Improved detection of blood stream pathogens by real-time PCR in severe sepsis.

OBJECTIVE: Evaluation of the technical and diagnostic feasibility of commercial multiplex real-time polymerase chain reaction (PCR) for detection of blood stream infections in a cohort of intensive care unit (ICU) patients with severe sepsis, performed in addition to conventional blood cultures. DESIGN: Dual-center cohort study. SETTING: Surgical ICU of two university hospitals. PATIENTS AND PARTICIPANTS: One hundred eight critically ill patients fulfilling the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) severe sepsis criteria were included. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: PCR results obtained in 453 blood samples from 108 patients were compared with corresponding blood culture results. PCR resulted in a twofold higher positivity rate when compared with conventional blood culture (BC) testing (114 versus 58 positive samples). In 40 out of 58 PCR positive assays the results of the corresponding blood cultures were identical to microorganisms detected by PCR. In 18 samples PCR and BC yielded discrepant results. Compared with conventional blood culture the sensitivity and specificity of PCR was 0.69 and 0.81, respectively. Further evaluation of PCR results against a constructed gold standard including conventional microbiological test results from other significant patient specimen (such as bronchio-alveolar lavage fluid, urine, swabs) and additionally generated clinical and laboratory information yielded sensitivity of 0.83 and specificity of 0.93. CONCLUSIONS: Our cohort study demonstrates improved pathogen detection using PCR findings in addition to conventional blood culture testing. PCR testing provides increased sensitivity of blood stream infection. Studies addressing utility including therapeutic decision-making, outcome, and cost-benefit following diagnostic application of PCR tests are needed to further assess its value in the clinical setting.

Mutational analyses of the BbCRASP-1 protein of Borrelia burgdorferi identify residues relevant for the architecture and binding of host complement regulators FHL-1 and factor H.

Borrelia burgdorferi exploits multiple strategies to evade host immune responses. One central immune escape mechanism is the inactivation of the host complement attack by acquisition host complement regulators FHL-1 and factor H via complement regulator-acquiring surface proteins (BbCRASPs). The BbCRASP-1 protein is the first bacterial factor H/FHL-1-binding protein for which the atomic structure has been solved. Previously, 3 regions including the C terminus were identified as putative contact sites for the two complement regulators by the pepspot analysis. Based on the crystallographic structure an in vitro mutagenesis approach was conducted to identify amino acid residues which are relevant for FHL-1 and factor H binding by exchanging single or multiple residues in region 1 and the C-terminally located region 3. Single changes at 4 positions in region 1 either reduced (Lys136, Lys141, Glu147) or completely eliminated (Leu146) binding of both complement regulators. Substitutions clustered within the C-terminal region decreased (Glu234, Lys238, Tyr239, Lys241, Asp244, Thr245) or abolished binding (Lys240, Asp242, Leu246) of both complement regulators. Mapping the mutations onto the atomic structure of BbCRASP-1 reveals that, in contrast to earlier assumption, the C-terminal mutations act indirectly on FHL-1 and factor H binding, whilst the region 1 mutations map the site of direct complement regulator interaction. The elucidation of BbCRASP-1 structure - function may allow development of novel therapeutic strategies against Lyme disease.

Identification and characterization of the factor H and FHL-1 binding complement regulator-acquiring surface protein 1 of the Lyme disease spirochete Borrelia spielmanii sp. nov.

