Bettina Wilske

European Borrelia burgdorferi isolated from humans and ticks culture conditions and antibiotic susceptibility

Growth of Borrelia burgdorferi in a modified Kelly-medium is described. Borrelia strains were isolated from patients (n = 11) and ticks I. ricinus (n = 19). The modified medium which contained Co-trimoxazole is a very effective medium for isolating and culturing of Borrelia sp. The susceptibility of 7 strains of B. burgdorferi to antibiotics was studied by the macrodilution and microdilution test. After preliminary testing for optimal conditions, we determined MICs in modified Kelly medium. The MIC concentration of each antibiotic was determined as the lowest concentration which completely inhibited growth of the tested organism. The B. burgdorferi was most susceptible to Erythromycin with MIC of less than or equal to 0.15 microgram/ml. Of the Penicillins tested, Ampicillin and Mezlocillin were more active than Penicillin G. The use of Tetracycline-HCl is recommended because of its low MIC in vitro its extra- and intracellular efficiency.

Antigenic Variability of Borrelia burgdorferi

Borrelia burgdorferi strains (six isolates from North America and 28 isolates from Europe) were analyzed by physicochemical and immunological methods. By SDS-PAGE, all Borrelia burgdorferi strains tested had two major proteins with constant molecular weights of 60 and 41 kDa and one, two, or three variable low molecular weight proteins (OspA = 30-32 kDa, OspB = 34-36 kDa, pC = 21-22 kDa). All combinations--except OspB alone or OspB/pC--were observed. Borrelia burgdorferi strains were different from relapsing fever borreliae by strong reactivity with OspA- and/or pC-specific polyclonal antibodies, whereas relapsing fever borreliae were only weakly reactive. Among 25 Borrelia burgdorferi isolates, seven different serotypes of Borrelia burgdorferi were defined according to their reactivity in the Western blot with three monoclonal OspA-specific antibodies (H5332, H3TS, and LA5), four OspA- or OspB-specific polyclonal antibodies, and 12 polyclonal antibodies against whole borreliae. Antigenic differences between European CSF and skin isolates were observed, all skin isolates (n = 11) belonging to serotype 2 in contrast to only two out of seven CSF isolates. CSF isolates were antigenically heterogenous (serotypes 1, 2, 3, 4, and 5). Serotypes 6 and 7 were represented by two tick isolates, and the other European tick isolates are not yet fully characterized. Antigenic differences between European and North American strains may play a role in differences in the clinical picture of Lyme borreliosis.

A new Borrelia species defined by multilocus sequence analysis of housekeeping genes.

ABSTRACT Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.

Survival of Borrelia burgdorferi in antibiotically treated patients with lyme borreliosis

SummaryThe persistence ofBorrelia burgdorferi in patients treated with antibiotics is described. The diagnosis of Lyme disease is based on clinical symptoms, epidemiology and specific IgG and IgM antibody titers toB. burgdorferi in serum. Antibiotic therapy may abrogate the antibody response to the infection as shown in our patients.B. burgdorferi may persist as shown by positive culture in MKP-medium; patients may have subclinical or clinical disease without diagnostic antibody titers toB. burgdorferi. We conclude that early stage of the disease as well as chronic Lyme disease with persistence ofB. burgdorferi after antibiotic therapy cannot be excluded when the serum is negative for antibodies againstB. burgdorferi.ZusammenfassungEs wird über die Persistenz vonBorrelia burgdorferi bei sechs Patienten berichtet. Nach dem Zecken- bzw. Insektenstich und Erythema migrans konnteB. burgdorferi noch Wochen nach der Antibiotikatherapie nachgewiesen werden. Serologische Befunde waren außer bei einem Patienten negativ. Diese Ergebnisse bestätigen unsere früheren Beobachtungen und sprechen dafür, dß die Antibiotikabehandlung die Antikörperbildung gegenB. burgdorferi beeinflussen kann. Ferner zeigen diese Ergebnisse und Beobachtungen, daß nicht nur im Frühstadium der Lyme Borreliose, sondern auch in chronischen Stadien bzw. bei Persistenz des Erregers der Nachweis von Antikörpern negativ bleiben kann.

