Staphylococcus epidermidis saeR is an effector of anaerobic growth and a mediator of acute inflammation.

The saeRS two-component regulatory system regulates transcription of multiple virulence factors in Staphylococcus aureus. In the present study, we demonstrated that the saePQRS region in Staphylococcus epidermidis is transcriptionally regulated in a temporal manner and is arranged in a manner similar to that previously described for S. aureus. Studies using a mouse foreign body infection model demonstrated that the virulence of strain 1457 and the virulence of a mutant, strain 1457 saeR, were statistically equivalent. However, histological analyses suggested that the polymorphonuclear neutrophil response at 2 days postinfection was significantly greater in 1457-infected mice than in 1457 saeR-infected mice, demonstrating that SaeR influences the early, acute phases of infection. Microarray analysis demonstrated that a saeR mutation affected the transcription of 65 genes (37 genes were upregulated and 28 genes were downregulated); in particular, 8 genes that facilitate growth under anaerobic conditions were downregulated in 1457 saeR. Analysis of growth under anaerobic conditions demonstrated that 1457 saeR had a decreased growth rate compared to 1457. Further metabolic experiments demonstrated that 1457 saeR had a reduced capacity to utilize nitrate as a terminal electron acceptor and exhibited increased production of lactic acid in comparison to 1457. These data suggest that in S. epidermidis SaeR functions to regulate the transition between aerobic growth and anaerobic growth. In addition, when grown anaerobically, 1457 saeR appeared to compensate for the redox imbalance created by the lack of electron transport-mediated oxidation of NADH to NAD+ by increasing lactate dehydrogenase activity and the subsequent oxidation of NADH.

The presence of icaADBC is detrimental to the colonization of human skin by Staphylococcus epidermidis.

Previous studies have demonstrated that Staphylococcus epidermidis isolates colonizing the skin of healthy humans do not typically encode icaADBC, the genes responsible for the production of polysaccharide intercellular adhesin or biofilms. It was therefore hypothesized that the presence of icaADBC was deleterious to the successful colonization of human skin by S. epidermidis. Using a human skin competition model, it was determined that the strong biofilm-producing S. epidermidis strain 1457 was outcompeted at 1, 3, and 10 days by an isogenic icaADBC mutant (1457 ica::dhfr), suggesting a fitness cost for carriage of icaADBC.

Initial characterizations of nontransformed and transformed [3H ]aldosterone-type I receptor complexes in brain cytosol.

Incubation of [3H]aldosterone-type I receptor complexes in mouse brain cytosol with the chaotropic anion thiocyanate increased the fraction of receptors retained by DNA-cellulose from less than 10% to over 40%, whereas it decreased the fraction retained by protamine sulfate from more than 90% to less than 10%. Thiocyanate-induced transformation to the DNA-binding species was also accompanied by a 2.1-fold decrease in the rate of [3H]aldosterone dissociation from type I receptors as well as by an increase in the apparent positive charge and hydrophobicity of the surface of these receptors, as revealed by DEAE Bio-Gel ion exchange and pentyl agarose hydrophobic interaction chromatography. Sucrose density gradient sedimentation and Sephacryl S-300 gel exclusion chromatography revealed a reduction in the sedimentation coefficient and Stokes radius of the steroid-receptor complex from 9.6S and 8.0 nm before to 4.7S and 6.1 nm after transformation, respectively. These changes in hydrodynamic parameters were found to correspond to a 2.8-fold reduction in the apparent molecular mass from 331,000 before to 120,000 after transformation. In view of these various findings as well as the known differential affinity of protamine sulfate for the 90K heat shock protein, we suggest that thiocyanate-induced transformation is initiated by the dissociation of two molecules of heat shock protein from each steroid/DNA-binding type I receptor subunit.

Aldosterone-stimulated down-regulation of both type I and type II adrenocorticosteroid receptors in mouse brain is mediated via type I receptors.

