Norbert Schnitzler

M‐related protein (Mrp) contributes to group A streptococcal resistance to phagocytosis by human granulocytes

The M protein has been postulated to be a major group A streptococcal (GAS) virulence factor because of its contribution to the bacterial resistance to opsono‐phagocytosis. Direct evidence of this was only provided for GAS strains which expressed a single M protein. The majority of GAS express additional, structurally similar M‐related proteins, Mrp and Enn, which have been described as IgG‐ and IgA‐binding proteins. To determine the involvement of Mrp and M protein in phagocytosis resistance, the mrp and emm genes from serotypes M2, M4, and M49 as well as from M‐untypeable strain 64/14 were insertionally inactivated. The mrp and emm mutants were subjected to direct bactericidal assays. As judged by numbers of surviving colony‐forming units, all mutant strains with the exception of the mrp4 mutant exhibited reduced multiplication factors as compared to the isogenic wild‐type strains. Subsequent analysis of phagocytosis by flow cytometry, measuring association of BCECF/AM‐labelled bacteria and granulocytes, paralleled the results from direct bactericidal assays regardless of whether isolated granulocytes or whole blood were utilized. Resistant wild‐type GAS strains bound to less than 24% of granulocytes, whereas phagocytosis‐sensitive controls attached to more than 90% of the white blood cells. 40 to 60% of the granulocytes associated with the mrp and emm mutants within 1 h of co‐incubation. Kinetic data suggested that attachment to granulocytes proceeds faster for emm mutants than for corresponding mrp mutants. By adding a dihydro‐rhodamine123 stain and measuring fluorescence induced by oxidative burst, the experimental data suggested that bacteria bound to granulocytes were also engulfed and integrated into phagolysosomes. Thus, these data indicated that, if present, both mrp and emm gene products contribute to phagocytosis resistance by decreasing bacterial binding to granulocytes.

A co-stimulatory signal through ICAM-β 2 integrin-bindingpotentiates neutrophil phagocytosis

The β2 integrin LFA–1 (lymphocyte function associated antigen; CD11a/CD18) is the common ligand for the intercellular adhesion molecules (ICAMs). Integrins support cell function by providing co-stimulatory second signals that are a precondition for full cell activation first described for ICAM–1–binding to LFA–1 in lymphocytes. Integrins can also serve to activate functions associated with distinct subunits of other integrins. In addition to LFA–1, neutrophils express the β2 integrin Mac–1 (CD11b/CD18; CR3) that apparently contains multiple sites that bind invading microbes directly or through surface–fixed C3 (ref. 6), resulting in activation of the phagocyte function. Expression of the LFA–1 counter–receptor ICAM–1 on endothelial cells occurs only at the site of inflammation. Therefore, in neutrophils, ICAM–1 ligand binding could, as with lymphocytes, also play a part as a co–stimulatory signal to induce full phagocytotic function. We show that in neutrophils, the LFA–1 ligand interaction is the stimulatory signal to express full phagocytotic activation. This is best demonstrated by the rapid association of Streptococcus pyogenes with neutrophils, followed by ingestion, strong oxidative–burst induction and enhanced killing of these bacteria, which are well–known for their resistance to human neutrophil defense. These findings may contribute to the development of therapeutic strategies targeting the modulation of ICAM–1–leukocyte interaction.

Recurrent bacteremia due to Brevibacterium casei in an immunocompromised patient

A case of an immunocompromised patient who experienced two episodes of septicemia caused by a coryneform bacterium is reported. Biochemical characteristics and analysis of cellular fatty acids and of cell wall components showed two identical strains ofBrevibacterium casei to be responsible for these infections. The lack of easy-to-perform methods for identification may have led, in the past, to an underestimation of the role of this bacterium, especially in immunocompromised patients.

