[Staphylococcal epidermal necrolysis occasionally affects children].
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Age Factors↗
Biomedical subjects
A Ljungh
Publications and source records attributed to A Ljungh.
Enterobacteriaceae found in high numbers in fish, minced meat and pasteurised milk or cream and the presence of toxin encoding genes.
Enterobacteriaceae were found in high numbers after storage at 7 degrees C in 6% of consumers packs of pasteurised milk or cream, in 31% of retailed fish and in 100% of retail packs of minced meat. Seventy two fresh-water fishes, 40 packs of minced meat and 430 milk packs were sampled. One hundred and eighty four isolates were randomly picked from Tryptone glucose extract (TGE) agar (30 degrees C for 3d) or Violet red bile glucose (VRBG) agar (37 degrees C for 1d). In minced meat, Serratia liquefaciens, Hafnia alvei, Rahnella aquatilis were frequently encountered. On fish, the most frequently found species were R. aquatilis, and in milk, the dominating species were S. liquefaciens, H. alvei and R. aquatilis. One to three isolates of Citrobacter freundii were found in all three food categories. Using a polymerase chain reaction (PCR) technique, the gene of Escherichia coli heat-labile toxin (lt) was indicated in one fish isolate of R. aquatilis whereas heat-stable toxin genes (s.t.) were indicated in four H. alvei isolates, two originating from fish and two from minced meat. Positive PCR-reaction for vero cytotoxin genes were found in one H. alvei strain originating from fish (vt1), in two S. liquefaciens strains from minced meat (vt2), and in a C. freundii reference strain. One of the st-positive H. alvei strains from meat harboured the eaeA gene involved in the attaching phenotype of enteropathogenic E. coli.
Animals↗
A new model to assess staphylococcal adhesion to intraocular lenses under in vitro flow conditions.
Adhesion of staphylococcal cells to intraocular lenses coated with heparin was studied under in vitro flow conditions (280 microl min(-1)) at 37 degrees C. The intraocular lenses were incubated with human cerebrospinal fluid for 1 h or with cerebrospinal fluid including 0.50% plasma for 12 h, prior to bacterial challenge. Two strains of Staphylococcus epidermidis selected for this study, were isolated from biomaterial-associated infections. Bacterial adhesion was quantitated by bioluminescence and visualized by fluorescence microscopy of acridine orange stained bacteria. Surface coating with heparin significantly decreased bacterial adhesion of both strains after incubation with cerebrospinal fluid including 0.50% plasma for 12 h (p = 0.0209). However, no difference in bacterial adhesion was obtained between intraocular lenses with and without heparin, after incubation with cerebrospinal fluid for 1 h (p = 0.327). Microscopy showed that more bacteria were present on intraocular lenses without heparin than on intraocular lenses with heparin. The results show that preincubation with a proteinaceous fluid influences subsequent bacterial adhesion to the polymer surface. The results suggest that IOLs with heparin coating may be less prone to bacterial adhesion under perfusion conditions after surface conditioning in human CSF with 0.50% plasma and a preincubation period of 12 h. Heparin coating might be a valuable tool to decrease implant-associated bacterial endophthalmitis.
Acridine Orange↗
Granulocyte-macrophage colony-stimulating factor (GM-CSF)-coated implants and their potential for reducing biomaterial-associated infection in neutropenic hosts.
The incidence of infections associated with the use of medical biomaterials is high for skin-penetrating devices, when microbes of the normal skin flora like coagulase-positive and coagulase-negative staphylococci dominate as causative organisms. The most serious ones are infections in immunocompromised individuals. A mouse model of subcutaneous staphylococcal infection yielding abscesses in cyclophosphamide-induced neutropenic mice implanted with heparinized polyethylene (H-PE) was used. The present study addresses the question of the effects of implant modification with recombinant granulocyte-macrophage stimulating factor (rGM-CSF) on the course of infection. Our findings demonstrate that such modification reduces the proliferation of bacteria within the abscess and as a consequence limits the dissemination of bacteria from the local infection induced in the neutropenic host.
Abscess↗
Quantitation of bacterial adhesion to polymer surfaces by bioluminescence.
