Differential diagnosis of Staphylococcus aureus from Staphylococcus epidermidis and Staphylococcus saprophyticus by alphazurine A dye

Staphylococcal bacterial suspensions were streaked on Trypticase soy agar with 5% sheep blood culture plates. Paper discs containing alphazurine A, a triphenylmethane dye, were placed on the inoculated plates which were incubated at 37 degrees C for 24 hours. A wide zone of inhibition of growth of Staphylococcus aureus was present around the paper discs. Growth of Staphylococcus epidermidis and Staphylococcus saprophyticus was not inhibited.     

Typing of Staphylococcus aureus and Staphylococcus epidermidis strains by PCR analysis of inter-IS256 spacer length polymorphisms

IS256 elements are present in multiple copies in the staphylococcal genome, either flanking the transposon Tn4001 or independent of it. PCR-based analysis of inter-IS256 spacer polymorphisms was developed for typing of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis strains. Using SmaI macrorestriction analysis resolved by pulsed-field gel electrophoresis (PFGE) as the reference method for MRSA typing, excellent reproducibility (100%), discriminatory power (97%), and in vivo stability were observed. Good concordance of the results with those of other molecular typing methods was found for two MRSA collections. Inter-IS256 PCR analysis of a U.S. collection of MRSA strains (n = 36), previously characterized by 15 typing methods, showed more limited discrimination. Agreement was 78% with PFGE analysis and 83% with ribotyping (HindIII). Analysis of a second set of Belgian MRSA strains (n = 17), categorized into two widespread epidemic clones by PFGE analysis, showed 65% agreement. For typing of S. epidermidis strains (n = 26), inter-IS256 PCR showed complete typeability (100%) and good discriminatory power (85%). Inter-IS256 PCR analysis is proposed as an efficient molecular typing assay for epidemiological studies of MRSA or S. epidermidis isolates.     

In vitro studies of pharmacodynamic properties of vancomycin against Staphylococcus aureus and Staphylococcus epidermidis

The bactericidal activities of vancomycin against two reference strains and two clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis were studied with five different concentrations ranging from 2x to 64x the MIC. The decrease in the numbers of CFU at 24 h was at least 3 log10 CFU/ml for all strains. No concentration-dependent killing was observed. The postantibiotic effect (PAE) was determined by obtaining viable counts for two of the reference strains, and the viable counts varied markedly: 1.2 h for S. aureus and 6.0 h for S. epidermidis. The determinations of the PAE, the postantibiotic sub-MIC effect (PA SME), and the sub-MIC effect (SME) for all strains were done with BioScreen C, a computerized incubator for bacteria. The PA SMEs were longer than the SMEs for all strains tested. A newly developed in vitro kinetic model was used to expose the bacteria to continuously decreasing concentrations of vancomycin. A filter prevented the loss of bacteria during the experiments. One reference strain each of S. aureus and S. epidermidis and two clinical isolates of S. aureus were exposed to an initial concentration of 10x the MIC of vancomycin with two different half-lives (t1/2s): 1 or 5 h. The post-MIC effect (PME) was calculated as the difference in time for the bacteria to grow 1 log10 CFU/ml from the numbers of CFU obtained at the time when the MIC was reached and the corresponding time for an unexposed control culture. The difference in PME between the strains was not as pronounced as that for the PAE. Furthermore, the PME was shorter when a t1/2 of 5 h (approximate terminal t1/2 in humans) was used. The PMEs at t1/2s of 1 and 5 h were 6.5 and 3.6 h, respectively, for S. aureus. The corresponding figures for S. epidermidis were 10.3 and less than 6 h. The shorter PMEs achieved with a t1/2 of 5 h and the lack of concentration-dependent killing indicate that the time above the MIC is the parameter most important for the efficacy of vancomycin.     

Clumping of Staphylococcus aureus by human fibronectin

Clumping of different staphylococci by fibronectin and other purified plasma proteins has been investigated. Purified fibronectin was capable of clumping Staphylococcus aureus strains in concentrations identical with concentrations of fibronectin in human plasma. S. epidermidis and S saprophyticus were not clumped by fibronectin. The binding of fibronectin to S. aureus was not mediated by protein-A, as a strain lacking protein-A clumped in the presence of fibronectin, and the presence of IgG could not inhibit the clumping of S. aureus strains. The fibronectin-binding component on the staphylococcal cell wall seems to be unrelated to the fibrinogen-binding clumping factor.     

