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Lyme borreliosis is a multisystem disorder with a diverse spectrum of clinical manifestations, caused by spirochaetes of the Borrelia burgdorferi sensu lato complex. It is an infectious disease that can be successfully cured by antibiotic therapy in the early stages; however, the possibility of the appearance of persistent signs and symptoms of disease following antibiotic treatment is recognized. It is known that Lyme borreliosis mimics multiple diseases that were never proven to have a spirochaete aetiology. Using complete modified Kelly-Pettenkofer medium we succeeded in cultivating live B. burgdorferi sensu lato spirochaetes from samples taken from people who suffered from undefined disorders, had symptoms not typical for Lyme borreliosis, but who had undergone antibiotic treatment due to a suspicion of having Lyme disease even though they were seronegative. We report the first recovery of live B. burgdorferi sensu stricto from residents of southeastern USA and the first successful cultivation of live Borrelia bissettii-like strain from residents of North America. Our results support the fact that B. bissettii is responsible for human Lyme borreliosis worldwide along with B. burgdorferi s.s. The involvement of new spirochaete species in Lyme borreliosis changes the understanding and recognition of clinical manifestations of this disease., (Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Bacterial biofilms are microbial communities held together by an extracellular polymeric substance matrix predominantly composed of polysaccharides, proteins and nucleic acids. We had previously shown that Borrelia burgdorferi sensu stricto, the causative organism of Lyme disease in the United States is capable of forming biofilms in vitro. Here, we investigated biofilm formation by B. afzelii and B. garinii, which cause Lyme disease in Europe. Using various histochemistry and microscopy techniques, we show that B. afzelii and B. garinii form biofilms, which resemble biofilms formed by B. burgdorferi sensu stricto. High-resolution atomic force microscopy revealed similarities in the ultrastructural organization of the biofilms form by three Borrelia species. Histochemical experiments revealed a heterogeneous organization of exopolysaccharides among the three Borrelia species. These results suggest that biofilm formation might be a common trait of Borrelia genera physiology., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Spirochetes of the Borrelia burgdorferi sensu lato group, the causative agents of Lyme borreliosis, exhibit a complex biology evolved in its zoonotic cycle. Cryo-electron tomography was used to investigate structural features of three species, B. burgdorferi, B. garinii and B. afzelii, known to cause different clinical manifestations in humans. All three organisms revealed an overall similar architecture and showed different numbers of periplasmic flagellar filaments, polar periplasmic void regions, vesicles budding from the outer membrane sheath, which was covered by an amorphous slime layer. The latter was shown to be distinct in its density when comparing the three human-pathogenic Lyme disease spirochetes and Borrelia hermsii, a species causing relapsing fever. Tomograms of dividing bacteria revealed vesicles near the site of division and new basal bodies that were attached at each end of newly establishing cytoplasmic cylinder poles, while periplasmic flagellar filaments still passed the impending site of division. Two different kinds of cytoplasmic filaments showed similarities to MreB or FtsZ filaments of other bacteria. The similar and distinct structural features of Borrelia and the previously investigated pathogenic and non-pathogenic Treponema species emphasize the importance of further studying phylogenetically distant spirochetes.

Purpose: First report of a patient with Borrelia-associated crystalline keratopathy with intracorneal evidence of Borrelia garinii by polymerase chain reaction (PCR) and electron microscopy (EM)., Methods: Report of a 67-year-old patient with medical history of recurrent iridocyclitis and arthritis presented with a bilateral, progressive, asymmetric crystalline keratopathy, which was particularly pronounced in the peripheral temporal superior cornea. After penetrating keratoplasty, crystalline keratopathy with stromal haziness recurred. Corneal regrafting was performed. The corneal specimen from the penetrating keratoplasty was examined by light and EM as well as by PCR., Results: In the explanted corneal graft, as well as retrospectively in the corneal specimen from the first keratoplasty, spirochetelike bodies and fragments were detected by light and EM. Borrelia burgdorferi sensu lato DNA was demonstrated by broad-range (16S rDNA) PCR. A more precise identification as Borrelia garinii serotype 5 was possible by analyses of the flaB and ospA gene sequences. Borrelia-specific serological tests showed borderline titers in immunofluorescence and weak reaction in immunoblot, respectively., Conclusions: This case illustrates that borreliae must be considered as a cause of crystalline keratopathy; Borrelia-specific serological tests can be false negative; explanted cornea specimens of etiologically unclear crystalline keratopathy should be analyzed by EM or PCR for detection of pathogens; and prolonged antibiotic treatment might be effective to prevent progression or recurrence of the disease.

Ehrlichiae are small gram-negative obligately intracellular bacteria that multiply within vacuoles of their host cells and are associated for a part of their life cycle with ticks, which serve as vectors for vertebrate hosts. Two morphologically and physiologically different ehrlichial cell types, reticulate cells (RC) and dense-cored cells (DC), are observed during experimental infection of cell cultures, mice, and ticks. Dense-cored cells and reticulate cells in vertebrate cell lines alternate in a developmental cycle. We observed ultrastructure of RC and DC of Ehrlichia muris in morulae in salivary gland cells and coinfection with Borrelia burgdorferi sensu lato (sl), "Candidatus Rickettsia tarasevichiae," and a flavivirus (presumably, tick-borne encephalitis virus [TBEV]) of Ixodes persulcatusticks collected in the Cis-Ural region of Russia. Polymerase chain reaction revealed 326 (81.5%) of 400 ticks carrying at least one infectious agent, and 41.5% (166 ticks) were coinfected with two to four agents. Ehrlichiae and rickettsiae were identified by sequencing of 359 bp of the 16S rRNA gene of E. muris and of 440 bp of the 16S rRNA gene and 385 bp of the gltA gene of "R. tarasevichiae." Different organs of the same tick harbored different microorganisms: TBEV in salivary gland and borreliae in midgut; E. muris in salivary gland; and "R. tarasevichiae" in midgut epithelium. Salivary gland cells contained both RC and DC, a finding that confirmed the developmental cycle in naturally infected ticks. Dense-cored cells in tick salivary glands were denser and of more irregular shape than DC in cell cultures. Ehrlichia-infected salivary gland cells had lysed cytoplasm, suggesting pathogenicity of E. muris for the tick host at the cellular level, as well as potential transmission during feeding. Rickettsiae in the midgut epithelial cells multiplied to significant numbers without altering the host cell ultrastructure. This is the first demonstration of E. muris, "R. tarasevichiae," and the ehrlichial developmental cycle in naturally infected I. persulcatus sticks.

