Evaluation of Screening and Commercial Methods for Detection of Methicillin Resistance in Coagulase-Negative Staphylococci

The National Committee for Clinical Laboratory Standards recommends 48 h of incubation by the oxacillin salt agar screen (OSAS) method for the detection of methicillin-resistant coagulase-negative staphylococci (CoNS). An earlier identification of methicillin resistance is desirable. The time to detection of the mecA gene by PCR was compared with the times to detection by OSAS, by the oxacillin disk diffusion (ODD) method, and with MicroScan Gram Positive Combo type 6 panels (MicroScan Inc. Sacramento, Calif.) and Vitek GPS-SA cards (bioMérieux Vitek Inc., Hazelwood, Mo.). The combination of the Vitek card and the ODD method detected 92 of 99 methicillin-resistant strains of CoNS at 24 h; however, 6 mecA-positive strains were phenotypically methicillin susceptible. We conclude that most methicillin-resistant CoNS can be detected and the results can be reported after overnight incubation by a combination of methods.     

Performance of the TechLab C. DIFF CHEK-60 enzyme immunoassay (EIA) in combination with the C. difficile Tox A/B II EIA kit, the Triage C. difficile panel immunoassay, and a cytotoxin assay for diagnosis of Clostridium difficile-associated diarrhea

We compared a recently marketed enzyme immunoassay for glutamate dehydrogenase (GDH), TechLab's C. DIFF CHEK-60 (TL-GDH), in combination with the C. difficile Tox A/B II enzyme immunoassay (Tox-A/B) with (i) the Triage C. difficile test, which detects both GDH (TR-GDH) and toxin A (TR-Tox-A); (ii) an in-house cytotoxin assay (C-Tox); and (iii) stool cultures for C. difficile. All C. difficile isolates were tested for the presence of the toxin genes by PCR. If a toxin gene-positive strain of Clostridium difficile was recovered and a toxin was detected by any method, the result was considered to be truly positive. Eighty-seven of 93 and 79 of 93 C. difficile culture-positive samples were also TL-GDH and TR-GDH positive, respectively. No test was able to detect toxin in all samples with true-positive results. Tox-A/B and TR-Tox-A in combination with the GDH detection tests and C-Tox were able to identify 52 and 50 samples with true-positive results. Tox-A/B and TR-Tox-A would have missed 15 and 31% of cases of C. difficile-associated diarrhea, respectively, if used alone.     

Relevance of apolipoprotein E polymorphism for coronary artery disease in the Saudi population

BACKGROUND: The apolipoprotein E alleles epsilon2 and epsilon4 have been reported as independent risk factors for coronary artery disease (CAD) and as predictors for the development of atherosclerosis. METHODS AND RESULTS: We determined by polymerase chain reaction the distribution of apolipoprotein E polymorphism in 320 Saudi blood donors (BD), 96 CAD patients, and 40 control subjects who had undergone angiography. Compared to controls, only epsilon4 was elevated in CAD patients. More than 61% (P <.0001) of the patients had angina, and 52.1% (P <.05) were diabetic; both of these factors were strongly associated with the presence of allele epsilon2. The epsilon2 allele was also associated with hypertension, elevated serum triglycerides, and total cholesterol. On the other hand, the allele epsilon4 appeared to be associated with increased risk of CAD and was also associated with hypertension, 3-vessel disease, and restenosis. CONCLUSIONS: Accordingly, epsilon4 may be associated with increased risk of CAD, whereas epsilon2 appears to be a predictor of several risk factors for atherosclerosis.     

Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells

Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin alpha(5)beta(1) (B. Sinha et al., Cell. Microbiol. 1:101-117, 1999). However, it is unknown whether this mechanism is sufficient for S. aureus invasion. To address this question, various S. aureus adhesins (FnBPA, FnBPB, and clumping factor [ClfA]) were expressed in Staphylococcus carnosus and Lactococcus lactis subsp. cremoris. Both noninvasive gram-positive microorganisms are genetically distinct from S. aureus, lack any known S. aureus surface protein, and do not bind fibronectin. Transformants of S. carnosus and L. lactis harboring plasmids coding for various S. aureus surface proteins (FnBPA, FnBPB, and ClfA) functionally expressed adhesins (as determined by bacterial clumping in plasma, specific latex agglutination, Western ligand blotting, and binding to immobilized and soluble fibronectin). FnBPA or FnBPB but not of ClfA conferred invasiveness to S. carnosus and L. lactis. Invasion of 293 cells by transformants was comparable to that of strongly invasive S. aureus strain Cowan 1. Binding of soluble and immobilized fibronectin paralleled invasiveness, demonstrating that the amount of accessible surface FnBPs is rate limiting. Thus, S. aureus FnBPs confer invasiveness to noninvasive, apathogenic gram-positive cocci. Furthermore, FnBP-coated polystyrene beads were internalized by 293 cells, demonstrating that FnBPs are sufficient for invasion of host cells without the need for (S. aureus-specific) coreceptors.     

Correlation of electrocorticographic to clinical seizure onset and interhemispheric propagation times in temporal lobe epilepsy

This study was performed to test the hypothesis that, in human temporal lobe epilepsy, electrocorticographic time factors involved in the ictal EEG to clinical ictal transition (electrocorticographic to clinical seizure onset time, ECOT) and the interhemispheric propagation of epileptic activity (interhemispheric propagation time, IHPT), which are independently correlated with temporal lobe epileptogenicity and predictive of seizure-free outcome following temporal lobectomy, are correlated with one another in a quantitative fashion. A series of 37 patients with medically intractable temporal lobe seizures was studied with long-term subdural videoelectroencephalographic monitoring. Temporal lobe seizure interhemispheric propagation time (IHPT) was found to be a negative, exponential function of electrocorticographic to clinical seizure onset time (ECOT) (f(x) = 8.201 × 10^−0.016x, r = 0.347, d.f. = 35, t = 2.19, p < 0.05, where f(x) = IHPT and x = ECOT). A small increase in ECOT was associated with a substantial decrease in IHPT and vice versa. The results suggest the electrophysiological time factor, ECOT, involved in the transition from ictal EEG seizure onset to clinical seizure onset, may determine the speed of interhemispheric propagation of established epileptic activity. The results suggest the interesting hypothesis that, in human temporal lobe epilepsy and, perhaps, under non-pathological circumstances, the human temporal lobe might possess a “time-labeling” function amenable to quantitative analysis.     