Tertiary structural changes and iron release from human serum transferrin

Guidelines for clinical practice are intended to suggest preferable approaches to particular medical problems as established by interpretation and collation of scientifically valid research, derived from extensive review of published literature. When data are not available that will withstand objective scrutiny, a recommendation may be made based on a consensus of experts. Guidelines are intended to apply to the clinical situation for all physicians without regard to specialty. Guidelines are intended to be flexible, not necessarily indicating the only acceptable approach, and should be distinguished from standards of care that are inflexible and rarely violated. Given the wide range of choices in any health care problem, the physician should select the course best suited to the individual patient and the clinical situation presented. These guidelines are developed under the auspices of the American College of Gastroenterology and its Practice Parameters Committee. These guidelines are also approved by the governing boards of American College of Gastroenterology and Practice Parameters Committee. Expert opinion is solicited from the outset for the document. Guidelines are reviewed in depth by the committee, with participation from experienced clinicians and others in related fields. The final recommendations are based on the data available at the time of the production of the document and may be updated with pertinent scientific developments at a later time. The following guidelines are intended for adults and not for pediatric patients.     

Inhibition of uptake of transferrin-bound iron by human hepatoma cells by nontransferrin-bound iron

The liver acquires iron from transferrin by transferrin receptor-mediated (TR) and transferrin receptor-independent pathways (NTR) and from nontransferrin-bound iron (NTB-Fe). Iron uptake by the NTR processes involves an iron-carrier mediated step. Experiments, using human hepatoma cells (HuH7) transfected with TR antisense (sense for control) RNA expression vectors to suppress TR expression, were performed to examine the effect of unlabeled NTB-Fe as iron citrate on the uptake of 59Fe-125I-transferrin. This was to determine if the uptake of transferrin-bound iron (Tf-Fe) and NTB-Fe uptake is mediated by a common iron-carrier. Iron citrate inhibited the uptake of 59Fe-transferrin (2.5 micromol/L Fe) in a concentration-dependent manner with a maximum effect when the citrate-iron:Tf-Fe molar ratio was 10:1. Transferrin uptake was not affected. At a lower Tf-Fe concentration of (0.125 micromol/L) when uptake of iron is TR-mediated, a 10-fold molar excess of iron citrate had no effect on Tf-Fe uptake by HuH7 TR antisense and sense cells. However, at a higher Tf-Fe concentration (2.5 micromol/L), when uptake occurs mainly by the NTR-mediated process, there was a 40% reduction in the membrane-bound and intracellular uptake of iron. Iron citrate did not affect the maximum rate (Vmax) of Tf-Fe uptake but the Michaelis-Menten constant (Km) for Tf-Fe uptake by the NTR-mediated process was increased, indicating there was competitive inhibition of Tf-Fe uptake by iron citrate. These results suggest that the uptake of NTB-Fe and Tf-Fe by the NTR- mediated process occurs by the same cellular pathway, using a common iron-carrier.     

Immunoglobulin production by human peripheral B cells against Staphylococcus aureus

Patterns of antiemetic therapy and its outcomes in patients undergoing high-dose antineoplastic therapy were studied. The study, conducted at a cancer center, included both a retrospective evaluation of patients undergoing highly emetogenic high-dose chemotherapy with peripheral blood stem-cell rescue between November 1994 and December 1995 and a concurrent evaluation of patients treated between January and May 1996. During the study period the recommended antiemetic regimen for highly emetogenic chemotherapy was a single dose of granisetron 1 mg i.v. daily 30 minutes before treatment on days of chemotherapy. Severity of nausea and vomiting during both the acute phase (from day 1 of chemotherapy to 24 hours after its completion) and delayed phase (from 24 hours to five days after the end of chemotherapy) was graded according to the Common Toxicity Criteria Grading Scale. A total of 59 patients were evaluable; 41 were reviewed retrospectively, and 18 were reviewed concurrently. On day 1 of the acute phase, 53 patients (90%) had no vomiting and 51 patients (86%) had no nausea. The frequency and severity of nausea and vomiting increased on successive acute-phase days, and it was necessary to add other antiemetics. Nausea and vomiting continued to be significant problems throughout the delayed phase; 32 (54%) of the patients had a maximum of grade 3 nausea, and 29 patients (49%) had a maximum of grade 2 vomiting. Substantial numbers of patients who received selective serotonin type 3 receptor antagonists before high-dose antineoplastic agents had significant nausea and vomiting that required the addition of other antiemetics.     