Immunochemical and immunological analysis of European Borrelia burgdorferi strains

Borrelia burgdorferi strains (n = 23), isolated from patients (n = 8) and ticks (n = 14), were analyzed by SDS PAGE and Western blotting with monoclonal antibodies and polyclonal sera (rabbit immune serum and sera from patients). Testing the 23 strains by SDS PAGE 9 different patterns of major protein bands were observed. In contrast to US strains some of our strains showed only weak or negative reactivity with the OspA specific monoclonal antibody H5332. Analysis with polyclonal sera gave further support that the OspA proteins of our strains have specific epitopes (recognized by patients sera) besides common ones (recognized by rabbit immune serum). Fifteen of the strains had another major protein in the 22K range without detectable antigenic variability. Patients with erythema migrans and lymphocytic meningitis had a good IgM- and IgG-immunoresponse to this protein. In contrast antibodies to the OspA were very rarely observed, probably due to the antigenetic heterogeneity of the causative agent on the one hand but also due to low response of the human immune system to the OspA on the other hand.

Active immunization with pC protein of Borrelia burgdorferi protects gerbils against B. burgdorferi infection

SummarySerious infection due toBorrelia burgdorferi and the disseminated infection characteristic of the disease possess unique treatment problems. The wide and still increasing incidence of Lyme borreliosis as well as the problems in treatment call for effective prevention strategies by active immunization. Vaccination experiments were done to determine if active immunization of gerbils with recombinant OspA and pC protects against infection with strains ofB. burgdorferi. Gerbils were vaccinated with recombinant OspA and pC (20 kDa protein) and challenged four weeks later with a clone (derived fromB. burgdorferi strain PKo) which expresses an abundant amount of pC but only little OspA. Non-immunized gerbils challenged with the sameB. burgdorferi strain were used as controls. Both groups of immunized gerbils developed antibodies against the recombinant vaccines. The pC vaccinated group was protected against infection, whereas the OspA vaccinated group showed signs of infection. The non-vaccinated group developed generalised infection. These results show that pC should be considered as a further vaccine candidate and probably needs to be combined with OspA for an efficient vaccine againstB. burgdorferi.ZusammenfassungWeite Verbreitung und zunehmende Inzidenz der Lyme Borreliose sowie Therapieprobleme bei schweren Erkrankungsformen besonders im Spätstdadium der Infektion sind Gründe für die Entwicklung einer möglichen Alternative zur Antiobitikatherapie wie zum Beispiel Schutzimpfung. Die Schutzwirkung einer aktiven Immunisierung mit rekombinanten OspA und pC gegen die Infektion mitBorrelia burgdorferi wurde bei Gerbils geprüft. Die Tiere wurden mit rekombinanten OspA und pC vomB. burgdorferi-Stamm PKo immunisiert; die Infektion erfolgte vier Wochen nach der Immunisierung mit dem PKo-Stamm (schwache OspA, gute pC Expression). Als Kontrollgruppe dienten infizierte, nicht immunisierte Gerbils. Die immunisierten Tiere bildeten Antikörper gegen rekombinante Vakzine. Die mit pC immunisierten Tiere waren vor der Infektion geschützt, die Kontrollgruppe zeigte eine generalisierte Infektion. Die Immunisierung mit OspA schützte nicht, die Tiere zeigten, wie die Kontrollgruppe Merkmale der Infektion. Die Ergebnisse dieser Studie zeigen, daß pC für eine Immunisierung in Frage kommt. Eine Kombination von OspA und pC scheint für eine effektive Vakzine gegenBorrelia burgdorferi nötig zu sein.

Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22kDa protein (pC) in Escherichia coli

We describe the cloning and expression of the pc gene which encodes a major immunodominant protein of Borrelia burgdorferi, the causative agent of Lyme borreliosis. The pC protein was purified from lysates of B. burgdorferi strain PKo. After tryptic digestion of the pC protein the resulting oligopeptides were applied to a gas‐phase sequenator. Thus partial amino acid sequences were obtained. The deduced oligonucleotides were used as hybridization probes. After Southern blotting a reactive band in the 3 kb range of PstI‐digested genomic DNA was detected. The insertion of these fragments into pUC vectors finally resulted in pc‐positive Escherichia coli clones. The gene (encoding a protein with 212 amino acids) was expressed in E. coli with varying deletions at the 5′ end. A sequence comparison with other outer membrane proteins of B. burgdorferi indicates a processing of pC that is similar to that of lipoproteins.

In vitro and in vivo susceptibility ofBorrelia burgdorferi

The antispirochetal activity in vitro and in vivo of several antibiotics against ten isolates ofBorrelia burgdorferi from human spinal fluids and skin biopsies was determined.Borrelia burgdorferi was most susceptible in vitro to erythromycin, ceftriaxone and cefotaxime (MIC90: 0.06, 0.06, 0.12 meg/ml respectively). Less activity was observed with tetracycline, amoxycillin and lincomycin (MIC90: 0.50 mcg/ml), imipenem and augmentin (MIC90. 0.25 mcg/ml), oxacillin (MIC90: 1 mcg/ml), ciprofloxacin (MIC90: 2 mcg/ml) and ofloxacin (MIC 90: 4 mcg/ml). Penicillin G, normally regarded as appropriate treatment for Lyme disease, had an MIC90 of only 4 mcg/ml. With the exception of erythromycin, activity in vitro corresponded to the activity in vivo. Erythromycin, however, was less active in vivo, and penicillin G showed poor activity both in vitro and in vivo.

Diversity of OspA and OspC among cerebrospinal fluid isolates ofBorrelia burgdorferi sensu lato from patients with neuroborreliosis in Germany

Neuroborreliosis is the most frequent manifestation of the second stage of Lyme borreliosis in Europe. However, only few isolates from the cerobrospinal 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.

Diagnosis of lyme borreliosis in Europe

In Europe, Lyme borreliosis is caused by at least three species, B. burgdorferi sensu stricto, B. afzelii and B. garinii. Thus microbiological diagnosis in European patients must consider the heterogeneity of Lyme disease borreliae for development of diagnostic tools such as PCR primers and diagnostic antigens. According to guidelines of the German Society of Hygiene and Microbiology, the serological diagnosis should follow the principle of a two-step procedure. A sensitive ELISA differentiating IgM and IgG is recommended as the first step. In case the ELISA is reactive, it is followed by immunoblots (IgM and IgG) as the second step. The reactive diagnostic bands should be clearly identified, which is easy if recombinant antigens are used. The sensitivity and standardization of immunoblots has been considerably enhanced by use of recombinant antigens instead of whole cell lysates. Improved sensitivity resulted from use of recombinant proteins that are expressed primarily in vivo (e.g., VlsE) and combination of homologous proteins from different strains of borrelia (e.g., DbpA). It also appears promising to use recombinant proteins (DbpA, VlsE, others) or synthetic peptides (the conserved C6 peptide derived from VlsE) as ELISA antigens. At present, detection rates for serum antibodies are 20-50% in stage I, 70-90% in stage II, and nearly 100% in stage III Lyme disease. The main goals for the future are to improve specificity in general and sensitivity for diagnosis of early manifestations (stage I and II). Detection of the etiological agent by culture or PCR should be confined to specific indications and specialised laboratories. Recommended specimens are skin biopsy specimens, CSF and synovial fluid. The best results are obtained from skin biopsies with culture or PCR (50-70%) and synovial tissue or fluid (50-70% with PCR). CSF yields positive results in only 10-30% of patients. Methods that are not recommended for diagnostic purposes are antigen tests in body fluids, PCR of urine, and lymphocyte transformation tests.