A selective antibiotic for Lyme disease.

Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment.

An rfuABCD-Like Operon and Its Relationship to Riboflavin Utilization and Mammalian Infectivity by Borrelia burgdorferi.

Riboflavin is an essential micronutrient, but its transport and utilization have remained largely understudied among pathogenic spirochetes. Here, we show that Borrelia burgdorferi, the zoonotic spirochete that causes Lyme disease, is able to import riboflavin via products of its rfuABCD-like operon as well as synthesize flavin mononucleotide and flavin adenine dinucleotide despite lacking canonical genes for their synthesis. Additionally, a mutant deficient in the rfuABCD-like operon is resistant to the antimicrobial effect of roseoflavin, a natural riboflavin analog, and is attenuated in a murine model of Lyme borreliosis. Our combined results indicate not only that are riboflavin and the maintenance of flavin pools essential for B. burgdorferi growth but also that flavin utilization and its downstream products (e.g., flavoproteins) may play a more prominent role in B. burgdorferi pathogenesis than previously appreciated.

Septic Arthritis: Diagnosis and Treatment.

Septic arthritis must be considered and promptly diagnosed in any patient presenting with acute atraumatic joint pain, swelling, and fever. Risk factors for septic arthritis include age older than 80 years, diabetes mellitus, rheumatoid arthritis, recent joint surgery, hip or knee prosthesis, skin infection, and immunosuppressive medication use. A delay in diagnosis and treatment can result in permanent morbidity and mortality. Physical examination findings and serum markers, including erythrocyte sedimentation rate and C-reactive protein, are helpful in the diagnosis but are nonspecific. Synovial fluid studies are required to confirm the diagnosis. History and Gram stain aid in determining initial antibiotic selection. Staphylococcus aureus is the most common pathogen isolated in septic arthritis; however, other bacteria, viruses, fungi, and mycobacterium can cause the disease. After synovial fluid has been obtained, empiric antibiotic therapy should be initiated if there is clinical concern for septic arthritis. Oral antibiotics can be given in most cases because they are not inferior to intravenous therapy. Total duration of therapy ranges from two to six weeks; however, certain infections require longer courses. Consideration for microorganisms such as Neisseria gonorrhoeae, Borrelia burgdorferi, and fungal infections should be based on history findings and laboratory results.

A Novel Laminin-Binding Protein Mediates Microbial-Endothelial Cell Interactions and Facilitates Dissemination of Lyme Disease Pathogens.

Borrelia burgdorferi conserved gene products BB0406 and BB0405, members of a common B. burgdorferi paralogous gene family, share 59% similarity. Although both gene products can function as potential porins, only BB0405 is essential for infection. Here we show that, despite sequence homology and coexpression from the same operon, both proteins differ in their membrane localization attributes, antibody accessibility, and immunogenicity in mice. BB0406 is required for spirochete survival in mammalian hosts, particularly for the disseminated infection in distant organs. We identified that BB0406 interacts with laminin, one of the major constituents of the vascular basement membrane, and facilitates spirochete transmigration across host endothelial cell barriers. A better understanding of how B. burgdorferi transmigrates through dermal and tissue vascular barriers and establishes disseminated infections will contribute to the development of novel therapeutics to combat early infection.

Prospective comparison of two enzyme-linked immunosorbent spot assays for the diagnosis of Lyme neuroborreliosis.

Commercial cellular tests are used to diagnose Lyme borreliosis (LB), but studies on their clinical validation are lacking. This study evaluated the utility of an in-house and a commercial enzyme-linked immunosorbent spot (ELISpot) assay for the diagnosis of Lyme neuroborreliosis (LNB). Prospectively, peripheral blood mononuclear cells (PBMCs) were isolated from patients and controls and analysed using an in-house Borrelia ELISpot assay and the commercial LymeSpot assay. B. burgdorferi B31 whole cell lysate and a mixture of outer surface proteins were used to stimulate the PBMCs and the numbers of interferon-gamma-secreting T cells were measured. Results were evaluated using receiver operating characteristic (ROC) curve analysis. Eighteen active and 12 treated LNB patients, 10 healthy individuals treated for an early (mostly cutaneous) manifestation of LB in the past and 47 untreated healthy individuals were included. Both assays showed a poor diagnostic performance with sensitivities, specificities, positive and negative predictive values ranging from 44.4-66.7%, 42.0-72.5%, 21.8-33.3% and 80.5-87.0%, respectively. The LymeSpot assay performed equally poorly when the calculation method of the manufacturer was used. Both the in-house and the LymeSpot assay are unable to diagnose active LNB or to monitor antibiotic treatment success.