Staphylococcus aureus binding to human nasal mucin

Colonization of human nasal mucosa with Staphylococcus aureus sets the stage for subsequent systemic infection. This study characterizes S. aureus adhesion to nasal mucosa in vitro and investigates the interaction of S. aureus with human nasal mucin. S. aureus binding to cell-associated and cell-free mucus was greater than to nonmucin-coated epithelial cells. Scanning electron microscopy of S. aureus incubated with human nasal mucosal tissue showed minimal binding to ciliated respiratory epithelium. In a solid-phase assay, S. aureus bound to purified human nasal mucin-coated wells significantly more than to bovine serum albumin-coated microtiter wells. Binding to mucin was saturable in a dose- and time-dependent fashion. Staphylococcal adherence to human nasal mucin was inhibited by bovine submaxillary mucin but not by fibrinogen. Pretreatment of mucin with periodate but not with pronase reduced adherence. Trypsin treatment of the bacteria significantly reduced adherence to mucin. 125I-labelled nasal mucin bound to two surface proteins (138 and 127 kDa) of lysostaphin-solubilized S. aureus. Binding to human nasal mucin occurs in part via specific adhesin-receptor interactions involving bacterial proteins and the carbohydrate moiety in mucin. These experiments suggest that S. aureus binding to mucin may be critical for colonization of the nasopharyngeal mucosa.     

Uptake of iron from transferrin by isolated rat-liver mitochondria mediated by phosphate compounds

1. Isolated rat-liver mitochondria accumulate iron from transferrin at neutral pH by a mechanism which is markedly stimulated by small-molecular-weight polyphosphate compounds. The efficiency of the phosphate compounds decreases in the order: pyrophosphate > ATP > GTP > 2,3-bis-(phospho)glycerate > phosphate. 2. The uptake has a very low energy dependence, and it does not depend on the hydrolysis of ATP or the saturation of transferrin, but it increases in parallel to the concentration of iron to reach a saturation level of 800-1200 pmol iron/mg protein. 3. Following a chase with unlabelled transferrin (125I)-labelled transferrin bound to the mitochondria remains constant, whereas the progressive uptake of 59Fe levels off. 4. During reincubation of iron-loaded mitochondria up to 30% of the iron is mobilized in the presence of ascorbate and ATP (or apotransferrin). 5. The results suggest that iron is mobilized from transferrin by the polyphosphate compounds outside the mitochondria in a subsequent reaction.     

Diagnostic molecular microbiology--identification of Staphylococcus epidermidis

The performance of two sandwich-type immunoassays for the determination of the tumour marker tetranectin using monoclonal antibodies Hyb 130-13 and 130-14 as catching layer was compared with the performance of a polyclonal assay. Sensitivities were 0.4-0.6 microg/l, and intra- and inter-assay coefficients of variation were < 10% in all assays. One-hundred-and-ten blood donors were examined, and women had higher concentrations of tetranectin in serum than men when measured with monoclonal assays (P < 0.05). In preoperative serum samples from 43 patients with ovarian cancer, tetranectin concentrations were reduced (P < 0.001), and the mean tetranectin concentration decreased with increasing FIGO stage of the patients (P < 0.05). In sera from patients with ovarian cancer, tetranectin concentrations were lower in the polyclonal assay than in the monoclonal assays. This could, hypothetically, be explained by ligand-binding or other conformational changes in tetranectin, influencing the antigenicity of the molecule.     

Identification of LytSR-regulated genes from Staphylococcus aureus

In this report, the characterization of a Staphylococcus aureus operon containing two LytSR-regulated genes, lrgA and lrgB, is described. Sequence and mutagenesis studies of these genes suggest that lrgA encodes a murein hydrolase exporter similar to bacteriophage holin proteins while lrgB may encode a protein having murein hydrolase activity.     

