Staphylococcal protein Ecb impairs complement receptor-1 mediated recognition of opsonized bacteria

Staphyloccus aureus is a major human pathogen leading frequently to sepsis and soft tissue infections with abscesses. Multiple virulence factors including several immune modulating molecules contribute to its survival in the host. When S. aureus invades the human body, one of the first line defenses is the complement system, which opsonizes the bacteria with C3b and attract neutrophils by release of chemotactic peptides. Neutrophils express Complement receptor-1 [CR1, CD35) that interacts with the C3b-opsonized particles and thereby plays an important role in pathogen recognition by phagocytic cells. In this study we observed that a fraction of S. aureus culture supernatant prevented binding of C3b to neutrophils. This fraction consisted of S. aureus leukocidins and Efb. The C-terminus of Efb is known to bind C3b and shares significant sequence homology to the extracellular complement binding protein [Ecb). Here we show that S. aureus Ecb displays various mechanisms to block bacterial recognition by neutrophils. The presence of Ecb blocked direct interaction between soluble CR1 and C3b and reduced the cofactor activity of CR1 in proteolytic inactivation of C3b. Furthermore, Ecb could dose-dependently prevent recognition of C3b by cell-bound CR1 that lead to impaired phagocytosis of NHS-opsonized S. aureus. Phagocytosis was furthermore reduced in the presence of soluble CR1 [sCR1). These data indicate that the staphylococcal protein Ecb prevents recognition of C3b opsonized bacteria by neutrophil CR1 leading to impaired killing by phagocytosis and thereby contribute to immune evasion of S. aureus.Peer reviewe

Certificatie van Salmonella in melkpoeder, een voorstudie

Abstract niet beschikbaarA feasibility study was undertaken for the certification of a reference material containing low numbers of Salmonella. A batch of 8800 capcules was prepared and eleven laboratories each enumerated 50 capsules from this batch, according to a standardized procedure. The results from four laboratories were excluded from the analysis of data after the first step of the statistical analysis based on their results found for homogeneity and mean contamination level. The remaining seven laboratories found contamination levels that did not differ significantly from each other. The mean contamination level found by these seven laboratories was 5.65 cfu/capsule. The homogeneity, based on all counts by these seven laboratories, was not fully conform to a Poisson distribution. A ratio of variance to mean of 1.18 was found, which is considered acceptable. The proportion of negative capsules found in the study was 0.58% and the upper limit of the corresponding confidence interval (one sided, (alpha = 0.05) was 1.8%. Concluded was that the batch prepared is of sufficient to be used for a certification study.EG/BCR HIGB VH

Intravital two-photon microscopy of host-pathogen interactions in a mouse model of Staphylococcus aureus skin abscess formation.

Staphylococcus (S.) aureus is a frequent cause of severe skin infections. The ability to control the infection is largely dependent on the rapid recruitment of neutrophils (PMN). To gain more insight into the dynamics of PMN migration and host-pathogen interactions in vivo, we used intravital two-photon (2-P) microscopy to visualize S. aureus skin infections in the mouse. Reporter S. aureus strains expressing fluorescent proteins were developed, which allowed for detection of the bacteria in vivo. By employing LysM-EGFP mice to visualize PMN, we observed the rapid appearance of PMN in the extravascular space of the dermis and their directed movement towards the focus of infection, which led to the delineation of an abscess within 1 day. Moreover, tracking of transferred labelled bone-marrow neutrophils showed that PMN localization to the site of infection is dependent on the presence of G-protein-coupled receptors on the PMN, whereas Interleukin-1 receptor was required on host cells other than PMN. Furthermore, the S. aureus complement inhibitor Ecb could block PMN accumulation at thesite of infection. Our results establish that 2-P microscopy is a powerful tool to investigate the orchestration of the immune cells, S. aureus location and gene expression in vivo on a single cell level

Detectie van Salmonella in de aanwezigheid van begeleidingsflora met behulp van referentiemonsters. BCR-FOOD Trial 2.

