Search Results (17)

Borrelia burgdorferi’s inosine-5′-monophosphate dehydrogenase (IMPDH, GuaB encoded by the guaB gene) is a potential therapeutic target. GuaB is necessary for B. burgdorferi replication in mammalian hosts but not in standard laboratory culture conditions. Therefore, we cannot test novel GuaB inhibitors against B. burgdorferi without utilizing mammalian infection models. This study aimed to evaluate modifications to a standard growth medium that may mimic mammalian conditions and induce the requirement of GuaB usage for replication. The effects of two GuaB inhibitors (mycophenolic acid, 6-chloropurine riboside at 125 μM and 250 μM) were assessed against B. burgdorferi (guaB+) grown in standard Barbour–Stoenner–Kelly-II (BSK-II) medium (6% rabbit serum) and BSK-II modified to 60% concentration rabbit serum (BSK-II/60% serum). BSK-II directly supplemented with adenine, hypoxanthine, and nicotinamide (75 μM each, BSK-II/AHN) was also considered as a comparison group. In standard BSK-II, neither mycophenolic acid nor 6-chloropurine riboside affected B. burgdorferi growth. Based on an ANOVA, a dose-dependent increase in drug effects was observed in the modified growth conditions (F = 4.471, p = 0.001). Considering higher drug concentrations at exponential growth, mycophenolic acid at 250 μM reduced spirochete replication by 48% in BSK-II/60% serum and by 50% in BSK-II/AHN (p < 0.001 each). 6-chloropurine riboside was more effective in both mediums than mycophenolic acid, reducing replication by 64% in BSK-II/60% serum and 65% in BSK-II/AHN (p < 0.001 each). These results demonstrate that modifying BSK-II medium with physiologically relevant levels of mammalian serum supports replication and induces the effects of GuaB inhibitors. This represents the first use of GuaB inhibitors against Borrelia burgdorferi, building on tests against purified B. burgdorferi GuaB. The strong effects of 6-chloropurine riboside indicate that B. burgdorferi can salvage and phosphorylate these purine derivative analogs. Therefore, this type of molecule may be considered for future drug development. Optimization of this culture system will allow for better assessment of novel Borrelia-specific GuaB inhibitor molecules for Lyme disease interventions. The use of GuaB inhibitors as broadcast sprays or feed baits should also be evaluated to reduce spirochete load in competent reservoir hosts. Full article

Lyme disease, a multisystemic infectious disorder caused by pathogenic spirochetes of the genus Borrelia transmitted by the bite of ticks, typically from the family Ixodidae, pose a significant public health issue worldwide. The Borrelia burgdorferi sensu lato (s.l.) group encompasses the Borrelia Lyme Group (LG), Borrelia Echidna-Reptile Group (REPG), and Borrelia Relapsing Fever Group (RFG), with some species remaining unclassified due to culturing challenges. Research into B. burgdorferi s.l. infection (Lyme Group) has intensified, focusing on its epidemiology, diagnosis, and treatment. Originally identified in North America and Europe, Lyme disease has now become a global concern, with Latin American countries reporting the microorganism, the disease, and/or its vectors. In Argentina, the presence of B. burgdorferi and Lyme disease has sparked significant scientific and medical debate. Ecological changes due to climate and habitat shifts have expanded the geographical distribution of these ticks. Argentina, with its diverse geography and climate, hosts various tick species that could potentially act as Lyme disease vectors, raising important public health questions. The confirmed presence of B. burgdorferi s.l. and Lyme disease in Argentina remains contentious but relevant, necessitating thorough scientific and medical examination. This work aims to enhance understanding and discussion of Lyme disease in Argentina by presenting clinical cases and their laboratory analyses, highlighting the disease’s presence and implications in the country. Through documenting suspected clinical cases and analyzing available data on B. burgdorferi and Lyme disease in Argentina, this study seeks to contribute to the understanding of the disease’s current status and inform future research, prevention, and control strategies in the region. The goal is to provide a basis for addressing Lyme disease’s public health impact in Argentina and promote further investigation into this evolving issue. Full article