Chelate mediated transfer of iron from transferrin to desferrioxamine

Desferrioxamine, widely used for the treatment of iron overload in Cooley's anaemia, binds iron so tightly that it should quantitatively remove iron from transferrin. Studies conducted in vivo and in vitro, however, have failed to demonstrate significant depletion of transferrin-bound iron by a stoichiometric excess of desferrioxamine. However, low molecular weight chelating agents, capable of forming ternary complexes with transferrin and ferric iron, can promote a rapid transfer of iron from transferrin to desferrioxamine. A possible mechanism for this facilitated exchange is offered.     

Trimethoprim resistance and susceptibility genes in Staphylococcus epidermidis

OBJECTIVES: This study was performed to assess the acute effects of the new angiotensin II antagonist, candesartan cilexetil, on systemic and renal haemodynamics in patients with sustained essential hypertension [diastolic blood pressure (DBP) 95-114 mmHg]. METHODS: After 4 weeks of placebo treatment, systemic and renal haemodynamics were investigated in 17 patients with a mean age of 62 years and a mean systolic and diastolic blood pressure of 170/98 mmHg, just before (baseline) and for 4 h after administration of a single oral dose of candesartan cilexetil, 16 mg. Plasma concentrations of candesartan (the active compound formed from the pro-drug candesartan cilexetil), angiotensin II (Ang II), as well as plasma renin activity (PRA), were measured before and after dosing. RESULTS: At 2, 3 h and 4 h after dosing with candesartan cilexetil, systolic blood pressure (SBP) and DBP, as well as mean arterial pressure (MAP), were significantly lower than at baseline. The mean reduction in MAP 4 h after dosing was 8.8 mmHg (-6.5%). This effect was due to a fall in total peripheral resistance (TPR), while heart rate (HR), stroke volume (SV) and cardiac output (CO) were virtually unchanged. After 4 h there was a marked reduction in renal vascular resistance (RVR) of 0.0273 mmHg x ml(-1) x min (-16%), resulting in an increased renal plasma flow of 64.9 ml x min(-1) (14%). The glomerular filtration rate was increased by 7.75 ml x min(-1) (8%), and the filtration fraction (FF) was not significantly changed. There was no apparent relationship between the changes observed in systemic and renal haemodynamic variables and plasma concentrations of candesartan. Plasma renin activity increased over the study period, but in general the patients had low PRA. Changes in plasma concentrations of angiotensin II were inconsistent between patients. CONCLUSION: A single oral tablet of candesartan cilexetil, 16 mg, induced systemic and renal arterial vasodilatation and blood pressure reduction, without compromising renal perfusion or filtration or affecting cardiac performance. Plasma renin activity which was low in general, increased over the study period, but changes in plasma concentrations of angiotensin II were inconsistent.     

Receptor-mediated folate uptake is positively regulated by disruption of the actin cytoskeleton

Receptor-mediated folate uptake is initiated by binding of ligand to a glycosyl phosphatidylinositol-anchored protein, folate receptor alpha (FR alpha). This receptor is expressed in a limited number of normal tissues but is overexpressed in a large number of epithelial malignancies. FR alpha synthesis, at least in part, is regulated by endogenous folate and by hormones in some cells, but much less is known about the control of function. Recently, we showed that phorbol 12-myristate 13-acetate increases the rate of receptor cycling, increases the rate of folate delivery, and causes the majority of the receptor to reside on the cell surface in nonmalignant cells in vitro (C. M. Lewis et al., Biochim. Biophys. Acta, 1401: 157-169, 1998). However, based upon effects (or lack of effects) of specific inhibitors of protein kinase C, the mechanism of action of phorbol 12-myristate 13-acetate is not likely via protein kinase C. Because exo- and endocytosis are controlled by the actin cytoskeleton, we tested cytochalasin D and latrunculin B, actin-disrupting agents, on FR alpha-mediated folate uptake. Disruption of the actin cytoskeleton reversibly increases the proportion of receptors on the cell surface and increases the rate of 5-methyltetrahydrofolate delivery. Disrupting microtubules with nocodazole had no effect. The increased rate of folate delivery caused by cytochalasin D is not observed in FR-negative cell lines. Although we have not yet identified the upstream effectors, likely candidates include small G-proteins such as rho, which are known to cause actin polymerization. In addition to identifying the machinery for receptor-mediated folate uptake, it may be important to integrate this new data into studies of FR alpha as a tumor antigen for imaging or delivering molecules via anti-FR antibodies or compounds coupled to folic acid.     

