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Concept: Spirochaetes

169

Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD). Recent studies have shown that recognition of the spirochete is mediated mainly by TLR2 and NOD2. The latter receptor has been associated with the induction of the intracellular degradation process called autophagy. The present study demonstrated for the first time the induction of autophagy by exposure to B. burgdorferi and that autophagy modulates the B. burgdorferi-dependent cytokine production. Human PBMCs treated with autophagy inhibitors showed an increased IL-1β and IL-6 production in response to the exposure of the spirochete, while TNFα production was unchanged. Autophagy induction against B. burgdorferi was dependent on reactive oxygen species (ROS) since cells from patients with chronic granulomatous disease (CGD), which are defective in ROS production, also produced elevated IL-1β. Further, the enhanced production of the pro-inflammatory cytokines was because of the elevated mRNA expression in the absence of autophagy. Our results thus demonstrate the induction of autophagy, which in-turn modulates cytokine production, by B. burgdorferi for the first time.

Concepts: Bacteria, Cytokine, Reactive oxygen species, Lyme disease, Borrelia burgdorferi, Borrelia, Spirochaetes, Spirochaete

169

Pathogenic Leptospira spp., the causative agents of leptospirosis, are slow-growing Gram-negative spirochetes. Isolation of Leptospira from clinical samples and testing of antimicrobial susceptibility are difficult and time-consuming. Here, we describe the development of a new solid medium that facilitates more-rapid growth of Leptospira spp. and the use of this medium to evaluate the Etest’s performance in determining antimicrobial MICs to drugs in common use for leptospirosis. The medium was developed by evaluating the effects of numerous factors on the growth rate of Leptospira interrogans strain NR-20157. These included the type of base agar, the concentration of rabbit serum (RS), and the concentration and duration of CO(2) incubation during the initial period of culture. The highest growth rate of NR-20157 was achieved using a Noble agar base supplemented with 10% RS (named LVW agar), with an initial incubation at 30°C in 5% CO(2) for 2 days prior to continuous culture in air at 30°C. These conditions were used to develop the Etest for three species, L. interrogans (NR-20161), L. kirschnerii (NR-20327), and L. borgpetersenii (NR-20151). The MICs were read on day 7 for all samples. The Etest was then performed on 109 isolates of pathogenic Leptospira spp. The MIC(90) values for penicillin G, doxycycline, cefotaxime, ceftriaxone, and chloramphenicol were 0.64 units/ml and 0.19, 0.047, 0.5, and 2 μg/ml, respectively. The use of LVW agar, which enables rapid growth, isolation of single colonies, and simple antimicrobial susceptibility testing for Leptospira spp., provides an opportunity for new areas of fundamental and applied research.

Concepts: Microbiology, Penicillin, Spirochaetes, Spirochaete, Leptospira, Leptospirosis, The Medium, Causative

146

Lyme disease is a tickborne illness that generates controversy among medical providers and researchers. One of the key topics of debate is the existence of persistent infection with the Lyme spirochete, Borreliaburgdorferi, in patients who have been treated with recommended doses of antibiotics yet remain symptomatic. Persistent spirochetal infection despite antibiotic therapy has recently been demonstrated in non-human primates. We present evidence of persistent Borrelia infection despite antibiotic therapy in patients with ongoing Lyme disease symptoms.

Concepts: Medicine, Bacteria, Lyme disease, Borrelia burgdorferi, Borrelia, Spirochaetes, Spirochaete, Tick-borne disease