Borrelia spielmanii, one of the etiological agents of Lyme disease found in Europe, evades host complement-mediated killing by recruitment of the immune regulators factor H and FHL-1 from human serum. Serum-resistant and intermediate serum-resistant isolates express up to 3 distinct complement regulator-acquiring surface proteins (CRASPs) that bind factor H and/or FHL-1. The present study describes identification and functional characterization of BsCRASP-1 as the dominant factor H and FHL-1 binding protein of B. spielmanii. BsCRASP-1 is a 27.7kDa outer surface lipoprotein, which after processing has a predicted mass of 24.9kDa. BsCRASP-1 is encoded by a single copy gene, cspA, that maps to a linear plasmid of approximately 55kb. Ligand affinity blot techniques revealed that both native and recombinant BsCRASP-1 from different isolates can strongly bind FHL-1, but only weakly factor H. Deletion mutants of recombinant BsCRASP-1 were generated and a high-affinity binding site for factor H and FHL-1 was mapped to its carboxy-terminal 10-amino-acid residue domain. Similarly, the dominant binding site of factor H and FHL-1 was localized to short consensus repeats (SCRs) 5-7. Factor H and FHL-1 maintained cofactor activity for factor I-mediated C3b inactivation when bound to full-length BsCRASP-1 but not to a deletion mutant lacking the carboxy-terminal 10-amino-acid residue domain. In conclusion, BsCRASP-1 binds the host immune regulators factor H and FHL-1, and is suggested to represent a key molecule of B. spielmanii for complement resistance. Thus, BsCRASP-1 most likely contributes to persistence of B. spielmanii and to pathogenesis of Lyme disease.

Deciphering the ligand-binding sites in the Borrelia burgdorferi complement regulator-acquiring surface protein 2 required for interactions with the human immune regulators factor H and factor H-like protein 1.

Borrelia burgdorferi, the etiologic agent of Lyme disease, employs sophisticated means to evade killing by its mammalian hosts. One important immune escape mechanism is the inhibition of complement activation mediated by interactions of the host-derived immune regulators factor H (CFH) and factor H-like protein 1 (CFHL1) with borrelial complement regulator-acquiring surface proteins (BbCRASPs). BbCRASP-2 is a distinctive CFH- and CFHL1-binding protein that is produced by serum-resistant B. burgdorferi strains. Here we show that binding of CFH by BbCRASP-2 is due to electrostatic as well as hydrophobic forces. In addition, 14 individual amino acid residues of BbCRASP-2 were identified as being involved in CFH and CFHL1 binding. Alanine substitutions of most of those residues significantly inhibited binding of CFH and/or CFHL1 by recombinant BbCRASP-2 proteins. To conclusively define the effects of BbCRASP-2 residue substitutions on serum sensitivity in the bacterial context, a serum-sensitive Borrelia garinii strain was transformed with plasmids that directed production of either wild-type or mutated BbCRASP-2 proteins. Critical amino acid residues within BbCRASP-2 were identified, with bacteria producing distinct mutant proteins being unable to bind either CFH or CFHL1, showing high levels of complement components C3, C6, and C5b-9 deposited on their surfaces and being highly sensitive to killing by normal serum. Collectively, we mapped a structurally sensitive CFH/CFHL1 binding site within borrelial BbCRASP-2 and identified single amino acid residues potentially involved in the interaction with both complement regulators.

Thymidine-dependent Staphylococcus aureus small-colony variants: human pathogens that are relevant not only in cases of cystic fibrosis lung disease.

We report the isolation of thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus from unusual infection sites of patients with chronic soft tissue infection, tympanitis, bronchitis, peritonitis, and septicemia. Furthermore, we provide evidence that the essential growth factor for TD-SCVs, i.e., thymidine, and its metabolite dTMP are present in various human specimens.

Systematic analysis highlights the key role of TLR2/NF-kappaB/MAP kinase signaling for IL-8 induction by macrophage-like THP-1 cells under influence of Borrelia burgdorferi lysates.

