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Concept: Allen Steere

170

Comparative analysis of ospC genes from 127 Borrelia burgdorferi sensu stricto strains collected in Lyme disease endemic and non-endemic European and North American regions revealed close relatedness of geographically distinct populations. OspC alleles A, B and L were detected on both continents in vectors and hosts including humans. Six ospC alleles, A, B, L, Q, R and V, were prevalent in Europe; 4 of them were detected in samples of human origin. Ten ospC alleles, A, B, D, E3, F, G, H, H3, I3 and M, were identified in the far-western U.S.A. Four ospC alleles, B, G, H and L, were abundant in the southeastern U.S.A. Here we present the first expanded analysis of ospC alleles of B. burgdorferi strains from the southeastern U.S.A with respect to their relatedness to strains from other North American and European localities. We demonstrate that ospC genotypes commonly associated with human Lyme disease in endemic European and North American regions were detected in B. burgdorferi strains isolated from non-human biting tick Ixodes affinis and rodent hosts in southeastern U.S.A. We discovered that some ospC alleles previously known only from Europe are widely distributed in the southeastern U.S.A., a finding that confirms the hypothesis of trans-oceanic migration of Borrelia species.

Concepts: Bacteria, Lyme disease, Ixodes scapularis, North America, Borrelia burgdorferi, Borrelia, Allen Steere, Willy Burgdorfer

29

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

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

19

The invasion of the blacklegged tick, Ixodes scapularis into Ontario, Canada poses a significant risk to public health because it is a vector for numerous pathogens, including Borrelia burgdorferi sensu stricto, the causative agent of Lyme disease. Baseline field sampling in 2014 and 2015 detected I. scapularis and B. burgdorferi at sites across southern, eastern and central Ontario, including a hot spot in eastern Ontario. A “speed of spread” model for I. scapularis developed by Leighton and colleagues (2012) estimated that the tick’s range was expanding northward at 46 km/year. In 2016, we revisited a subset of sites sampled in 2014 and 2015 to understand the changing nature of risk, and assess whether the rate of tick invasion is consistent with the speed of spread estimate. Ticks were collected via tick dragging at 17 out of 36 sites, 5 of which were new sites for I. scapularis. Samples were positive for B. burgdorferi at 8 sites. No other I. scapularis-borne pathogens were detected. Centrographic statistics revealed an increase in the dispersion of I. scapularis positive sites in eastern Ontario. Field data for each site were then compared to the model’s predicted year of establishment for each census subdivision. Our findings illustrate that the range expansion of I. scapularis and the emergence of B. burgdorferi is ongoing, and provide short timescale evidence of the processes associated with I. scapularis spread. The range front appears to be moving at a rate of ~46 km/year, with colonization of the tick behind this range front occurring at a slower and heterogeneous rate. Assessment of site-level ecological factors did not provide any insight into the underlying processes that may be influencing the colonization of I. scapularis in specific areas. Ongoing field sampling is needed to monitor this dynamic process. This study highlights the current geographic risk associated with Lyme disease, which can be used to target public health interventions to the areas of greatest risk.

Concepts: Bacteria, Lyme disease, Tick, Ixodes scapularis, Borrelia burgdorferi, Borrelia, Babesiosis, Allen Steere

18

The white-footed mouse (Peromyscus leucopus) is an important reservoir host for Borrelia burgdorferi, the pathogen responsible for Lyme disease, and its distribution is expanding northward. We used an Ecological Niche Factor Analysis to identify the climatic factors associated with the distribution shift of the white-footed mouse over the last 30 years at the northern edge of its range, and modeled its current and potential future (2050) distributions using the platform BIOMOD. A mild and shorter winter is favouring the northern expansion of the white-footed mouse in Québec. With more favorable winter conditions projected by 2050, the distribution range of the white-footed mouse is expected to expand further northward by 3° latitude. We also show that today in southern Québec, the occurrence of B. burgdorferi is associated with high probability of presence of the white-footed mouse. Changes in the distribution of the white-footed mouse will likely alter the geographical range of B. burgdorferi and impact the public health in northern regions that have yet to be exposed to Lyme disease.

Concepts: Bacteria, Climate, Lyme disease, Borrelia burgdorferi, Borrelia, Allen Steere, Willy Burgdorfer, White-footed Mouse

17

Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10-20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under experimental stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that appear resistant in vitro to customary first-line antibiotics for Lyme disease. To identify more effective drugs with activity against the round body form of B. burgdorferi, we established a round body persister model induced by exposure to amoxicillin (50 μg/ml) and then screened the Food and Drug Administration drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven individual drugs scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. In this amoxicillin-induced round body model, some drug candidates such as daptomycin and clofazimine also displayed enhanced activity which was similar to a previous screen against stationary phase B. burgdorferi persisters not exposure to amoxicillin. Additional candidate drugs active against round bodies identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against amoxicillin-induced round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi persisters in vitro, even when pre-treated with amoxicillin. These findings may have implications for improved treatment of Lyme disease.