Postoperative enteritis caused by methicillin-resistant Staphylococcus aureus

We examined the clinical features of 14 men (mean age 72 years) with postoperative enteritis caused by methicillin-resistant Staphylococcus aureus (MRSA). The patients had all undergone surgery for the treatment of digestive diseases and had received antibiotic prophylaxis consisting of an extended-spectrum cephem. Diarrhea appeared a mean of 3.3 days postoperatively and lasted for 5 days on average. In severe cases organ insufficiency was involved. Coagulate-positive staphylococci were the predominant organisms isolated from watery diarrhea. In 13 of 14 patients, coagulase type II isolates producing enterotoxins A, C and toxic shock syndrome toxin-1 (TSST-1) with enterotoxin A, C, and 1st genes were isolated. These strains were sensitive to vancomycin and arbekacin; however, they were highly resistant to many other antibiotics. We also investigated the effects of a glucocorticoid hormone and gamma globulin on production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2) obtained from healthy volunteers. TNF-alpha and IL-2 production was enhanced by TSST-1 and the supernatant of Iscove-modified dulbecco medium, in which coagulase type II isolates producing enterotoxins A, C and TSST-1 with enterotoxin A, C were cultured for 24 h. Both glucocorticoid hormone and gamma globulin suppressed TNF-alpha and IL-2 production, thus suggesting that these drugs may be effective in treating postoperative MRSA enteritis.     

Identification of atypical strains of Staphylococcus epidermidis by use of molecular tools

Four atypical coagulase-negative staphylococcal (CNS) isolates from clinical sources were compared with Staphylococcus epidermidis strains by ribotyping. The ribotypes of the four strains shared close rDNA restriction profiles with those of the S. epidermidis strains used. The DNA sequence encoding 16S rRNA demonstrated 99.9% homology with S. epidermidis. S1 nuclease experiments showed that these atypical strains formed a homogeneous genomic group. DNA-DNA homologies between the S. epidermidis type strain CCM 2124 and the four CNS isolates ranged from 70 to 89%. The guanine-plus-cytosine content of the deoxyribonucleic acid of the four strains ranged from 31 to 32 mol%.     

Absorption of various actinomycins by Staphylococcus aureus cells

The absorption of actinomycin D by the cell suspension of Staphylococcus aureus via diffusion linearly depended on the antibiotic concentration in the suspension within the ranges of 2 to 15 micrograms/ml. The absorption of active actinomycins C2, C3 and Au6 was the same as that of actinomycin D. The Staphylococcus intact membranes limited the inlet of the actinomycins to the cells since the membranotropic substances such as gramicidin S and its derivatives and thyrocidin increased their absorption by 30-70 per cent. The absorption of a low active actinomycin D0 and inactive actinomycinic acid even after the exposure to the membranotropic substances was not detectable. These compounds did not form any complexes with DNA. The level of the absorption of the actinomycins by the cells was likely defined by their ability to complex with DNA.     

trans-complementation of a Staphylococcus aureus agr mutant by Staphylococcus lugdunensis agr RNAIII

RNAIII from Staphylococcus lugdunensis (RNAIII-sl) in a Staphylococcus aureus agr mutant partially restored the Agr phenotype. A chimeric construct consisting of the 5' end of RNAIII-sl and the 3' end of RNAIII from S. aureus restored the Agr phenotype to a greater extent, suggesting the presence of independent regulatory domains.     

Formation of extracellular protein A by Staphylococcus aureus

Staphylococcus aureus contains cell wall protein A as well as extracellular protein A. The two types of protein A have very similar amino acid compositions, electrophoretic mobilities and sizes. The release of extracellular protein A from exponentially growing bacteria is dependent on protein synthesis de novo and protein A is released directly after being synthesized on the ribosomes. Bacteria in the stationary phase, however, release protein A as a result of cell lysis. Protoplasts have been isolated which produce protein A as extensively as the intact bacteria but because of the absence of of the formation of extracellular protein A is observed from cells also producing cell wall protein A.