A chemosensory-like histidine kinase is dispensable for chemotaxis in vitro but regulates the virulence of Borrelia burgdorferi through modulating the stability of RpoS.

As an enzootic pathogen, the Lyme disease bacterium Borrelia burgdorferi possesses multiple copies of chemotaxis proteins, including two chemotaxis histidine kinases (CHK), CheA1 and CheA2. Our previous study showed that CheA2 is a genuine CHK that is required for chemotaxis; however, the role of CheA1 remains mysterious. This report first compares the structural features that differentiate CheA1 and CheA2 and then provides evidence to show that CheA1 is an atypical CHK that controls the virulence of B. burgdorferi through modulating the stability of RpoS, a key transcriptional regulator of the spirochete. First, microscopic analyses using green-fluorescence-protein (GFP) tags reveal that CheA1 has a unique and dynamic cellular localization. Second, loss-of-function studies indicate that CheA1 is not required for chemotaxis in vitro despite sharing a high sequence and structural similarity to its counterparts from other bacteria. Third, mouse infection studies using needle inoculations show that a deletion mutant of CheA1 (cheA1mut) is able to establish systemic infection in immune-deficient mice but fails to do so in immune-competent mice albeit the mutant can survive at the inoculation site for up to 28 days. Tick and mouse infection studies further demonstrate that CheA1 is dispensable for tick colonization and acquisition but essential for tick transmission. Lastly, mechanistic studies combining immunoblotting, protein turnover, mutagenesis, and RNA-seq analyses reveal that depletion of CheA1 affects RpoS stability, leading to reduced expression of several RpoS-regulated virulence factors (i.e., OspC, BBK32, and DbpA), likely due to dysregulated clpX and lon protease expression. Bulk RNA-seq analysis of infected mouse skin tissues further show that cheA1mut fails to elicit mouse tnf-α, il-10, il-1ß, and ccl2 expression, four important cytokines for Lyme disease development and B. burgdorferi transmigration. Collectively, these results reveal a unique role and regulatory mechanism of CheA1 in modulating virulence factor expression and add new insights into understanding the regulatory network of B. burgdorferi.

Whole genome sequencing of human Borrelia burgdorferi isolates reveals linked blocks of accessory genome elements located on plasmids and associated with human dissemination.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

Borrelia burgdorferi Outer Surface Protein C Is Not the Sole Determinant of Dissemination in Mammals.

Lyme disease in the United States is most often caused by Borrelia burgdorferi sensu stricto. After a tick bite, the patient may develop erythema migrans at that site. If hematogenous dissemination occurs, the patient may then develop neurologic manifestations, carditis, or arthritis. Host-pathogen interactions include factors that contribute to hematogenous dissemination to other body sites. Outer surface protein C (OspC), a surface-exposed lipoprotein of B. burgdorferi, is essential during the early stages of mammalian infection. There is a high degree of genetic variation at the ospC locus, and certain ospC types are more frequently associated with hematogenous dissemination in patients, suggesting that OspC may be a major contributing factor to the clinical outcome of B. burgdorferi infection. In order to evaluate the role of OspC in B. burgdorferi dissemination, ospC was exchanged between B. burgdorferi isolates with different capacities to disseminate in laboratory mice, and these strains were then tested for their ability to disseminate in mice. The results indicated that the ability of B. burgdorferi to disseminate in mammalian hosts does not depend on OspC alone. The complete genome sequences of two closely related strains of B. burgdorferi with differing dissemination phenotypes were determined, but a specific genetic locus that could explain the differences in the phenotypes could not be definitively identified. The animal studies performed clearly demonstrated that OspC is not the sole determinant of dissemination. Future studies of the type described here with additional borrelial strains will hopefully clarify the genetic elements associated with hematogenous dissemination.

Breast Ultrasound Volume Sweep Imaging: A New Horizon in Expanding Imaging Access for Breast Cancer Detection.

