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Biomedical subjects

A Ljungh

Publications and source records attributed to A Ljungh.

At least 19 recordsLinked to original sources
PubMed1998

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↗
PubMed1998

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↗
PubMed1998

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↗
PubMed1998

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↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

Vitronectin-binding staphylococci enhance surface-associated complement activation.

Coagulase-negative staphylococci are well recognized in medical device-associated infections. Complement activation is known to occur at the biomaterial surface, resulting in unspecific inflammation around the biomaterial. The human serum protein vitronectin (Vn), a potent inhibitor of complement activation by formation of an inactive terminal complement complex, adsorbs to biomaterial surfaces in contact with blood. In this report, we discuss the possibility that surface-immobilized Vn inhibits complement activation and the effect of Vn-binding staphylococci on complement activation on surfaces precoated with Vn. The extent of complement activation was measured with a rabbit anti-human C3c antibody and a mouse anti-human C9 antibody, raised against the neoepitope of C9. Our data show that Vn immobilized on a biomaterial surface retains its ability to inhibit complement activation. The additive complement activation-inhibitory effect of Vn on a heparinized surface is very small. In the presence of Vn-binding strain, Staphylococcus hemolyticus SM131, complement activation on a surface precoated with Vn occurred as it did in the absence of Vn precoating. For S. epidermidis 3380, which does not express binding of Vn, complement activation on a Vn-precoated surface was significantly decreased. The results could be repeated on heparinized surfaces. These data suggest that Vn adsorbed to a biomaterial surface may serve to protect against surface-associated complement activation. Furthermore, Vn-binding staphylococcal cells may enhance surface-associated complement activation by blocking the inhibitory effect of preadsorbed Vn.

Animals↗
PubMed1997

Immunoblot assay for serodiagnosis of Helicobacter pylori infections.

An immunoblot assay for the serological diagnosis of Helicobacter pylori infection was evaluated. Serum samples from patients whose gastric biopsy specimens were known to be positive or negative for H. pylori on culture were used to establish interpretive criteria for the immunoblot assay. A panel of sera from patients with diseases other than H. pylori infection and sera from healthy blood donors were included to validate these criteria. All sera were initially assessed in an enzyme immunoassay (Ge-EIA), based on acid glycine-extracted cell surface proteins of H. pylori NCTC 11637. The same antigen extract was used in the immunoblot assay. In addition, the Ge-EIA and the immunoblot assay were compared with a commercially available EIA (Seradyn, Color Vue Pylori). Bands of 110/120 kDa and/or two of five low-molecular-mass proteins (26, 29, 30, 31, and 33 kDa, in any combination) showed a strong correlation with the H. pylori culture-positive patients (97.5%) compared to the correlation obtained with the EIA results (Ge-EIA, 87.5%; Seradyn EIA, 92.5%), and the antibody responses to these proteins were considered specific reactions. In 37 of 40 serum samples from culture-negative patients and also in sera from patients with other disorders, a moderate antibody reactivity to the medium-size proteins (43 to 66 kDa) was observed, and these were considered not valuable for a specific immunoblot assay. Among sera from culture-positive patients, 39 of 40 serum samples were defined to be immunoblot positive, and from among sera from culture-negative patients, 3 of 40 serum samples were defined to be immunoblot positive. The use of sera from patients with negative cultures for H. pylori as negative controls may decrease the sensitivity due to sampling error and false-negative culture results. Immunoblot assay-positive results were detected among 10% of sera from patients with other diseases, whereas they were detected among 42.5% of sera by the Ge-EIA and 47.5% of sera by the Seradyn-EIA. The higher number of EIA-positive sera in this group reflects a possible cross-reactivity (false-positive EIA result). Of the blood donors, representing asymptomatic but possibly colonized subjects, 24% were immunoblot positive. In conclusion, our data indicate that immunoblotting is more sensitive as well as more specific than EIA. Moreover, it permits detection of antibody responses to specific antigens, e.g., the cytotoxin-associated CagA protein, which may have pathological implications.

Adult↗
PubMed1997

Altered immune response to staphylococcal antigens in long-lasting implanted mice.

Staphylococcal infections constitute one of the main problems associated with clinical applications of various prosthetic medical devices (biomaterials). As the magnitude of the infection risk depends often on the duration of device installation, and the incidence of infections is higher in skin-penetrating devices, we studied some parameters of specific immune response to staphylococcal antigens in mice subcutaneously (s.c.) implanted for three months with heparinized polyethylene (H-PE). Three weeks before the evaluation of immune response, mice (implanted and non-implanted) were s.c. infected with 10(7) of Staphylococcus aureus Cowan 1. The proliferation of lymph node cells was determined on the basis of 3H-thymidine incorporation in 3-days cultures stimulated with: staphylococcal lipoteichoic acid (LTA), protein A (SpA), alpha-toxin, or with phytohemagglutinin (PHA). Moreover, the levels of specific antibodies to staphylococcal antigens were determined in serum samples (ELISA against: LTA, SpA, alpha-toxin). The data obtained indicate that long-lasting implantation caused evident changes in proliferative activity of lymphocytes and humoral response to staphylococcal antigens. It enhances alpha-toxin and LTA stimulated proliferation of lymph node lymphocytes in vitro. In contrast, H-PE-implanted animals demonstrated a significant decrease in the production of anti-SpA IgG2a and IgG2b and increase in the synthesis of anti-LTA IgG1 antibodies.

Animals↗
PubMed1996

Protein adsorption and bacterial adhesion to biliary stent materials.

Four biliary stents inserted for relief of jaundice in patients with biliary obstruction due to carcinoma of pancreatic head were examined for the adsorption of biliary proteins and bacterial colonization. Fibronectin and vitronectin (S-protein) were found to be the two main proteins adsorbed on the inner surface of the stents. Biliary isolates included Escherichia coli, Enterococcus faecalis, and Candida albicans. In vitro studies were performed to clarify the kinetics of biliary protein and bacterial adhesion. Biliary drains of polytetrafluorethylene, polyethylene, polyurethane, and rubber were placed in a flow cell and perfused with human bile at 37% C for 24 hr. The materials were subjected to either detection of adsorbed biliary proteins or perfusion with 3 H-labeled E. coli cells (1 X 10 6 cfu/ml). The results show that the adsorbed biliary proteins were detectable on the surface of biliary stents and able to enhance bacterial adhesion to the surface in the first 24 hrs after the exposure of stent materials to bile, and that both the adsorption of biliary proteins and the adhesion of bacterial cells were material- and strain-dependent. Furthermore, there was a clear correlation between the amount of adsorbed fibronectin and the number of adherent bacteria. The results indicate that, in the clinical situation, biliary proteins may be adsorbed on the surface of inserted stents within a short time after insertion, and some of them may be used by bacterial cells as receptors for adhesion to the surface, and thus involved in the process of bacterial adhesion.

Adsorption↗