Lyme borreliosis is a spirochetal infection caused by the Borrelia burgdorferi sensu lato complex that can proceed towards an inflammatory joint manifestation known as Lyme arthritis. Production of chemokines orchestrating neutrophil infiltration is supposed to be key to early arthritic pathogenesis. Using PMA-differentiated macrophage-like THP-1 (mTHP-1) cells we identified by antibody array methodology or mRNA analysis IL-8, GRO-alpha, NAP-2, and SDF-1alpha as being among those chemokines that are upregulated by bacterial lysates obtained from B. burgdorferi. Based on these observations, we set out to characterize in detail mechanisms mediating IL-8 release in this cellular model. TLR2 blocking antibodies, analysis of p65 translocation, and electromobility-shift analysis revealed activation of the TLR2/NF-kappaB axis by B. burgdorferi. The functional importance of this pathway was substantiated by suppression of IL-8 after inhibition of IkappaB kinase. Notably, MAP kinases, specifically the MEK1/2-ERK1/2 pathway, were essential for IL-8 secretion. Those data were confirmed by using freshly isolated adherent peripheral blood mononuclear cells. On the contrary, B. burgdorferi-induced IL-8 in mTHP-1 was unlikely related to flagellin, alpha3beta1-integrin signaling, lipopolysaccharide, bacterial DNA, NOD1/NOD2 agonists, or to intermediate production of IL-1beta and TNF-alpha. Induction of IL-8 by B. burgdorferi was not due to amplification of constitutive AP-1 DNA-binding activity detectable in mTHP-1 cells. Data presented herein validate that TLR2, particularly on mTHP-1 cells, holds a central position in mediating IL-8 secretion associated with extracellular B. burgdorferi and beyond that suggest inhibition of IkappaB kinase and MEK1/2 kinases as promising pharmacological strategies aiming at IL-8 in early Lyme arthritis.

The thymidine-dependent small-colony-variant phenotype is associated with hypermutability and antibiotic resistance in clinical Staphylococcus aureus isolates.

Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus can be isolated from the airway secretions of patients suffering from cystic fibrosis (CF) and are implicated in persistent and treatment-resistant infections. These characteristics, as well as the variety of mutations in the thymidylate synthase-encoding thyA gene which are responsible for thymidine dependency, suggest that these morphological variants are hypermutable. To prove this hypothesis, we analyzed the mutator phenotype of different S. aureus phenotypes, in particular CF-derived TD-SCVs, CF-derived isolates with a normal phenotype (NCVs), and non-CF NCVs. The comparative analysis revealed that the CF isolates had significantly higher mutation rates than the non-CF isolates. The TD-SCVs, in turn, harbored significantly more strong hypermutators (mutation rate > or = 10(-7)) than the CF and non-CF NCVs. In addition, antimicrobial resistance to non-beta-lactam antibiotics, including gentamicin, ciprofloxacin, erythromycin, fosfomycin, and rifampin, was significantly more prevalent in TD-SCVs than in CF and non-CF NCVs. Interestingly, macrolide resistance, which is usually mediated by mobile genetic elements, was conferred in half of the macrolide-resistant TD-SCVs by the point mutation A2058G or A2058T in the genes encoding the 23S rRNA. Sequence analysis of mutS and mutL, which are involved in DNA mismatch repair in gram-positive bacteria, revealed that in hypermutable CF isolates and especially in TD-SCVs, mutL was often truncated due to frameshift mutations. In conclusion, these data provide direct evidence that TD-SCVs are hypermutators. This hypermutability apparently favors the acquisition of antibiotic resistance and facilitates bacterial adaptation during long-term persistence.

Borrelia burgdorferi complement regulator-acquiring surface proteins (BbCRASPs): Expression patterns during the mammal-tick infection cycle.

Host complement is widely distributed throughout mammalian body fluids and can be activated immediately as part of the first line of defense against invading pathogens. The agent of Lyme disease, Borrelia burgdorferi sensu lato (s.l.), is naturally resistant to that innate immune defense system of its hosts. One resistance mechanism appears to involve binding fluid-phase regulators of complement to distinct borrelial outer surface molecules known as CRASPs (complement regulator acquiring surface proteins). Using sensitive molecular biology techniques, expression patterns of all three classes of genes encoding the CRASPs of B. burgdorferi sensu stricto (BbCRASPs) have been analyzed throughout the natural tick-mammal infection cycle. Each class shows a different expression profile in vivo and the results are summarized herein. Studies on the expression of B. burgdorferi genes using animal models of infection have advanced our knowledge on the ability of the causative agent to circumvent innate immune defenses, the contributions of CRASPs to spirochete infectivity, and the pathogenesis of Lyme disease.