OBJECTIVE: The majority of people in the world lack basic access to breast diagnostic imaging resulting in delay to diagnosis of breast cancer. In this study, we tested a volume sweep imaging (VSI) ultrasound protocol for evaluation of palpable breast lumps that can be performed by operators after minimal training without prior ultrasound experience as a means to increase accessibility to breast ultrasound. METHODS: Medical students without prior ultrasound experience were trained for less than 2 hours on the VSI breast ultrasound protocol. Patients presenting with palpable breast lumps for standard of care ultrasound examination were scanned by a trained medical student with the VSI protocol using a Butterfly iQ handheld ultrasound probe. Video clips of the VSI scan imaging were later interpreted by an attending breast imager. Results of VSI scan interpretation were compared to the same-day standard of care ultrasound examination. RESULTS: Medical students scanned 170 palpable lumps with the VSI protocol. There was 97% sensitivity and 100% specificity for a breast mass on VSI corresponding to 97.6% agreement with standard of care (Cohen's κ = 0.95, P < .0001). There was a detection rate of 100% for all cancer presenting as a sonographic mass. High agreement for mass characteristics between VSI and standard of care was observed, including 87% agreement on Breast Imaging-Reporting and Data System assessments (Cohen's κ = 0.82, P < .0001). CONCLUSIONS: Breast ultrasound VSI for palpable lumps offers a promising means to increase access to diagnostic imaging in underserved areas. This approach could decrease delay to diagnosis for breast cancer, potentially improving morbidity and mortality.

Akkermansia muciniphila and Its Pili-Like Protein Amuc_1100 Modulate Macrophage Polarization in Experimental Periodontitis.

Periodontitis is a chronic inflammatory disease triggered by dysbiosis of the oral microbiome. Porphyromonas gingivalis is strongly implicated in periodontal inflammation, gingival tissue destruction, and alveolar bone loss through sustained exacerbation of the host response. Recently, the use of other bacterial species, such as Akkermansia muciniphila, has been suggested to counteract inflammation elicited by P. gingivalis In this study, the effects of A. muciniphila and its pili-like protein Amuc_1100 on macrophage polarization during P. gingivalis infection were evaluated in a murine model of experimental periodontitis. Mice were gavaged with P. gingivalis alone or in combination with A. muciniphila or Amuc_1100 for 6 weeks. Morphometric analysis demonstrated that the addition of A. muciniphila or Amuc_1100 significantly reduced P. gingivalis-induced alveolar bone loss. This decreased bone loss was associated with a proresolutive phenotype (M2) of macrophages isolated from submandibular lymph nodes as observed by flow cytometry. Furthermore, the expression of interleukin 10 (IL-10) at the RNA and protein levels was significantly increased in the gingival tissues of the mice and in macrophages exposed to A. muciniphila or Amuc_1100, confirming their anti-inflammatory properties. This study demonstrates the putative therapeutic interest of the administration of A. muciniphila or Amuc_1100 in the management of periodontitis through their anti-inflammatory properties.

The Lyme Disease Biobank: Characterization of 550 Patient and Control Samples from the East Coast and Upper Midwest of the United States.

Lyme disease (LD) is an increasing public health problem. Current laboratory testing is insensitive in early infection, the stage at which appropriate treatment is most effective in preventing disease sequelae. The Lyme Disease Biobank (LDB) collects samples from individuals with symptoms consistent with early LD presenting with or without erythema migrans (EM) or an annular, expanding skin lesion and uninfected individuals from areas of endemicity. Samples were collected from 550 participants (298 cases and 252 controls) according to institutional review board-approved protocols and shipped to a centralized biorepository. Testing was performed to confirm the presence of tick-borne pathogens by real-time PCR, and a subset of samples was tested for Borrelia burgdorferi by culture. Serology was performed on all samples using the CDC's standard two-tiered testing algorithm (STTTA) for LD. LD diagnosis was supported by laboratory testing in 82 cases, including positive results by use of the STTTA, PCR, or culture or positive results by two enzyme-linked immunosorbent assays for cases presenting with EM lesion sizes of >5 cm. The remaining 216 cases had negative laboratory testing results. For the controls, 43 were positive by at least one of the tiers and 6 were positive by use of the STTTA. The results obtained with this collection highlight and reinforce the known limitations of serologic testing in early LD, with only 29% of individuals presenting with EM lesion sizes of >5 cm yielding a positive result using the STTTA. Aliquots of whole blood, serum, and urine from clinically characterized patients with and without LD are available to investigators in academia and industry for evaluation or development of novel diagnostic assays for LD, to continue to improve upon currently available methods.