M Group A Streptococci Are Phagocytized and Killed in Whole Blood by C5a-Activated Polymorphonuclear Leukocytes

ABSTRACT Historically, resistance to phagocytosis has been determined by incubating group A streptococci in human blood and comparing the numbers of CFU before and after incubation. Utilizing a flow cytometry-based technique, we have investigated the phagocytosis of M+ group A streptococci by polymorphonuclear leukocytes (PMNs) in heparinized human peripheral whole blood. Intracellular labeling of streptococci with a nontoxic fluorescent dye allowed us to quantify the association and phagocytosis of M+ streptococci by PMNs in whole blood in the presence or absence of C5a, a physiologically important chemotactic activator of PMNs. We found that wild-type strains of group A streptococci that are resistant to phagocytosis (determined by the classical Lancefield method) readily associate with C5a-activated whole-blood PMNs. In the absence of opsonizing M-type-specific antibodies, the M+ streptococci associated with PMNs are phagocytized and killed. In addition, blockade of the β2 integrin, CD11b/CD18, with anti-human CD11b monoclonal antibody inhibited association between M+ streptococci and C5a-activated PMNs. These findings establish a new relationship between M+ streptococci and PMNs, in which C5a-activated PMNs have the capacity to kill M+ streptococci in whole blood through a receptor-mediated phagocytic mechanism.

Measuring resistance to phagocytosis of group A and G streptococci: comparison of direct bactericidal assay and flow cytometry

M protein is thought to contribute to the ability of non-opsonized group A and group G streptococci (GAS and GGS, respectively) to resist phagocytosis by polymorphonuclear leukocytes. In previous studies, correlation between M protein expression and phagocytosis was determined by incubating these pathogens in human blood and comparing colony-forming bacterial counts prior to and after exposure to blood (direct bactericidal assay; DBA). Here, we report the application of flow cytometry to measure GAS and GGS resistance to phagocytosis. The results of the assays were in complete agreement with those from DBAs. Nevertheless, flow cytometry was regarded as superior to DBA because of its speed and potential uses for quantitative studies. In addition, the use of anti-CD1 1b monoclonal antibody for granulocyte staining guaranteed a non-compromized granulocyte function. The optimized protocol for flow cytometry presented here could be utilized to directly measure the involvement of individual protein types in bacterial resistance to phagocytosis

Simultaneous measurement of biopolymer-mediated Mac-1 up-regulation and adherence of neutrophils: a novel flow cytometric approach for predicting initial inflammatory interaction with foreign materials

Implantation of any medical device normally causes an inflammatory cell interaction with the foreign material. In vitro cell activation of human neutrophils (Mac-1 upregulation) has been taken as one measure to assess the attributable risk of inflammation due to biopolymers before their clinical application. Mac-1 expression has generally been measured by flow cytometric assays, whereas quantification of neutrophil adhesion to the biopolymer surfaces has been performed by separate and time-consuming assays, e.g. microscopically by differential cell counting. However, due to an increasing number of surface-modified novel biopolymers entering clinical usage, effective testing of their inflammatory potential is now mandatory. To facilitate these analyses, we have developed a novel flow cytometric assay permitting simultaneous measurement of biopolymer-mediated neutrophil activation and adhesion. The biopolymers were used as beads (diameter 25+/-10 microm), and were demonstrated to be non-phagocytosable and non-fluorescent before being co-incubated with whole human blood (range of ratio granulocytes/beads from 5:1 to 1:1). Besides flow cytometric measurement of Mac-1 up-regulated neutrophils as fluorescing events, a fluorescence of the bead population indicates the adherence of activated neutrophils to the biopolymer surface.After establishing this assay, we evaluated it by comparing six different biopolymers. We observed high levels of Mac-1 expression (>70% of positive control) accompanied by increased adhesiveness (>60% of neutrophils) for polyurethane (PUR), polymethylmetacrylate (PMMA), and poly-DL-lactide (PDLLA) beads. Low Mac-1 expression levels (<10%) accompanied by a low percentage of adhering neutrophils (<10%) were observed for polyethylene (PE), polyisoprene (PI), and silicone (SI) beads.

