Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation.

Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms.

Is bacteriologic surveillance in endoscope reprocessing stringent enough?

Endoscopes, including duodenoscopes, are medical devices that are frequently associated with outbreaks of nosocomial infections. We investigated an outbreak of multidrug-resistant PSEUDOMONAS AERUGINOSA sepsis affecting three patients after endoscopic retrograde cholangiopancreaticography (ERCP). Epidemiologic investigation supplemented by molecular typing revealed that one ERCP scope was the source of infection with P. AERUGINOSA. No contamination with this microorganism was found after screening of washer-disinfectors, connecting tubes, and environmental surfaces in the endoscopy center. PSEUDOMONAS isolates from blood and endoscope channels before gas sterilization with ethylene oxide (ETO) were characterized by molecular typing as "linked isolates". Though the current surveillance system did not prevent the infections in three patients, our microbiological surveillance protocol with routine culturing of endoscopes was helpful in detecting the source of contamination and probably avoided numerous cross-contaminations in other patients who underwent ERCP procedures with endoscopes.

Endoscope disinfection and its pitfalls--requirement for retrograde surveillance cultures.

BACKGROUND AND STUDY AIMS: Several endoscopy-related outbreaks of infection have been reported in recent years. For early recognition of inadequate disinfection of endoscopes we designed a microbiological surveillance system to evaluate the efficacy of the cleaning and disinfection procedure, and to trace disinfection problems to individual endoscopes or washer-disinfectors. METHODS: Our surveillance protocol included anterograde and retrograde sampling, a decision algorithm, genetic fingerprinting, and scanning electron microscopy. RESULTS: Over a period of 29 months we found an increasing number of patient-ready endoscopes testing positive for Candida species other than albicans, especially C. parapsilosis. These yeasts were also isolated from the washer-disinfectors. The number of positive tests for Candida species varied from 1 out of 21 to 14 out of 27 samples from nine frequently used endoscopes. The number of colony-forming units per milliliter ranged from 1 - 10 to 3000 for endoscopes and 0.002 to 0.06 for the washer disinfectors. DNA fingerprinting was not able to discriminate different strains within C. parapsilosis. CONCLUSIONS: Our protocol was able to detect a structural problem in the endoscope disinfection process. Retrograde sampling was crucial for this purpose, because it has much higher sensitivity than anterograde sampling. Endoscopes with damaged working channels are probably the source of the contamination problem with Candida species.

Massive right atrial myxoma causing exertional dyspnoea.

Metastatic tumours are the commonest cardiac tumours being found in 1-3% of patients dying of cancer while primary tumours are unusual and have an incidence of 0.02-0.5%. The majority (80%) of all primary cardiac tumours are benign with myxomas accounting for 50%. Myxomas arising from the right atrium are uncommon. We present the case of a 39-year-old female with a 4-month history of progressive exertional dyspnoea accompanied by symptoms of palpitations and presyncope. Transthoracic echocardiography showed an extremely large right atrial myxoma prolapsing into the right ventricle and obstructing the tricuspid valve. We demonstrate how intraoperative transoesophageal echocardiography, prior to sternotomy, was useful in providing information about the myxoma which clearly displayed its attachment and anatomical relationship in the planning of the 'safe' surgical excision.

HDV ribozymes.

The self-cleaving RNA sequences, or ribozymes, in the genomic and antigenomic strands of hepatitis delta virus (HDV) RNA fold into structures that are similar to each other but distinct from those of small ribozymes associated with the RNA replicons that infect plants. HDV ribozymes have provided a tractable system for studying the mechanism of catalytic RNA, and results of biochemical and structural studies on the HDV ribozymes, from a number of labs, have enhanced our understanding and expanded our thinking about the potential for catalytic roles of RNA side chains. The results of these studies are consistent with models suggesting that both an active-site cytosine and a divalent metal ion have catalytic roles in facilitating the cleavage reaction in the HDV ribozymes. Despite recent advances, details about the catalytic mechanism of the HDV ribozyme continue to be debated, and these ribozymes should serve as a good system for further study.