Akkermansia muciniphila reduces Porphyromonas gingivalis-induced inflammation and periodontal bone destruction.

AIM: Akkermansia muciniphila is a beneficial gut commensal, whose anti-inflammatory properties have recently been demonstrated. This study aimed to evaluate the effect of A. muciniphila on Porphyromonas gingivalis elicited inflammation. MATERIAL AND METHODS: In lean and obese mice, A. muciniphila was administered in P. gingivalis-induced calvarial abscess and in experimental periodontitis model (EIP). Bone destruction and inflammation were evaluated by histomorphometric analysis. In vitro, A. muciniphila was co-cultured with P. gingivalis, growth and virulence factor expression was evaluated. Bone marrow macrophages (BMMϕ) and gingival epithelial cells (TIGK) were exposed to both bacterial strains, and the expression of inflammatory mediators, as well as tight junction markers, was analysed. RESULTS: In a model of calvarial infection, A. muciniphila decreased inflammatory cell infiltration and bone destruction. In EIP, treatment with A. muciniphila resulted in a decreased alveolar bone loss. In vitro, the addition of A. muciniphila to P. gingivalis-infected BMMϕ increased anti-inflammatory IL-10 and decreased IL-12. Additionally, A. muciniphila exposure increases the expression of junctional integrity markers such as integrin-ß1, E-cadherin and ZO-1 in TIGK cells. A. muciniphila co-culture with P. gingivalis reduced gingipains mRNA expression. DISCUSSION: This study demonstrated the protective effects of A. muciniphila administration and may open consideration to its use as an adjunctive therapeutic agent to periodontal treatment.

Bacterial Content of Fortified and Unfortified Holder Pasteurized Donor Human Milk During Prolonged Refrigerated Storage.

OBJECTIVE: To assess the microbial growth in unfortified and fortified Holder pasteurized donor human milk (HPDHM) during 96 hours of refrigerated storage in a clinical setting. METHODS: Thirty-six unfortified samples and 77 fortified samples of HPDHM were prepared in a neonatal intensive care milk preparation room and stored in the NICU refrigerator at 4°C to simulate a real-life feeding environment. One milliliter aliquots were removed at 24-hour intervals and cultured in duplicate for bacterial growth on solid blood agar medium. Viable bacterial colonies were characterized using standard microbiological methods. RESULTS: 96.5% of milk samples manipulated in a vertical laminar flow hood were negative for bacterial growth. In the remainder 3.5% of the samples, the maximum growth was 1 colony forming unit/0.1 ml plated. Higher colony counts were observed when the laminar hood was not used. In all cases, the colonies represented common skin bacteria and demonstrated an inconsistent and unsustained growth. Fortifier status and storage time were not significantly associated with increased bacterial growth (P > 0.05). CONCLUSIONS: Unfortified and fortified HPDHM remain largely free of bacterial growth for up to 96 hours of refrigerated storage in NICU settings. Sample handling techniques are important for preventing microbial contamination.

Stage-specific global alterations in the transcriptomes of Lyme disease spirochetes during tick feeding and following mammalian host adaptation.