Quantitation of microbes adhering to a surface is commonly used in studies of microbial adhesion to different surfaces. We have quantified different staphylococcal strains adhering to polymer surfaces by measuring bacterial ATP (adenosine triphosphate) by bioluminescence. The method is sensitive, having a detection limit of 10(4) bacterial cells. Viable counting of bacterial cells may yield falsely low results due to the presence of "dormant" and adherent bacteria. By using bioluminescence, this can be avoided. Cells of different bacterial species and cells of strains of the same species were shown to differ significantly in their basal ATP content (8.7 x 10(-13) - 5.2 x 10(-22) MATP). The size of adherent and planktonic bacteria decreased with time (0.7 micron-->0.3 micron, 20 days). During incubation in nutrient-poor buffer ("starvation"), the ATP content of adherent bacteria decreased after 24-96 h whereas that of planktonic bacteria was stable over 20 days. The presence of human serum or plasma did not interfere significantly with the test results. Since the ATP concentration of bacterial strains of different species varies and is also influenced by the growth conditions of bacteria (solid or liquid culture medium), a species-specific standard curve has to be established for bacteria grown under the same culture conditions. We conclude that the method is a sensitive tool to quantify adherent bacteria during experiments lasting for less than 6 h and constitutes a valuable method to be used in conjunction with different microscopical techniques.
Adenosine Triphosphate↗
In vitro anti-staphylococcal activity of heparinized biomaterials bonded with combinations of rifampicin.
Biomaterial implants in various human body tissues are highly susceptible to bacterial colonization. We report here on the coating of heparinized biomaterials with heparin binding extracellular matrix proteins giving special regard to the efficient adsorption and slow release of antibiotics. Heparin was partially degraded and the resulting fragments were covalently end-point attached to 0.5 cm long silicone biomaterial surface. Collagen type I was immobilized on the heparinized biomaterials and then cross-linked with acyl-azide or carbodiimide. Finally, the resulting biosurfaces were exposed to antibiotics, i.e. rifampicin in combination with cefuroxime, fusidic acid, ofloxacin or vancomycin, respectively. The antibiotic bonded biomaterials were evaluated for their anti-staphylococcal activity after elution in NaCl, serum or blood by measuring the zones of inhibition for S. epidermidis strain RP12. Furthermore, we examined the in-vitro colonization resistance to S. epidermidis RP12 for these combinations of rifampicin-bonded biomaterials by an ATP bioluminescence assay. The ATP measurements showed that initially adherent bacteria were eradicated from the polymer surface, for at least 24 or 48 h (fusidic acid > cefuroxime > vancomycin > ofloxacin). The anti-staphylococcal activity of rifampicin-fusidic acid bonded heparinized biomaterials seems of sufficient duration and efficacy to merit testing in an animal model.
Anti-Bacterial Agents↗
Binding of immobilized fibronectin by biliary drain isolates.
Occlusion of biliary stents, as the result of bacterial adhesion and colonization onto biliary stents, still remains a major problem. Biliary proteins, such as fibronectin (Fn) and vitronectin (Vn), have been presumed to be involved in the process of bacterial adhesion to biliary biomaterial. In the present study, Fn binding by 5 strains of E. coli isolated from biliary drains or from bile was studied. All strains did not bind detectable amounts of soluble Fn but bound to immobilized plasma Fn. Adhesion of four strains of E. coli to ovalbumin was reduced by periodate treatment of ovalbumin, but adhesion to Fn was unaffected. Adhesion was inhibited by mannose-containing saccharides, trypsin treatment of the protein, and protease treatment of the bacterial cells. Autoradiography showed that components of cell extracts from three E. coli strains bind 125I-Fn but not a 150 kD Fn fragment. The findings indicate that the adhesion of these bacteria to Fn is a protein-protein interaction, inhibited by D-mannose, and possibly mediated by fimbrial components.
Animals↗
The lactosylceramide binding specificity of Helicobacter pylori.
The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.
Animals↗
Plasminogen binding and activation at the surface of Helicobacter pylori CCUG 17874.
The binding of iodine-labelled plasminogen to Helicobacter pylori CCUG 17874 was characterized. Inhibition of the binding was observed after preincubation of H. pylori cells with nonradiolabelled plasminogen, lysine, or the lysine analogue epsilon-aminocaproic acid. Fragments of plasminogen, kringles 1 to 3, kringle 4, and mini-plasminogen, were also studied as potential inhibitors. Mini-plasminogen caused total inhibition of the plasminogen binding, while the other fragments caused only partial inhibition. These findings suggest that H. pylori binds specifically the fifth kringle structure of the plasminogen molecule. Plasminogen binding to H. pylori seems to be independent of culture media and independent of the presence of the cytotoxin-associated CagA antigen. Immunoblot analysis identified two plasminogen binding proteins of 57 and 42 kDa. Scatchard plot analysis revealed one binding mechanism with a Kd value of 7 x 10(-7) M. Conversion of H. pylori cell-bound plasminogen to plasmin in the presence of a tissue-type plasminogen activator was demonstrated by digestion of the chromogenic substrate S-2251. No activation was noted when plasminogen or tissue-type plasminogen activator was incubated with H. pylori cells alone. Formation of H. pylori cell surface-bound plasmin may be important to provide a powerful proteolytic mechanism for gastric tissue penetration in type B gastritis and peptic ulcer disease, since plasmin degrades not only fibrin but also extracellular matrix proteins such as various collagens and fibronectin.