Linezolid resistance in Staphylococcus aureus: gene dosage effect, stability, fitness costs, and cross-resistances.

Linezolid resistance in Staphylococcus aureus is typically associated with mutations in the 23S rRNA gene. Here we show that the accumulation of a single point mutation, G2576T, in the different copies of this gene causes stepwise increases in resistance, impairment of the biological fitness, and cross-resistance to quinupristin-dalfopristin and chloramphenicol.

Borrelia burgdorferi complement regulator-acquiring surface protein 2 (CspZ) as a serological marker of human Lyme disease.

Serological diagnosis of Lyme disease may be complicated by antigenic differences between infecting organisms and those used as test references. Accordingly, it would be helpful to include antigens whose sequences are well conserved by a broad range of Lyme disease spirochetes. In the present study, line blot analyses were performed using recombinant complement regulator-acquiring surface protein 2 (BbCRASP-2) from Borrelia burgdorferi sensu stricto strain B31 and serum samples from human Lyme disease patients from throughout the United States and Germany. The results indicated that a large proportion of the patients had produced antibodies recognizing recombinant BbCRASP-2. In addition, Lyme disease spirochetes isolated from across North America and Europe were found to contain genes encoding proteins with high degrees of similarity to the B. burgdorferi type strain B31 BbCRASP-2, consistent with the high percentage of serologically positive patients. These data indicate that BbCRASP-2 may be valuable for use in a widely effective serological assay.

Influence of dTMP on the phenotypic appearance and intracellular persistence of Staphylococcus aureus.

Thymidine-dependent small-colony variants (SCVs) of Staphylococcus aureus are frequently associated with persistent and recurrent infections in cystic fibrosis patients. The phenotypic appearance of S. aureus SCVs or normal-colony variants (NCVs) is postulated to be affected by the intracellular amount of dTMP. This hypothesis was proven by metabolic pathway assays revealing altered intracellular dTMP concentrations, followed by investigation of the associated phenotype. Inhibition of the staphylococcal thymidylate synthase, which generated intracellular dTMP from dUMP, using 5-fluorouracil and co-trimoxazole resulted in an SCV phenotype. Inhibition of a nucleoside transporter, which provided the bacterial cell with extracellular thymidine, caused growth inhibition of SCVs. In turn, reversion of SCVs to NCVs was achieved by supplying extracellular dTMP. High-performance liquid chromatography additionally confirmed the intracellular lack of dTMP in SCVs, in contrast to NCVs. Moreover, the dTMP concentration is postulated to influence the intracellular persistence of S. aureus. Cell culture experiments with cystic fibrosis cells revealed that clinical and co-trimoxazole-induced SCVs with a diminished amount of dTMP showed significantly better intracellular persistence than NCVs. In conclusion, these results show that the dTMP concentration plays a key role in both the phenotypic appearance and the intracellular persistence of S. aureus.

Release of latent ClyA cytolysin from Escherichia coli mediated by a bacteriophage-associated putative holin (BlyA) from Borrelia burgdorferi.

Introduction of the Borrelia burgdorferi blyAB locus into Escherichia coli has recently been reported to cause a hemolytic phenotype that is dependent on the E. coli clyA (hlyE, sheA) gene (a cytolysin gene present in many E. coli strains, including E. coli K-12, which is repressed under standard in vitro growth conditions). The blyA gene product has been suggested to be a prophage-encoded holin, but the processes triggered in E. coli by the expression of blyA and/or blyB, which lead to the hemolytic phenotype, remained unclear. Here we show that expression of blyA in E. coli causes damage to the E. coli cell envelope and a clyA-dependent hemolytic phenotype, regardless whether blyB is present or absent. The expression of blyB in E. coli, on the other hand, did not have obvious phenotypic effects. Transcriptional studies demonstrated that the clyA gene is not induced in E. coli cells expressing blyA. Furthermore, protein analyses suggested that the impairment of the E. coli cell envelope by BlyA is responsible for the emergence of the hemolytic activity as it allows latent intracellular ClyA protein, derived from basal-level expression of the clyA gene, to leak into the medium and to lyse erythrocytes. These findings are compatible with the presumption that BlyA functions as a membrane-active holin.