Early experience with a helical coronary thrombectomy device in patients with acute coronary thrombosis.

OBJECTIVE: To report our experience with a new thrombectomy device (X-SIZER(TM)) in patients with angiographically visible thrombus or total coronary occlusion in the setting of acute coronary syndromes. DESIGN: A safety and feasibility study in the use of the X-SIZER during intervention in patients with acute coronary syndromes. PATIENTS: 35 patients, age range 31 to 83 years (mean 60). SETTING: University Hospitals of Coventry and Warwickshire NHS Trust (tertiary referral centre). INTERVENTIONS: The indication for intervention was primary or salvage percutaneous coronary intervention for acute myocardial infarction in 17 of the 35 patients; unstable angina or non-ST-elevation myocardial infarction in 10; and unstable postinfarct angina in eight. Abciximab was given in 11 patients. MAIN OUTCOME MEASURES: Device success (successful deployment of the device at the site of the lesion with resultant improvement in TIMI flow); clinical success (no residual stenosis at the end of the procedure with no in-hospital major adverse coronary events). RESULTS: Successful use of the device was achieved in 26 of the 35 cases. It failed to cross the lesion in five and failed to improve TIMI flow despite crossing the lesion in four. Clinical success was achieved in 30 of the 35 cases. Device related complications occurred in two cases (vessel perforation) and there was one intraprocedural death (acute myocardial infarction with cardiogenic shock). CONCLUSIONS: Thrombectomy with the X-SIZER catheter system appears promising in percutaneous coronary intervention where thrombus extraction is considered necessary before stent implantation.

Ribozyme activity in the genomic and antigenomic RNA strands of hepatitis delta virus.

In the hepatitis delta virus, ribozymes are encoded in both the genomic strand RNA and its complement, the antigenomic strand. The two ribozymes are similar in sequence and structure, are most active in the presence of divalent cation and catalyze RNA cleavage reactions which generate a 5'-hydroxyl group and a 2',3'-cyclic phosphate group. Recent progress has been made in understanding the catalytic mechanism. One key was a crystal structure of the genomic ribozyme that revealed a specific cytosine positioned to act as a general acid-base catalyst. The folding of the ribozyme in the context of the longer viral RNA is another area of interest. The biology requires that each ribozyme act only once, and mechanisms proposed for regulation of ribozyme activity sometimes invoke alternative RNA structures. Likewise, interference of ribozyme function by polyadenylation of the antigenomic RNA strand could be controlled through alternative structures, and a model for such control is proposed.

Energetic contribution of non-essential 5' sequence to catalysis in a hepatitis delta virus ribozyme.

Hepatitis delta virus (HDV) ribozymes employ multiple catalytic strategies to achieve overall rate enhancement of RNA cleavage. These strategies include general acid-base catalysis by a cytosine side chain and involvement of divalent metal ions. Here we used a trans-acting form of the antigenomic ribozyme to examine the contribution of the 5' sequence in the substrate to HDV ribozyme catalysis. The cleavage rate constants increased for substrates with 5' sequence alterations that reduced ground-state binding to the ribozyme. Quantitatively, a plot of activation free energy of chemical conversion versus Gibb's free energy of substrate binding revealed a linear relationship with a slope of -1. This relationship is consistent with a model in which components of the substrate immediately 5' to the cleavage site in the HDV ribozyme-substrate complex destabilize ground-state binding. The intrinsic binding energy derived from the ground-state destabilization could contribute up to 2 kcal/mol toward the total 8.5 kcal/mol reduction in activation free energy for RNA cleavage catalyzed by the HDV ribozyme.

Evaluation of patient characteristics and utilisation of invasive cardiac procedures in a UK ethnic population with unstable angina pectoris.