Borrelia burgdorferi protein interactions critical for microbial persistence in mammals.

Borrelia burgdorferi is the causative agent of Lyme disease that persists in a complex enzootic life cycle, involving Ixodes ticks and vertebrate hosts. The microbe invades ticks and vertebrate hosts in spite of active immune surveillance and potent microbicidal responses, and establishes long-term infection utilising mechanisms that are yet to be unravelled. The pathogen can cause multi-system disorders when transmitted to susceptible mammalian hosts, including in humans. In the past decades, several studies identified a limited number of B. burgdorferi gene-products critical for pathogen persistence, transmission between the vectors and the host, and host-pathogen interactions. This review will focus on the interactions between B. burgdorferi proteins, as well as between microbial proteins and host components, protein and non-protein components, highlighting their roles in pathogen persistence in the mammalian host. A better understanding of the contributions of protein interactions in the microbial virulence and persistence of B. burgdorferi would support development of novel therapeutics against the infection.

Generality of Post-Antimicrobial Treatment Persistence of Borrelia burgdorferi Strains N40 and B31 in Genetically Susceptible and Resistant Mouse Strains.

A basic feature of infection caused by Borrelia burgdorferi, the etiological agent of Lyme borreliosis, is that persistent infection is the rule in its many hosts. The ability to persist and evade host immune clearance poses a challenge to effective antimicrobial treatment. A link between therapy failure and the presence of persister cells has started to emerge. There is growing experimental evidence that viable but noncultivable spirochetes persist following treatment with several different antimicrobial agents. The current study utilized the mouse model to evaluate if persistence occurs following antimicrobial treatment in disease-susceptible (C3H/HeJ [C3H]) and disease-resistant (C57BL/6 [B6]) mouse strains infected with B. burgdorferi strains N40 and B31 and to confirm the generality of this phenomenon, as well as to assess the persisters' clinical relevance. The status of infection was evaluated at 12 and 18 months after treatment. The results demonstrated that persistent spirochetes remain viable for up to 18 months following treatment, as well as being noncultivable. The phenomenon of persistence in disease-susceptible C3H mice is equally evident in disease-resistant B6 mice and not unique to any particular B. burgdorferi strain. The results also demonstrate that, following antimicrobial treatment, both strains of B. burgdorferi, N40 and B31, lose one or more plasmids. The study demonstrated that noncultivable spirochetes can persist in a host following antimicrobial treatment for a long time but did not demonstrate their clinical relevance in a mouse model of chronic infection. The clinical relevance of persistent spirochetes beyond 18 months following antimicrobial treatment requires further studies in other animal models.

Molecular Testing of Serial Blood Specimens from Patients with Early Lyme Disease during Treatment Reveals Changing Coinfection with Mixtures of Borrelia burgdorferi Genotypes.

Borrelia burgdorferi is the etiological agent of Lyme disease. In the current study, we used direct-detection PCR and electrospray ionization mass spectrometry to monitor and genotype B. burgdorferi isolates from serially collected whole-blood specimens from patients clinically diagnosed with early Lyme disease before and during 21 days of antibiotic therapy. B. burgdorferi isolates were detected up to 3 weeks after the initiation of antibiotic treatment, with ratios of coinfecting B. burgdorferi genotypes changing over time.

Azithromycin nanocrystals for dermal prevention of tick bite infections.

Azithromycin was optimized as nanocrystals with a drug content of 10.0 % (w/w) and a surfactant D-α -tocopheryl polyethylenglycol 1000 succinate (TPGS) content of 1.0 % (w/w) using bead milling for 10 min. The photon correlation spectroscopy (PCS) diameter of the bulk population was 189 nm, laser diffraction (LD) diameter 90 % was 370 nm. Spherical morphology of the optimal nanocrystals was observed by transmission electron microscope (TEM). They were stable over 1 year of storage at 4 °C with the particle size within the nanometer range which was confirmed by PCS, LD and light microscope. An acceptable physical stability of 2 years was also obtained when stored at 4 °C. No microbial attack to the nanocrystals was observed before 3 years storage at 4 °C. The saturation solubility of the nanocrystals was up to triple compared to the raw drug powder (RDP) in water. When incorporated into the gel base, highest penetration efficacy was achieved by the optimal nanocrystals compared to 1) the clinically effective ethanol-solution-gel, 2) the gel with propylene glycol and 3) the gel with RDP in the ex vivo porcine ear penetration study. Even though propylene glycol improved saturation solubility of nanocrystals, it could not bring benefit to nanocrystals in the penetration study. Based on these optimized azithromycin nanocrystals, topical administration for enhanced dermal bioavailability of azithromycin seems to be feasible.