In this study the new ketolide telithromycin was tested in vitro against motile and cystic forms of Borrelia afzelii, one of the species of Borrelia burgdorferi sensu lato. Acridine orange staining, dark field microscopy, and transmission electron microscopy were the techniques used to study the influence of telithromycin on the bacteria. The activity was unexpectedly high, 0.0003 microg/ml < MBC < or = 0.0006 microg/ml for the mobile forms after 7 days of incubation at 34 degrees C. MIC was < 0.00015 microg/ml. It is likely that the agent works bacteriostatically and kills in a time-dependent and concentration-independent way, by binding tightly to the ribosomes. The agent was not able to prevent cyst formation, and the cysts were not affect ed at an in vivo achievable concentration. Electron microscopy also supports the hypothesis of telithromycin being an effective agent against the mobile bacteria.

Apart from its devastating impact on individuals and their families, multiple sclerosis (MS) creates a huge economic burden for society by mainly afflicting young adults in their most productive years. Although effective strategies for symptom management and disease modifying therapies have evolved, there exists no curative treatment yet. Worldwide, MS prevalence parallels the distribution of the Lyme disease pathogen Borrelia (B.) burgdorferi, and in America and Europe, the birth excesses of those individuals who later in life develop MS exactly mirror the seasonal distributions of Borrelia transmitting Ixodes ticks. In addition to known acute infections, no other disease exhibits equally marked epidemiological clusters by season and locality, nurturing the hope that prevention might ultimately be attainable. As minocycline, tinidazole and hydroxychloroquine are reportedly capable of destroying both the spirochaetal and cystic L-form of B. burgdorferi found in MS brains, there emerges also new hope for those already afflicted. The immunomodulating anti-inflammatory potential of minocycline and hydroxychloroquine may furthermore reduce the Jarisch Herxheimer reaction triggered by decaying Borrelia at treatment initiation. Even in those cases unrelated to B. burgdorferi, minocycline is known for its beneficial effect on several factors considered to be detrimental in MS. Patients receiving a combination of these pharmaceuticals are thus expected to be cured or to have a longer period of remission compared to untreated controls. Although the goal of this rational, cost-effective and potentially curative treatment seems simple enough, the importance of a scientifically sound approach cannot be overemphasised. A randomised, prospective, double blinded trial is necessary in patients from B. burgdorferi endemic areas with established MS and/or Borrelia L-forms in their cerebrospinal fluid, and to yield reasonable significance within due time, the groups must be large enough and preferably taken together in a multi-centre study.

The diagnosis of Lyme disease in 18 patients has been proved by detection of Borrelia burgdorferi sensu lato when using immunoelectron microscopy or detecting its nucleic acid by PCR in the plasma or the cerebrospinal fluid. The positive results occurred in the plasma or in the cerebrospinal fluid in the period of 4-68 months after an antibiotic treatment. The typical clinical manifestations of Lyme disease were observed in 9 patients and non-specific symptoms in another 9 patients. According to presented results we can recommend repeated examination using PCR of the plasma and other biological specimens in the individuals with persistent or recurring complaints after an acute form of Lyme disease and its antibiotic treatment. Also examination of the cerebrospinal fluid with non-specific symptoms and simultaneously displayed pathology electroencephalogram and/or magnetic resonance imaging findings can be advantageous.

Little is known to date about the in vitro activity of fluoroquinolones against Borrelia species. Our study aimed at determining the in vitro activities of 15 quinolones against nine isolates of the Borrelia burgdorferi sensu lato complex in addition to one Borrelia valaisiana and one Borrelia bissettii tick isolate. For the determination of MICs, a standardized colorimetric microdilution method was applied. Determination of minimal borreliacidal concentrations providing 100% killing of the final inoculum (MBCs) after 72 h and time-kill experiments were performed by conventional culture in Barbour-Stoenner-Kelly medium in combination with dark-field microscopy. The rank order of potency on a microgram-per-milliliter basis for the substances with in vitro activity against B. burgdorferi was gemifloxacin (MIC at which 90% of the isolates tested are inhibited [MIC(90)], 0.12 microg/ml) > sitafloxacin (MIC(90), 0.5 microg/ml), grepafloxacin (MIC(90), 0.5 microg/ml) > gatifloxacin (MIC(90), 1 microg/ml), sparfloxacin (MIC(90), 1 microg/ml), trovafloxacin (MIC(90), 1 microg/ml) > moxifloxacin (MIC(90), 2 microg/ml), ciprofloxacin (MIC(90), 2 microg/ml) > levofloxacin (MIC(90), 4 microg/ml) > ofloxacin (MIC(90), 8 microg/ml), norfloxacin (MIC(90), 8 microg/ml) > fleroxacin (MIC(90), >16 microg/ml), and pefloxacin (MIC(90), 32 microg/ml) > nalidixic acid (MIC(90), 256 microg/ml). After 72 h of exposure, gemifloxacin was borreliacidal (100% killing) against the isolates investigated at a median MBC of 4 microg/ml. In the other compounds tested, median MBCs were higher (> or =8 microg/ml). Results of electron microscopy and time-kill studies clearly support an in vitro activity of some fluoroquinolones against borreliae. Our study demonstrates for the first time the enhanced in vitro effectiveness of some of the recently introduced 4-quinolones against B. burgdorferi.

The objective of this study was to visualize borreliae directly in whole-body sections of Ixodes ricinus by fluorescence in situ hybridization (FISH). Borrelia afzelii mono-infected or Borrelia burgdorferi sensu stricto (ss)/B. afzelii double-infected nymphs were fixed, embedded in cold polymerizing resin and sectioned. The same sample processing was applied to skin biopsies taken from a Mongolian gerbil after an infectious tick-bite. FISH was carried out using 16S-rRNA-directed, fluorescence-labelled oligonucleotide probes specific for the genus Borrelia and specific within the group of Lyme borreliosis-associated genospecies B. afzelii, B. burgdorferi ss, Borrelia garinii and Borrelia valaisiana. Sensitivity and specificity of the newly designed probes were evaluated using PCR, dot-blot hybridizations and FISH. Despite significant autofluorescence of certain tick tissues (which allowed good histological orientation within the sections), borreliae showing the typical spirochaetal morphotype were clearly visible in five out of six putatively infected ticks. These findings were confirmed by electron microscopy of ticks from the same infected batch as used for FISH. Attempts to produce ticks infected by two different Borrelia genospecies were not successful. FISH on whole-body sections of resin-embedded ticks offers the possibility of visualizing and identifying borreliae within tick tissues. This technique has great potential for the investigation of the transmission of bacteria or other micro-organisms by arthropod vectors. Furthermore, clear visualization of single spirochaetes distributed along subcutaneous fat cell membranes in gerbil skin biopsies suggests that FISH might also be suitable for the detection of borreliae in clinical tissue specimens.