The diagnostic tests available to identify vector-borne pathogens have major limitations. Clinicians must consider an assortment of often diverse symptoms to decide what pathogen or pathogens to suspect and test for. Even then, there are limitations to the currently available indirect detection methods, such as serology, or direct detection methods such as molecular tests with or without culture enrichment. Bartonella spp., which are considered stealth pathogens, are particularly difficult to detect and diagnose. We present a case report of a patient who experienced a spider bite followed by myalgia, lymphadenopathy, and trouble sleeping. She did not test positive for Bartonella spp. through clinically available testing. Her symptoms progressed and she was told she needed a double hip replacement. Prior to the surgery, her blood was submitted for novel molecular testing, where Bartonella spp. was confirmed, and a spirochete was also detected. Additional testing using novel methods over a period of five years found Bartonella henselae and Borrelia burgdorferi in her blood. This patient’s case is an example of why new diagnostic methods for vector-borne pathogens are urgently needed and why new knowledge of the variable manifestations of Bartonellosis need to be provided to the medical community to inform and heighten their index of suspicion. Full article

Human Lyme borreliosis (LB) represents a multisystem disorder that can progress in stages. The causative agents are transmitted by hard ticks of the Ixodes ricinus complex that have been infected with the spirochete Borrelia burgdorferi sensu lato. Today, LB is considered the most important human tick-borne illness in the Northern Hemisphere. The causative agent was identified and successfully isolated in 1982 and, shortly thereafter, antibiotic treatment was found to be safe and efficacious. Since then, various in vitro studies have been conducted in order to improve our knowledge of the activity of antimicrobial agents against B. burgdorferi s. l. The full spectrum of in vitro antibiotic susceptibility has still not been defined for some of the more recently developed compounds. Moreover, our current understanding of the in vitro interactions between B. burgdorferi s. l. and antimicrobial agents, and their possible mechanisms of resistance remains very limited and is largely based on in vitro susceptibility experiments on only a few isolates of Borrelia. Even less is known about the possible mechanisms of the in vitro persistence of spirochetes exposed to antimicrobial agents in the presence of human and animal cell lines. Only a relatively small number of laboratory studies and cell culture experiments have been conducted. This review summarizes what is and what is not known about the in vitro susceptibility of B. burgdorferi s. l. It aims to shed light on the known unknowns that continue to fuel current debates on possible treatment resistance and mechanisms of persistence of Lyme disease spirochetes in the presence of antimicrobial agents. Full article

Round bodies in spirochete cultures have been a controversial subject since their description seven decades ago. We report the existence of round bodies (spherical cells) in cultures of Mucispirillum schaedleri, a spiral bacterium phylogenetically distant from spirochetes. Furthermore, when grown in biofilms, M. schaedleri demonstrates a unique morphology known as cording, which has been previously described only in mycobacteria. Thus, M. schaedleri has two distinct features, each previously thought to be unique to two different phylogenetically distant groups of bacteria. Full article

Borrelia burgdorferi, the causative agent of Lyme disease, has a highly reduced genome and relies heavily on glycolysis for carbon metabolism. As such, established inhibitors of lactate dehydrogenase (LDH) were evaluated in cultures to determine the extent of their impacts on B. burgdorferi growth. Both racemic and enantiopure (AT-101) gossypol, as well as oxamate, galloflavin, and stiripentol, caused the dose-dependent suppression of B. burgdorferi growth in vitro. Racemic gossypol and AT-101 were shown to fully inhibit spirochetal growth at concentrations of 70.5 and 187.5 μM, respectively. Differences between racemic gossypol and AT-101 efficacy may indicate that the dextrorotatory enantiomer of gossypol is a more effective inhibitor of B. burgdorferi growth than the levorotatory enantiomer. As a whole, LDH inhibition appears to be a promising mechanism for suppressing Borrelia growth, particularly with bulky LDH inhibitors like gossypol. Full article

Saprophytic leptospires are spirochetes enclosed within the non-pathogenic clade of the genus Leptospira, which in turn is subdivided into two subclades S1 and S2. To date, the microorganisms included in these subclades have been isolated from the environment in various parts of the world, and are believed to have no known animal reservoirs. After a case of Leptospira interrogans serovar Pomona was notified to the owner of a farm in Sardinia, all of the farm animals (11 pigs and 3 donkeys) were examined for the presence of Leptospira. Sera of all tested animals resulted positive for antibodies to Leptospira using a microscopic agglutination test (MAT). Moreover, nine (82%) kidney samples from pigs and three urine samples collected from donkeys (100%) tested positive for Leptospira DNA after qPCR. Results obtained after MLST analysis and sequencing of rrs, rpoB, and secY genes, performed on six Leptospira strains isolated in culture, revealed the presence of the genomospecies L. interrogans serovar Pomona in the kidney samples. Conversely, whole-genome sequencing combined with mean nucleotide identity revealed the presence of the saprophytic L. montravelensis in the urine samples. Our results report, for the first time, the isolation of a saprophytic species from mammalian urine, suggesting a new ecological specialization for these bacteria, with a possible transition from free-living to a symbiotic lifestyle. Further studies will have to be conducted to understand the evolution of virulence of these bacteria, potential infectivity, and possible public health implications. Full article