Borrelia burgdorferi, the agent of Lyme disease, is maintained in nature within an enzootic cycle involving a mammalian reservoir and an Ixodes sp. tick vector. The transmission, survival and pathogenic potential of B. burgdorferi depend on the bacterium's ability to modulate its transcriptome as it transits between vector and reservoir host. Herein, we employed an amplification-microarray approach to define the B. burgdorferi transcriptomes in fed larvae, fed nymphs and in mammalian host-adapted organisms cultivated in dialysis membrane chambers. The results show clearly that spirochetes exhibit unique expression profiles during each tick stage and during cultivation within the mammal; importantly, none of these profiles resembles that exhibited by in vitro grown organisms. Profound shifts in transcript levels were observed for genes encoding known or predicted lipoproteins as well as proteins involved in nutrient uptake, carbon utilization and lipid synthesis. Stage-specific expression patterns of chemotaxis-associated genes also were noted, suggesting that the composition and interactivities of the chemotaxis machinery components vary considerably in the feeding tick and mammal. The results as a whole make clear that environmental sensing by B. burgdorferi directly or indirectly drives an extensive and tightly integrated modulation of cell envelope constituents, chemotaxis/motility machinery, intermediary metabolism and cellular physiology. These findings provide the necessary transcriptional framework for delineating B. burgdorferi regulatory pathways throughout the enzootic cycle as well as defining the contribution(s) of individual genes to spirochete survival in nature and virulence in humans.

Borrelia burgdorferi induces a type I interferon response during early stages of disseminated infection in mice.

BACKGROUND: Lyme borrelia genotypes differ in their capacity to cause disseminated disease. Gene array analysis was employed to profile the host transcriptome induced by Borrelia burgdorferi strains with different capacities for causing disseminated disease in the blood of C3H/HeJ mice during early infection. RESULTS: B. burgdorferi B515, a clinical isolate that causes disseminated infection in mice, differentially regulated 236 transcripts (P < 0.05 by ANOVA, with fold change of at least 2). The 216 significantly induced transcripts included interferon (IFN)-responsive genes and genes involved in immunity and inflammation. In contrast, B. burgdorferi B331, a clinical isolate that causes transient skin infection but does not disseminate in C3H/HeJ mice, stimulated changes in only a few genes (1 induced, 4 repressed). Transcriptional regulation of type I IFN and IFN-related genes was measured by quantitative RT-PCR in mouse skin biopsies collected from the site of infection 24 h after inoculation with B. burgdorferi. The mean values for transcripts of Ifnb, Cxcl10, Gbp1, Ifit1, Ifit3, Irf7, Mx1, and Stat2 were found to be significantly increased in B. burgdorferi strain B515-infected mice relative to the control group. In contrast, transcription of these genes was not significantly changed in response to B. burgdorferi strain B331 or B31-4, a mutant that is unable to disseminate. CONCLUSIONS: These results establish a positive association between the disseminating capacity of B. burgdorferi and early type I IFN induction in a murine model of Lyme disease.

Detection of Borrelia burgdorferi nucleic acids after antibiotic treatment does not confirm viability.

The persistence of dormant, noncultivable Borrelia burgdorferi after ceftriaxone treatment was examined. B. burgdorferi isolates were cultivated in Barbour-Stoenner-Kelly medium in the presence or absence of ceftriaxone, and cultures were monitored for up to 56 days. Viability of B. burgdorferi was assessed by subculture, growth, morphology, and pH (as a surrogate for metabolic activity). In addition, the presence of B. burgdorferi DNA and mRNA was assayed by PCR and by real-time reverse transcription (RT)-PCR, respectively. Spirochetes could not be successfully subcultured by day 3 after exposure to ceftriaxone. In cultures treated with ceftriaxone, the pH of the culture medium did not change through day 56, whereas it declined by at least 1 pH unit by 14 days in untreated cultures. These results suggest that B. burgdorferi viability is rapidly eliminated after antibiotic treatment. Nevertheless, DNA was detected by B. burgdorferi-specific PCR for up to 56 days in aliquots from both ceftriaxone-treated and untreated cultures. In addition, although ceftriaxone treatment resulted in a reduction in the quantities of transcript for ospC, ospA, flaB, and pfk, certain mRNAs could be detected through day 14. Transcript for all 4 genes was essentially undetectable after 28 days of treatment. Taken together, the results suggest that B. burgdorferi DNA and mRNA can be detected in samples long after spirochetes are no longer viable as assessed by classic microbiological parameters. PCR positivity in the absence of culture positivity following antibiotic treatment in animal and human studies should be interpreted with caution.