62

Although most Lyme disease patients can be cured with antibiotics doxycycline or amoxicillin using 2-4 week treatment durations, some patients suffer from persistent arthritis or post-treatment Lyme disease syndrome. Why these phenomena occur is unclear, but possibilities include host responses, antigenic debris, or B. burgdorferi organisms remaining despite antibiotic therapy. In vitro, B. burgdorferi developed increasing antibiotic tolerance as morphology changed from typical spirochetal form in log phase growth to variant round body and microcolony forms in stationary phase. B. burgdorferi appeared to have higher persister frequencies than E. coli as a control as measured by SYBR Green I/propidium iodide (PI) viability stain and microscope counting. To more effectively eradicate the different persister forms tolerant to doxycycline or amoxicillin, drug combinations were studied using previously identified drugs from an FDA-approved drug library with high activity against such persisters. Using a SYBR Green/PI viability assay, daptomycin-containing drug combinations were the most effective. Of studied drugs, daptomycin was the common element in the most active regimens when combined with doxycycline plus either beta-lactams (cefoperazone or carbenicillin) or an energy inhibitor (clofazimine). Daptomycin plus doxycycline and cefoperazone eradicated the most resistant microcolony form of B. burgdorferi persisters and did not yield viable spirochetes upon subculturing, suggesting durable killing that was not achieved by any other two or three drug combinations. These findings may have implications for improved treatment of Lyme disease, if persistent organisms or detritus are responsible for symptoms that do not resolve with conventional therapy. Further studies are needed to validate whether such combination antimicrobial approaches are useful in animal models and human infection.

Concepts: Bacteria, Antibiotic resistance, Lyme disease, Antibiotic, Borrelia burgdorferi, Borrelia, Spirochaetes, Spirochaete

38

Is chronic illness in patients with Lyme disease caused by persistent infection? Three decades of basic and clinical research have yet to produce a definitive answer to this question. This review describes known and suspected mechanisms by which spirochetes of the Borrelia genus evade host immune defenses and survive antibiotic challenge. Accumulating evidence indicates that Lyme disease spirochetes are adapted to persist in immune competent hosts, and that they are able to remain infective despite aggressive antibiotic challenge. Advancing understanding of the survival mechanisms of the Lyme disease spirochete carry noteworthy implications for ongoing research and clinical practice.

Concepts: Immune system, Medicine, Bacteria, Lyme disease, Borrelia burgdorferi, Borrelia, Spirochaetes, Spirochaete

32

The agent of Lyme borreliosis, Borrelia burgdorferi, evades host immunity and establishes persistent infections in its varied mammalian hosts. This persistent biology may pose challenges to effective antibiotic treatment. Experimental studies in dogs, mice, and non-human primates have found persistence of B. burgdorferi DNA following treatment with a variety of antibiotics, but persisting spirochetes are non-cultivable. Persistence of B. burgdorferi DNA has been documented in humans following treatment, but the significance remains unknown. The present study utilized a ceftriaxone treatment regimen in the C3H mouse model that resulted in persistence of non-cultivable B. burgdorferi in order to determine their long-term fate, and to examine their effects on the host. Results confirmed previous studies, in which B. burgdorferi could not be cultured from tissues, but low copy numbers of B. burgdorferi flaB DNA were detectable in tissues at 2, 4 and 8 months after completion of treatment, and the rate of PCR-positive tissues appeared to progressively decline over time. However, there was resurgence of spirochete flaB DNA in multiple tissues at 12 months, with flaB DNA copy levels nearly equivalent to those found in saline-treated mice. Despite the continued non-cultivable state, RNA transcription of multiple B. burgdorferi genes was detected in host tissues, flaB DNA was acquired by xenodiagnostic ticks, and spirochetal forms could be visualized within ticks and mouse tissues by immunofluorescence and immunohistochemistry, respectively. A number of host cytokines were up- or down-regulated in tissues of both saline- and antibiotic-treated mice in the absence of histopathology, indicating host response to the presence of non-cultivable, despite the lack of inflammation in tissues.

Concepts: Gene, Bacteria, Lyme disease, Borrelia burgdorferi, Borrelia, Spirochaetes, Spirochaete, Allen Steere

28

The Lyme borreliosis complex is a heterogeneous group of tick-borne spirochaetes of the genus Borrelia (Spirochaetales: Spirochaetaceae) that are distributed all over the temperate zone of the northern hemisphere. Due to the usage of new methods for phylogenetic analysis, this group has expanded rapidly during the past 5 years. Along with this development, the number of Borrelia spp. regarded as pathogenic to humans also increased. Distribution areas as well as host and vector ranges of Lyme borreliosis agents turned out to be much wider than previously thought. Furthermore, there is evidence that ticks, reservoir hosts, and patients can be coinfected with multiple Borrelia spp. or other tick-borne pathogens, which indicates a need to establish new and well-defined diagnostic and therapeutic standards for Lyme borreliosis. This review gives a broad overview on the occurrence of Lyme borreliosis spirochaetes worldwide with particular emphasis on their vectors and vertebrate hosts as well as their pathogenic potential and resultant problems in diagnosis and treatment. Against the background that many issues regarding distribution, species identity, ecology, pathogenicity, and coinfections are still unsolved, the purpose of this article is to reveal directions for future research on the Lyme borreliosis complex.