Borreliella (syn. Borrelia) burgdorferi is a spirochete bacterium that causes tick-borne Lyme disease. Along its lifecycle B. burgdorferi develops several pleomorphic forms with unclear biological and medical relevance. Surprisingly, these morphotypes have never been compared at the global transcriptome level. To fill this void, we grew B. burgdorferi spirochete, round body, bleb, and biofilm-dominated cultures and recovered their transcriptomes by RNAseq profiling. We found that round bodies share similar expression profiles with spirochetes, despite their morphological differences. This sharply contrasts to blebs and biofilms that showed unique transcriptomes, profoundly distinct from spirochetes and round bodies. To better characterize differentially expressed genes in non-spirochete morphotypes, we performed functional, positional, and evolutionary enrichment analyses. Our results suggest that spirochete to round body transition relies on the delicate regulation of a relatively small number of highly conserved genes, which are located on the main chromosome and involved in translation. In contrast, spirochete to bleb or biofilm transition includes substantial reshaping of transcription profiles towards plasmids-residing and evolutionary young genes, which originated in the ancestor of Borreliaceae. Despite their abundance the function of these Borreliaceae-specific genes is largely unknown. However, many known Lyme disease virulence genes implicated in immune evasion and tissue adhesion originated in this evolutionary period. Taken together, these regularities point to the possibility that bleb and biofilm morphotypes might be important in the dissemination and persistence of B. burgdorferi inside the mammalian host. On the other hand, they prioritize the large pool of unstudied Borreliaceae-specific genes for functional characterization because this subset likely contains undiscovered Lyme disease pathogenesis genes. Full article

Uptake of the Lyme disease spirochete by its tick vector requires not only chemical signals present in the tick’s saliva but a responsive phenotype by the Borrelia burgdorferi living in the mammalian host. This is the principle behind xenodiagnosis, wherein pathogen is detected by vector acquisition. To study migration of B. burgdorferi toward Ixodes scapularis tick saliva, with the goal of identifying chemoattractant molecules, we tested multiple assays and compared migration of host-adapted spirochetes to those cultured in vitro. We tested mammalian host-adapted spirochetes, along with those grown in culture at 34 °C, for their relative attraction to tick saliva or the nutrient N-acetyl-D-glucosamine (D-GlcNAc) and its dimer chitobiose using two different experimental designs. The host-adapted B. burgdorferi showed greater preference for tick saliva over the nutrients, whereas the cultured incubator-grown B. burgdorferi displayed no significant attraction to saliva versus a significant response to the nutrients. Our results not only describe a validated migration assay for studies of the Lyme disease agent, but provide a further understanding of how growth conditions and phenotype of B. burgdorferi are related to vector acquisition. Full article

Equine recurrent uveitis (ERU) causes painful inflammatory attacks and oftentimes blindness in the affected eyes. The disease is considered a late sequela of systemic leptospirosis. The most effective therapy is the surgical removal of the vitreous (vitrectomy), which is not only therapeutic, but provides vitreous material that can be assessed diagnostically. For example, the lipL32 gene, culturable Leptospira spp., and anti-Leptospira antibodies have all been detected in vitreous samples obtained from eyes with chronic ERU. Despite this clear evidence of leptospiral involvement, the systemic administration of antibiotics in infected horses is ineffective at resolving ERU. This syndrome of chronic recurrent inflammation, which is unresponsive to antibiotic therapy, combined with apparent bacteria evading the immune response, is consistent with a biofilm-associated infection. The purpose of this study, therefore, was to detect the in vivo biofilm formation of Leptospira spp. in vitreous samples collected during vitrectomy and examined using a Warthin-Starry silver stain and immunohistochemistry. All known steps of biofilm formation were visualized in these samples, including individual Leptospira spp., leptospiral microcolonies and dense roundish accumulations of Leptospira spp. In many instances spirochetes were surrounded by an extracellular substance. Taken together, data from the present study show that ERU is a biofilm-associated intraocular leptospiral infection, which best explains the typical clinical course. Full article