Short-Chain Fatty Acids Alter Metabolic and Virulence Attributes of Borrelia burgdorferi.

Borrelia burgdorferi responds to a variety of host-derived factors and appropriately alters its gene expression for adaptation under different host-specific conditions. We previously showed that various levels of acetate, a short-chain fatty acid (SCFA), altered the protein profile of B. burgdorferi In this study, we determined the effects of other physiologically relevant SCFAs in the regulation of metabolic/virulence-associated proteins using mutant borrelial strains. No apparent increase in the synthesis of outer surface protein C (OspC) was noted when a carbon storage regulator A (csrA of B. burgdorferi, or csrABb ) mutant (mt) was propagated within dialysis membrane chambers implanted within rat peritoneal cavity, while the parental wild type (wt; B31-A3 strain) and csrABb cis-complemented strain (ct) had increased OspC with a reciprocal reduction in OspA levels. Growth rates of wt, mt, ct, 7D (csrABb mutant lacking 7 amino acids at the C terminus), and 8S (csrABb with site-specific changes altering its RNA-binding properties) borrelial strains were similar in the presence of acetate. Increased levels of propionate and butyrate reduced the growth rates of all strains tested, with mt and 8S exhibiting profound growth deficits at higher concentrations of propionate. Transcriptional levels of rpoS and ospC were elevated on supplementation of SCFAs compared to those of untreated spirochetes. Immunoblot analysis revealed elevated levels of RpoS, OspC, and DbpA with increased levels of SCFAs. Physiological levels of SCFAs prevalent in select human and rodent fluids were synergistic with mammalian host temperature and pH to increase the levels of aforementioned proteins, which could impact the colonization of B. burgdorferi during the mammalian phase of infection.

Identifying Vancomycin as an Effective Antibiotic for Killing Borrelia burgdorferi.

Borrelia burgdorferi is the causative agent of Lyme borreliosis. Antibiotic therapy of early acute infection is effective for most patients, but 10 to 20% go on to develop posttreatment Lyme disease syndrome (PTLDS). The nature of PTLDS remains unknown, but currently approved antibiotics for the treatment of Lyme disease do not appear to impact these symptoms after they have developed. We reason that minimizing the time the pathogen interacts with the host will diminish the probability of developing PTLDS, irrespective of its nature. This calls for an efficient eradication of the pathogen during acute infection. In search of a superior killing antibiotic, we examined approved antibiotics for their ability to kill B. burgdorferi Vancomycin proved more effective in killing the pathogen in vitro than ceftriaxone, the standard of care for disseminated B. burgdorferi infection. Both compounds were also the most effective in killing stationary-phase cells. This is surprising, given that inhibitors of cell wall biosynthesis are known to only kill growing bacteria. We found that peptidoglycan synthesis continues in stationary-phase cells of B. burgdorferi, explaining this paradox. A combination of vancomycin and gemifloxacin sterilized a stationary-phase culture of B. burgdorferi Examination of the action of antibiotics in severe combined immunodeficient (SCID) mice showed that doxycycline, a standard of care for uncomplicated acute infection, did not clear the pathogen. In contrast, both ceftriaxone and vancomycin cleared the infection. A trial examining the early use of more potent antibiotics on the development of PTLDS may be warranted.

Fluorescent Proteins, Promoters, and Selectable Markers for Applications in the Lyme Disease Spirochete Borrelia burgdorferi.

