The genes coding for the outer surface protein A (OspA) of 19 different Borrelia burgdorferi strains belonging to the seven OspA-serotypes 1-7, previously described [Wilske et al. (1993) J Clin Microbiol, 31: 340-350], have been investigated. B. burgdorferi sensu lato strains were chosen from various biological sources (ticks, human skin and cerebrospinal fluid) as well as different geographical origins (Germany, Slovenia, Austria, United States). The open reading frames of all ospA genes consist of 819-825 nucleotides corresponding to proteins of approximately 30 kDa. The ospA sequences obtained in this study and previous published studies were compared with the results from OspA serotyping with monoclonal antibodies. The classification into the seven OspA serotypes could be confirmed on a genetic basis (ospA genotypes 1-7) for all strains analyzed so far (n = 29). In addition, one strain without OspA expression could be assigned to ospA genotype 2. Genetic stability could be proven for the ospA gene of B. burgdorferi strain PWudI after inocculation and reisolation from a gerbil. However, we found evidence for intragenic recombination by cluster analysis of ospA sequence data. Accordance of ospA genotype 1 strains with B. burgdorferi sensu stricto and ospA genotype 2 strains with B. afzelii, as well as the ospA genotype strains 3-7 with B. garinii was confirmed by pulsed-field gel electrophoresis of MluI-digested genomic DNA. B. garinii is not only more heterogeneous in respect to the OspA-encoding genes, but shows moreover major subgroups formed by genotypes 4, 5 and 6 and genotypes 3 and 7, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

25 skin biopsy isolates of Borrelia burgdorferi sensu lato, mainly from German patients with erythema migrans [EM], borrelial lymphocytoma [BL] and acrodermatitis chronica atrophicans [ACA], were species-differentiated using pulsed-field gel electrophoresis (PFGE). The isolates revealed 15 B. afzelii, 8 B. garinii and 2 B. burgdorferi sensu stricto species according to Miu I digestion. All 6 ACA isolates were identified as B. afzelii species, whereas the 2 borrelial lymphocytoma isolates were grouped into B. garinii species. 12 B. afzelii strains, including 4 ACA isolates, were further investigated by digestion with 5 restriction enzymes. Most of the strains revealed an individual pattern. No specific characteristically large restriction fragment pattern (LRFP) could be detected within the ACA and/or EM group. The ACA isolates could not be clearly differentiated from the EM isolates according to the LRFP patterns. PFGE is thus a highly effective method for detecting differences among B. afzelii isolates.

Neuroborreliosis is the most frequent manifestation of the second stage of Lyme borreliosis in Europe. However, only few isolates from the cerebrospinal fluid (CSF) have been characterized with controversial results. A large panel of 36 CSF isolates isolated over a 10-year period in Munich has now been analyzed for their OspA and OspC type, resulting in at least eight different types, respectively. Representatives of the different types cultivated from CSF in Munich have also been isolated from other geographical regions in Europe from CSF or ticks, suggesting a widespread distribution of pathogenic strains. A certain OspA type (type 4) was frequently observed in adults but rarely in children or ticks. Since OspA and OspC are the most promising candidates for a Borrelia vaccine, the considerable heterogeneity found among CSF isolates has important implications for development of a vaccine in Europe.

A total of 36 European Borrelia burgdorferi sensu lato cerebrospinal fluid isolates (mainly from southern Germany) were analyzed by pulsed-field gel electrophoresis (PFGE) for large restriction fragment pattern (LRFP) and linear plasmid profiles. Analyzing this large panel of isolates, we detected all three species of B. burgdorferi sensu lato pathogenic for humans in cerebrospinal fluid from patients with Lyme neuroborreliosis by PFGE typing after MluI digestion: 21 B. garinii (58%), 10 B. afzelii (28%), and 4 B. burgdorferi sensu stricto (11%) strains as well as 1 isolate with bands characteristic of both B. afzelii and B. garinii. Species classification by PFGE typing was confirmed by 16S rRNA-specific PCR. Eighteen isolates (11 B. garinii, 6 B. afzelii, and 1 B. burgdorferi sensu stricto isolate) were further characterized by LRFP with four different restriction enzymes (ApaI, KspI, SmaI, and XhoI). All B. afzelii isolates showed identical patterns for each restriction enzyme group. Considerable heterogeneity was demonstrated within the B. garinii group. Subsequent analysis of plasmid profiles revealed only marginal differences for B. afzelii strains but different patterns for B. garinii isolates. In one B. afzelii strain we found a linear plasmid of about 110 kbp not described before. LRFP analysis by PFGE is a suitable tool for the molecular characterization of B. burgdorferi sensu lato strains and allows determination not only of the species but also of the subtypes within B. garinii.

As clinical persistence of Borrelia burgdorferi in patients with active Lyme borreliosis occurs despite obviously adequate antibiotic therapy, in vitro investigations of morphological variants and atypical forms of B. burgdorferi were undertaken. In an attempt to learn more about the variation of B. burgdorferi and the role of atypical forms in Lyme borreliosis, borreliae isolated from antibiotically treated and untreated patients with the clinical diagnosis of definite and probable Lyme borreliosis and from patient specimens contaminated with bacteria were investigated. Furthermore, the degeneration of the isolates during exposure to penicillin G in vitro was analysed. Morphological analysis by darkfield microscopy and scanning electron microscopy revealed diverse alterations. Persisters isolated from a great number of patients (60-80%) after treatment with antibiotics had an atypical form. The morphological alterations in culture with penicillin G developed gradually and increased with duration of incubation. Pleomorphism, the presence of elongated forms and spherical structures, the inability of cells to replicate, the long period of adaptation to growth in MKP-medium and the mycoplasma-like colonies after growth in solid medium (PMR agar) suggest that B. burgdorferi produce spheroplast-L-form variants. With regard to the polyphasic course of Lyme borreliosis, these forms without cell walls can be a possible reason why Borrelia survive in the organism for a long time (probably with all beta-lactam antibiotics) [corrected] and the cell-wall-dependent antibody titers disappear and emerge after reversion.