Leptospirosis is a re-emerging form of zoonosis that is caused by the spirochete pathogen Leptospira. Extracellular proteins play critical roles in the pathogenicity and survival of this pathogen in the host and environment. Extraction and analysis of extracellular proteins is a difficult task due to the abundance of enrichments like serum and bovine serum albumin in the culture medium, as is distinguishing them from the cellular proteins that may reach the analyte during extraction. In this study, extracellular proteins were separated as secretory proteins from the culture supernatant and surface proteins were separated during the washing of the cell pellet. The proteins identified were sorted based on the proportion of the cellular fractions and the extracellular fractions. The results showed the identification of 56 extracellular proteins, out of which 19 were exclusively extracellular. For those proteins, the difference in quantity with respect to their presence within the cell was found to be up to 1770-fold. Further, bioinformatics analysis elucidated characteristics and functions of the identified proteins. Orthologs of extracellular proteins in various Leptospira species were found to be closely related among different pathogenic forms. In addition to the identification of extracellular proteins, this study put forward a method for the extraction and identification of extracellular proteins. Full article

Borrelia burgdorferi, the causative agent of Lyme disease, has been recently shown to form biofilm structures in vitro and in vivo. Biofilms are tightly clustered microbes characterized as resistant aggregations that allow bacteria to withstand harsh environmental conditions, including the administration of antibiotics. Novel antibiotic combinations have recently been identified for B. burgdorferi in vitro, however, due to prohibiting costs, those agents have not been tested in an environment that can mimic the host tissue. Therefore, researchers cannot evaluate their true effectiveness against B. burgdorferi, especially its biofilm form. A skin ex vivo model system could be ideal for these types of experiments due to its cost effectiveness, reproducibility, and ability to investigate host–microbial interactions. Therefore, the main goal of this study was the establishment of a novel ex vivo murine skin biopsy model for B. burgdorferi biofilm research. Murine skin biopsies were inoculated with B. burgdorferi at various concentrations and cultured in different culture media. Two weeks post-infection, murine skin biopsies were analyzed utilizing immunohistochemical (IHC), reverse transcription PCR (RT-PCR), and various microscopy methods to determine B. burgdorferi presence and forms adopted as well as whether it remained live in the skin tissue explants. Our results showed that murine skin biopsies inoculated with 1 × 10⁷ cells of B. burgdorferi and cultured in BSK-H + 6% rabbit serum media for two weeks yielded not just significant amounts of live B. burgdorferi spirochetes but biofilm forms as well. IHC combined with confocal and atomic force microscopy techniques identified specific biofilm markers and spatial distribution of B. burgdorferi aggregates in the infected skin tissues, confirming that they are indeed biofilms. In the future, this ex vivo skin model can be used to study development and antibiotic susceptibility of B. burgdorferi biofilms in efforts to treat Lyme disease effectively. Full article

Canine cases of relapsing fever (RF) borreliosis have been described in Israel and the USA, where two RF species, Borrelia turicatae and Borrelia hermsii, can cause similar clinical signs to the Borrelia persica in dogs and cats reported from Israel, including fever, lethargy, anorexia, thrombocytopenia, and spirochetemia. In this report, we describe the first clinical cases of two dogs and a cat from Spain (Cordoba, Valencia, and Seville) caused by the RF species Borrelia hispanica. Spirochetes were present in the blood smears of all three animals, and clinical signs included lethargy, pale mucosa, anorexia, cachexia, or mild abdominal respiration. Laboratory findings, like thrombocytopenia in both dogs, may have been caused by co-infecting pathogens (i.e., Babesia vogeli, confirmed in one dog). Anemia was noticed in one of the dogs and in the cat. Borrelia hispanica was confirmed as an infecting agent by molecular analysis of the 16S rRNA locus. Molecular analysis of housekeeping genes and phylogenetic analyses, as well as successful in vitro culture of the feline isolate confirmed the causative agent as B. hispanica. Full article