The concentrations of type I and type II adrenocorticosteroid receptors in brain cytosol obtained from adrenalectomized-ovariectomized female mice were measured with five different assay conditions. Among the five brain regions studied, hippocampus had the highest concentration of type I receptors, whereas cerebral cortex had the highest concentration of type II receptors. The value of properly correcting for dexamethasone cross-binding to type I receptors when type II receptors are being assayed was demonstrated using the type II receptor-selective ligand RU28362. A time-course study revealed a transient up-regulation of both receptor classes in most brain regions after adrenalectomy-ovariectomy, with maximal values achieved 3-5 days postsurgery and a reduction to near-intact levels by 16 days postsurgery. A single sc injection of aldosterone given to adrenalectomized-ovariectomized mice produced a profound down-regulation of type I receptors in hippocampal, cerebral cortex, hypothalamic, brain stem, and cerebellar samples, whereas it down-regulated type II receptors only in hippocampal and cerebral cortical samples. A similar injection of RU28362 failed to down-regulate type I receptors in any brain region, but it did reduce the concentration of type II receptors in all brain regions except cerebellum. The actions of aldosterone appear to be mediated solely through type I receptors, since injections of the type I receptor antagonist RU26752 prevented aldosterone-induced down-regulation of both type I and type II receptors, whereas RU26752 had no effect on the down-regulatory actions of RU28362. The ability of aldosterone to down-regulate type I, but not type II, receptors in hypothalamic, brain stem, and cerebellar samples suggests that type I and type II receptors are concentrated in separate populations of cells in these brain regions, whereas in hippocampus and cerebral cortex there is a sufficient degree of colocalization to permit type II receptor down-regulation via the action of aldosterone-type I receptor complexes. We speculate that this action is mediated at least in part at the genomic level by the suppression of type I and type II receptor mRNA synthesis brought about by the interactions of transformed aldosterone-type I receptor complexes with the DNA regulatory elements upstream from the genes for these receptors.

High affinity binding and regulatory actions of dexamethasone-type I receptor complexes in mouse brain.

It is often implied that the various molecular, physiological, and behavioral responses to the glucocorticoid dexamethasone (DEX) are mediated in brain exclusively via the interactions of this synthetic steroid with the classical glucocorticoid (type II) receptor. The results reported in this study, however, suggest this generalization may, at least for the female mouse, be too restrictive. In the first experiment we compared the efficacy of the mineralocorticoid aldosterone (ALDO) with that of DEX to measure the classical mineralocorticoid (type I) receptor in brain cytosol. Since both of these steroids also bind to type II receptors, our assays included the type II receptor-selective ligand, RU26988. Whereas the specific binding of ALDO to type I receptors was largely unaffected by a 10-fold increase in the concentration of RU26988 (50- vs. 500-fold excess), there was a dramatic reduction in the specific binding of DEX. In a follow-up experiment, Scatchard analyses were used to confirm the differential affinity of RU26988 for DEX- vs. ALDO-type I receptor-binding sites and to reveal that the affinity of type I receptors for DEX (Kd approximately 0.83 nM) was nearly as high as it was for ALDO (Kd approximately 0.46 nM). A series of competition studies indicated that the competitive affinity (Kdc) of DEX for the ALDO-binding site was equivalent to the Kd computed in the saturation analyses, thus suggesting that the high affinity binding sites for DEX and ALDO on type I receptors may be equivalent or at least overlapping. The binding of DEX to these high affinity sites may prove to be important, since the systemic administration of this steroid was found to down-regulate both type I and type II receptors in a number of brain regions. Because coadministration of the type I receptor antagonist RU26752 was shown to block these actions on type I, but not type II receptors, the formation of the DEX-type I receptor complex appears to be required for DEX-induced type I receptor down-regulation. An analysis of the in vitro efficacy of ALDO- vs. DEX-type I receptor transformation suggests that whereas there is a significant increase in the binding of both complexes to DNA-cellulose after treatment with thiocyanate, there is also a dramatic decrease in the stability of DEX- but not ALDO-type I receptor binding. We contend that it is this decrease in binding stability that mediates the DEX-induced down-regulation of type I receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Measles pneumonia. Treatment of a near-fatal case with corticosteroids and vitamin A.