Human pathogenic Borrelia spielmanii sp. nov. resists complement-mediated killing by direct binding of immune regulators factor H and factor H-like protein 1.

Borrelia spielmanii sp. nov. has recently been shown to be a novel human pathogenic genospecies that causes Lyme disease in Europe. In order to elucidate the immune evasion mechanisms of B. spielmanii, we compared the abilities of isolates obtained from Lyme disease patients and tick isolate PC-Eq17 to escape from complement-mediated bacteriolysis. Using a growth inhibition assay, we show that four B. spielmanii isolates, including PC-Eq17, are serum resistant, whereas a single isolate, PMew, was more sensitive to complement-mediated lysis. All isolates activated complement in vitro, as demonstrated by covalent attachment of C3 fragments; however, deposition of the later activation products C6 and C5b-9 was restricted to the moderately serum-resistant isolate PMew and the serum-sensitive B. garinii isolate G1. Furthermore, serum adsorption experiments revealed that all B. spielmanii isolates acquired the host alternative pathway regulators factor H and factor H-like protein (FHL-1) from human serum. Both complement regulators retained their factor I-mediated C3b inactivation activities when bound to spirochetes. In addition, two distinct factor H and FHL-1 binding proteins, BsCRASP-1 and BsCRASP-2, were identified, which we estimated to be approximately 23 to 25 kDa in mass. A further factor H binding protein, BsCRASP-3, was found exclusively in the tick isolate, PC-Eq17. This is the first report describing an immune evasion mechanism utilized by B. spielmanii sp. nov., and it demonstrates the capture of human immune regulators to resist complement-mediated killing.

Binding of human factor H-related protein 1 to serum-resistant Borrelia burgdorferi is mediated by borrelial complement regulator-acquiring surface proteins.

BACKGROUND: Isolates of Borrelia burgdorferi, the causative agent of Lyme disease, express up to 5 distinct complement regulator-acquiring surface proteins (CRASP-1, -2, -3, -4, and -5). METHODS: By use of ligand affinity blotting, enzyme-linked immunosorbent assay, surface plasmon resonance, and functional complement assays, we have identified factor H-related protein 1 (FHR-1) as a novel protein that binds to the bacterium via CRASP-3, -4, and -5. RESULTS: When incubated in serum, serum-resistant Borrelia burgdorferi strain LW2 bind FHR-1, an additional member of the human factor H protein family, and, similarly, 2 mouse FHR proteins bind to the surface. Recombinant FHR-1 binds to 3 borrelial surface proteins (CRASP-3, -4, and -5) but not to CRASP-1 and -2. A comparative analysis of the individual CRASPs revealed common as well as distinct binding profiles for the 3 human regulators. FHR-1 binds to 3 CRASPs, and factor H binds to all 5 CRASPs. In addition, factor H-like protein 1 interacts with CRASP-1 and -2 but with no other borrelial proteins. CONCLUSIONS: Thus, by expressing multiple surface proteins with different binding properties, the pathogen can attach a unique combination of host complement regulators to its surface. For the pathogen, this type of surface decoration and specific acquisition of different host plasma proteins allows fine-tuning of the host immune attack.

Coordinated expression of Borrelia burgdorferi complement regulator-acquiring surface proteins during the Lyme disease spirochete's mammal-tick infection cycle.