OBJECTIVES: To evaluate patient characteristics and utilisation of invasive cardiac procedures in a UK ethnic population with unstable angina pectoris (UAP). DESIGN: Retrospective, observational study. SETTING: Tertiary referral cardiology centre in the United Kingdom serving a large Asian ethnic population. SUBJECTS: White and Asian patients undergoing PTCA for UAP over a 2.5-year period at a UK referral cardiology centre from a comprehensive PTCA database. Data were also collated for all emergency admissions with unstable angina, or angina (type unspecified), to our institute. MAIN OUTCOME MEASURES: Demographic and angiographic characteristics of patients undergoing PTCA. Frequency of usage of invasive cardiac procedures was determined in emergency angina admissions. RESULTS: From January 1997 to July 1999, 435 White and 36 Asian patients underwent PTCA for UAP at our institute. Asian patients were on average 4.4 years younger (P=0.015), had 19.3% more diabetes (P=0.003) and 19.7% less smoking (P=0.007). Trends to more single vessel disease in Whites and more double vessel disease in Asians were observed, with similar rates of triple vessel disease. Interestingly, Asians also had trends towards more left-sided coronary artery disease and revascularisation, smaller vessels and less bail-out stenting. Asian patients were significantly less likely to undergo coronary angiography, OR 0.64 (CI 0.45-0.91, P=0.012), during the index admission, and showed a trend to less PTCA. CONCLUSIONS: Asian patients with unstable angina pectoris appear to have clinical and angiographic differences from their White counterparts, and are less likely to have invasive cardiac procedures deployed. The reasons for these observations require elucidation and the prognostic significance of these findings is uncertain.

Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage.

Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76 Delta) ribozymes in D(2)O and H(2)O. In both reactions, a similar kinetic isotope effect and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76 Delta mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under k(cat)/K(M) conditions, correlated with the pKa of the base. From these data a Brønsted coefficient (beta) of 0.51 was determined for the base-rescued reaction of C76 Delta. This value is consistent with that expected for proton transfer in the transition state. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage.

A pH-sensitive RNA tertiary interaction affects self-cleavage activity of the HDV ribozymes in the absence of added divalent metal ion.

Self-cleavage of the genomic and antigenomic ribozymes from hepatitis delta virus (HDV) requires divalent cation for optimal activity. Recently, the HDV genomic ribozyme has been shown to be active in NaCl in the absence of added divalent metal ion at low pH (apparent pKa 5.7). However, we find that the antigenomic ribozyme is 100 to 1000-fold less active under similar conditions. With deletion of a three-nucleotide sequence (C41-A42-A43) unique to the genomic ribozyme, the rate constant for cleavage decreased substantially, while activity of the antigenomic ribozyme was enhanced by introducing a CAA sequence. From the crystal structure, it has been proposed that C41 in this sequence is protonated. To investigate a possible connection between activity at low pH and protonation of C41, mutations were made that were predicted to either eliminate protonation or alter the nature of the tertiary interaction upon protonation. In the absence of added Mg2+, these mutations reduced activity and eliminated the observed pH-rate dependence. Thermal denaturation studies revealed a pH-sensitive structural feature in the genomic ribozyme, while unfolding of the mutant ribozymes was pH-independent. We propose that, in the absence of added Mg2+, protonation of C41 contributes to enhanced activity of the HDV genomic ribozyme at low pH.

Multiple coronary aneurysms in a patient with neurofibromatosis type 1: case report and intravascular ultrasound of aneurysm.

A 54 year old woman with neurofibromatosis type 1 (NF-1) was found to have multiple coronary aneurysms. Intraoperative intravascular ultrasound (IVUS) revealed severe coronary disease proximal to the aneurysm that had not been apparent angiographically. An IVUS picture of one of the giant coronary aneurysms is also shown. The vascular manifestations of neurofibromatosis and the causes of coronary aneurysms are reviewed.

Kinetic scheme for intermolecular RNA cleavage by a ribozyme derived from hepatitis delta virus RNA.