Whether Borrelia burgdorferi, the causative agent of Lyme disease, can persist for long periods in the human body has been a controversial question. The objective of this study was to see if we could find B. burgdorferi in a Lyme disease patient after a long clinical course and after long-term antibiotic treatment. Therefore, we investigated the potential presence of B. burgdorferi antigens and DNA in human autopsy tissues from a well-documented serum-, PCR-, and culture-positive Lyme disease patient, a 53-year-old female from northern Westchester County in the lower Hudson Valley Region of New York State, who had received extensive antibiotic treatments during extensive antibiotic treatments over the course of her 16-year-long illness. We also asked what form the organism might take, with special interest in the recently found antibiotic-resistant aggregate form, biofilm. We also examined the host tissues for the presence of inflammatory markers such as CD3+ T lymphocytes. Autopsy tissue sections of the brain, heart, kidney, and liver were analyzed by histological and immunohistochemical methods (IHC), confocal microscopy, fluorescent in situ hybridization (FISH), polymerase chain reaction (PCR), and whole-genome sequencing (WGS)/metagenomics. We found significant pathological changes, including borrelial spirochetal clusters, in all of the organs using IHC combined with confocal microscopy. The aggregates contained a well-established biofilm marker, alginate, on their surfaces, suggesting they are true biofilm. We found B. burgdorferi DNA by FISH, polymerase chain reaction (PCR), and an independent verification by WGS/metagenomics, which resulted in the detection of B. burgdorferi sensu stricto specific DNA sequences. IHC analyses showed significant numbers of infiltrating CD3+ T lymphocytes present next to B. burgdorferi biofilms. In summary, we provide several lines of evidence that suggest that B. burgdorferi can persist in the human body, not only in the spirochetal but also in the antibiotic-resistant biofilm form, even after long-term antibiotic treatment. The presence of infiltrating lymphocytes in the vicinity of B. burgdorferi biofilms suggests that the organism in biofilm form might trigger chronic inflammation. Full article

Lyme disease is the most common vector borne-disease in the United States (US). While the majority of the Lyme disease patients can be cured with 2–4 weeks antibiotic treatment, about 10–20% of patients continue to suffer from persisting symptoms. While the cause of this condition is unclear, persistent infection was proposed as one possibility. It has recently been shown that B. burgdorferi develops dormant persisters in stationary phase cultures that are not killed by the current Lyme antibiotics, and there is interest in identifying novel drug candidates that more effectively kill such forms. We previously identified some highly active essential oils with excellent activity against biofilm and stationary phase B. burgdorferi. Here, we screened another 35 essential oils and found 10 essential oils (Allium sativum L. bulbs, Pimenta officinalis Lindl. berries, Cuminum cyminum L. seeds, Cymbopogon martini var. motia Bruno grass, Commiphora myrrha (T. Nees) Engl. resin, Hedychium spicatum Buch.-Ham. ex Sm. flowers, Amyris balsamifera L. wood, Thymus vulgaris L. leaves, Litsea cubeba (Lour.) Pers. fruits, Eucalyptus citriodora Hook. leaves) and the active component of cinnamon bark cinnamaldehyde (CA) at a low concentration of 0.1% have strong activity against stationary phase B. burgdorferi. At a lower concentration of 0.05%, essential oils of Allium sativum L. bulbs, Pimenta officinalis Lindl. berries, Cymbopogon martini var. motia Bruno grass and CA still exhibited strong activity against the stationary phase B. burgdorferi. CA also showed strong activity against replicating B. burgdorferi, with a MIC of 0.02% (or 0.2 μg/mL). In subculture studies, the top five essential oil hits Allium sativum L. bulbs, Pimenta officinalis Lindl. berries, Commiphora myrrha (T. Nees) Engl. resin, Hedychium spicatum Buch.-Ham. ex Sm. flowers, and Litsea cubeba (Lour.) Pers. fruits completely eradicated all B. burgdorferi stationary phase cells at 0.1%, while Cymbopogon martini var. motia Bruno grass, Eucalyptus citriodora Hook. leaves, Amyris balsamifera L. wood, Cuminum cyminum L. seeds, and Thymus vulgaris L. leaves failed to do so as shown by visible spirochetal growth after 21-day subculture. At concentration of 0.05%, only Allium sativum L. bulbs essential oil and CA sterilized the B. burgdorferi stationary phase culture, as shown by no regrowth during subculture, while Pimenta officinalis Lindl. berries, Commiphora myrrha (T. Nees) Engl. resin, Hedychium spicatum Buch.-Ham. ex Sm. flowers and Litsea cubeba (Lour.) Pers. fruits essential oils all had visible growth during subculture. Future studies are needed to determine if these highly active essential oils could eradicate persistent B. burgdorferi infection in vivo. Full article