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Staphylococcus schleiferi subsp. schleiferi is a coagulase-negative staphylococcus, usually present as a contaminant in human specimens. A near relative, S. schleiferi subsp. coagulans, possesses coagulase activity but has not been reported from humans. We here describe three isolates of pseudocoagulase-positive S. schleiferi subsp. schleiferi and one isolate of S. schleiferi subsp. coagulans from human patients. The pseudocoagulase from the S. schleiferi subsp. schleiferi isolates differs from S. aureus staphylocoagulase by being sensitive to a combination of protease inhibitors (aprotinin, N-ethylmaleimide, and heparin). These isolates could all easily be confused with S. aureus in a typical clinical laboratory, since they all possess a heat-stable DNase and promote clotting formation. Moreover, S. schleiferi subsp. coagulans produces protein A, and S. schleiferi subsp. schleiferi expresses a clumping factor (fibrinogen affinity factor). Southern blot hybridization with an S. aureus coa-specific probe revealed no sequence related to the coa gene in any of the S. schleiferi isolates, and their riboprobe profiles and biochemical characteristics were typical of S. schleiferi subspecies, not of S. aureus. This study demonstrates that both subspecies of S. schleiferi can promote clotting of rabbit plasma in the standard tube test for coagulase.

Several components of Klebsiella pneumoniae including a membrane proteoglycan (Kp-MPG) were reported to activate macrophages and to induce T-independent polyclonal activation of mouse B cells. Chemically defined derivatives of Kp-MPG were prepared and characterized, enabling us to approach the molecular substructures involved in the binding to lymphocytes and the activation of B cells. Five derivatives were characterized: (i) an acylpoly(1,3)galactoside containing ester-linked fatty acids (EFA-APG) which was obtained by mild alkaline hydrolysis, (ii) a polymer of EFA-APG (APG pol1), (iii) a preparation obtained by drastic alkaline hydrolysis and delipidation which removed the esterified fatty acids (APG), (iv) a polymer of the latter compound (APG pol2), and (v) an APG preparation submitted to mild acid hydrolysis which removed all fatty acids but left the galactose chain of APG (GC-APG) intact. The derivatives were studied for their capacity to bind to and to activate mouse splenocytes. Binding was investigated on BALB/c and C3H/HeJ splenocytes by indirect immunofluorescence using biotinylated F(ab')2 of anti-Kp-MPG antibodies and the streptavidin-phycoerythrin amplification system in flow cytometry and by competition of unlabeled APG with biotinylated APG. Activation was studied by measuring (i) [3H]thymidine incorporation into spleen cells from BALB/c, C3H/HeJ, nude (nu+/nu+) mouse strains, and purified B cells of BALB/c; (ii) immunoglobulin secretion in culture supernatants; and (iii) blastogenesis. The results demonstrate a specific uptake of EFA-APG and APG by T cells as well as by B cells and exclude a contribution of the polygalactose part of the APG molecule (GC-APG) to the binding to spleen lymphocytes. Unlike LPS from the same strain of K. pneumoniae, APG pol1 stimulated B cell activation in the LPS-resistant C3H/HeJ strain as well as in BALB/c mice. The compounds did not activate T cells and were T-independent B cell activators, stimulating nu+/nu+ spleen cells and inducing primarily IgM and IgG3 synthesis. Polymers were more potent activators than monomers and removal of ester-linked fatty acids completely abrogated B cell-activating properties. The monomer APG antagonized B cell activation by Kp-MPG, LPS from K. pneumoniae, and APG pol1. The data indicate that within the EFA-APG molecule, distinct substructures are required for binding and for triggering B cell response.

The stimulating activity of several preparations isolated from a membrane proteoglycan of a nonencapsulated smooth strain of Klebsiella pneumoniae (Kp-MPG) on the oxidative burst of human blood monocytes was assessed by luminol-enhanced chemiluminescence (CL). Five Kp derivatives were studied: a 34-kd acylpoly(1,3)galactoside (APG), obtained by drastic alkaline hydrolysis and purified by chromatography; an APG preparation subjected to acid hydrolysis that removed the core part and all fatty acids, leaving intact the galactose chain of APG (GC-APG); an APG preparation subjected to mild oxidation (ox APG); a preparation obtained by mild alkaline hydrolysis of Kp-MPG, containing additional ester-linked C14 and C16 fatty acids bound to the APG molecule (EFA-APG); and a polymer of the latter compound, APG pol. EFA-APG directly stimulated monocyte CL, whereas Kp-MPG, APG pol, and the whole bacterial cells had little or no activity. APG itself and ox APG induced a weaker response than EFA-APG. Polymyxin B sulfate completely inhibited the CL response to bacterial lipopolysaccharide (LPS) but not to EFA-APG. The stimulating action of EFA-APG on blood monocytes was dependent on the extracellular levels of both calcium and magnesium. Preincubation of monocytes with monoclonal antibody anti-Mac-1 directed against CD11b, the alpha chain of complement receptor type 3 (CR3; CD11b/CD18), strongly inhibited CL activation by EFA-APG and to a lesser extent CL activation by unopsonized zymosan and rough LPS. Altogether, these findings provide indirect evidence for the contribution of the CD11b/CD18 integrin in the functional interaction of EFA-APG with monocyte membranes. They demonstrate the role of fatty acids in the triggering of monocyte oxidative burst, while the polysaccharide chain itself does not contribute to induction of the CL response in this model. In keeping with the effects of EFA-APG and APG, we show that the monocyte CL response was triggered by bacterial LPS from the rough strain of Salmonella minnesota Re 595 and its lipid A, but not by LPS from smooth strains, again suggesting a critical role for the lipid moiety.

The capacity of a K. pneumoniae membrane proteoglycan (Kp-MPG) and four of its chemically defined derivatives to activate human monocytes was studied by measuring immunoreactive IL-1 beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha) in culture supernatants. Monocyte culture supernatants were also tested for their comitogenic activity on concanavalin A-stimulated thymocytes and for their cytotoxic activity on the mouse fibroblastic L929 cell line. The four Kp-MPG derivatives were: (i) an acylpoly(1-3)galactoside (APG); (ii) an APG preparation submitted to acid hydrolysis which removed all fatty acids but left intact the galactose chain of APG (GC-APG); (iii) a preparation obtained by mild alkaline hydrolysis, containing additional ester-linked C14 and C16 fatty acids bound to the APG molecule (EFA-APG); and (iv) a polymer of the latter compound (APG pol). Kp-MPG induced the synthesis of IL-1 beta, IL-6 and TNF-alpha with dose-responses and kinetics similar to those of Salmonella minnesota lipopolysaccharide (Sm-Re-LPS). APG pol and EFA-APG induced the secretion of the three cytokines with lower potency than Kp-MPG or Sm-Re-LPS. APG did not trigger any detectable cytokine production and GC-APG induced only borderline and inconsistent responses. Our data demonstrate the critical role of ester-linked C14 and C16 fatty acids in the triggering of monocyte response to Kp-MPG derivatives.