A minimal kinetic mechanism for a trans-acting ribozyme derived from the HDV antigenomic RNA self-cleaving element was established from steady-state, pre-steady-state, single-turnover, and binding kinetics. Rate constants for individual steps, including substrate binding and dissociation, cleavage, and product release and binding, were measured at 37 degrees C at pH 8.0 in 10 mM Mg(2+) using oligonucleotides as either substrates, noncleavable analogues or 3' product mimics. A substrate containing a normal 3',5'-linkage was cleaved with a first-order rate constant (k(2)) of 0.91 min(-)(1). The association rate constant for the substrate to the ribozyme (2.1 x 10(7) M(-)(1) min(-)(1)) was at the lower range of the expected value for RNA duplex formation, and the substrate dissociated with a rate constant (1.4 min(-)(1)) slightly faster than that for cleavage. Thus the binary complex was not at equilibrium with free enzyme and substrate prior to the cleavage step. Following cleavage, product release was kinetically ordered in that the 5' product was released rapidly (>12 min(-)(1)) relative to the 3' product (6.0 x 10(-)(3) min(-)(1)). Rapid 5' product release and lack of a demonstrable binding site for the 5' product could contribute to the difficulty in establishing the ribozyme-catalyzed reverse reaction (ligation). Slow release of the 3' product was consistent with the extremely low turnover under steady-state conditions as 3' product dissociation was rate-limiting. The equilibrium dissociation constant for the substrate was 24-fold higher than that of the 3' cleavage product. A substrate with a 2',5'-linkage at the cleavage site was cleaved with a rate constant (k(2)) of 1.1 x 10(-)(2) min(-)(1). Thus, whereas cleavage of a 3',5'-linkage followed a Briggs-Haldane mechanism, 2', 5' cleavage followed a Michaelis-Menten mechanism.

Imidazole rescue of a cytosine mutation in a self-cleaving ribozyme.

Ribozymes use a number of the same catalytic strategies as protein enzymes. However, general base catalysis by a ribozyme has not been demonstrated. In the hepatitis delta virus antigenomic ribozyme, imidazole buffer rescued activity of a mutant with a cytosine-76 (C76) to uracil substitution. In addition, a C76 to adenine substitution reduced the apparent pKa (where Ka is the acid constant) of the self-cleavage reaction by an amount consistent with differences in the pKa values of these two side chains. These results suggest that, in the wild-type ribozyme, C76 acts as a general base. This finding has implications for potential catalytic functions of conserved cytosines and adenines in other ribozymes and in ribonuclear proteins with enzymatic activity.

Ribozyme cleavage of a 2,5-phosphodiester linkage: mechanism and a restricted divalent metal-ion requirement.

The natural substrate cleaved by the hepatitis delta virus (HDV) ribozyme contains a 3',5'-phosphodiester linkage at the cleavage site; however, a 2',5'-linked ribose-phosphate backbone can also be cleaved by both trans-acting and self-cleaving forms of the HDV ribozyme. With substrates containing either linkage, the HDV ribozyme generated 2',3'-cyclic phosphate and 5'-hydroxyl groups suggesting that the mechanisms of cleavage in both cases were by a nucleophilic attack on the phosphorus center by the adjacent hydroxyl group. Divalent metal ion was required for cleavage of either linkage. However, although the 3',5'-linkage was cleaved slightly faster in Ca2+ than in Mg2+, the 2',5'-linkage was cleaved in Mg2+ (or Mn2+) but not Ca2+. This dramatic difference in metal-ion specificity is strongly suggestive of a crucial metal-ion interaction at the active site. In contrast to the HDV ribozymes, cleavage at a 2',5'-phosphodiester bond was not efficiently catalyzed by the hammerhead ribozyme. The relaxed linkage specificity of the HDV ribozymes may be due in part to lack of a rigid binding site for sequences 5' to the cleavage site.