Lyme borreliosis is a multisystemic disorder primarily affecting the skin, nervous system, and joints. It is caused by the spirochete Borrelia burgdorferi sensu lato and is transmitted via ticks of the Ixodidae family. Persistence of borreliae within macrophages has been implicated in the often chronic history of borreliosis. The uptake of B. burgdorferi by professional phagocytes occurs predominantly by coiling phagocytosis, a host cell-driven process in which single pseudopods wrap around and engulf the spirochetes. In the present study, we investigated the molecular machinery and the signal transduction pathways controlling the formation of these unique uptake structures. We found that the phagocytosis of borreliae by primary human macrophages is accompanied by the formation of f-actin-rich structures, which in their morphological organization correspond well to the earlier described coiling pseudopods. Further experiments revealed that Wiskott-Aldrich Syndrome protein and Arp2/3 complex, major regulators of actin polymerization, are also recruited to these sites of actin accumulation. In addition, inhibition of an upstream regulator of Wiskott-Aldrich Syndrome protein, the Rho-family GTPase CDC42Hs, greatly inhibited the occurrence of borrelia-induced phagocytic uptake structures. Inhibition of Rac1, another Rho family GTPase, had a less-pronounced inhibitory effect, while blocking of Rho activity showed no discernible influence. These results suggest that basic mechanisms of actin polymerization that control other types of phagocytosis are also functional in the formation of the morphologically unique uptake structures in coiling phagocytosis. Our findings should enhance the understanding of the infection process of B. burgdorferi and contribute to devising new strategies for countering Lyme disease.

Objective: To develop a novel 3-dimensional (3-D) in vitro model of Lyme arthritis to use in the study of the interactions between Borrelia burgdorferi (Bb) and human synovial host cells with respect to phagocytosis and potential persistence of Bb as well as the induction of proinflammatory cytokines and chemokines., Methods: Two distinct culture systems, consisting of synovial membrane explants or interactive synovial cells embedded in 3-D fibrin matrices, were chosen. Both systems were artificially infected with Bb, and the interactions between Bb and synovial tissue/cells were studied by histology, immunohistochemistry, and electron microscopy. Functional analyses included the induction/secretion of cytokines by Bb in the model system., Results: Both culture systems proved to be stable and reproducible. The host cells and spirochetes showed high levels of viability and maintained their physiologic shape for >3 weeks. Bb invaded the synovial tissue and the artifical matrix in a time-dependent manner. Host cells were activated by Bb, as indicated by the induction of interleukin-1beta and tumor necrosis factor alpha. Electron microscopic analysis revealed Bb intracellularly within macrophages as well as synovial fibroblasts, suggesting that not only professional phagocytes, but also resident synovial cells are capable of phagocytosing Bb. Most interestingly, the uptake of the spirochetes appeared to cause severe damage of the synovial fibroblasts, since the majority of these cells displayed ultrastructural features of disintegration., Conclusion: A novel 3-D in vitro model has been established that allows the study of distinct aspects of Lyme arthritis under conditions that resemble the pathologic condition in humans. This reproducible, standardized model supplements animal studies and conventional 2-D cultures. The disintegration of synovial fibroblasts containing Bb or Bb fragments challenges the concept of an intracellular persistence of Bb and may instead reflect a mechanism that contributes to the inflammatory processes characteristic of Lyme arthritis.

The ultrastructural features of Borrelia garinii found in the ear tissues of the vole, Clethrionomys rufocanus, are described. The spirochetes were observed in the interstitium of connective tissue or in contact with fibroblasts and were occasionally situated in the endoneurium of the peripheral nerves. The spirochetes did not injure or enter into the fibroblasts or Schwann cells. The cytotoxicity and migration of the spirochetes are also discussed.

Borrelia burgdorferi s. lato in naturally infected females of tick Ixodes persulcatus were examined by transmission electron microscopy. The Borreliae were found in midgut and ovary. Location and ultrastructure of bacteria indicate extracellular migration through the midgut epithelium as a preferential way. In gonad, the borreliae intracellular situate in ovarian epithelium and oocytes before and at the beginning of vitellogenesis. The demonstration of numerous spirochetes in the oocytes provides the support for transovarial transmission of the agent. Two morphological types of borreliae were observed.

A growth inhibition assay (GIA) and an immunofluorescence test detecting deposited complement components C6 and C9 were compared for their ability to classify Borrelia isolates with respect to their resistance to non-immune human serum (NHS). In both assays a total of 34 Borrelia isolates of all three human pathogenic genospecies were tested. Interestingly, 95% of the serum-sensitive or intermediate serum-sensitive isolates belonged to the genospecies B. burgdorferi s. s. and B. garinii, whereas most B. afzelii isolates (83%) proved serum-resistant. Consequently, a strong correlation between the assignment of the isolates to the different genospecies and their degree of serum sensitivity was seen. These findings were supported strongly by the quantitative analysis of the deposited complement components and the location of the terminal complement complex on the bacterial surface as detected by means of immunoelectron microscopy. The GIA displayed an obvious lack of sensitivity to slow growing isolates, whereas the IFA allowed classification of all Borrelia isolates. Discrimination between serum-sensitive and serum-resistant isolates in the IFA was the most specific provided that the detection of C6 and C9 was incorporated into the final classification of isolates. Accordingly, both assays, turned out to be effective and reliable tools for the investigation of borrelial serum sensitivity. The IFA, however, is regarded as superior to the GIA owing to the obvious ease of performance and its rapid capability for the classification of even very slow growing isolates.