Lyme disease is the most widely reported vector-borne disease in the United States. Its incidence is rapidly increasing, and disease symptoms can be debilitating. The need to understand the biology of the disease agent, the spirochete Borrelia burgdorferi, is thus evermore pressing. Despite important advances in B. burgdorferi genetics, the array of molecular tools available for use in this organism remains limited, especially for cell biological studies. Here, we adapt a palette of bright and mostly monomeric fluorescent proteins for versatile use and multicolor imaging in B. burgdorferi We also characterize two novel antibiotic selection markers and establish the feasibility of their use in conjunction with extant markers. Last, we describe a set of promoters of low and intermediate strengths that allow fine-tuning of gene expression levels. These molecular tools complement and expand current experimental capabilities in B. burgdorferi, which will facilitate future investigation of this important human pathogen. To showcase the usefulness of these reagents, we used them to investigate the subcellular localization of BB0323, a B. burgdorferi lipoprotein essential for survival in the host and vector environments. We show that BB0323 accumulates at the cell poles and future division sites of B. burgdorferi cells, highlighting the complex subcellular organization of this spirochete.IMPORTANCE Genetic manipulation of the Lyme disease spirochete B. burgdorferi remains cumbersome, despite significant progress in the field. The scarcity of molecular reagents available for use in this pathogen has slowed research efforts to study its unusual biology. Of interest, B. burgdorferi displays complex cellular organization features that have yet to be understood. These include an unusual morphology and a highly fragmented genome, both of which are likely to play important roles in the bacterium's transmission, infectivity, and persistence. Here, we complement and expand the array of molecular tools available for use in B. burgdorferi by generating and characterizing multiple fluorescent proteins, antibiotic selection markers, and promoters of varied strengths. These tools will facilitate investigations in this important human pathogen, as exemplified by the polar and midcell localization of the cell envelope regulator BB0323, which we uncovered using these reagents.

Vancomycin Reduces Cell Wall Stiffness and Slows Swim Speed of the Lyme Disease Bacterium.

Borrelia burgdorferi, the spirochete that causes Lyme disease, is a tick-transmitted pathogen that requires motility to invade and colonize mammalian and tick hosts. These bacteria use a unique undulating flat-wave shape to penetrate and propel themselves through host tissues. Previous mathematical modeling has suggested that the morphology and motility of these spirochetes depends crucially on the flagellar/cell wall stiffness ratio. Here, we test this prediction using the antibiotic vancomycin to weaken the cell wall. We found that low to moderate doses of vancomycin (≤2.0 µg/mL for 24 h) produced small alterations in cell shape and that as the dose was increased, cell speed decreased. Vancomycin concentrations >1.0 µg/mL also inhibited cell growth and led to bleb formation on a fraction of the cells. To quantitatively assess how vancomycin affects cell stiffness, we used optical traps to bend unflagellated mutants of B. burgdorferi. We found that in the presence of vancomycin, cell wall stiffness gradually decreased over time, with a 40% reduction in the bending stiffness after 36 h. Under the same conditions, the swimming speed of wild-type B. burgdorferi slowed by ∼15%, with only marginal changes to cell morphology. Interestingly, our biophysical model for the swimming dynamics of B. burgdorferi suggested that cell speed should increase with decreasing cell stiffness. We show that this discrepancy can be resolved if the periplasmic volume decreases as the cell wall becomes softer. These results provide a testable hypothesis for how alterations of cell wall stiffness affect periplasmic volume regulation. Furthermore, since motility is crucial to the virulence of B. burgdorferi, the results suggest that sublethal doses of antibiotics could negatively impact spirochete survival by impeding their swim speed, thereby enabling their capture and elimination by phagocytes.

Outer Membrane Proteins BB0405 and BB0406 Are Immunogenic, but Only BB0405 Is Required for Borrelia burgdorferi Infection.

We recently identified the Borrelia burgdorferi outer membrane protein (OMP) BB0406 and found that the gene encoding this OMP was cotranscribed with the gene encoding the OMP BB0405. Interestingly, BB0405 and BB0406 share 59% similarity and are grouped into the same B. burgdorferi paralogous gene family. Given their overall similarity, it is plausible that both OMPs have similar or overlapping functions in this pathogenic spirochete. BB0405 was recently shown to be required for mammalian infection despite the observations that BB0405 is poorly immunogenic and not recognized during mouse or human infection. BB0405 orthologs have also been shown to bind the complement regulator protein factor H. Therefore, to better elucidate the role of BB0405 and its paralog BB0406 during infection and in serum resistance, we examined both proteins in animal infection, factor H binding, and serum sensitivity assays. Our combined results suggest that BB0405- and BB0406-specific antibodies are borreliacidal and that both OMPs are immunogenic during nonhuman primate infection. Additionally, while BB0405 was found to be required for establishing mouse infection, BB0406 was not found to be essential for infectivity. In contrast to data from previous reports, however, neither OMP was found to bind human factor H or to be required for enhancing serum resistance of B. burgdorferi in vitro.

Sleeper cells: the stringent response and persistence in the Borreliella (Borrelia) burgdorferi enzootic cycle.

