Antepartum fibrinogen concentration as a predictor of bleeding complications.

OBJECTIVE: The objective of this study was to examine the predictive value of fibrinogen concentration for bleeding complications among women presenting for delivery and for whom a fibrinogen level was measured before delivery. STUDY DESIGN: This was a nested case-control study using a cohort of all women who delivered at our institution from October 2001 to July 2016 and in whom a fibrinogen concentration was obtained within 48 hours before delivery. We identified all cases that had one or more of the following events: (1) postpartum hemorrhage; (2) postpartum hysterectomy; (3) transfusion of select blood products; or (4) a ≥ 33% decrease in hematocrit from the first hematocrit measured during the hospital stay to any subsequent hematocrit value drawn either simultaneously with or following the fibrinogen concentration measurement. We included the first case or control delivery for a given woman. Controls were the next one or two consecutive deliveries without a bleeding complication and matched for number of fetuses. We used logistic regression to calculate the odds ratio and 95% confidence intervals and calculated the area under the receiver operating characteristic curve. RESULTS: We identified 424 cases and 801 controls. The mean predelivery fibrinogen concentration was significantly lower in cases (425 ± 170 mg/dL) than controls (523 ± 122 ng/mL) for all case types combined (p < .001) and for each case type individually (all p < .001). For every 100-mg/dL decrease in fibrinogen, the odds of a bleeding complication increased 1.63 times (95% confidence interval: 1.48-1.80). However, the area under the receiver operating characteristic curve was poor (0.69; 95% confidence interval: 0.65-0.72). Below 300 mg/dL there were 104 (24.5%) cases and 31 (3.9%) controls, yielding high specificity (96.1%) but extremely low sensitivity (24.5%). We could not identify a cutoff value that yielded acceptable values of both sensitivity and specificity. CONCLUSIONS: Antepartum fibrinogen concentration was significantly lower among women who developed bleeding complications, though these differences may not be large enough to provide clinically meaningful critical values. Nevertheless, a higher threshold for the critical value during pregnancy should be considered.

Pseudomonas aeruginosa AlgR controls cyanide production in an AlgZ-dependent manner.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic infections in individuals suffering from the genetic disorder cystic fibrosis. In P. aeruginosa, the transcriptional regulator AlgR controls a variety of virulence factors, including alginate production, twitching motility, biofilm formation, quorum sensing, and hydrogen cyanide (HCN) production. In this study, the regulation of HCN production was examined. Strains lacking AlgR or the putative AlgR sensor AlgZ produced significantly less HCN than did a nonmucoid isogenic parent. In contrast, algR and algZ mutants showed increased HCN production in an alginate-producing (mucoid) background. HCN production was optimal in a 5% O2 environment. In addition, cyanide production was elevated in bacteria grown on an agar surface compared to bacteria grown in planktonic culture. A conserved AlgR phosphorylation site (aspartate at amino acid position 54), which is required for surface-dependent twitching motility but not alginate production, was found to be critical for cyanide production. Nuclease protection mapping of the hcnA promoter identified a new transcriptional start site required for HCN production. A subset of clinical isolates that lack this start site produced small amounts of cyanide. Taken together, these data show that the P. aeruginosa hcnA promoter contains three transcriptional start sites and that HCN production is regulated by AlgZ and AlgR and is maximal under microaerobic conditions when the organism is surface attached.

Pseudomonas aeruginosa AlgR represses the Rhl quorum-sensing system in a biofilm-specific manner.