Concepts: Bacteria, Lyme disease, Borrelia burgdorferi, Borrelia, Allen Steere, Willy Burgdorfer

17

Doxycycline is a commonly used antibiotic to treat Lyme disease and other bacterial infections. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for Borrelia burgdorferi have been investigated by different groups, but are experimentally established here as a function of input cell density. We demonstrate that B. burgdorferi treated in the stationary phase have a higher probability of regrowth following removal of antibiotic. In addition, we determine experimentally and mathematically that the spirochetes which persist post-treatment do not have a longer lag phase, but exhibit a slower growth rate than untreated spirochetes. Finally, we demonstrate that treating the spirochetes by pulse-dosing was not found to eliminate growth or reduce the persister population in vitro. From these data, we propose that B. burgdorferi persister development is stochastic and driven by slowed growth.

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

14

Out of 20 spirochete species from Borrelia burgdorferi sensu lato (s.l.) complex recognized to date some are considered to have a limited distribution, while others are worldwide dispersed. Among those are Borrelia burgdorferi sensu stricto (s.s.) and Borrelia bissettii which are distributed both in North America and in Europe. While B. burgdorferi s.s. is recognized as a cause of Lyme borreliosis worldwide, involvement of B. bissettii in human Lyme disease was not so definite yet.

Concepts: Bacteria, United States, Lyme disease, Borrelia burgdorferi, Borrelia, Spirochaetes, Allen Steere, Willy Burgdorfer

12

Lyme disease is a tick-borne, multi-systemic disease, caused by the bacterium Borrelia burgdorferi. Though antibiotics are used as a primary treatment, relapse often occurs after the discontinuation of antimicrobial agents. The reason for relapse remains unknown, however previous studies suggest the possible presence of antibiotic resistant Borrelia round bodies, persisters and attached biofilm forms. Thus, there is an urgent need to find antimicrobial agents suitable to eliminate all known forms of B. burgdorferi. In this study, natural antimicrobial agents such as Apis mellifera venom and a known component, melittin, were tested using SYBR Green I/PI, direct cell counting, biofilm assays combined with LIVE/DEAD and atomic force microscopy methods. The obtained results were compared to standalone and combinations of antibiotics such as Doxycycline, Cefoperazone, Daptomycin, which were recently found to be effective against Borrelia persisters. Our findings showed that both bee venom and melittin had significant effects on all the tested forms of B. burgdorferi. In contrast, the control antibiotics when used individually or even in combinations had limited effects on the attached biofilm form. These findings strongly suggest that whole bee venom or melittin could be effective antimicrobial agents for B. burgdorferi; however, further research is necessary to evaluate their effectiveness in vivo, as well as their safe and effective delivery method for their therapeutic use.

Concepts: Bacteria, Antibiotic resistance, Effectiveness, Lyme disease, Borrelia burgdorferi, Borrelia, Allen Steere, Willy Burgdorfer

10

Two-tier serology testing is most frequently used for the diagnosis of Lyme borreliosis (LB); however, a positive result is no proof of active disease. To establish a diagnosis of active LB, better diagnostics are needed. Tests investigating the cellular immune system are available, but studies evaluating the utility of these tests on well-defined patient populations are lacking. Therefore, we investigated the utility of an enzyme-linked immunospot (ELISpot) assay to diagnose active Lyme neuroborreliosis. Peripheral blood mononuclear cells (PBMCs) of various study groups were stimulated by using Borrelia burgdorferi strain B31 and various recombinant antigens and subsequently, the number of Borrelia-specific interferon-gamma (IFN-γ) secreting T-cells was measured.We included 33 active and 37 treated Lyme neuroborreliosis patients, 28 healthy individuals treated for an early manifestation of LB in the past and 145 untreated healthy individuals. The median number of B. burgdorferi B31-specific IFN-γ secreting T-cells/2.5x105 PBMCs did not differ between active Lyme neuroborreliosis patients, treated Lyme neuroborreliosis patients and treated healthy individuals (6.0, interquartile range (IQR): 0.5 - 14.0; 4.5, IQR: 2.0 - 18.6; 7.4, IQR: 2.3 - 14.9; respectively) (p 1.000); however, the median number of B. burgdorferi B31-specific IFN-γ secreting T-cells/2.5x105 PBMCs among untreated healthy individuals was lower (2.0, IQR: 0.5 - 3.9) (p ≤0.016).We conclude that the Borrelia ELISpot, measuring the number of B. burgdorferi B31-specific INF-γ secreting T-cells/2.5x105 PBMCs, correlates with exposure to the Borrelia bacterium, but cannot be used for the diagnosis of active Lyme neuroborreliosis.

Concepts: Immune system, Bacteria, Lyme disease, Borrelia burgdorferi, Borrelia, Allen Steere, Willy Burgdorfer, Neuroborreliosis