It has been demonstrated previously that motile Borrelia burgdorferi cells transform into non-motile cyst-forms when incubated for several weeks in BSKII (a complex medium) lacking rabbit serum. B. burgdorferi cells cannot synthesize fatty acids de novo and serum is thought to provide a source of fatty acids and lipids. When B. burgdorferi cells were serum-starved in defined RPMI medium, -90% of the cells formed spherical cysts within 48 h. Cyst formation was inhibited by tetracycline. Cyst opening and recovery of vegetative cells was rapidly induced by the addition of either BSKII or rabbit serum. The percentage of viable cells recovered from cysts ranged from 2.9% to 52-5%. Viability was inversely proportional to cyst age. Protein synthesis by B. burgdorferi during serum starvation was examined by labelling cells with Tran35S-Label and analysing the labelled proteins by two-dimensional gel electrophoresis and fluorography. The synthesis of over 20 proteins was induced during serum starvation. Western blots of proteins from vegetative cells and cysts probed with sera from either B. burgdorferi-infected humans or monkeys revealed that several cyst proteins were antigenic. These data suggest that cells of B. burgdorferi, although possessing a small genome and extremely limited biosynthetic capabilities, rapidly respond to conditions of serum starvation by inducing changes in protein synthesis and cell morphology. This study may help explain how cells of B. burgdorferi can survive periods of nutrient deprivation in different hosts and host tissues.

We have recovered a DNase-protected, chloroform-resistant molecule of DNA from the cell-free supernatant of a Borrelia burgdorferi culture. The DNA is a 32-kb double-stranded linear molecule that is derived from the 32-kb circular plasmids (cp32s) of the B. burgdorferi genome. Electron microscopy of samples from which the 32-kb DNA molecule was purified revealed bacteriophage particles. The bacteriophage has a polyhedral head with a diameter of 55 nm and appears to have a simple 100-nm-long tail. The phage is produced constitutively at low levels from growing cultures of some B. burgdorferi strains and is inducible to higher levels with 10 microg of 1-methyl-3-nitroso-nitroguanidine (MNNG) ml(-1). In addition, the prophage can be induced with MNNG from some Borrelia isolates that do not naturally produce phage. We have isolated and partially characterized the phage associated with B. burgdorferi CA-11.2A. To our knowledge, this is the first molecular characterization of a bacteriophage of B. burgdorferi.

The aim of this study was to examine the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole. Because B. burgdorferi is a microaerobic bacterium like Helicobacter pylori, metronidazole (MZ) was chosen in the susceptibility test. For both microaerobic and aerobic incubation the normal mobile spirochetes were resistant to this antibiotic with an MBC > or = 512 microg/ml. Conversion of mobile spirochetes to cystic forms was not observed when they were incubated with MZ. When they were incubated under microaerobic conditions, the biologically active cystic forms had an MBC > or = 4 microg/ml, but the MBC was > or = 32 microg/ml with aerobic incubation at 37 degrees C. Staining with acridine orange (AO), dark field microscopy (DFM), and transmission electron microscopy (TEM) revealed that the contents of the cysts were degraded when the concentration of MZ was > or = MBC. Some cysts were also ruptured. When incubated with a sufficient concentration of MZ, core structures did not develop inside the cysts, and AO revealed less RNA in the cysts. Our observations may help efforts to treat resistant infections caused by B. burgdorferi with a combination of MZ and other antibiotics in order to eradicate both cystic and mobile forms of B. burgdorferi.

Mobile Borrelia burgdorferi were transferred to distilled water (10(6) per ml). The cultures were observed by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). 95% of the spirochetes were converted to cysts after 1 min, and after 4 h no normal mobile borreliae were observed. When transferred to growth medium (BSK-H), the cysts became smaller and more irregular, and were filled with organic substances. After 1 day, 1-5 thin structures sprouted from the cysts. They continued to grow in both length and thickness until they attained a normal spirochetal structure. Finally, these new-born spirochetes detached from the cysts, by which time their mobility had become normal. The present method for producing large amounts of cystic forms of B. burgdorferi is well suited for further studies of this unique microbe.

Since culture of Borrelia burgdorferi from patients with chronic Lyme disease has been an extraordinarily rare event, clarification of the nature of the illness and proving its etiology as infectious have been difficult. A method for reliably and reproducibly culturing B. burgdorferi from the blood of patients with chronic Lyme disease was therefore sought by making a controlled blood culture trial studying 47 patients with chronic Lyme disease. All had relapsed after long-term oral and intravenous antibiotics. 23 patients with other chronic illness formed the control group. Positive cultures were confirmed by fluorescent antibody immuno-electron microscopy using monoclonal antibody directed against Osp A, and Osp A PCR. 43/47 patients (91%) cultured positive. 23/23 controls (100%) cultured negative. Although persistent infection has been, to date, strongly suggested in chronic Lyme disease by positive PCR and antigen capture, there are major problems with these tests. This new method for culturing B. burgdorferi from patients with chronic Lyme disease certainly defines the nature of the illness and establishes that it is of chronic infectious etiology. This discovery should help to reestablish the gold standard in laboratory diagnosis of Lyme disease.

The purpose of this study was to examine the structural alterations of Borrelia burgdorferi when exposed to spinal fluid. Normal, mobile spirochetes were inoculated into spinal fluid, and the spirochetes were converted to cysts (spheroplast L-forms) after 1-24 h. When these cystic forms were transferred to a rich BSK-H medium, the cysts were converted back to normal, mobile spirochetes after incubation for 9 to 17 days. The cultures were examined by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). When neuroborreliosis is suspected, it is necessary to realize that B. burgdorferi can be present in a cystic form, and these cysts have to be recognized by microscopy. This study may also explain why cultivation of spinal fluid often is negative with respect to B. burgdorferi.

The role of outer surface proteins (Osp) A and B and length of time in culture on the adhesion and cytotoxicity of Borrelia burgdorferi to C6 glioma and PC-12 pheochromocytoma cells was investigated using 6 different spirochete strains in an ELISA. Statistically significant differences in adhesion between OspB mutants and parental isolates were not seen, yet clear differences in adhesion were noted between low- and high-passage isolates. Polar adhesion and penetration by the tips of spirochetes resulted in the formation of surface cavities and blebs. Adhesion of spirochetes to C6 and to undifferentiated PC-12 cells did not result in significant cytotoxicity, but adhesion of spirochetes to PC-12 cells differentiated with nerve growth factor resulted in a loss of confluence of the monolayer and cytotoxicity at high spirochete-to-cell ratios. These results demonstrate that B. burgdorferi can induce damage to neural cells directly.

Borrelia burgdorferi has demonstrated a capacity to resist the in vitro effects of powerful eukaryotic and prokaryotic metabolic inhibitors. However, treatment of laboratory cultures on Barbour-Stoenner-Kelly medium with melittin, a 26-amino acid peptide contained in honeybee venom, showed immediate and profound inhibitory effects when they were monitored by dark-field microscopy, field emission scanning electron microscopy, and optical density measurements. Furthermore, at melittin concentrations as low as 100 microg/mL, virtually all spirochete motility ceased within seconds of inhibitor addition. Ultrastructural examination of these spirochetes by scanning electron microscopy revealed obvious alterations in the surface envelope of the spirochetes. The extraordinary sensitivity of B. burgdorferi to mellitin may provide both a research reagent useful in the study of selective permeability in microorganisms and important clues to the development of effective new drugs against lyme disease.