A 29-year-old woman experienced overwhelming rubeola pneumonia requiring endotracheal intubation and mechanical ventilation. Treatment with high-dose corticosteroids and vitamin A was accompanied by a prompt clinical response. Further investigation of this novel therapy is needed.

Central venous catheter-related Corynebacterium minutissimum bacteremia.

Although Corynebacterium minutissimum is well-known as the cause of erythrasma, it is noted as the etiologic agent of nondermatologic disease only rarely. We document this organism as a cause of central venous catheter-associated bacteremia and report the use of pulsed-field gel electrophoresis to characterize its molecular epidemiology.

Environmental contamination with vancomycin-resistant enterococci in an outpatient setting.

Eleven cancer patients colonized with vancomycin-resistant enterococci (VRE) were followed as outpatients. Environmental cultures were obtained from clinic rooms before and after patients care. Environmental contamination occurred in 29% of encounters. Superficial disinfection did not eradicate contamination, although more thorough cleaning did. We conclude that environmental VRE contamination occurs in the outpatient setting. Infection control practices, similar to those used in the inpatient setting, may be necessary for outpatient clinics.

Outbreak of vancomycin-resistant Enterococcus faecium in a neonatal intensive care unit.

An outbreak of vancomycin-resistant Enterococcus faecium involving 28 infants in a neonatal intensive care unit was observed. Successful control of the outbreak was achieved following use of patient and staff cohorting, contact isolation precautions, patient and environmental surveillance cultures, environmental decontamination, molecular typing, introduction of an alcohol-based hand disinfectant, and decreased use of vancomycin.

Ischemia induces a selective biphasic response in brain mitochondrial mRNA levels.

We determined the independent effects of hypoxia, glucose deprivation and ischemia (hypoxia plus glucose deprivation) on steady-state levels of mRNA coding for specific nuclear and mitochondrially encoded enzymes of oxidative metabolism in cultured rat neurons and glia. Neither hypoxia nor low glucose alone changed steady-state message levels for any transcript. However, ischemia induced a biphasic effect on mitochondrially encoded transcripts for cytochrome oxidase subunit two (CO2) and the subunits 8 and 6 of ATPase (A 8/6), initially decreasing and then increasing mRNA levels to or above the levels recorded prior to ischemia. In contrast, three nuclear encoded transcripts for mitochondrial proteins were decreased by ischemia. These data demonstrate a lack of coordination between the expression of nuclear and mitochondrial genes in the initial response to ischemia and suggest that a selective, primary reaction to brain cell insults exists within the mitochondrion.

Genetic and phenotypic analysis of biofilm phenotypic variation in multiple Staphylococcus epidermidis isolates.

Production of biofilm in Staphylococcus epidermidis is mediated through enzymes produced by the four-gene operon ica and is subject to phenotypic variation. The purpose of these experiments was to investigate the regulation of ica and icaR transcription in phenotypic variants produced by multiple unrelated isolates of S. epidermidis. Ten isolates were chosen for the study, four of which contained IS256. IS256 mediates a reversible inactivation of ica in approximately 30 % of phenotypic variants. All ten strains produced at least two types of phenotypic variant (intermediate and smooth) in which biofilm formation was significantly impaired. Reversion studies indicated that all phenotypic variants were stable after overnight growth, but began to revert to other phenotypic forms after 5 days of incubation at 37 degrees C. ica transcriptional analysis was performed on phenotypic variants from three IS256-negative isolates; 1457, SE5 and 14765. This analysis demonstrated that ica transcription was significantly reduced in the majority of phenotypic variants, although two variants from SE5 and 1457 produced wild-type quantities of ica transcript. Analysis of seven additional phenotypic variants from SE5 revealed that ica expression was only reduced in three. Expression of icaR transcript was unaffected in all smooth phenotypic variants. Mutations within ica were identified in two SE5 variants with wild-type levels of ica transcription. It is concluded that mutation and transcriptional regulation of ica are the primary mechanisms that govern phenotypic variation of biofilm formation within IS256-negative S. epidermidis.