AlgR controls numerous virulence factors in Pseudomonas aeruginosa, including alginate, hydrogen cyanide production, and type IV pilus-mediated twitching motility. In this study, the role of AlgR in biofilms was examined in continuous-flow and static biofilm assays. Strain PSL317 (DeltaalgR) produced one-third the biofilm biomass of wild-type strain PAO1. Complementation with algR, but not fimTU-pilVWXY1Y2E, restored PSL317 to the wild-type biofilm phenotype. Comparisons of the transcriptional profiles of biofilm-grown PAO1 and PSL317 revealed that a number of quorum-sensing genes were upregulated in the algR deletion strain. Measurement of rhlA::lacZ and rhlI::lacZ promoter fusions confirmed the transcriptional profiling data when PSL317 was grown as a biofilm, but not planktonically. Increased amounts of rhamnolipids and N-butyryl homoserine lactone were detected in the biofilm effluent but not the planktonic supernatants of the algR mutant. Additionally, AlgR specifically bound to the rhlA and rhlI promoters in mobility shift assays. Moreover, PAO1 containing a chromosomal mutated AlgR binding site in its rhlI promoter formed biofilms and produced increased amounts of rhamnolipids similarly to the algR deletion strain. These observations indicate that AlgR specifically represses the Rhl quorum-sensing system during biofilm growth and that such repression is necessary for normal biofilm development. These data also suggest that AlgR may control transcription in a contact-dependent or biofilm-specific manner.

The transcriptional regulator AlgR controls cyanide production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis (CF) patients. One characteristic of P. aeruginosa CF isolates is the overproduction of the exopolysaccharide alginate, controlled by AlgR. Transcriptional profiling analyses comparing mucoid P. aeruginosa strains to their isogenic algR deletion strains showed that the transcription of cyanide-synthesizing genes (hcnAB) was approximately 3-fold lower in the algR mutants. S1 nuclease protection assays corroborated these findings, indicating that AlgR activates hcnA transcription in mucoid P. aeruginosa. Quantification of hydrogen cyanide (HCN) production from laboratory isolates revealed that mucoid laboratory strains made sevenfold more HCN than their nonmucoid parental strains. In addition, comparison of laboratory and clinically derived nonmucoid strains revealed that HCN was fivefold higher in the nonmucoid CF isolates. Moreover, the average amount of cyanide produced by mucoid clinical isolates was 4.7 +/- 0.85 micromol of HCN/mg of protein versus 2.4 +/- 0.40 micromol of HCN/mg of protein for nonmucoid strains from a survey conducted with 41 P. aeruginosa CF isolates from 24 patients. Our data indicate that (i) mucoid P. aeruginosa regardless of their origin (laboratory or clinically derived) produce more cyanide than their nonmucoid counterparts, (ii) AlgR regulates HCN production in P. aeruginosa, and (iii) P. aeruginosa CF isolates are more hypercyanogenic than nonmucoid laboratory strains. Taken together, cyanide production may be a relevant virulence factor in CF lung disease, the production of which is regulated, in part, by AlgR.

Identification of AlgR-regulated genes in Pseudomonas aeruginosa by use of microarray analysis.

The Pseudomonas aeruginosa transcriptional regulator AlgR controls a variety of different processes, including alginate production, type IV pilus function, and virulence, indicating that AlgR plays a pivotal role in the regulation of gene expression. In order to characterize the AlgR regulon, Pseudomonas Affymetrix GeneChips were used to generate the transcriptional profiles of (i) P. aeruginosa PAO1 versus its algR mutant in mid-logarithmic phase, (ii) P. aeruginosa PAO1 versus its algR mutant in stationary growth phase, and (iii) PAO1 versus PAO1 harboring an algR overexpression plasmid. Expression analysis revealed that, during mid-logarithmic growth, AlgR activated the expression of 58 genes while it repressed the expression of 37 others, while during stationary phase, it activated expression of 45 genes and repression of 14 genes. Confirmatory experiments were performed on two genes found to be AlgR repressed (hcnA and PA1557) and one AlgR-activated operon (fimU-pilVWXY1Y2). An S1 nuclease protection assay demonstrated that AlgR repressed both known hcnA promoters in PAO1. Additionally, direct measurement of hydrogen cyanide (HCN) production showed that P. aeruginosa PAO1 produced threefold-less HCN than did its algR deletion strain. AlgR also repressed transcription of two promoters of the uncharacterized open reading frame PA1557. Further, the twitching motility defect of an algR mutant was complemented by the fimTU-pilVWXY1Y2E operon, thus identifying the AlgR-controlled genes responsible for this defect in an algR mutant. This study identified four new roles for AlgR: (i) AlgR can repress gene transcription, (ii) AlgR activates the fimTU-pilVWXY1Y2E operon, (iii) AlgR regulates HCN production, and (iv) AlgR controls transcription of the putative cbb3-type cytochrome PA1557.