A distinctive renal lesion consisting of glomerulonephritis, diffuse tubular necrosis with regeneration, and interstitial inflammation was found in 49 biopsy/necropsy cases obtained from 1987 to 1992. This lesion is manifested clinically as a rapidly progressive glomerular disease that was uniformly fatal. Immune-mediated membranoproliferative glomerulonephritis predominated (43/49, 88%). Membranous glomerulonephritis (5/49, 10%) and amyloidosis (1/49, 2%) were also noted. Subendothelial deposits, IgG, IgM, and C3 were present along glomerular basement membranes. IgA was absent. The exact cause of the tubular necrosis is unknown. Affected dogs were significantly younger (5.6 +/- 2.6 years) than dogs with other forms of glomerulonephritis (7.1 +/- 3.6 years) and amyloidosis (7.8 +/- 3.5 years) both in the studied population for the same period and in the reported canine population. Labrador and Golden retrievers were 6.4 and 4.9 times more likely, respectively, to develop this lesion. This is the first report of a breed predilection for spontaneous canine glomerulonephritis. Previous reports have associated this lesion with Borrelia burgdorferi exposure. All dogs in this study were from Lyme disease-endemic areas. Of 18 dogs serologically tested, all were positive for exposure. Silver stain examination of kidneys revealed rare spirochetes, suggesting that the presence of spirochetes in the kidney is apparently unrelated to lesion development. The role of vaccination in development of the renal lesion is undetermined. The association of this histologically and clinically unique lesion, Lyme nephritis, with Borrelia burgdorferi infection is significant because it is the only fatal form of canine Lyme borreliosis.

Coiling phagocytosis has been observed previously only by chance, and there has been no systematic investigation of this uptake mechanism. Therefore, a comparative electron microscopical study was performed. Different human and murine cell populations, phagocytes from various vertebrate and invertebrate species, and predatory amoebae were incubated with Borrelia burgdorferi, one of the microbes known to induce coiling phagocytosis, to study the uptake mechanisms used. In this model, coiling phagocytosis was observed with both vertebrate and invertebrate species but not with amoebae. With cells from humans and mice, this uptake mechanism was restricted to phagocytic cells of myeloid origin. The coiled membrane gaps did not give rise to phagosomes; instead, membrane fusion was followed by membrane dissipation. Thus, coiling of B. burgdorferi apparently is an alternative uptake mechanism used by metazoan phagocytes, involving special membrane processing. However, coiling phagocytosis may show different features with different microbes.

Spirochetes are an evolutionary and structurally unique group of bacteria. Outermost is a membrane sheath (OS), and within this sheath are the protoplasmic cell cylinder (PC) and periplasmic flagella (PFs). The PFs are attached at each end of the PC and, depending on the species, may or may not overlap in the center of the cell. The precise location of the PFs within the spirochetal cells is unknown. The PFs could lie along the cell axis. Alternatively, the PFs could wrap around the PC in either a right- or a left-handed sense. To understand the factors that cause the PFs to influence cell shape and allow the cells to swim, we determined the precise location of the PFs in the Leptospiraceae (Leptonema illini) and Borrelia burgdorferi. Our approach was to use high-voltage electron microscopy and analyze the three-dimensional images obtained from thick sections of embedded cells. We found that a single PF in L. illini is located in a central channel 29 nm in diameter running along the helix axis of the right-handed PC. The presence of the PFs is associated with the end being hook shaped. The results obtained agree with the current model of Leptospiraceae motility. In B. burgdorferi, which forms a flattened wave, the relationship between the PFs and the PC is more complicated. A multistrand ridge 67 nm in diameter, which was shown to be composed of PFs by cross-sectional and mutant analysis, was found to extend along the entire length of the cell. We found that the PFs wrapped around the PC in a right-handed sense. However, the PFs formed a left-handed helix in space. The wavelength of the cell body and the helix pitch of the PFs were found to be identical (2.83 microm). The results obtained were used to propose a model of B. burgdorferi motility whereby backward-propagating waves, which gyrate counterclockwise as viewed from the back of the cell, are generated by the counterclockwise rotation of the internal PFs. Concomitant with this motion, the cell is believed to rotate clockwise about the body axis as shown for the Leptospiraceae.

To perform the first systematic electronmicroscopic (EM) and immunoelectron microscopy (IEM) study of the pathological changes and the evidence of spirochete presence in synovial membranes and synovial fluid (SF) cells of patients with chronic Lyme arthritis. EM examination was performed on four synovial membrane and eight SF cell samples from eight patients with chronic Lyme disease. Spirochetal antigens in the samples were sought by IEM using monoclonal antibody to Borrelia burgdorferi outer surface protein A (OspA) as the immunoprobe. Prominent ultrastructural findings were surface fibrin-like material, thickened synovial lining cell layer and signs of vascular injury. Borrelia-like structures were identified in all four synovial membranes and in two of eight SF cell samples. The presence of spirochetal antigens was confirmed by IEM in all four samples studied (one synovial membrane and three SF cell samples). OspA labelling was in perivascular areas, deep synovial stroma among collagen bundles, and in vacuoles of fibroblasts in synovial membranes; and in cytophagosomes of mononuclear cells in SF cell samples. Electron microscopy adds further evidence for persistence of spirochetal antigens in the joint in chronic Lyme disease. Locations of spirochetes or spirochetal antigens both intracellulary and extracellulary in deep synovial connective tissue as reported here suggest sites at which spirochaetes may elude host immune response and antibiotic treatment.

Bacterial pathogens have evolved various strategies to acquire iron from the iron-restricted environment found in mammalian hosts. Borrelia burgdorferi should be no different with regard to its requirement for ferric iron, and previous studies have suggested that transferrin (Tf) may be a source of iron in vivo. By probing blots with Tf conjugated to horseradish peroxidase, we have identified an outer membrane protein (28 kDa) from B. burgdorferi B31 that bound holo-Tf but not apo-Tf. The 28-kDa protein bound human, rat, or mouse Tf and was produced only by low-passage (less than passage 5), virulent isolates of strain B31. In addition, the Tf-binding protein (Tbp) from strain B31 retained the ability to bind Tf after treatment with 2% sodium dodecyl sulfate-1% beta-mercaptoethanol and heating to 100 degrees C for 5 min. These properties are remarkably similar to those of the Tbp of Staphylococcus aureus and Tbp2 from Neisseria meningitidis. B. burgdorferi Sh-2-82 produced an outer membrane protein different in size, i.e., 26 kDa, but with properties similar to those of to the protein from strain B31, suggesting variation in B. burgdorferi Tbps. The exact role of the 28-kDa protein in iron acquisition by B. burgdorferi remains to be determined.