Abstract niet beschikbaarThe detection of Salmonella in the presence of competitive microorganisms was tested with the use of reference samples. Thirtyeight laboratories investigated 25 samples of which 5 were negative for salmonella. The Rappaport-Vassiliadis broth was used as reference (RV- method) and compared with a selective enrichment broth chosen by the laboratory itself (OWN-method). The Salmonella samples had a contamination level of 5.48 salmonellae/capsule, the competitive microorganisms of about 2x10 cfu/capsule. The strains used for competitors were: Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis. Significant differences were found between laboratories and between the RV and OWN method. With the RV method 42% of the labs found 19 or 20 positive isolations ; the percentage positive isolations (averaged over all labs) was 81.5%. With the OWN method only 19% of the labs found 19 or 20 positives, the percentage positive isolations was 62.3%. Eight laboratories found one or more of the Salmonella negative samples positive for Salmonella. The competitive microorganisms reference samples is mutable to test the isolation procedure for Salmonella under more realistic circumstances.EG/BCR VHI HIG

Telling van Salmonella in referentie monsters, een ringonderzoek met twaalf laboratoria

De telling van Salmonella in referentiemonsters volgens een methode ontwikkeld door het RIVM werd getest door 12 europese laboratoria. De methode bestaat uit drie stappen: - oplossen van de capsule in physiologische zoutoplossing - herstel van sublethaal beschadigde cellen in Plate Count Agar - selectieve groei in Violet Rood Gal Glucose Agar. Elk laboratorium ontving 50 Salmonella referentiemonsters tesamen met een gedetailleerde beschrijving van de methode. Analyse van de data met behulp van Gegeneraliseerde Lineaire Regressie gaf aan dat na het weglaten van de resultaten van een laboratorium er geen verschil in het besmettingsniveau tussen de laboratoria kon worden aangetoond. Drie laboratoria vonden een significant grotere variatie in het aantal kiemen tussen de capsules dan verwacht kon worden volgens een Poisson verdeling. Alle kolonies die werden bevestigd bleken Salmonella te zijn. Geconcludeerd werd dat deze ontwikkelde quantitatieve methode geschikt is voor de certificering van deze referentiemonster.Abstract not availableEG/BCR VHI HI

Detectie van Listeria monocytogenes in de aanwezigheid van competatieve micro-organismen met gebruik van referentiematerialen, BCR-Food Trial 4

Abstract niet beschikbaarA trial with 42 laboratories participating, was organized in May 1990 to test the L.monocytogenes reference materials in combination with reference materials containing competitive microorganisms. The materials were tested by 2 methods, one standardized for all laboratories (SM method) and the other method chosen by the laboratories themselves (OWN method). Thirty samples were tested by each method of which nine were negative for Listeria. The contamination level of the L.monocytogenes capsules was 4.9 cfu/capsule. Each laboratory was expected to find the organism in 18 or more out of the 21 L.monocytogens contaminated samples. The total contamination of microorganisms was 2.9 x 10-4 cfu/capsule. Using the standard method Listeria monocytogenes was detected in 79.3% of the contaminated samples, and 45% of the participating laboratories made the expected number of isolations. With the OWN method these percentages were 78.1% and 52.5% respectively. Concluded was that the materials tested are suitable to test the performance of the detection method for Listeria in a laboratory.EG/BCR VHI HIG

Staphylococcal complement inhibitor modulates phagocyte responses by dimerization of convertases.

The human pathogen Staphylococcus aureus produces several complement-evasion molecules that enable the bacterium to withstand the host immune response. The human-specific staphylococcal complement inhibitor (SCIN) blocks the central C3 convertase enzymes that trigger critical complement functions, such as C3b deposition, phagocytosis, and C5a generation. SCIN effectively blocks the conversion of C3 by alternative pathway C3 convertases (C3bBb), but also induces dimerization of these enzymes. In this study, we show that formation of dimeric convertases by SCIN is important for S. aureus immune evasion because it modulates complement recognition by phagocytic receptors. Dimeric, but not monomeric, SCIN convertases showed an impaired binding to complement receptor 1 and the complement receptor of the Ig superfamily. The dimerization site of SCIN is essential for its strong antiphagocytic properties. These studies provide critical insights into the unique immune-evasion strategies used by S. aureus

Stabiliteit en homogeniteit van microbiologisch referentiemateriaal: enkele statistische modellen

Microbiological reference materials are being developed by the RIVM since several years. These materials consist of capsules filled with milkpowder artificially contaminated with a bacteria test strain of choice (i.e. Escherichia coli, Enterobacter cloacae, Salmonella typhimurium). Both from longterm stability tests, and from short-term challenge tests at different storage temperatures, it is clear that these materials cannot survive forever. Standard statistical techniques for quality control, assuming both a stable process and the Normal distribution cannot be used. In this report we will deal with these non-standard problems.EG/BCRHIMHVH