Infections with tick-transmitted Borreliella (Borrelia) burgdorferi, the cause of Lyme disease, represent an increasingly large public health problem in North America and Europe. The ability of these spirochetes to maintain themselves for extended periods of time in their tick vectors and vertebrate reservoirs is crucial for continuance of the enzootic cycle as well as for the increasing exposure of humans to them. The stringent response mediated by the alarmone (p)ppGpp has been determined to be a master regulator in B. burgdorferi. It modulates the expression of identified and unidentified open reading frames needed to deal with and overcome the many nutritional stresses and other challenges faced by the spirochete in ticks and animal reservoirs. The metabolic and morphologic changes resulting from activation of the stringent response in B. burgdorferi may also be involved in the recently described non-genetic phenotypic phenomenon of tolerance to otherwise lethal doses of antimicrobials and to other antimicrobial activities. It may thus constitute a linchpin in multiple aspects of infections with Lyme disease borrelia, providing a link between the micro-ecological challenges of its enzootic life-cycle and long-term residence in the tissues of its animal reservoirs, with the evolutionary side effect of potential persistence in incidental human hosts.

Borrelia burgdorferi glycosaminoglycan-binding proteins: a potential target for new therapeutics against Lyme disease.

The spirochete bacterium Borrelia burgdorferi sensu lato is the causative agent of Lyme disease, the most common vector-borne disease in Europe and the United States. The spirochetes can be transmitted to humans via ticks, and then spread to different tissues, leading to arthritis, carditis and neuroborreliosis. Although antibiotics have commonly been used to treat infected individuals, some treated patients do not respond to antibiotics and experience persistent, long-term arthritis. Thus, there is a need to investigate alternative therapeutics against Lyme disease. The spirochete bacterium colonization is partly attributed to the binding of the bacterial outer-surface proteins to the glycosaminoglycan (GAG) chains of host proteoglycans. Blocking the binding of these proteins to GAGs is a potential strategy to prevent infection. In this review, we have summarized the recent reports of B. burgdorferi sensu lato GAG-binding proteins and discussed the potential use of synthetic and semi-synthetic compounds, including GAG analogues, to block pathogen interaction with GAGs. Such information should motivate the discovery and development of novel GAG analogues as new therapeutics for Lyme disease. New therapeutic approaches should eventually reduce the burden of Lyme disease and improve human health.

Effects of dexamethasone and meloxicam on Borrelia burgdorferi-induced inflammation in glial and neuronal cells of the central nervous system.

BACKGROUND: Lyme neuroborreliosis (LNB), caused by the spirochete Borrelia burgdorferi (Bb), affects both the central and peripheral nervous systems. Previously, we reported that in a model of acute LNB in rhesus monkeys, treatment with the anti-inflammatory drug dexamethasone significantly reduced both pleocytosis and levels of cerebrospinal fluid (CSF) immune mediators that were induced by Bb. Dexamethasone also inhibited the formation of inflammatory, neurodegenerative, and demyelinating lesions in the brain and spinal cord of these animals. In contrast, these signs were evident in the infected animals that were left untreated or in those that were treated with meloxicam, a non-steroidal anti-inflammatory drug. METHODS: To address the differential anti-inflammatory effects of dexamethasone and meloxicam in the central nervous system (CNS), we evaluated the potential of these drugs to alter the levels of Bb-induced inflammatory mediators in culture supernatants of rhesus frontal cortex (FC) explants, primary rhesus astrocytes and microglia, and human oligodendrocytes. We also ascertained the potential of dexamethasone to modulate Bb-induced apoptosis in rhesus FC explants. As meloxicam is a known COX-2 inhibitor, we evaluated whether meloxicam altered the levels of COX-2 as induced by live Bb in cell lysates of primary rhesus astrocytes and microglia. RESULTS: Dexamethasone but not meloxicam significantly reduced the levels of several Bb-induced immune mediators in culture supernatants of FC explants, astrocytes, microglia, and oligodendrocytes. Dexamethasone also had a protective effect on Bb-induced neuronal and oligodendrocyte apoptosis in rhesus FC explants. Further, meloxicam significantly reduced the levels of Bb-induced COX-2 in microglia, while both Bb and meloxicam were unable to alter the constitutive levels of COX-2 in astrocytes. CONCLUSIONS: These data indicate that dexamethasone and meloxicam have differential anti-inflammatory effects on Bb-induced inflammation in glial and neuronal cells of the CNS and help explain the in vivo findings of significantly reduced inflammatory mediators in the CSF and lack of inflammatory neurodegenerative lesions in the brain and spinal cord of Bb-infected animals that were treated with dexamethasone but not meloxicam. Signaling cascades altered by dexamethasone could serve as possible therapeutic targets for limiting CNS inflammation and tissue damage in LNB.