The Lyme disease spirochete, Borrelia burgdorferi, is ingested rapidly by mouse macrophages in vitro. Spirochetes attach by their ends and become progressively coiled as they move deeper into cells. From the earliest measurements, spirochetes colocalize with a marker of endosomes and lysosomes, and degradation of spirochetes occurs within lysosomes.

The monoclonal antibody LA7 was raised against the species-specific Borrelia burgdorferi lipoprotein P22 (= IPLA7), which induces antibody formation in patients with Lyme arthritis. It is composed of 194 amino acids with a calculated molecular mass of 21.8 kDa. Its gene on the linear chromosome is 582 nucleotides in length. The aim of this study was to localize the protein P22 by immune electron microscopy. Immunolabeling of Borrelia burgdorferi with LA7 and an anti-mouse immunogold conjugate proved that P22 is an outer membrane protein. This finding was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis of the outer envelope fraction, which contained 99% of the P22 proteins.

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.

In an open non-comparative clinical trial with the aim of evaluating the clinical efficacy and safety of a 14 day course of 2 g ceftriaxone once daily, 46 patients with neuroborreliosis were entered at the Infectious Diseases Teaching Hospital in Prague 8. In 39 patients the diagnosis was early Lyme neuroborreliosis. Seven patients suffered from late stage disease. Clinical results were 30% of patients cured at the end of treatment and 85% after 9 months in early stage disease. In late stage disease two patients out of seven were cured and four had improved after 12 months. One patient died because of cardiac infarction. In no patient had treatment to be discontinued because of adverse reactions to antibiotics.

A critical issue regarding the molecular architectures of Treponema pallidum and Borrelia burgdorferi, the agents of venereal syphilis and Lyme disease, respectively, concerns the membrane topologies of their major lipoprotein immunogens. A related question is whether these lipid-modified membrane proteins form intramembranous particles during freeze fracture electron microscopy. To address these issues, native borrelial and treponemal lipoproteins were reconstituted into liposomes of diverse composition. The importance of the covalently associated lipids for membrane association of lipoproteins was revealed by the observation that nonlipidated recombinant forms of both B. burgdorferi OspA and the T. pallidum 47-kDa immunogen (Tpp47) showed very weak or no binding to model bilayer vesicles. In contrast to control liposomes reconstituted with bacteriorhodopsin or bovine rhodopsin, two well-characterized transmembrane proteins, none of the lipoprotein-liposomes contained particles when examined by freeze fracture electron microscopy. To extend these findings to prokaryotic lipoproteins with relatively amphiphilic polypeptides, similar experiments were conducted with a recombinant nonlipidated form of Escherichia coli TraT, a lipoprotein which has putative transmembrane domains. The nonlipidated TraT oligomers bound vesicles derived from E. coli lipids but, surprisingly, did not form particles in the freeze-fractured liposomes. These findings support (i) a proposed topology of spirochetal lipoproteins in which the polypeptide is extrinsic to the membrane surface and (ii) the contention that particles visualized in freeze-fractured spirochetal membranes represent poorly characterized transmembrane proteins.

The lack of methods for isolating Borrelia burgdorferi outer membranes (OMs) has hindered efforts to characterize borrelial surface-exposed proteins. Here we isolated OMs by immersion of motile spirochetes in hypertonic sucrose followed by isopycnic ultracentrifugation of the plasmolyzed cells. The unilamellar vesicles thus obtained were shown to be OMs by the following criteria: (i) they contained OspA and OspB; (ii) they did not contain flagellin, NADH oxidase activity, or the 60-kDa heat shock protein; and (iii) their morphology by freeze-fracture electron microscopy was identical to that of OMs of intact organisms. Consistent with previous studies which employed immunoelectron microscopy and detergent-based solubilization of B. burgdorferi OMs, only small proportions of the total cellular content of OspA or OspB were OM associated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fluorography of OMs from spirochetes metabolically radiolabeled with [3H]palmitate or 35S-amino acids demonstrated that the OMs contained both nonlipidated and lipidated proteins. This fractionation procedure was also used to isolate OMs from virulent and avirulent isolates of the well-characterized B. burgdorferi N40 strain. SDS-PAGE fluorography revealed that OMs from the two isolates differed with respect to both nonlipoprotein and lipoprotein constituents. When whole cells, protoplasmic cylinders, and OMs were immunoblotted against sera from mice persistently infected with B. burgdorferi N40, the majority of antibody reactivity was directed against intracellular proteins. The availability of isolated OMs should facilitate efforts to elucidate the complex relationship(s) between B. burgdorferi membrane composition and Lyme disease pathogenesis.

Antibiotic therapy with penicillin, doxycycline, and ceftriaxone has proven to be effective for the treatment of Lyme borreliosis. In some patients, however, it was noticed that borreliae can survival in the tissues in spite of seemingly adequate therapy. For a better understanding of this phenomenon, we investigated the different modes of degeneration of Borrelia burgdorferi suspensions during a 96-h exposure to various antibiotics. By dark-field microscopy and ultrastructural investigations, increasing blebbing and the gradual formation of granular and cystic structures could be followed during the exposure time. Although antibiotic concentrations at the MIC at which 90% of organisms are inhibited after 72 h were 80% or even greater, motile organisms were still present after incubation with penicillin and doxycycline but not after incubation with ceftriaxone. By transmission electron microscopy, intact spirochetal parts, mostly situated in cysts, were seen up to 96 h after exposure with all three antibiotics tested. According to experiences from studies with other spirochetes it is suggested that encysted borreliae, granules, and the remaining blebs might be responsible for the ongoing antigenic stimulus leading to complaints of chronic Lyme borreliosis.