Correlation of oxacillin MIC with mecA gene carriage in coagulase-negative staphylococci

The National Committee for Clinical Laboratory Standards has recently changed the oxacillin breakpoint from >/=4 mg/liter to >/=0. 5 mg/liter to detect methicillin-resistant coagulase-negative staphylococci (CoNS) because the previous breakpoint lacked sensitivity. To determine the correlation between the new oxacillin breakpoint and the presence of the mecA gene, 493 CoNS of 11 species were tested. The presence of the mecA gene was determined by PCR, and oxacillin susceptibility was determined by the agar dilution method with Mueller-Hinton agar containing 2% NaCl and oxacillin (0. 125 to 4.0 mg/liter). The new breakpoint correctly classified all CoNS strains with mecA as methicillin resistant and strains of Staphylococcus epidermidis, S. haemolyticus, and S. hominis without mecA as methicillin susceptible. The breakpoint of >/=0.5 mg/liter was not specific for S. cohnii, S. lugdunensis, S. saprophyticus, S. warneri, and S. xylosus, in that it categorized 70 of 74 strains of these species without mecA (94.6%) as methicillin resistant. The results of this study indicate that the new oxacillin breakpoint accurately identifies strains of CoNS with mecA but is not specific for strains of certain species of CoNS without mecA.     

Binding of nucleosomes to a cell surface receptor: Redistribution and endocytosis in the presence of lupus antibodies

In the present study, we sought evidence for a surface nucleosome receptor in the fibroblastic cell line CV-1, and questioned whether anti-double-stranded (ds)DNA and/or anti-histone autoantibodies could recognized and influence the fate of cell surface-bound nucleosomes. ¹²⁵I-labeled mononucleosomes were shown to bind to the cell layer in a specific, concentration-dependent and a saturable manner. Scatchard analysis revealed the presence of two binding sites: a high-affinity site with a Kd of ～ 7nM and a low-affinity site (Kd ～ 400 nM) with a high capacity of 9 × 10⁷ sites. Visualization of bound mononucleosomes by fluorescence revealed staining on both the cell surface and the extracellular matrix (ECM). Purified mononucleosome-derived dsDNA (180–200 bp) was found to compete for binding of ¹²⁵I-mononucleosomes on the low-affinity site, to stain exclusively the ECM in immunofluorescence, and to precipitate three specific proteins of 43, 180 and 240 kDa from ¹²⁵-I-labeled cell lysates. Nucleosomes were found to precipitate not only the 180-kDa dsDNA-reactive component, but also a unique protein of 50 kDa, suggesting that this protein is a cell surface receptor for nucleosomes on these fibroblasts. Once bound on the cell surface, mononucleosomes were recognized and secondarily complexed by lupus anti-dsDNA or anti-histone antibodies (i.e. anti-nucleosome antibodies), thus forming immune complexes in situ. The presence of these complexing auto-antibodies was found dramatically to enhance the kinetics of mononucleosome internalization. Following the internalization of the nucleosome-anti-nucleosome complexes by immunofluorescence, we observed the formation of vesicles at the edge of the cells by 5–10 min which moved toward the perinuclear region by 20–30 min. By means of double-fluorescence labeling and proteolytic treatment, these fluorescent vesicles were shown to be in the cytoplasm, suggesting true endocytosis of nucleosome-anti-nucleosome immune complexes. As shown by confocal microscopy, at no stage of this endocytic process was there any indication that coated pits or coated vesicles participated. Co-distribution of the endocytic vesicles with regions rich in actin filaments and inhibition of endocytosis of nucleosome-anti-nucleosome complexes by disruption of the micro-filament network with cytochalasin D suggest a mechanism mediated by the cytoskeleton. Taken together, our data provide evidence for the presence of a surface nucleosome receptor. We also show that anti-dsDNA and anti-histone antibodies can form nucleosome-anti-nucleosome immune complexes in situ at the cell surface, and thus dramatically enhance the kinetics of nucleosome endocytosis.     

Osteogenic Cells Derived From Embryonic Stem Cells Produced Bone Nodules in Three-Dimensional Scaffolds

An approach for 3D bone tissue generation fromembryonic stem (ES) cells was investigated. The ES cells wereinduced to differentiate into osteogenic precursors, capable ofproliferating and subsequently differentiating into bone-formingcells. The differentiated cells and the seeded scaffolds werecharacterized using von Kossa and Alizarin Red staining, electronmicroscopy, and RT-PCR analysis. The results demonstrated thatES-derived bone-forming cells attached to and colonized thebiocompatible and biodegradable scaffolds. Furthermore, thesecells produced bone nodules when grown for 3–4 weeks inmineralization medium containing ascorbic acid andbeta-glycerophosphate both in tissue culture plates and inscaffolds. The differentiated cells also expressed osteospecificmarkers when grown both in the culture plates and in 3Dscaffolds. Osteogenic cells expressed alkaline phosphatase,osteocalcin, and osteopontin, but not an ES cell-specific marker,oct-4. These findings suggest that ES cell can be usedfor in vitro tissue engineering and cultivation of graftable skeletal structures.