Borrelia burgdorferi requires glycerol for maximum fitness during the tick phase of the enzootic cycle.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is a vector-borne pathogen that cycles between a mammalian host and tick vector. This complex life cycle requires that the spirochete modulate its gene expression program to facilitate growth and maintenance in these diverse milieus. B. burgdorferi contains an operon that is predicted to encode proteins that would mediate the uptake and conversion of glycerol to dihydroxyacetone phosphate. Previous studies indicated that expression of the operon is elevated at 23°C and is repressed in the presence of the alternative sigma factor RpoS, suggesting that glycerol utilization may play an important role during the tick phase. This possibility was further explored in the current study by expression analysis and mutagenesis of glpD, a gene predicted to encode glycerol 3-phosphate dehydrogenase. Transcript levels for glpD were significantly lower in mouse joints relative to their levels in ticks. Expression of GlpD protein was repressed in an RpoS-dependent manner during growth of spirochetes within dialysis membrane chambers implanted in rat peritoneal cavities. In medium supplemented with glycerol as the principal carbohydrate, wild-type B. burgdorferi grew to a significantly higher cell density than glpD mutant spirochetes during growth in vitro at 25°C. glpD mutant spirochetes were fully infectious in mice by either needle or tick inoculation. In contrast, glpD mutants grew to significantly lower densities than wild-type B. burgdorferi in nymphal ticks and displayed a replication defect in feeding nymphs. The findings suggest that B. burgdorferi undergoes a switch in carbohydrate utilization during the mammal to tick transition. Further, the results demonstrate that the ability to utilize glycerol as a carbohydrate source for glycolysis during the tick phase of the infectious cycle is critical for maximal B. burgdorferi fitness.

Whole genome sequencing of Borrelia burgdorferi isolates reveals linked clusters of plasmid-borne accessory genome elements associated with virulence.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 patient-derived B. burgdorferi sensu stricto ( Bbss ) isolates from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bbss isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bbss isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ∻800 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, are associated with increased rates of dissemination. OspC type A strains possess a unique constellation of strongly linked genetic changes including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. The patterns of OspC type A strains typify a broader paradigm across Bbss isolates, in which genetic structure is defined by correlated groups of strain-variable genes located predominantly on plasmids, particularly for expression of surface-exposed lipoproteins. These clusters of genes are inherited in blocks through strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

No sonographer, no radiologist: Assessing accuracy of artificial intelligence on breast ultrasound volume sweep imaging scans.