The rationale and method for constructing internal control DNA used in pertussis polymerase chain reaction

The inclusion of an appropriate internal control DNA in polymerase chain reaction (PCR) is a rapid and simple method for the detection of PCR failure. Two PCR coamplification internal control DNAs (ICD I and ICD II) with the same primer-binding sequences as the target DNA for the detection of Bordetella pertussis and Bordetella parapertussis were produced using an overlap extension technique and a PCR MIMIC construction kit, respectively. The ICD II was further evaluated in a prospective clinical study in 360 patients with a clinical diagnosis of pertussis. From 360 nasopharyngeal swabs the internal control was positive in 318 (88%) samples, but was negative in 42 (12%). After phenol-chloroform extraction an additional 10 internal controls became positive. For the detection of PCR failure, the use of internal control DNA is highly recommended for PCR-based identification of B. pertussis and B. parapertussis organisms from nasopharyngeal swabs and aspirates.

ACTIVATION OF GRANULOCYTES BY PHORBOL-12-MYRISTATE-14-ACETATE (PMA) ENHANCES PHAGOCYTOSIS OF Streptococcus pyogenes

The expression of the M protein is thought to be responsible for the ability of Streptococcus pyogenes to resist phagocytosis by human polymorphonuclear leukocytes (PMNL)6. Recently M-like proteins (Mrp) were shown to contribute to this major virulence mechanism10.

Negative Impact of Linezolid on Human Neutrophil Functions in vitro

Background/Aims: In a recent phase III clinical trial on linezolid, more patients in the linezolid treatment arm acquired Gram-negative catheter-related bloodstream infections despite the adequate therapy of infections caused by Gram-negative bacteria. We tested our hypothesis that linezolid impairs phagocytosis and the killing of Gram-negative bacteria by polymorphonuclear leukocytes (PMN). Methods: The influence of clinically relevant concentrations (5, 20 and 50 mg/l) of linezolid on granulocyte function in vitro was tested. Phagocytosis was determined by flow cytometry, and killing of bacteria was evaluated by plate counting. Chemotaxis was examined by an under-agarose cell migration assay. Gram-positive and Gram-negative bacteria were used. Results: Linezolid significantly impaired phagocytosis of a specific Escherichia coli strain in a concentration-dependent manner, whereas the effect on Pseudomonas aeruginosa was less prominent. No such effects were observed with a different E. coli strain or Staphylococcus aureus. Neither killing nor the chemotactic behaviour of PMN was significantly affected by linezolid. Conclusions: The observed concentration-dependent impairment of the phagocytic function might contribute to the higher frequency of catheter-related Gram-negative bloodstream infections in patients treated with linezolid. Individual patient risk may also depend on the causative Gram-negative strain.

CD66b Overexpression and Loss of C5a Receptors as Surface Markers for Staphylococcus aureus-Induced Neutrophil Dysfunction

Neutrophil granulocytes constitute the main component of innate immunity in the clearance of bacterial infections. However, during systemic inflammation, immunoparalysis may occur resulting in neutrophil dysfunction. This study presents a new in vitro model for analyzing the dysfunction of human peripheral blood neutrophils resulting from the interaction with Staphylococcus aureus components in whole blood. After induction of a massive complement activation by S. aureus supernatant, the neutrophils exhibit a reduced phagocytic capacity resulting in a dramatic reduction of the antibacterial activity similar to that of neutrophils isolated from septic patients. The number of phagocytozing neutrophils is drastically reduced as well as the phagocytic capacity designated by a significantly lower number of ingested microbes. This dysfunction correlates with the loss of complement component 5a receptor 1 from the neutrophil cell surface and can be further characterized by a C5a-induced CD66b overexpression. The presented in vitro model offers a new platform for preclinical testing of immunosuppressive drugs and delivers new information for the understanding of neutrophil dysfunctions under the conditions described.