Aminocaproic Acid↗
Virulence properties of Escherichia coli isolated from Ethiopian patients with acute or persistent diarrhoea.
Escherichia coli strains isolated from faecal specimens of 108 Ethiopian patients with acute watery diarrhoea (n = 30), acute bloody (n = 9), and persistent (n = 25) diarrhoea, and from 44 patients who recently had recovered from diarrhoea were analyzed for the presence of virulence factors using DNA probes, and for adhesion to HeLa cells. Eighty-two patients were under five years of age. Enterotoxigenic E.coli (ETEC) were most frequently isolated (63 patients, 58%). Eighteen of the ETEC strains also hybridized with probes for EPEC adherence factor (EAF) and Enteroaggregative (EAgg) adherence. Enteroaggregative E. coli (EAggEC) were more frequently isolated than EAF positive E.coli, and more frequently from patients with persistent diarrhoea (10/25) than from patients with acute diarrhoea (11/39). In total, 103 of the patients harboured faecal E. coli which hybridized with one or more of the virulence probes. Haemagglutination of one or more erythrocyte species was expressed by 65/70 strains. Using monoclonal antibodies to Colonization Factor Antigen I and Coli Surface antigens 1-5, only 18/66 strains were found to produce one or more of these adhesions and no more than 15 of 43 ETEC strains were agglutinated by the antisera to these adhesins. Forty-nine strains adhered to HeLa cells in autoaggregative (23 strains), localized (17 strains) or diffuse (9 strains) pattern. The study shows that E.coli strains carrying genes for the different virulence factors are prevalent in Ethiopia. Testing for the presence of these virulence factors, as well as for putative colonization factor antigens, should be included in epidemiological studies in this area.
Acute Disease↗
Adhesion of staphylococci to polymers with and without immobilized heparin in cerebrospinal fluid.
Infections of cerebrospinal fluid (CSF) shunts constitute a serious clinical problem. The role of adhesion by coagulase negative staphylococci, the most common etiological agent, was examined in vitro to polyvinyl chloride (PVC), silicone, and to PVC and silicone with end-point attached (EPA) heparin. These are flexible materials commonly used in neurosurgical implants. Bacterial adhesion was quantitated by bioluminescence. The bacterial adhesion to biomaterial surfaces increased with increasing concentrations of bacterial cells. Scatchard plot analysis showed continuous negative (concave) slopes, indicating multiple interactions between biomaterial and bacteria. The thermodynamic studies showed a positive value of the standard entropy change at 37 degrees C, which indicates that hydrophobic interactions are important in bacterial adhesion to polymers. Incubation with CSF for 1 h decreased bacterial adhesion in 75% of the samples compared to incubation in buffer. Thus, the contribution of CSF proteins, like fibronectin, for the initial bacterial adhesion might be small. Heparinization of silicone and PVC decreased the numbers of adhered bacteria by 23 to 54% and 0 to 43% compared to unheparinized surfaces. Among putative inhibitors tested, suramin, chondroitin sulfate, and fucoidan inhibited adhesion to 81 +/- 19, 78 +/- 22, and 64 +/- 7%, respectively. These findings indicate that hydrophobic interactions play an important role, and heparinization rendering the biomaterial surface hydrophilic is therefore effective to reduce bacterial adhesion. Heparinized polymers incubated with putative inhibitors may be the optimal way to prevent shunt infections.
Bacterial Adhesion↗
Protein depositions on one hydrocephalus shunt and on fifteen temporary ventricular catheters.
Biomaterials are commonly used in modern medicine. Proteins are adsorbed to the surface of the biomaterial immediately after insertion. This report demonstrates the presence of adsorbed proteins in one infected cerebrospinal shunt from a child with hydrocephalus and on fifteen temporary ventricular catheters from adult patients with spontaneous or traumatic brain injuries. Depositions of vitronectin, fibrinogen and thrombospondin-fibronectin to some extent--on the shunt surface was imaged by field-emission scanning electron microscopy. Vitronectin, fibronectin, fibrinogen, and thrombospondin on the ventricular catheters were shown with radio-actively labelled antibodies. Furthermore, protein adsorption from human cerebrospinal fluid to heparinized and unheparinized polymers was studied under flowing conditions in vitro. On heparinized polymer, significantly reduced levels of vitronectin, fibronectin, and thrombospondin were exposed, as measured after 4 hours in vitro perfusion. After 24 hours perfusion, the differences in protein exposition between heparinized and unheparinized polymers were substantially reduced.