Breast ultrasound provides a first-line evaluation for breast masses, but the majority of the world lacks access to any form of diagnostic imaging. In this pilot study, we assessed the combination of artificial intelligence (Samsung S-Detect for Breast) with volume sweep imaging (VSI) ultrasound scans to evaluate the possibility of inexpensive, fully automated breast ultrasound acquisition and preliminary interpretation without an experienced sonographer or radiologist. This study was conducted using examinations from a curated data set from a previously published clinical study of breast VSI. Examinations in this data set were obtained by medical students without prior ultrasound experience who performed VSI using a portable Butterfly iQ ultrasound probe. Standard of care ultrasound exams were performed concurrently by an experienced sonographer using a high-end ultrasound machine. Expert-selected VSI images and standard of care images were input into S-Detect which output mass features and classification as "possibly benign" and "possibly malignant." Subsequent comparison of the S-Detect VSI report was made between 1) the standard of care ultrasound report by an expert radiologist, 2) the standard of care ultrasound S-Detect report, 3) the VSI report by an expert radiologist, and 4) the pathological diagnosis. There were 115 masses analyzed by S-Detect from the curated data set. There was substantial agreement of the S-Detect interpretation of VSI among cancers, cysts, fibroadenomas, and lipomas to the expert standard of care ultrasound report (Cohen's κ = 0.73 (0.57-0.9 95% CI), p<0.0001), the standard of care ultrasound S-Detect interpretation (Cohen's κ = 0.79 (0.65-0.94 95% CI), p<0.0001), the expert VSI ultrasound report (Cohen's κ = 0.73 (0.57-0.9 95% CI), p<0.0001), and the pathological diagnosis (Cohen's κ = 0.80 (0.64-0.95 95% CI), p<0.0001). All pathologically proven cancers (n = 20) were designated as "possibly malignant" by S-Detect with a sensitivity of 100% and specificity of 86%. Integration of artificial intelligence and VSI could allow both acquisition and interpretation of ultrasound images without a sonographer and radiologist. This approach holds potential for increasing access to ultrasound imaging and therefore improving outcomes related to breast cancer in low- and middle- income countries.

BB0844, an RpoS-regulated protein, is dispensable for Borrelia burgdorferi infectivity and maintenance in the mouse-tick infectious cycle.

The genome of Borrelia burgdorferi, the causative agent of Lyme disease, is comprised of a large linear chromosome and numerous smaller linear and circular plasmids. B. burgdorferi exhibits substantial genomic variation, and previous studies revealed genotype-specific variation at the right chromosomal telomere. A correlation has also been established between genotype and invasiveness. The correlation between chromosome length and genotype and between genotype and invasiveness suggested that a gene(s) at the right chromosome telomere may be required for virulence. Of particular interest was bb0844, an RpoS-regulated gene at the right telomere, the expression of which is induced when the spirochete undergoes adaptation to the mammalian host. The structure of the right chromosomal telomere was examined in 53 B. burgdorferi clinical isolates of various genotypes. Four distinct patterns were observed for bb0844: (i) chromosomal localization, (ii) plasmid localization, (iii) presence on both chromosome and plasmid, and (iv) complete absence. These patterns correlated with the B. burgdorferi genotype. On the basis of available sequence data, we propose a mechanism for the genomic rearrangements that accounts for the variability in bb0844 genomic localization. To further explore the role of BB0844 in the spirochete life cycle, a bb0844 deletion mutant was constructed by allelic exchange, and the viability of wild-type and bb0844 deletion mutants was examined in an experimental mouse-tick infection model. The bb0844 mutant was fully infectious in C3H/HeJ mice by either needle inoculation or tick transmission with B. burgdorferi-infected Ixodes scapularis larvae. Naïve larval ticks acquired both wild-type and mutant spirochetes with equal efficiency from B. burgdorferi-infected mice. The results demonstrate that BB0844 is not required for spirochete viability, pathogenicity, or maintenance in the tick vector or the mammalian host. At present, a defined role for BB0844 in B. burgdorferi cannot be ascertained.

Improving the yield of blood cultures from patients with early Lyme disease.

Approximately 45% of untreated United States patients with early Lyme disease associated with erythema migrans have a positive blood culture based on microscopic detection of Borrelia burgdorferi in Barbour-Stoenner-Kelly medium after 2 to 12 weeks of incubation. In this study we demonstrate that the yield of blood cultures can be significantly increased to 70.8% by the use of a combined culture-quantitative PCR technique and that among those patients found to have a positive blood culture, positivity was detected in over 90% within just 7 days of incubation. Patients with multiple erythema migrans were almost uniformly culture positive by this technique.