Concepts: Bacteria, Species, Lyme disease, Tick, Borrelia burgdorferi, Borrelia, Spirochaetes, Spirochaete

27

In the early 1980s, Ixodes spp. ticks were implicated as the key North American vectors of Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt and Brenner) (Spirocheatales: Spirochaetaceae), the etiological agent of Lyme disease. Concurrently, other human-biting tick species were investigated as potential B. burgdorferi vectors. Rashes thought to be erythema migrans were observed in patients bitten by Amblyomma americanum (L.) (Acari: Ixodidae) ticks, and spirochetes were visualized in a small percentage of A. americanum using fluorescent antibody staining methods, sparking interest in this species as a candidate vector of B. burgdorferi. Using molecular methods, the spirochetes were subsequently described as Borrelia lonestari sp. nov. (Spirocheatales: Spirochaetaceae), a transovarially transmitted relapsing fever Borrelia of uncertain clinical significance. In total, 54 surveys from more than 35 research groups, involving more than 52,000 ticks, have revealed a low prevalence of B. lonestari, and scarce B. burgdorferi, in A. americanum. In Lyme disease-endemic areas, A. americanum commonly feeds on B. burgdorferi-infected hosts; the extremely low prevalence of B. burgdorferi in this tick results from a saliva barrier to acquiring infection from infected hosts. At least nine transmission experiments involving B. burgdorferi in A. americanum have failed to demonstrate vector competency. Advancements in molecular analysis strongly suggest that initial reports of B. burgdorferi in A. americanum across many states were misidentified B. lonestari, or DNA contamination, yet the early reports continue to be cited without regard to the later clarifying studies. In this article, the surveillance and vector competency studies of B. burgdorferi in A. americanum are reviewed, and we conclude that A. americanum is not a vector of B. burgdorferi.

Concepts: Bacteria, Lyme disease, Tick, Ixodidae, Borrelia burgdorferi, Borrelia, Spirochaetes, Amblyomma americanum

27

Connective tissues are the most common area of colonization for the Lyme disease spirochete Borrelia burgdorferi. Colonization is aided by the interaction between numerous bacterial adhesins with components of the extracellular matrix (ECM). Here we describe a novel interaction between B. burgdorferi and the major ECM proteoglycan found in joints, aggrecan. Using affinity chromatography and mass spectrometry we identify two borrelial aggrecan-binding proteins: the known ECM ligand Bgp (BB0588) and an uncharacterized protease BbHtrA (BB0104). Proteinase K studies demonstrate that BbHtrA is surface exposed. Immunoblots using sera from patients with both early and late Lyme disease establish that BbHtrA is expressed during human disease, immunogenic, and conserved in the three major Lyme disease spirochete species. Consequences of the interaction between aggrecan and BbHtrA were examined by proteolysis assays. BbHtrA cleaves aggrecan at a site known to destroy aggrecan function and which has been previously observed in the synovial fluid of patients with Lyme arthritis. These data demonstrate that B. burgdorferi possess aggrecan-binding proteins which may provide the organism with additional capability to colonize connective tissues. Moreover, our studies provide the first evidence that B. burgdorferi possess proteolytic activity which may contribute to the pathogenesis of Lyme arthritis.

Concepts: Bacteria, Lyme disease, Protease, Borrelia burgdorferi, Borrelia, Spirochaetes, Spirochaete, Allen Steere

24

Lyme disease is a vector-associated infectious disease, caused by the agent, spirochete Borrelia burgdorferi. Neurologic findings are observed in approximately 12% of the cases and termed Lyme neuroborreliosis (LNB). Lyme neuroborreliosis may manifest with different clinical neurologic manifestations.

Concepts: Medicine, Infectious disease, Lyme disease, Borrelia burgdorferi, Borrelia, Spirochaetes, Spirochaete, Neuroborreliosis