdorferi Isolated from Erythema Migrans Lesions: Interrelationship of Three Molecular Typing Methods

Abstract

Genetic diversity among Borrelia burgdorferi isolates recovered from the skin of Lyme disease patients was assessed by ribosomal DNA (rDNA) spacer restriction fragment length polymorphism analysis, genomic restriction site polymorphism analysis, and plasmid content analysis. There was a significant association between the three rDNA spacer types, the six pulsed-field gel types, and plasmid content (P < 0.001). The association between distinct chromosomal and plasmid markers implies a clonal origin for each genotype.Lyme disease, a disorder which may affect multiple organ systems, results from infection with the spirochete Borrelia burgdorferi. Between 1989 and 1997, 114,314 cases of Lyme disease were reported to the Centers for Disease Control and Prevention, making Lyme disease the most common vectorborne disease in the United States (16,21). Early Lyme disease is manifested by a characteristic skin rash, erythema migrans (EM), and is frequently accompanied by systemic symptoms (14, 15). Molecular analysis of B. burgdorferi isolates has resulted in the differentiation of B. burgdorferi sensu lato into 10 distinct species (23). B. burgdorferi sensu stricto is the only known Lyme disease borrelia infecting patients in North America. Significant genetic heterogeneity among B. burgdorferi sensu stricto isolates in North America has been reported (1,6,8,13). The type strain of B. burgdorferi sensu stricto, B31 has a large linear genome of 910,725 bp and 21 linear and circular plasmids with a total size of 610,694 bp (3, 7).It has long been recognized that the clinical expression of B. burgdorferi infection is diverse (15). Possible explanations for this variability are differences in the genotypes of the infecting strain of B. burgdorferi. We developed a typing method based on restriction fragment length polymorphism (RFLP) in the 16S-23S ribosomal DNA (rDNA) spacer of B. burgdorferi and used it to analyze clinical isolates obtained from early Lyme disease patients (9). A highly significant association was found between the infecting RFLP type in the skin and the presence of spirochetemia or multiple EM lesions (25), suggesting that differences in the clinical presentations of Lyme disease patients may, indeed, be related to B. burgdorferi genotype. rDNA spacer analysis provides genetic typing information for a single genomic locus and interrogates a chromosomal region which is unlikely to have a direct role in pathogenesis. In order to obtain additional, broader typing information, 48 cutaneous isolates from early Lyme disease patients were analyzed by pulsed-field gel electrophoresis (PFGE).Subjects, skin biopsy, and cultivation. All subjects were adults with EM enrolled in a prospective study at the Lyme Disease Diagnostic Center of the Westchester Medical Center, Valhalla, N.Y. Skin biopsy specimens (2 mm) were obtained from the leading edge of primary EM lesions and cultured in BSK-II medium as described previously (19).PFGE. Spirochetes were harvested by centrifugation and washed twice with sterile phosphate-b...

Detection of Established Virulence Genes and Plasmids To Differentiate Borrelia burgdorferi Strains

“…(ii) A restriction fragment length polymorphism (RFLP) between the 16S and 23S rRNA genes has been used to categorize isolates into four ribosomal spacer types (RSTs) called RST1, RST2, RST3A, and RST3B. The RST1, RST2, and RST3B groups have been shown to be significantly associated with invasiveness and disease in the mouse model of infection and in humans (46,49,57,58,60,61,103,104,107,108). However, these methods alone cannot always be used to distinguish various B. burgdorferi strains, and these classifications do not contribute at all to determination of the overall plasmid content of the strains.…”

“…Before the completion of the sequence of B31, pulsed-field gel electrophoresis (PFGE) was primarily employed in determining the plasmid content of various spirochete strains, even though large circular plasmids are not resolved well in such gels and different linear plasmids often have very similar sizes in the 25-to 30-kb range (8,63,111,112). PFGE is still used to discriminate the plasmid profiles of B. burgdorferi clinical isolates (45,46,84,85), although plasmid loss in culture (see below) can make it an unreliable indicator of relatedness.…”

Identification of Three Clinically Relevant Borrelia burgdorferi Sensu Lato Genospecies by PCR-Restriction Fragment Length Polymorphism Analysis of 16S-23S Ribosomal DNA Spacer Amplicons

“…Also in this study, the 16S-23S rDNA nested-PCR-RFLP typing method, originally used for the typing of B. burgdorferi in clinical specimens from early LB patients (12,17), was extended to include the typing of B. garinii and B. afzelii species directly in field-collected ticks. Only a brief mention of such an approach was found in the literature (17).…”

Comparative Analysis of the Properties and Ligand Binding Characteristics of CspZ, a Factor H Binding Protein, Derived fromBorrelia burgdorferiIsolates of Human Origin

“…Isolates were cultivated in BSK-H complete media (Sigma-Aldrich) at 33°C in sealed bottles under 5% CO 2 and were harvested by centrifugation (14,000 ϫ g, 4°C, 20 min). The isolates were obtained from human Lyme disease patients residing in New York and Maryland and have been characterized in previous studies (1,11,22,23,47). Brief descriptions of the isolates employed in this study and their genotypic properties and FH binding abilities are shown in Table 1.…”