Bicarbonate enhances expression of the endocarditis and biofilm associated pilus locus, ebpR-ebpABC, in Enterococcus faecalis

<p>Abstract</p> <p>Background</p> <p>We previously identified <it>ebpR</it>, encoding a potential member of the AtxA/Mga transcriptional regulator family, and showed that it is important for transcriptional activation of the <it>Enterococcus faecalis </it><b>e</b>ndocarditis and <b>b</b>iofilm associated <b>p</b>ilus operon, <it>ebpABC</it>. Although <it>ebpR </it>is not absolutely essential for <it>ebpABC </it>expression (100-fold reduction), its deletion led to phenotypes similar to those of an <it>ebpABC </it>mutant such as absence of pili at the cell surface and, consequently, reduced biofilm formation. A non-piliated <it>ebpABC </it>mutant has been shown to be attenuated in a rat model of endocarditis and in a murine urinary tract infection model, indicating an important participation of the <it>ebpR-ebpABC </it>locus in virulence. However, there is no report relating to the environmental conditions that affect expression of the <it>ebpR-ebpABC </it>locus.</p> <p>Results</p> <p>In this study, we examined the effect of CO₂/HCO₃^-, pH, and the Fsr system on the <it>ebpR-ebpABC </it>locus expression. The presence of 5% CO₂/0.1 M HCO₃^-increased <it>ebpR-ebpABC </it>expression, while the Fsr system was confirmed to be a weak repressor of this locus. The mechanism by which the Fsr system repressed the <it>ebpR-ebpABC </it>locus expression appears independent of the effects of CO₂^-bicarbonate. Furthermore, by using an <it>ebpA</it>::<it>lacZ </it>fusion as a reporter, we showed that addition of 0.1 M sodium bicarbonate to TSBG (buffered at pH 7.5), but not the presence of 5% CO₂, induced <it>ebpA </it>expression in TSBG broth. In addition, using microarray analysis, we found 73 genes affected by the presence of sodium bicarbonate (abs(fold) > 2, <it>P </it>< 0.05), the majority of which belong to the PTS system and ABC transporter families. Finally, pilus production correlated with <it>ebpA </it>mRNA levels under the conditions tested.</p> <p>Conclusions</p> <p>This study reports that the <it>ebp </it>locus expression is enhanced by the presence of bicarbonate with a consequential increase in the number of cells producing pili. Although the molecular basis of the bicarbonate effect remains unclear, the pathway is independent of the Fsr system. In conclusion, <it>E. faecalis </it>joins the growing family of pathogens that regulates virulence gene expression in response to bicarbonate and/or CO₂.</p

Importance of the ebp (endocarditis- and biofilm-associated pilus) locus in the pathogenesis of Enterococcus faecalis ascending urinary tract infection.

BACKGROUND: We recently demonstrated that the ubiquitous Enterococcus faecalis ebp (endocarditis- and biofilm-associated pilus) operon is important for biofilm formation and experimental endocarditis. Here, we assess its role in murine urinary tract infection (UTI) by use of wild-type E. faecalis OG1RF and its nonpiliated, ebpA allelic replacement mutant (TX5475). METHODS: OG1RF and TX5475 were administered transurethrally either at an ~1 : 1 ratio (competition assay) or individually (monoinfection). Kidney pairs and urinary bladders were cultured 48 h after infection. These strains were also tested in a peritonitis model. RESULTS: No differences were observed in the peritonitis model. In mixed UTIs, OG1RF significantly outnumbered TX5475 in kidneys (P=.0033) and bladders (P\u3c or =.0001). More OG1RF colony-forming units were also recovered from the kidneys of monoinfected mice at the 4 inocula tested (P=.015 to P=.049), and 50% infective doses of OG1RF for kidneys and bladder (9.1x10(1) and 3.5x10(3) cfu, respectively) were 2-3 log(10) lower than those of TX5475. Increased tropism for the kidney relative to the bladder was observed for both OG1RF and TX5475. CONCLUSION: The ebp locus, part of the core genome of E. faecalis, contributes to infection in an ascending UTI model and is the first such enterococcal locus shown to be important in this site

Adherence to host extracellular matrix and serum components by Enterococcus faecium isolates of diverse origin.