The Lyme disease spirochete, Borrelia burgdorferi, is largely resistant to being killed by its hosts' alternative complement activation pathway. One possible resistance mechanism of these bacteria is to coat their surfaces with host complement regulators, such as factor H. Five different B. burgdorferi outer surface proteins having affinities for factor H have been identified: complement regulator-acquiring surface protein 1 (BbCRASP-1), encoded by cspA; BbCRASP-2, encoded by cspZ; and three closely related proteins, BbCRASP-3, -4, and -5, encoded by erpP, erpC, and erpA, respectively. We now present analyses of the recently identified BbCRASP-2 and cspZ expression patterns throughout the B. burgdorferi infectious cycle, plus novel analyses of BbCRASP-1 and erp-encoded BbCRASPs. Our results, combined with data from earlier studies, indicate that BbCRASP-2 is produced primarily during established mammalian infection, while BbCRASP-1 is produced during tick-to-mammal and mammal-to-tick transmission stages but not during established mammalian infection, and Erp-BbCRASPs are produced from the time of transmission from infected ticks into mammals until they are later acquired by other feeding ticks. Transcription of cspZ and synthesis of BbCRASP-2 were severely repressed during cultivation in laboratory medium relative to mRNA levels observed during mammalian infection, and cspZ expression was influenced by culture temperature and pH, observations which will assist identification of the mechanisms employed by B. burgdorferi to control expression of this borrelial infection-associated protein.

Molecular analysis of the thymidine-auxotrophic small colony variant phenotype of Staphylococcus aureus.

Thymidine-auxotrophic small colony variants (SCVs) of Staphylococcus aureus are frequently isolated from the chronically infected airways of patients suffering from cystic fibrosis. To date, little is known regarding the molecular mechanisms leading to the formation of this special phenotype, but the auxotrophism for thymidine suggests that impaired thymidine metabolism might play a major role. Sequence analysis of the thymidylate synthase-encoding thyA gene of six clinical thymidine-auxotrophic S. aureus SCVs revealed that all isolates had mutations within thyA. In five isolates the function of the thymidylate synthase was definitely impaired: three of them showed a truncation of the thyA coding sequence by nonsense or frame-shift mutations, in one further isolate the active site of the enzyme was affected by an internal 12-bp deletion, and another isolate had a 173-bp deletion spanning the 5'-terminal region of thyA and the preceding DNA sequence. The sixth isolate showed two amino acid substitutions within the thyA gene product. To confirm the importance of impaired thymidylate synthase synthesis or activity for the formation of the thymidine-auxotrophic SCV phenotype, we constructed a thyA knock-out mutant of a wild-type S. aureus strain. This mutant showed all characteristics of clinical SCVs, such as slow growth, decreased pigment production, reduced hemolytic activity, auxotrophism for thymidine, resistance to trimethoprim/sulfamethoxazol, and reduced plasma coagulase activity. Complementation of the thyA knock-out mutant with intact thyA in trans nearly restored the normal phenotype. In conclusion, these data confirm at the molecular level that impaired thymidylate synthase function is causative for the formation of the thymidine-auxotrophic SCV phenotype in S. aureus.

First case of human babesiosis in Germany - Clinical presentation and molecular characterisation of the pathogen.

Babesiosis is a common infection of animals and is gaining increasing attention as an emerging tick-borne zoonosis of humans in Europe. Here we report on the first case of human babesiosis in Germany in a 63-year-old splenectomised German patient with a relapse of nodular lymphocyte-predominant Hodgkin's lymphoma. After treatment with a chimeric anti-CD20 antibody preparation (Rituximab), the patient was hospitalised because of anaemia and dark urine from haemoglobinuria. Presumptive diagnosis of babesiosis was made based on piriform parasitic erythrocytic inclusions in peripheral blood smears and confirmed by Babesia-specific 18S rDNA PCR. Sequence analysis revealed a >99% homology of the amplicon with the recently described EU1 organism clustering within the Babesia divergens/Babesia odocoilei complex. Despite treatment with quinine and clindamycin the patient relapsed and developed chronic parasitaemia requiring re-treatment and long-term maintenance therapy with atovaquone before he eventually seroconverted and the parasite was cleared. Our findings suggest that human babesiosis occurs in Germany and can take a chronic course in immunocompromised individuals.