Treatment of mouse brain cytosol with dextran-coated charcoal and high salt does not reveal a new glucocorticoid binder.

Incubation of steroid-free whole mouse brain cytosol from adrenalectomized-ovariectomized mice with saturating concentrations of tritiated dexamethasone was found to label all Type I as well as all Type II adrenocorticosteroid receptors. The quantitative and brain regional distribution of residual dexamethasone binding in cytosols pre-treated with dextran-coated charcoal (DCC) and 300 mM KCl was indistinguishable from that for tritiated aldosterone-Type I receptor complexes under the same conditions. We therefore conclude that the dexamethasone binding sites remaining after DCC and KCl treatment of steroid-free brain cytosol are due to the presence of Type I receptors. The differential sensitivity of Type I and Type II receptors to the DCC/KCl treatment paradigm may be useful in the purification of Type I receptors.

Differential up- and down-regulation of type I and type II receptors for adrenocorticosteroid hormones in mouse brain.

Adult female mice were adrenalectomized and ovariectomized and the concentration of Type I and Type II receptors in whole brain, kidney, and liver cytosol determined at various time thereafter by incubation with [3H]aldosterone (+ RU 26988 to prevent binding to Type II receptors) or [3H]dexamethasone, respectively. Type I receptor binding in brain was found to undergo a dramatic biphasic up-regulation, with levels six times that of intact levels by 24 h post-surgery and a doubling again by 4-8 days post-surgery. By 16 days, however, Type I specific binding had returned to intact levels. Similar, but less dramatic fluctuations were seen in kidney and liver, whereas much smaller fluctuations were seen for Type II receptors in all three tissues. In a follow-up study with Scatchard analyses we observed a similar transient up- and down-regulation in maximal binding for Type I, and to a lesser extent Type II receptors in all three tissues. As expected, the apparent binding affinity for both receptors increased after surgical removal of competing endogenous steroids. Radioimmunoassays revealed that plasma concentrations of corticosterone were reduced to near undetectable levels by 24 h post-surgery. A direct comparison of male and female mice revealed no sex-related differences in Type I receptor binding capacity fluctuations in brain cytosol after adrenalectomy-gonadectomy. Lastly, treatment with exogenous aldosterone or corticosterone was found to prevent adrenalectomy-gonadectomy-induced up-regulation of Type I and, to a lesser extent, Type II receptors in brain. Somewhat surprisingly, the potency of these two adrenocorticosteroids appeared to be very similar for both receptor types.

Automated homologue matching of human G-banded chromosomes.

Two sets of human G-banded chromosomes were employed to test a computer algorithm for homologue matching. One set was produced at the Rigshospitalet, Copenhagen, and the other at the University of Texas M. D. Anderson Hospital. Employing a cross-correlation measure to select candidate homologue mates for each chromosome resulted in correct matches in 91.1% of the cases in the Anderson set and 93.1% in the Denmark set. To identify each chromosome and to give a measure of classification accuracy when performed by humans, five cytogeneticists were asked to independently karyotype the 49 cells in the Anderson set. Agreement between two cytogeneticists was measured by the kappa statistic. If all chromosomes that could not be identified by any given cytogeneticist were removed from the comparison, kappa values were found to be in the vicinity of 0.95. With such unidentifiable chromosomes included as a separate class, the kappa values were closer to 0.89.

Inappropriate intravascular device use: a prospective study.