Borrelia burgdorferi bba74 is expressed exclusively during tick feeding and is regulated by both arthropod- and mammalian host-specific signals.

Although BBA74 initially was described as a 28-kDa virulence-associated outer-membrane-spanning protein with porin-like function, subsequent studies revealed that it is periplasmic and downregulated in mammalian host-adapted spirochetes. To further elucidate the role of this protein in the Borrelia burgdorferi tick-mammal cycle, we conducted a thorough examination of its expression profile in comparison with the profiles of three well-characterized, differentially expressed borrelial genes (ospA, ospC, and ospE) and their proteins. In vitro, transcripts for bba74 were expressed at 23 degrees C and further enhanced by a temperature shift (37 degrees C), whereas BBA74 protein diminished at elevated temperatures; in contrast, neither transcript nor protein was expressed by spirochetes grown in dialysis membrane chambers (DMCs). Primer extension of wild-type B. burgdorferi grown in vitro, in conjunction with expression analysis of DMC-cultivated wild-type and rpoS mutant spirochetes, revealed that, like ospA, bba74 is transcribed by sigma(70) and is subject to RpoS-mediated repression within the mammalian host. A series of experiments utilizing wild-type and rpoS mutant spirochetes was conducted to determine the transcriptional and translational profiles of bba74 during the tick-mouse cycle. Results from these studies revealed (i) that bba74 is transcribed by sigma(70) exclusively during the larval and nymphal blood meals and (ii) that transcription of bba74 is bracketed by RpoS-independent and -dependent forms of repression that are induced by arthropod- and mammalian host-specific signals, respectively. Although loss of BBA74 does not impair the ability of B. burgdorferi to complete its infectious life cycle, the temporal compartmentalization of this gene's transcription suggests that BBA74 facilitates fitness of the spirochete within a narrow window of its tick phase. A reexamination of the paradigm for reciprocal regulation of ospA and ospC, performed herein, revealed that the heterogeneous expression of OspA and OspC displayed by spirochete populations during the nymphal blood meal results from the intricate sequence of transcriptional and translational changes that ensue as B. burgdorferi transitions between its arthropod vector and mammalian host.

Identification of a Kavain Analog with Efficient Anti-inflammatory Effects.

Kavain, a compound derived from Piper methysticum, has demonstrated anti-inflammatory properties. To optimize its drug properties, identification and development of new kavain-derived compounds was undertaken. A focused library of analogs was synthesized and their effects on Porphyromonas gingivalis (P. gingivalis) elicited inflammation were evaluated in vitro and in vivo. The library contained cyclohexenones (5,5-dimethyl substituted cyclohexenones) substituted with a benzoate derivative at the 3-position of the cyclohexanone. The most promising analog identifed was a methylated derivative of kavain, Kava-205Me (5,5-dimethyl-3-oxocyclohex-1-en-1-yl 4-methylbenzoate.) In an in vitro assay of anti-inflammatory effects, murine macrophages (BMM) and THP-1 cells were infected with P. gingivalis (MOI = 20:1) and a panel of cytokines were measured. Both cell types treated with Kava-205Me (10 to 200 µg/ml) showed significantly and dose-dependently reduced TNF-α secretion induced by P. gingivalis. In BMM, Kava-205Me also reduced secretion of other cytokines involved in the early phase of inflammation, including IL-12, eotaxin, RANTES, IL-10 and interferon-γ (p < 0.05). In vivo, in an acute model of P. gingivalis-induced calvarial destruction, administration of Kava-205Me significantly improved the rate of healing associated with reduced soft tissue inflammation and osteoclast activation. In an infective arthritis murine model induced by injection of collagen-antibody (ArthriomAb) + P. gingivalis, administration of Kava-205Me was able to reduce efficiently paw swelling and joint destruction. These results highlight the strong anti-inflammatory properties of Kava-205Me and strengthen the interest of testing such compounds in the management of P. gingivalis elicited inflammation, especially in the management of periodontitis.