Enterococcus faecium has emerged as an important cause of nosocomial infections over the last two decades. We recently demonstrated collagen type I (CI) as a common adherence target for some E. faecium isolates and a significant correlation was found to exist between acm-mediated CI adherence and clinical origin. Here, we evaluated 60 diverse E. faecium isolates for their adherence to up to 15 immobilized host extracellular matrix and serum components. Adherence phenotypes were most commonly observed to fibronectin (Fn) (20% of the 60 isolates), fibrinogen (17%) and laminin (Ln) (13%), while only one or two of the isolates adhered to collagen type V (CV), transferrin or lactoferrin and none to the other host components tested. Adherence to Fn and Ln was almost exclusively restricted to clinical isolates, especially the endocarditis-enriched nosocomial genogroup clonal complex 17 (CC17). Thus, the ability to adhere to Fn and Ln, in addition to CI, may have contributed to the emergence and adaptation of E. faecium, in particular CC17, as a nosocomial pathogen

Evaluation of Standard- and High-Dose Daptomycin versus Linezolid against Vancomycin-Resistant Enterococcus Isolates in an In Vitro Pharmacokinetic/Pharmacodynamic Model with Simulated Endocardial Vegetations

Daptomycin MICs for enterococci are typically 1- to 2-fold higher than those for Staphylococcus aureus, and there is an imminent need to establish the optimal dose for appropriate treatment of enterococcal infections. We investigated the bactericidal activity of daptomycin at various dose exposures compared to that of linezolid against vancomycin-resistant enterococcus (VRE) in an in vitro pharmacokinetic/pharmacodynamic model utilizing simulated endocardial vegetations over 96 h. Daptomycin at doses of 6, 8, 10, and 12 mg/kg of body weight/day and linezolid at a dose of 600 mg every 12 h were evaluated against two clinical vancomycin-resistant Enterococcus faecium strains (EFm11499 and 09-184D1051), one of which was linezolid resistant (09-184D1051), and one clinical vancomycin-resistant Enterococcus faecalis strain (EFs11496). Daptomycin MICs were 4, 2, and 0.5 μg/ml for EFm11499, 09-184D1051, and EFs11496, respectively. Bactericidal activity, defined as a ≥3 log10 CFU/g reduction from the initial colony count, was demonstrated against all three isolates with all doses of daptomycin; however, bactericidal activity was not sustained with the daptomycin 6- and 8-mg/kg/day regimens. Linezolid was bacteriostatic against EFm11499 and displayed no appreciable activity against 09-184D1051 or EFs11496. Concentration-dependent killing was displayed with more sustained reduction in colony count (3.58 to 6.46 and 5.89 to 6.56 log10 CFU/g) at 96 h for the simulated regimen of daptomycin at doses of 10 and 12 mg/kg/day, respectively (P ≤ 0.012). No E. faecium mutants with reduced susceptibility were recovered at any dosage regimen; however, the E. faecalis strain developed reduced daptomycin susceptibility with daptomycin at 6, 8, and 10 but not at 12 mg/kg/day. Daptomycin displayed a dose-dependent response against three VRE isolates, with high-dose daptomycin producing sustained bactericidal activity. Further research is warranted.This investigator-initiated study was funded by a research grant from Cubist Pharmaceuticals. We are grateful to Audrey Wanger for providing isolate 09-184D1051 and to Marcus Zervos for providing isolates SF11496 and SF11499. M.J.R. has received grant support, has served as a consultant, or has participated as a speaker for Astellas, Cubist, Forest Laboratories, Pfizer, Rib-X, and Novartis and is supported in part by grant R21AI092055 from the NIAID. C.A.A. has received lecture fees, research support, and consulting fees from Pfizer, Inc., and Cubist and has served as a speaker for Novartis. C.A.A. is supported in part by NIH grants R00 AI72961 and R01 AI093749 from the NIAID. B.E.M. has received grant support from Johnson & Johnson, Astellas, Palumed, Intercell, and Cubist, has served as a consultant for Astellas, Cubist, The Medicines Company, Pfizer, Rib-X, AstraZeneca, and Duranta Therapeutics, and was supported in part by NIH grant R01 AI72961

Specific roles for the GATA transcription factors end-1 and end-3 during C. elegans E-lineage development

Abstractend-1 and end-3 are GATA transcription factors important for specifying endoderm cell fate in Caenorhabditis elegans. Deletion of both factors together results in larval arrest, 0% survival and a fate change in the endoderm-specifying E lineage. Individual deletions of either factor, however, result in the development of viable, fertile adults, with 100% of worms developing to adults for end-1(−) and 95% for end-3(−). We sought to quantify the variable phenotypes seen in both deletions using automated cell lineaging. We quantified defects in cell lifetime, cell movement and division axis in end-3(−) embryos, while quantifying perturbations in downstream reporter gene expression in strains with homozygous deletions for either gene, showing that each deletion leads to a unique profile of downstream perturbations in gene expression and cellular phenotypes with a high correlation between early and late defects. Combining observations in both cellular and gene expression defects we found that misaligned divisions at the E2 stage resulted in ectopic expression of the Notch target ref-1 in end-3(−) embryos. Using a maximum likelihood phylogenetic approach we found end-1 and end-3 split to form two distinct clades within the Caenorhabditis lineage with distinct DNA-binding structures. These results indicate that end-1 and end-3 have each evolved into genes with unique functions during endoderm development, that end-3(−) embryos have a delay in the onset of E lineage cell fate and that end-1 has only a partially penetrant ability to activate E lineage fate

Maternal plasma DHA levels increase prior to 29 days post-LH surge in women undergoing frozen embryo transfer: a prospective, observational study of human pregnancy