Despite the clinical significance of complications due to intravascular catheters, the inappropriate use of intravascular catheters in hospitalised patients has not been adequately characterised. The objective of this prospective observational study was to develop definitions for appropriate intravascular device use, to estimate the frequency of inappropriate use of intravascular devices, and to examine risk factors and outcomes associated with inappropriate use in hospitalised patients. Among 436 patients admitted between October and December 2007, a total of 2909 hospitalisation days and use of 876 intravascular devices was observed. Of the 3806 total catheter-days recorded, 1179 (31%) were found to be inappropriate based on the study criteria. Logistic regression analysis indicated that age, total number of catheters used and total duration of catheterisation were risk factors for inappropriate device use (P<0.05). Inappropriate usage was strongly associated with increased intensive care unit admission (P<0.05) and length of hospital stay (4.9±4.3 days for appropriate vs 8.5±12.6 days for inappropriate; P<0.05). Use of central venous catheters was not a predictor for inappropriate device use. Inappropriate intravascular device use is a very common phenomenon in hospitalised patients and is strongly linked to adverse device-related outcomes. These results may be used to develop strategies to systematically reduce excessive intravascular device use which would be expected to reduce adverse events associated with morbidity, mortality, and excess healthcare costs.

SigmaB and SarA independently regulate polysaccharide intercellular adhesin production in Staphylococcus epidermidis.

The production of polysaccharide intercellular adhesin (PIA) is an essential process in foreign body infections mediated by Staphylococcus epidermidis. Transcriptional regulation of the icaADBC operon, the genes responsible for production of enzymes that synthesize PIA, is multi-factorial and involves at least SarA and sigmaB. Transcriptional and promoter fusion studies revealed that the decreased transcription of the icaADBC operon observed in a S. epidermidis 1457 sigB mutant is not mediated through a direct interaction of sigmaB-RNA polymerase at the icaADBC promoter region but instead through the upregulation of IcaR, a known repressor of icaADBC transcription. Transcriptional analysis of a 1457 sigB-icaR double mutant confirmed that the decreased icaADBC transcript in 1457 sigB is IcaR dependent. Furthermore, primer extension studies suggest that the icaR promoter appears to be sigmaA dependent, suggesting that sigmaB indirectly controls icaR transcription through an unknown pathway. In addition, it was confirmed that the loss of SarA results in the loss of icaADBC transcription and PIA production in S. epidermidis. It was further demonstrated, through the over-production of SarA in 1457 sigB, that the loss of sarP1 promoter activity in 1457 sigB has little or no effect on the loss of PIA production in this mutant. Finally, it was demonstrated that PIA production could be restored in both 1457 sigB and 1457 sarA by complementing these mutants with a full-length icaADBC operon controlled by a cadmium-inducible noncognate promoter. It is concluded that sigmaB and SarA operate independently of each other to regulate PIA production and biofilm development in S. epidermidis.

Adherence of Staphylococcus epidermidis to biomaterials is augmented by PIA.

Staphylococcus epidermidis is the most common cause of orthopaedic prosthetic device infections. Polysaccharide intercellular adhesin (PIA) is important in the pathogenesis of intravascular catheter-associated infection, and has an essential role in cellular aggregation and biofilm formation. However, the role of PIA in orthopaedic infections is less well understood. We used genetically defined strains of S. epidermidis in an in vitro adherence assay to assess the importance of PIA in the adherence to various orthopaedic biomaterials. On all biomaterials tested (zirconia, ultra-high molecular weight polyethylene, polymethylmethacrylate, cobalt chromium, titanium, stainless steel, and silastic), PIA-positive S. epidermidis 1457 exhibited greater levels of adherence thanS. epidermidis 1457 M10, an isogenic icaA Tn917 mutant. PIA appears to play a critical role in the adherence of S. epidermidis to orthopaedic biomaterials, and may serve as an important virulence determinant in orthopaedic prosthetic device infections.