Characterization of the inner membrane protein BB0173 from Borrelia burgdorferi.

BACKGROUND: The bacterial spirochete Borrelia burgdorferi is the causative agent of the most commonly reported arthropod-borne illness in the United States, Lyme disease. A family of proteins containing von Willebrand Factor A (VWFA) domains adjacent to a MoxR AAA+ ATPase have been found to be highly conserved in the genus Borrelia. Previously, a VWFA domain containing protein of B. burgdorferi, BB0172, was determined to be an outer membrane protein capable of binding integrin α3ß1. In this study, the characterization of a new VWFA domain containing membrane protein, BB0173, is evaluated in order to define the location and topology of this multi-spanning membrane protein. In addition, functional predictions are made. RESULTS: Our results show that BB0173, in contrast to BB0172, is an inner membrane protein, in which the VWFA domain is exposed to the periplasmic space. Further, BB0173 was predicted to have an aerotolerance regulator domain, and expression of BB0173 and the surrounding genes was evaluated under aerobic and microaerophilic conditions, revealing that these genes are downregulated under aerobic conditions. Since the VWFA domain containing proteins of B. burgdorferi are highly conserved, they are likely required for survival of the pathogen through sensing diverse environmental oxygen conditions. CONCLUSIONS: Presently, the complex mechanisms that B. burgdorferi uses to detect and respond to environmental changes are not completely understood. However, studying the mechanisms that allow B. burgdorferi to survive in the highly disparate environments of the tick vector and mammalian host could allow for the development of novel methods of preventing acquisition, survival, or transmission of the spirochete. In this regard, a putative membrane protein, BB0173, was characterized. BB0173 was found to be highly conserved across pathogenic Borrelia, and additionally contains several truly transmembrane domains, and a Bacteroides aerotolerance-like domain. The presence of these functional domains and the highly conserved nature of this protein, strongly suggests a required function of BB0173 in the survival of B. burgdorferi.

In vitro susceptibility of Borrelia burgdorferi isolates to three antibiotics commonly used for treating equine Lyme disease.

BACKGROUND: Lyme disease in humans is predominantly treated with tetracycline, macrolides or beta lactam antibiotics that have low minimum inhibitory concentrations (MIC) against Borrelia burgdorferi. Horses with Lyme disease may require long-term treatment making frequent intravenous or intramuscular treatment difficult and when administered orally those drugs may have either a high incidence of side effects or have poor bioavailability. The aim of the present study was to determine the in vitro susceptibility of three B. burgdorferi isolates to three antibiotics of different classes that are commonly used in practice for treating Borrelia infections in horses. RESULTS: Broth microdilution assays were used to determine minimum inhibitory concentration of three antibiotics (ceftiofur sodium, minocycline and metronidazole), for three Borrelia burgdorferi isolates. Barbour-Stoner-Kelly (BSK K + R) medium with a final inoculum of 106 Borrelia cells/mL and incubation periods of 72 h were used in the determination of MICs. Observed MICs indicated that all isolates had similar susceptibility to each drug but susceptibility to the tested antimicrobial agents varied; ceftiofur sodium (MIC = 0.08 µg/ml), minocycline hydrochloride (MIC = 0.8 µg/ml) and metronidazole (MIC = 50 µg/ml). CONCLUSIONS: The MIC against B. burgorferi varied among the three antibiotics with ceftiofur having the lowest MIC and metronidazole the highest MIC. The MIC values observed for ceftiofur in the study fall within the range of reported serum and tissue concentrations for the drug metabolite following ceftiofur sodium administration as crystalline-free acid. Minocycline and metronidazole treatments, as currently used in equine practice, could fall short of attaining MIC concentrations for B. burgdorferi.

Bactericidal Activity of Octenidine to Various Genospecies of Borrelia burgdorferi, Sensu Lato Spirochetes in Vitro and in Vivo.

The aim of our studies was to invent a reliable method for detection of the bactericidal activity of disinfectants against Borrelia burgdorferi in suspension (in vitro) and in cell line cultures (in vivo). In the suspension method, 0.01% octenidine at 20°C and 35°C was bactericidal to Borrelia afzeli; Borrelia garini, B. burgdorferi sensu stricto after 5 minutes treatment. Increase of the temperature to 35°C speed up the bactericidal effect to 1 minute. The bactericidal action of octenidine towards B. burgdorferi spirochetes growing in fibroblasts was less effective and needed a longer time to kill them than in the suspension.