Lyme borreliosis is a newly recognized systemic infection with protean clinical manifestations. Because the localization of the causative spirochete (Borrelia burgdorferi) in infected tissues is unknown, we used electron microscopy to find spirochetes in the hearts of chronically infected mice. There were three predominant locations for the spirochete in the hearts. In mice infected for one month or less, the spirochetes were mostly in or around blood vessels. They were either in the lumen or in the perivascular space. Mice infected for more than one month had B. burgdorferi in cardiac myocytes as well, often with clear spaces around them. The third area in which spirochetes were common was collagen fibers; the borreliae were wrapped around fibers with their long axis parallel to the fibers. The number of spirochetes was relatively low, but there was no appreciable decrease in numbers of spirochetes with increasing time postinfection. Inflammatory infiltrates were primarily in the endocardium and pericardium, but spirochetes were generally not in or near areas of inflammation. These data are consistent with previously published information that have identified the heart as a site of chronic infection and inflammation in the mouse. The studies extend our understanding of the behavior of the spirochete in vivo by identifying common locations of B. burgdorferi and by noting the disparity between infection and inflammation.

The aim of this study was to investigate the morphological changes of Borrelia burgdorferi associated with penicillin treatment. An isolate of B. burgdorferi from an erythema migrans lesion was cultivated in BSK II medium and exposed to increasing concentrations (0.0625 mg/l-2 mg/l) of penicillin G for 5 days. The in vitro minimal inhibitory concentration (MIC) was determined to be 0.5 mg/l by broth dilution method. The morphological structures of untreated spirochetes, as well as their characteristic ultrastructural changes when exposed to penicillin, were observed by electron microscopy. The following alterations were discovered: (i) Numerous outer sheath blebs at a penicillin concentration of 0.0625 mg/l. (ii) A characteristic irregular waveform of the borrelial cells and complete loss of the outer sheath at a penicillin concentration of 0.125 mg/l. (iii) The presence of "spheroplasts" at the same concentration. (iv) Structural changes of the protoplasmic cylinder complex which showed an irregular pattern at a penicillin concentration of 0.125 mg/l. (v) Disruption of the protoplasmic cylinder complex into several parts at penicillin concentrations of 0.25 mg/l and 0.5 mg/l. (vi) Severe cytolysis at penicillin concentrations of 1 mg/l and 2 mg/l.

Subcellular locations and chaperone functions of Hsp60 and Hsp70 with flagellin were investigated in Borrelia burgdorferi. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis of fractionated cells showed Hsp60 to be present in the soluble fractions and the Triton X-100 detergent-soluble membrane fraction at growth temperatures ranging from 20 to 37 degrees C. The relative amount of Hsp60 associated with the membrane increased with growth temperature. Hsp70 was found in soluble fractions at growth temperatures between 28 and 37 degrees C, but at 20 degrees C it was also present in the Triton X-100-insoluble membrane fraction. Immunoelectron microscopy revealed that the majority of Hsp60 was localized in the cytoplasm but a detectable fraction (approximately 30%) was associated with the cell envelope. The chaperone functions of Hsp60 and Hsp70 were analyzed by immunoprecipitation of [35S]methionine-labeled cell lysates under nondenaturing conditions in the presence or absence of ATP. Hsp70 was found to bind flagellin at all temperatures tested between 33 and 41 degrees C. This association could be decreased with ATP when cells had been incubated at 41 degrees C during radioactive labeling but not at lower temperatures. Both flagellin and Hsp70 were found to associate with Hsp60, forming a complex of the three proteins. Hsp70 association with this complex could be decreased with ATP, but flagellin binding to Hsp60 was ATP independent at all temperatures studied. Both Hsp70 and flagellin were inaccessible to monoclonal antibodies against them when bound to Hsp60. These studies suggest that in B. burgdorferi, a major function of Hsp60 and Hsp70 is in the molecular processing of flagellin.

Interaction of B. burgdorferi organisms with mouse bone marrow-derived macrophages (BMM phi) leads to phagocytosis of microorganisms, induction of nitric oxide (NO) and superoxide radicals (O2-) by BMM phi and killing of spirochetes. Destruction of spirochetes by BMM phi was quantified by a new method based on the release of radioactivity from spirochetes pre-labelled with [3H]adenine. Uptake of B. burgdorferi by BMM phi, which mainly occurs by coiling phagocytosis, generation of NO and O2- radicals as well as killing of spirochetes were significantly enhanced by pre-opsonization of spirochetes with monoclonal antibodies (mAb) to the outer surface proteins A and B but not with those to the periplasmic flagellin. Addition of inhibitors specific for NO and O2- radical synthesis either separately or together to cultures of BMM phi and spirochetes resulted in only partial reduction of the killing potential of effector cells. The data indicate that NO and O2- radicals are necessary, but not sufficient, for complete elimination of B. burgdorferi by macrophages. Together with previous findings that protection against B. burgdorferi infection is conveyed by humoral immune responses the present data indicate that one of the important functions of specific antibodies is their participation in macrophage-mediated control of spirochetes.

To investigate dermal and epidermal involvement in the presence of Borrelia burgdorferi and to analyze the role of Langerhans cells and keratinocytes, 14 cases of erythema chronicum migrans and two controls were studied by means of electron microscopy, using negative staining and sectioning techniques. Using immunoelectron microscopy and histochemistry, positive results for B. burgdorferi were disclosed in 5 cases of erythema chronicum migrans and 3 cases of neuroborreliosis which were confirmed by cultivation. We cultured 4 stains of B. burgdorferi from the skin, 1 from blood and 2 from cerebrospinal fluid in BSK medium. Near to the centre of erythema chronicum migrans with focal necrosis were both a dissolved basal membrane and keratinocyte desmosomes surrounding damaged B. burgdorferi cells in the epidermis. Markedly oedematous keratinocytes and Langerhans cells with B. burgdorferi were released into lymphocyte infiltrates. At the periphery of all erythema chronicum migrans lesions, keratinocytes were well preserved while all dendritic cells seemed to be vacuolated. Above foci of B. burgdorferi located perivascular or among collagen fibers, Langerhans cells were frequent and more granulated. The possible role of Langerhans cells in the identification and elimination of B. burgdorferi is discussed.