Adsorption↗
Fibronectin exposes different domains after adsorption to a heparinized and an unheparinized poly(vinyl chloride) surface.
The adsorption of fibronectin to poly(vinyl chloride) catheters with end-point attached (EPA) heparin and tridodecylmethylammonium chloride-heparinized poly(vinyl chloride) was compared to that of unheparinized poly(vinyl chloride) using antibodies directed against four different domains of the protein. After perfusion of human plasma on the EPA-heparinized surface, the exposure of the N-terminal 29-kD fragment increased during the first 5 h of perfusion. Also, the exposure of the 30-kD gelatin-binding and 65-kD cell-binding fragments increased with time, but at a lower level. On the unheparinized catheter, low levels of antibodies bound to the different domains, and the binding showed little variation during the 5 h of plasma perfusion, indicating that the fibronectin molecule does not change configuration to a significant extent on this surface after the initial adsorption. When the EPA-heparinized surface was preabsorbed with human fibrinogen before incubation with fibronectin, significantly less of the 29-kD (fibrin-binding) domain was exposed, and the 30-kD domain was not exposed. Exposure of the 31- and 65-kD domains increased after preadsorption of fibrinogen to the surface. Since fibronectin has heparin-binding domains, it adsorbs differently to a heparinized versus an unheparinized surface. This will influence subsequent binding of other proteins to the surface, as well as potential binding of microbes. The use of antibodies to defined domains of the fibronectin molecule provides a powerful tool in studies of configurational changes of fibronectin after adsorption to different surfaces.
Adsorption↗
Ribotype delineation and description of Staphylococcus sciuri subspecies and their potential as reservoirs of methicillin resistance and staphylolytic enzyme genes.
Three subspecies of Staphylococcus sciuri, S. sciuri subsp. sciuri Kloos, Schleifer, and Smith 1976, 23AL emend. Kloos et al. 1997 [corrected], S. sciuri subsp. carnaticus subsp. nov., and S. sciuri subsp. rodentium subsp. nov., are described on the basis of their ribotype patterns, DNA-DNA liquid hybridization data, and phenotypic characteristics. Normalized ribotyping subdivided the S. sciuri patterns into three blocks of patterns, each corresponding to a subspecies. Each subspecies formed a separate, well-defined DNA similarity group when DNA-DNA hybridizations were conducted under stringent (70 degrees C) reassociation conditions. S. sciuri subsp. sciuri could be distinguished from the other subspecies on the basis of its ability to produce acid from D-cellobiose, alkaline phosphatase activity, and inability to produce either clumping factor or protein A. S. sciuri subsp. carnaticus could be distinguished by its ability to produce acid aerobically from D-xylose and maltose, inability to produce acid from D-melezitose, and smaller colony size on P agar. S. sciuri subsp. rodentium could be distinguished by its positive reaction in the latex agglutination test for clumping factor and/or protein A and generally higher frequencies and levels of oxacillin and methicillin resistance. All 40 strains of S. sciuri tested (including representatives of all three subspecies) hybridized with the mecA gene probe. All strains of S. sciuri subsp. sciuri, 79% of the strains of S. sciuri subsp. carnaticus and 89% of the strains of S. sciuri subsp. rodentium exhibited extracellular, staphylolytic enzyme activity. This activity was associated with an enzyme(s) that immunoblotted with a lysostaphin-specific monoclonal antibody; however, only three strains hybridized with a lysostaphin (end) gene probe. The type strain of S. sciuri subsp. carnaticus is DD 791 (= ATCC 700058), and the type strain of S. sciuri subsp. rodentium is DD 4761 (= ATCC 700061).
Bacterial Typing Techniques↗
Vitronectin may mediate staphylococcal adhesion to polymer surfaces in perfusing human cerebrospinal fluid.
