Antimicrobial Activity of Bee Venom and Melittin against Borrelia burgdorferi

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

Influence of Tick and Mammalian Physiological Temperatures on Borrelia Burgdorferi Biofilms

The spirochaete bacterium Borrelia burgdorferi sensu lato is the aetiologic agent of Lyme disease. Borrelia is transmitted to mammals through tick bite and is adapted to survive at tick and mammalian physiological temperatures. We have previously shown that B. burgdorferi can exist in different morphological forms, including the antibiotic-resistant biofilm form, in vitro and in vivo. B. burgdorferi forms aggregates in ticks as well as in humans, indicating potential of biofilm formation at both 23 and 37 °C. However, the role of various environmental factors that influence Borrelia biofilm formation remains unknown. In this study, we investigated the effect of tick (23 °C), mammalian physiological (37 °C) and standard in vitro culture (33 °C) temperatures with the objective of elucidating the effect of temperature on Borrelia biofilm phenotypes invitro using two B. burgdorferi sensu stricto strains (B31 and 297). Our findings show increased biofilm quantity, biofilm size, exopolysaccharide content and enhanced adherence as well as reduced free spirochaetes at 37 °C for both strains, when compared to growth at 23 and 33 °C. There were no significant variations in the biofilm nano-topography and the type of extracellular polymeric substance in Borrelia biofilms formed at all three temperatures. Significant variations in extracellular DNA content were observed in the biofilms of both strains cultured at the three temperatures. Our results indicate that temperature is an important regulator of Borrelia biofilm development, and that the mammalian physiological temperature favours increased biofilm formation in vitro compared to tick physiological temperature and in vitro culture temperature

Effectiveness of Stevia Rebaudiana Whole Leaf Extract Against the Various Morphological Forms of Borrelia Burgdorferi in Vitro

Lyme disease is a tick-borne multisystemic disease caused by Borrelia burgdorferi. Administering antibiotics is the primary treat-ment for this disease; however, relapse often occurs when antibiotic treatment is discontinued. The reason for relapse remains un-known, but recent studies suggested the possibilities of the presence of antibiotic resistant Borreliapersister cells and biofilms.In this study, we evaluated the effectiveness of whole leaf Stevia extract against B. burgdorferispirochetes, persisters, and bio-film forms in vitro. The susceptibility of the different forms was evaluated by various quantitative techniques in addition to differ-ent microscopy methods. The effectiveness of Stevia was compared to doxycycline, cefoperazone, daptomycin, and their combina-tions. Our results demonstrated that Stevia had significant effect in eliminating B. burgdorferi spirochetes and persisters. Sub-culture experiments with Stevia and antibiotics treated cells were established for 7 and 14 days yielding, no and 10% viable cells, respectively compared to the above-mentioned antibiotics and antibiotic combination. When Stevia and the three antibiotics weretested against attached biofilms, Stevia significantly reduced B. burgdorferiforms. Results from this study suggest that a natural product such as Stevia leaf extract could be considered as an effective agent against B. burgdorferi

Evidence of in Vivo Existence of Borrelia Biofilm in Borrelial Lymphocytomas

Lyme borreliosis, caused by the spirochete Borrelia burgdorferi sensu lato, has grown into a major public health problem. We recently identified a novel morphological form of B. burgdorferi, called biofilm, a structure that is well known to be highly resistant to antibiotics. However, there is no evidence of the existence of Borrelia biofilm in vivo; therefore, the main goal of this study was to determine the presence of Borrelia biofilm in infected human skin tissues. Archived skin biopsy tissues from borrelial lymphocytomas (BL) were reexamined for the presence of B. burgdorferi sensu lato using Borrelia-specific immunohistochemical staining (IHC), fluorescent in situ hybridization, combined fluorescent in situ hybridization (FISH)—IHC, polymerase chain reaction (PCR), and fluorescent and atomic force microscopy methods. Our morphological and histological analyses showed that significant amounts of Borrelia-positive spirochetes and aggregates exist in the BL tissues. Analyzing structures positive for Borrelia showed that aggregates, but not spirochetes, expressed biofilm markers such as protective layers of different mucopolysaccharides, especially alginate. Atomic force microscopy revealed additional hallmark biofilm features of the Borrelia/alginate-positive aggregates such as inside channels and surface protrusions. In summary, this is the first study that demonstrates the presence of Borrelia biofilm in human infected skin tissues

Large-Scale Sequencing of <i>Borreliaceae</i> for the Construction of Pan-Genomic-Based Diagnostics

The acceleration of climate change has been associated with an alarming increase in the prevalence and geographic range of tick-borne diseases (TBD), many of which have severe and long-lasting effects—particularly when treatment is delayed principally due to inadequate diagnostics and lack of physician suspicion. Moreover, there is a paucity of treatment options for many TBDs that are complicated by diagnostic limitations for correctly identifying the offending pathogens. This review will focus on the biology, disease pathology, and detection methodologies used for the Borreliaceae family which includes the Lyme disease agent Borreliella burgdorferi. Previous work revealed that Borreliaceae genomes differ from most bacteria in that they are composed of large numbers of replicons, both linear and circular, with the main chromosome being the linear with telomeric-like termini. While these findings are novel, additional gene-specific analyses of each class of these multiple replicons are needed to better understand their respective roles in metabolism and pathogenesis of these enigmatic spirochetes. Historically, such studies were challenging due to a dearth of both analytic tools and a sufficient number of high-fidelity genomes among the various taxa within this family as a whole to provide for discriminative and functional genomic studies. Recent advances in long-read whole-genome sequencing, comparative genomics, and machine-learning have provided the tools to better understand the fundamental biology and phylogeny of these genomically-complex pathogens while also providing the data for the development of improved diagnostics and therapeutics

Effectiveness of Stevia rebaudiana

Lyme disease is a tick-borne multisystemic disease caused by Borrelia burgdorferi. Administering antibiotics is the primary treat-ment for this disease; however, relapse often occurs when antibiotic treatment is discontinued. The reason for relapse remains un-known, but recent studies suggested the possibilities of the presence of antibiotic resistant Borreliapersister cells and biofilms.In this study, we evaluated the effectiveness of whole leaf Stevia extract against B. burgdorferispirochetes, persisters, and bio-film forms in vitro. The susceptibility of the different forms was evaluated by various quantitative techniques in addition to differ-ent microscopy methods. The effectiveness of Stevia was compared to doxycycline, cefoperazone, daptomycin, and their combina-tions. Our results demonstrated that Stevia had significant effect in eliminating B. burgdorferi spirochetes and persisters. Sub-culture experiments with Stevia and antibiotics treated cells were established for 7 and 14 days yielding, no and 10% viable cells, respectively compared to the above-mentioned antibiotics and antibiotic combination. When Stevia and the three antibiotics weretested against attached biofilms, Stevia significantly reduced B. burgdorferiforms. Results from this study suggest that a natural product such as Stevia leaf extract could be considered as an effective agent against B. burgdorferi