Freeze-fracture electron microscopy was used to investigate the membrane architectures of high-passage Borrelia burgdorferi B31 and low- and high-passage isolates of B. burgdorferi N40. In all three organisms, fractures occurred almost exclusively through the outer membrane (OM), and the large majority of intramembranous particles were distributed randomly throughout the concave OM leaflet. The density of intramembranous particles in the concave OM leaflet of the high-passage N40 isolate was significantly greater than that in the corresponding leaflet of the low-passage N40 isolate. Also noted in the OMs of all three organisms were unusual structures, designated linear bodies, which typically were more or less perpendicular to the axis of the bacterium. A comparison of freeze-fractured B. burgdorferi and Treponema pallidum, the syphilis spirochete, revealed that the OM architectures of these two pathogens differed markedly. All large membrane blebs appeared to be bounded by a membrane identical to the OM of B. burgdorferi whole cells; in some blebs, the fracture plane also revealed a second bilayer closely resembling the B. burgdorferi cytoplasmic membrane. Aggregation of the lipoprotein immunogens outer surface protein A (OspA) and OspB on the bacterial surface by incubation of B. burgdorferi B31 with specific polyclonal antisera did not affect the distribution of OM particles, supporting the contention that lipoproteins do not form particles in freeze-fractured OMs. The expression of poorly immunogenic, surface-exposed proteins as virulence determinants may be part of the parasitic strategy used by B. burgdorferi to establish and maintain chronic infection in Lyme disease.

Two types of tailed bacteriophages were detected by electron microscopy in a Borrelia burgdorferi strain which had been isolated from infected human skin and exposed to the gyrase inhibitor ciprofloxacin. One of these phages displayed an isometric 30 nm head, a neck and a contractile tail 50-64 nm in length with a baseplate, according to an A-1 morphology. The other phage showing a B-1 morphology had a 30 nm isometric head as well and a long non-contractile straight tail 115-130 nm in length without neck and baseplate. Both types of phages could be found together within one plasmolysed spirochetal cell. We conclude that we are dealing with a lysogenic strain of Borrelia burgdorferi carrying at least two different prophages inducible by ciprofloxacin.

This paper analyzed morphological characteristics of Lyme disease spirochetes (Borrelia burgdorferi), isolated from Ixodes persulcatus from different areas of China, with scanning and transmissive electron microscopies. Cells of 8 strains were 8.4-36.0 microns long and 0.12-0.35 micron wide. There were 1-9 left-handed spires with wavelength of 1.09-4.30 microns and amplitude of 0.38-2.10 microns. Ends of the cells were pointed or spindle. 7, 8 or 9 flagella were inserted subterminally at each end of the cell. Inner structures of cells were no tubles. A few cells were at stage of division. These results showed that Ixodes persulcatus spirochetes possessed morphological characteristics of Borrelia burgdorferi and there were many types of morphological characteristics.

The gene of the immunodominant major protein pC of Borrelia burgdorferi was previously cloned and sequenced (R. Fuchs, S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, and E. Soutschek, Mol. Microbiol. 6:503-509, 1992). pC is abundantly expressed on the outer surface of B. burgdorferi, as demonstrated by immunoelectron microscopy with monoclonal antibody L22 1F8. Accordingly, pC is renamed OspC, by analogy to the outer surface proteins OspA and OspB. Western immunoblot analysis of 45 B. burgdorferi isolates with monoclonal antibodies revealed that OspC is immunologically heterogeneous. Partial sequence analysis of the ospC gene confirmed the protein heterogeneity at the genetic level. We found that the degree of identity between the ospC partial sequences of five strains representing different OspA serotypes was only 63.3 to 85.4%. Immunological heterogeneity was also observed among representatives of the three newly designated genospecies of B. burgdorferi sensu lato, B. burgdorferi sensu stricto, B. garinii, and group VS461. Heterogeneity was confirmed for B. garinii at the genetic level. The ospC gene was also cloned from strains that did not express OspC, and antibody-reactive OspC was expressed in Escherichia coli. In addition, OspC-expressing variants were obtained from a nonexpressing strain by plating single colonies on solid medium. These findings confirm that the ospC gene is also present in nonexpressing strains. Because OspC is an immunodominant protein for the early immune response in Lyme borreliosis and was effective as a vaccine in an animal model, the immunological and molecular polymorphisms of ospC and OspC have important implications for the development of diagnostic reagents and vaccines.

Borrelia burgdorferi, a spirochete and the causative agent of Lyme disease, has been reported to be susceptible to a variety of antimicrobial agents. In this investigation, the action of vancomycin, a glycopeptide antibiotic not previously known to have activity against spirochetes, against borrelias was examined. The in vitro activity of vancomycin against a variety of strains of B. burgdorferi and one strain of Borrelia hermsii was determined by use of a microdilution MIC method (L.L. Dever, J.H. Jorgensen, and A.G. Barbour, J. Clin. Microbiol. 30:2692-2697, 1992). MICs ranged from 0.5 to 2 micrograms/ml. MICs of the glycopeptides ristocetin and teicoplanin and the lipopeptide daptomycin against strain B31 of B. burgdorferi were all > or = 8 micrograms/ml. Subsurface plating, time-kill studies, synergy studies, and electron microscopy were used to investigate further the activity of vancomycin against B31. The MBC of vancomycin was 2 micrograms/ml. Time-kill curves demonstrated > or = 3-log10-unit (99.9%) killing of the final inoculum after 72 h by vancomycin concentrations twice the MIC. Synergy between vancomycin and penicillin was demonstrated at concentrations one-fourth the MIC of each drug. In electron microscopy, B31 cells exposed to vancomycin showed a disruption of cellular integrity and were indistinguishable from those exposed to penicillin. These studies demonstrate another class of microorganisms susceptible in vitro to vancomycin.

The ability of Borrelia burgdorferi to attach to and invade human fibroblasts was investigated by scanning electron and confocal microscopy. By scanning electron microscopy, B. burgdorferi were tightly adherent to fibroblast monolayers after 24-48 h but were eliminated from the cell surface by treatment with ceftriaxone (1 microgram/mL) for 5 days. Despite the absence of visible spirochetes on the cell surface after antibiotic treatment, viable B. burgdorferi were isolated from lysates of the fibroblast monolayers. B. burgdorferi were observed in the perinuclear region within human fibroblasts by laser scanning confocal microscopy. Intracellular spirochetes specifically labeled with monoclonal anti-flagellin antibody were also identified by fluorescent laser scanning confocal microscopy. These observations suggest that B. burgdorferi can adhere to, penetrate, and invade human fibroblasts in organisms that remain viable.

We observed Lyme borrelia by electron microscopy in the tissues of the ticks, Ixodes persulcatus, which were indicated positive for borreliae by BSK cultures of their internal organs. Borreliae (0.25 micron in diameter) were found only in the lumen of the midgut. They were closely associated with the microvilli on the midgut epithelium but never penetrated into the epithelial cells. Ultrastructural features common to Lyme borreliae., i.e., the three-layered membranes surrounding the cytoplasm and orientation of the flagella insertions, were obviously confirmed. The present results are useful to understand tick tissue-borrelia interface.