Prosthetic devices are frequently used for temporary or permanent drainage of cerebrospinal fluid (CSF), i.e., ventricular catheters with or without external monitoring devices and shunts. Infections constitute a serious complication in the use of biomaterials in contact with CSF; coagulase-negative staphylococci (CNS) are the most common aetiological agents. In the present study, polyvinylchloride (PVC) and PVC with endpoint-attached heparin were exposed to human CSF under perfusion to mimic conditions in vivo. Adhesion of strains of CNS isolated from patients with or without biomaterial-associated infection was determined: (i) after pre-incubation with fibronectin (Fn) or vitronectin (Vn) to block bacterial surface binding structures; and (ii) after preincubation of biomaterials with antibodies to Fn or Vn to block exposure of bacteria-binding domains on these host proteins. Pre-incubation of bacterial cells with Vn significantly reduced subsequent adhesion to polystyrene precoated with Vn 0.5 microg/well. When PVC pre-exposed to CSF was incubated with antibodies to Vn, subsequent bacterial adhesion of a Vn-binding strain, S. epidermidis 5703, was significantly reduced. The study shows that Vn may mediate adhesion of CNS in the presence of CSF. However, strains retrieved from biomaterials did not express binding of Vn or Fn to a higher extent than non-biomaterial-associated strains. Expression of heparin binding under static conditions did not correlate with staphylococcal adhesion to heparinised polymers under perfusion with CSF. The extent of adhesion of staphylococci to heparinised PVC was either reduced or the same as to unheparinised PVC.
Antibodies↗
Specific immune response to staphylococcal antigens during long-lasting biomaterial implantation.
Biomaterial-associated infections caused by staphylococci are one of the main therapeutic problems in modern medicine. There is no doubt that local disfunction of polymorphonuclear leukocytes and macrophages predisposes to such infections. However, it is not clear how implantation of a foreign body influences the antibacterial immune response. We analyzed some parameters of the specific immune response to staphylococcal antigens, in mice implanted for 3 months with heparinized polyethylene. Three weeks before the evaluation of the immune response, mice (implanted and non-implanted) were infected i.p. with 2 x 10(7) cells of Staphylococcus aureus Cowan 1. The proliferation of splenocytes was determined on the basis of [3H]thymidine incorporation in cultures stimulated with staphylococcal lipoteichoic acid, protein A, alpha-toxin, or phytohemagglutinin. Moreover, the level of specific antibodies to staphylococcal antigens was determined in serum samples (ELISA with the antigens lipoteichoic acid, protein A, and alpha-toxin). The data obtained indicate that long-lasting implantation caused evident changes in proliferative activity of lymphocytes and in humoral response to staphylococcal antigens. It enhanced spontaneous and lipoteichoic acid- or alpha-toxin-stimulated proliferation of splenocytes, in vitro. In contrast, heparinized polyethylene-implanted animals showed a significant decrease in the production of anti-protein A IgG2b and anti-alpha-toxin IgG2a and IgG2b.
Animals↗
Fibronectin binding by Propionibacterium acnes.
Strains of Propionibacterium acnes, isolated from different kinds of orthopaedic and biomaterial-associated infections and from skin flora were shown to express binding of soluble as well as immobilized fibronectin. Among these 7 strains isolated from orthopaedic infections, 2 from breast prostheses, and 9 skin isolates, 2, 2, and 5 strains respectively bound immobilized fibronectin. The fibronectin binding was sensitive to protease and heat treatment, and was inhibited by a cell surface extract from one of the binding strains. In SDS-PAGE and autoradiography of cell surface extracts, a band corresponding to a MW of about 80 kD reacted with fibronectin and the 150 kD fragment of fibronectin. Binding to fibronectin and the 150 kD fragment of fibronectin could be inhibited with heparin. We thus present a first Fn binding protein of P. acnes, a surface exposed protein of 80 kD. None of the strains bound soluble collagen, and only one strain expressed weak binding of vitronectin and bone sialoprotein II.
Adhesins, Bacterial↗
A case control study of chorioamniotic infection and histological chorioamnionitis in stillbirth.
In order to elucidate the role and aetiology of chorioamnionitis in stillbirth a case referent study was carried out in 58 pregnant women with late foetal death (cases) and in 58 pregnant women at term with live foetus (referents) matched for age and parity in Maputo Mozambique. Samples from women, stillborns and liveborns, were collected for microbiological and histological assessment. Histological chorioamnionitis was diagnosed in 96% of the cases and in 67% of the referents (OR = 13.5; 95% CI: 2.9-123.9). Escherichia coli was the species most frequently isolated in stillborns; in 14/16 (88%) cases it was isolated from intracardiac fluid. E. coli was associated with chorioamnionitis in 28% of the stillborns as compared to 5% of the referents (OR = 6.9; 95% CI: 1.4-65.4). No group B streptococci were recovered from any placenta or newborn. Vasculitis was present in 12 (21%) cases and in 3 (5%) referents (OR = 4.8; 95%, CI: 1.2-27.7). Histological chorioamnionitis was thus associated with stillbirth. E. coli was common in stillborns. The presence of vasculitis in one fifth of the stillborns indicated that the foetus was alive at the onset of infection.
Age Factors↗