Prevalence and clinical significance of Staphylococcus aureus small-colony variants in cystic fibrosis lung disease.

Small-colony variants (SCVs) of Staphylococcus aureus can be isolated from the chronically infected airways of patients suffering from cystic fibrosis (CF). These slow-growing morphological variants have been associated with persistent and antibiotic-resistant infections, such as osteomyelitis and device-related infections, but no information is available to date regarding the clinical significance of this special phenotype in CF lung disease. We therefore investigated the prevalence of S. aureus SCVs in CF lung disease in a 12-month prospective study and correlated the microbiological culture results with the patients' clinical data. A total of 252 patients were screened for the presence of SCVs. The prevalence rate was determined to be 17% (95% confidence interval, 10 to 25%) among S. aureus carriers. S. aureus isolates with the SCV phenotype showed significantly higher antibiotic resistance rates than those with the normal phenotype. Patients positive for SCVs were significantly older (P = 0.0099), more commonly cocolonized with Pseudomonas aeruginosa (P = 0.0454), and showed signs of more advanced disease, such as lower forced expiratory volume in 1 s (P = 0.0148) than patients harboring S. aureus with a solely normal phenotype. The logistic regression model determined lower weight (P = 0.016), advanced age (P = 0.000), and prior use of trimethoprim-sulfamethoxazole (P = 0.002) as independent risk factors for S. aureus SCV positivity. The clinical status of CF patients is known to be affected by multiple parameters. Nonetheless, the independent risk factors determined here point to the impact of S. aureus SCVs on chronic and persistent infections in advanced CF lung disease.

Antimicrobial susceptibility of Borrelia burgdorferi sensu lato: what we know, what we don't know, and what we need to know.

Human Lyme borreliosis is a multisystem disorder that can progress in stages and is transmitted by ticks of the Ixodes ricinus complex infected with the spirochete Borrelia burgdorferi sensu lato. Today, Lyme borreliosis is regarded as the most important human tickborne illness in the northern hemisphere. Soon after the causative agent was correctly identified and successfully isolated in 1982, antibiotic treatment was shown to be effective and since then a variety of in vitro and in vivo studies have been performed to further characterize the activity of antimicrobial agents against B. burgdorferi s.l. Although many antimicrobial agents have been tested for their in vitro activity against borreliae, the full spectrum of antibiotic susceptibility in B. burgdorferi s.l. has not been defined for many compounds. Moreover, our current understanding of possible antimicrobial resistance mechanisms in B. burgdorferi s.l. is limited and is largely founded on in vitro experiments on relatively few borrelial isolates. This review will summarize what is and what is not known about antimicrobial resistance in B. burgdorferi s.l. and will discuss open questions that continue to fuel the current debate on treatment-resistant Lyme borreliosis.

Binding of human complement regulators FHL-1 and factor H to CRASP-1 orthologs of Borrelia burgdorferi.

The complement regulator-acquiring surface protein (CRASP)-1 is a member of the paralogous gene family gbb54 and the dominant FHL-1 and factor H binding protein of Borrelia burgdorferi sensu stricto (s.s.). It was shown recently that expression of BbCRASP-1 directly correlates with serum resistance of B. burgdorferi s.s. isolates. In the present study we have elucidated the putative potential of other members of the gbb54 paralogous family, including orthologs ZSA66, ZSA69, ZSA70, ZSA71, ZSA72 and ZSA73 of the European B. burgdorferi s.s. strain ZS7, to bind human FHL-1 and factor H. In spite of their overall similarity in protein sequence, between 47% and 67%, and the fact that the C-terminal region of ZSA69 shows 70% similarity with BbCRASP-1, none of the orthologous proteins was able to bind human FHL-1 and/or factor H. BbCRASP-1 is the only member of the paralogous gene family gbb54 that binds to human complement regulators, supporting the notion that BbCRASP-1 plays a critical role in evasion of complement by B. burgdorferi s.s. and thus may be helpful in the development of novel therapeutic strategies against Lyme borreliosis.