Context: Docosahexaenoic acid (DHA) is an important fatty acid required for neurological development but its importance during early fetal neurological organogenesis is unknown. Objective: To assess plasma fatty acid changes in early pregnancy in women undergoing natural cycle-frozen embryo transfer as a means of achieving accurately-timed periconceptual sampling. Design: Women undergoing frozen embryo transfer were recruited and serial fasting blood samples were taken pre-luteinising hormone (LH) surge, and at days 18, 29 and 45 post-LH surge and fatty acids were analysed using gas chromatography. Setting: Assisted Conception Unit, Glasgow Royal Infirmary, Scotland Main outcome measures: Plasma fatty acid concentrations, influence of twin pregnancies on DHA plasma concentration. Results: In pregnant women, there was a rapid, early increase in the maternal rate of change of plasma DHA concentration observed by 29 days post-LH surge (mean±SD, from 0.1±1.3 to 1.6±2.9 nmol DHA per mL plasma per day). This early pressure to increase plasma DHA concentration was further emphasised in twin pregnancies where the increase in DHA concentration over 45 days was two-fold higher than in singleton pregnancies (mean±SD increase, 74±39 nmol/mL versus 36±40 nmol/mL). An index of delta-6 desaturase activity increased 30% and positively correlated with the rate of change of DHA concentration between day 18 and 29-post LH surge (R-squared adjusted = 41%, P=0.0002). DHA was the only fatty acid with a continual accelerated increase in plasma concentration and a positive incremental area under the curve (mean±SD, 632±911 nmol/mL x day) over the first 45 days of gestation. Conclusions: An increase in maternal plasma DHA concentration is initiated in human pregnancy prior to neural tube closure which occurs at 28 days' gestation

Analysis of clonality and antibiotic resistance among early clinical isolates of Enterococcus faecium in the United States.

BACKGROUND: The Enterococcus faecium genogroup, referred to as clonal complex 17 (CC17), seems to possess multiple determinants that increase its ability to survive and cause disease in nosocomial environments. METHODS: Using 53 clinical and geographically diverse US E. faecium isolates dating from 1971 to 1994, we determined the multilocus sequence type; the presence of 16 putative virulence genes (hyl(Efm), esp(Efm), and fms genes); resistance to ampicillin (AMP) and vancomycin (VAN); and high-level resistance to gentamicin and streptomycin. RESULTS: Overall, 16 different sequence types (STs), mostly CC17 isolates, were identified in 9 different regions of the United States. The earliest CC17 isolates were part of an outbreak that occurred in 1982 in Richmond, Virginia. The characteristics of CC17 isolates included increases in resistance to AMP, the presence of hyl(Efm) and esp(Efm), emergence of resistance to VAN, and the presence of at least 13 of 14 fms genes. Eight of 41 of the early isolates with resistance to AMP, however, were not in CC17. CONCLUSIONS: Although not all early US AMP isolates were clonally related, E. faecium CC17 isolates have been circulating in the United States since at least 1982 and appear to have progressively acquired additional virulence and antibiotic resistance determinants, perhaps explaining the recent success of this species in the hospital environment

Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium.

Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a \u27ladder\u27 pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpC(fm) is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpA(fm)), fms5 (ebpB(fm)) and ebpC(fm) are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen

Criteria for the diagnosis of corticobasal degeneration

Current criteria for the clinical diagnosis of pathologically confirmed corticobasal degeneration (CBD) no longer reflect the expanding understanding of this disease and its clinicopathologic correlations. An international consortium of behavioral neurology, neuropsychology, and movement disorders specialists developed new criteria based on consensus and a systematic literature review. Clinical diagnoses (early or late) were identified for 267 nonoverlapping pathologically confirmed CBD cases from published reports and brain banks. Combined with consensus, 4 CBD phenotypes emerged: corticobasal syndrome (CBS), frontal behavioral-spatial syndrome (FBS), nonfluent/agrammatic variant of primary progressive aphasia (naPPA), and progressive supranuclear palsy syndrome (PSPS). Clinical features of CBD cases were extracted from descriptions of 209 brain bank and published patients, providing a comprehensive description of CBD and correcting common misconceptions. Clinical CBD phenotypes and features were combined to create 2 sets of criteria: more specific clinical research criteria for probable CBD and broader criteria for possible CBD that are more inclusive but have a higher chance to detect other tau-based pathologies. Probable CBD criteria require insidious onset and gradual progression for at least 1 year, age at onset ≥50 years, no similar family history or known tau mutations, and a clinical phenotype of probable CBS or either FBS or naPPA with at least 1 CBS feature. The possible CBD category uses similar criteria but has no restrictions on age or family history, allows tau mutations, permits less rigorous phenotype fulfillment, and includes a PSPS phenotype. Future validation and refinement of the proposed criteria are needed

Cryo-electron Microscopy Structure of the 70S Ribosome from Enterococcus faecalis

Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalisnow extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen