[Massive haemolysis by Clostridium perfringens: quick diagnosis in a patient with severe sepsis]. / Hémolyse massive par Clostridium perfringens: diagnostic rapide chez une patiente présentant un sepsis sévère.

Septicaemia by clostridia is a rare but nearly always fatal disease. We report a case of mortal massive haemolysis, with virtually no circulating erythrocytes, in a patient with septicaemia by Clostridium perfringens. The early finding of Clostridium perfringens in the direct Gram stain of patient's peripheral blood allowed a quick diagnosis.

In vitro evaluation of the antibacterial and osteogenic activity promoted by selenium-doped calcium phosphate coatings.

Selenium is an essential trace element present in 25 selenoenzymes, playing critical roles in a variety of physiological processes, such as anti-oxidative defense and the modulation of cell proliferation and differentiation. This paper characterizes selenium-doped calcium phosphate coatings and evaluates their effects on the osteogenic activity, the proliferation of osteosarcoma cells and biofilm formation. To do so, the structure and elemental composition of the obtained coatings were analyzed, in addition to their thicknesses, and they were compared to pure calcium phosphate coatings. Moreover, the dose-effect ratio of two coatings with the lower (0.6 at%) and the higher (2.7 at%) selenium content was studied in terms of osteogenic, anti-biofilm and cancerous anti-proliferative properties. The results showed the incorporation of selenium in the form of selenite groups into the hydroxyapatite structure, with a similar crystalline pattern to the latter and increased roughness of the coatings. The calcium phosphate coatings with 2.7 at% of selenium resulted in significant osteogenic activity (p < 0.01) of healthy pre-osteoblasts (MC3T3-E1) over long periods of incubation, a significant anti-proliferative effect (p < 0.01) on cancerous osteoblasts (MG63) in a preliminary study, and anti-biofilm properties (p < 0.01) against Staphylococcus epidermidis and Staphylococcus aureus bacterial strains, which are responsible for most infections after orthopedic surgeries.

Cooperation between the components of the meningococcal transferrin receptor, TbpA and TbpB, in the uptake of transferrin iron by the 37-kDa ferric-binding protein (FbpA).

Meningococcal TbpAB complexes TbpA, TbpB and FbpA were purified and used to study their role in the uptake of iron from transferrin to FbpA. Purification was achieved by affinity chromatography techniques, yielding homogeneous, non-denatured and functional material. TbpA could not be separated from TbpB and had to be purified from a TbpB-defective mutant strain. FbpA was able to bind iron from transferrin only when TbpAB complexes, TbpA and/or TbpB, were also present during the interaction. The highest uptake efficiences were obtained with TbpAB complexes or TbpA/TbpB mixtures. We conclude that the TbpA and TbpB molecules form true functional transferrin receptors, that FbpA is able to take iron directly from transferrin when in the presence of the components of the receptor, and that both Tbps are necessary for an optimal operation of the uptake system.

Characterization of the transferrin-iron uptake system in Neisseria meningitidis.

The transferrin-iron uptake system of six Neisseria meningitidis strains was characterized using 125I-transferrin in receptor assays and 55Fe-loaded transferrin in uptake assays. Receptors for transferrin varied among the strains both in number (from 700 to 4700 receptors per cell) and in their affinity constants for the protein (Ka ranged from 0.7 x 10(7) to 4.0 x 10(7) l mol-1). Neither receptor numbers nor affinity constants were significantly different in carrier and invasive strains, although the Ka seem to be somewhat higher in the latter. Iron uptake from transferrin was also variable among the strains, but showed the same lack of correlation with their origin.

Analysis of the molecular mass heterogeneity of the transferrin receptor in Neisseria meningitidis and commensal Neisseria.

Peroxidase-conjugated transferrin was used to detect transferrin receptors both in intact outer membrane vesicles (OMVs) from Neisseria species in a dot blot assay, and in SDS-PAGE-separated OMV proteins after transferring to nitrocellulose membranes. All N. meningitidis strains produced transferrin receptors after culturing in either iron sufficiency or iron restriction although expression was higher in the latter case, whereas only six N. lactamica and two N. sicca (among 20 commensal species) were able to bind transferrin. Molecular mass (MM) of the receptors were mainly between 78 kDa and 85 kDa (87.5% of strains), 12.5% had receptors with MM close to 70 kDa, and 5% showed receptors with MM over 85 kDa. Our results confirm the molecular mass heterogeneity of the transferrin receptors in N. meningitidis, completely disagree with the 'universal' 98 kDa receptor proposed by some authors, and show a low expression of the receptor in commensal Neisseria.

Purification of the Neisseria meningitidis transferrin binding protein-2 (TBP2) to homogeneity using column chromatography.

A method for purifying TBP2 from N. meningitidis has been developed using affinity chromatography on Tf-agarose followed by ion exchange chromatography; the Tf-binding activity of fractions was evaluated by a dot-blot technique. This method allowed the purification of the TBP2 in milder conditions than those used previously and to a high degree of homogeneity as was demonstrated by Coomassie brilliant blue or Silver staining after SDS-PAGE electrophoresis. The SDS-PAGE profile of the material obtained in the Tf-agarose affinity chromatography step shows only two detectable proteins, identified as the TBP1 and the TBP2, with a small amount of contamination. Passage through a MonoQ HR anion exchange column, allowed the isolation of TBP2 in the absence of TBP1. Our results demonstrate the conservation of the antigenic reactivity of this protein, which produces monospecific serum with the antibodies elicited reacting exclusively with the TBP2 in whole outer membrane vesicles.

Analysis of the expression of the putatively virulence-associated neisserial protein RmpM (class 4) in commensal Neisseria and Moraxella catarrhalis strains.

The RmpM protein has been reported to be present only in pathogenic Neisseria species. In the present study we demonstrate that this protein is also present at least in N. lactamica and N. sicca strains. The N. lactamica protein reacts with a RmpM-specific monoclonal antibody (185,H-8), having a molecular mass ( approximately 31 kDa) slightly lower than that of the meningococcal RmpM, and mouse antibodies from sera against outer membrane vesicles from both N. lactamica and N. sicca strains cross-react with the meningococcal RmpM. PCR and hybridization experiments with a complete rmpM probe agree with the immunodetection experiments. Our results strongly suggest that the meningococcal RmpM should not be considered a virulence marker, and the presence of this protein in the commensal species agrees with its role as a structural protein, proposed for the RmpM, which should be considerably conserved in the Neisseria species.

Iron and outer membrane proteins in the susceptibility of Neisseria meningitidis to human serum.

The proportion of carrier-isolated Neisseria meningitidis strains sensitive to human serum (37.2%) was found to be significantly higher than that of case-isolated ones (4.1%), although the difference is too low to consider serum-resistance responsible for invasion in this microorganism. Serum-susceptibility was not related to the existence of specific outer membrane proteins, as is the case of N. gonorrhoeae. Iron restriction induced iron-regulated outer membrane proteins in each strain (but not the same proteins in all strains) but without any detectable effect on serum-susceptibility. Iron excess was also unable to induce changes in the susceptibility of N. meningitidis to human serum.

Surface free energy and interaction of Staphylococcus epidermidis with biomaterials.

The adhesion of twenty nine Staphylococcus epidermidis strains to teflon, polyethylene, polycarbonate and bovine pericardium was studied in vitro and examined in relation to the surface free energies of both bacteria and biomaterials. All S. epidermidis strains had similar surface free energies, close to that of water, and adhered better to the materials with analogous surface free energies. There was a significant correlation (Kendall's Tau B = 1000) of biomaterial's surface free energy with the number of adhering bacteria. This correlation is inverse (Kendall's Tau B = -1000) when surface hydrophobicity is considered instead of surface free energy. This indicates that in Staphylococcus epidermidis adherence to biomaterials is inversely correlated to the surface hydrophobicity of the last, being so just the opposite of that occurring with other bacteria.

Analysis of the expression of outer-membrane proteins in Neisseria meningitidis in iron-replete and iron-deficient media.

A total of 103 Neisseria strains, including 42 carrier and 40 invasive N. meningitidis and 21 commensal species were studied. Outer-membrane proteins from carrier and invasive N. meningitidis showed similar molecular mass distributions (except in the range from 78 to 82 kDa in which 90% of the invasive but only 47.6% of the carrier strains expressed proteins), many strains showing neither class II (41 kDa) nor class III (38 kDa) proteins. When grown in iron-restricted conditions proteins were induced mainly in the ranges from 62 to 92 kDa with no significant differences between groups. Commensal species, both in normal and in iron-restricted conditions, showed outer-membrane protein patterns different from those of N. meningitidis in several molecular mass ranges. Cluster analysis based on expression of principal outer-membrane proteins allowed the differentiation of commensal species and N. meningitidis, although not that of the invasive and carrier groups within the latter.

Analysis of TbpA and TbpB functionality in defective mutants of Neisseria meningitidis.

Iron uptake analysis suggested that the Neisseria meningitidis transferrin (Tf) binding proteins, TbpA and TbpB, form only one type of receptor complex. Mutants defective in the synthesis of either TbpA or TbpB, but not defective in both proteins, can bind Tf, suggesting that both proteins are surface exposed and function in Tf binding. Also, iron uptake from Tf into the meningococci did not require the presence of both Tbps. The TbpB-defective mutant incorporated c. 37% of the iron taken up by the wild-type strain, but this was insufficient for bacterial growth. The TbpA-defective mutant incorporated c. 50% of the iron taken up by the wild-type strain and was able to grow with Tf as the only iron source. Mouse antibodies specific for TbpA were able to block c. 70% of the iron uptake from Tf in the wild-type strain, whereas they blocked only 22% of iron uptake in the TbpB-defective mutant and did not block uptake in the TbpA-defective strain. These results emphasise that TbpA should be considered in future vaccine trials in which iron-restricted proteins are to be included in the vaccine formulation.

Blocking of iron uptake by monoclonal antibodies specific for the Neisseria meningitidis transferrin-binding protein 2.

The existence of epitopes common to different strains in the Neisseria meningitidis transferrin (Tf)-binding protein 2 (TBP2), combined with the ability of polyclonal anti-TBP2 antibodies to inhibit Tf binding and block iron uptake in this species, led to this study on the effect of anti-TBP1+2 monoclonal antibodies (MAbs) to determine the presence of epitopes inside the Tf-binding region. All MAbs used reacted exclusively with the homologous strain when tested by dot-blots of outer membrane vesicles, with the reaction being specific for TBP2 after SDS-PAGE and electroblotting. In contrast, ELISA and iron-uptake blocking assays were also positive with heterologous strains belonging to Rokbi's group II (high mol.wt TBP2). The results confirmed the two group classification proposed by Rokbi and, in contrast to other studies, indicated the existence of epitopes in the Tf-binding region that are common only to strains of Rokbi's group II. These epitopes may become denatured after drying for dot-blot assays or after SDS-PAGE and electroblotting.

In vivo human immune response to transferrin-binding protein 2 and other iron-regulated proteins of Neisseria meningitidis.

When grown under iron restriction, Neisseria meningitidis expresses new outer-membrane proteins, some of which are antigenic and potentially useful as vaccine components. This is particularly relevant to N. meningitidis serogroup B, against which neither polysaccharide nor conjugate vaccines are effective. We investigated recognition of N. meningitidis serogroup B outer-membrane antigens by three sera from patients recovered from meningitis. Recognition of antigens from the homologous strain provided information on in vivo expression during infection and immunogenicity, while cross-reactivity with outer membrane proteins from the other two strains and from another five strains in our collection allowed evaluation of antigenic heterogeneity. Our results demonstrate that transferrin-binding protein 2 (TBP2) is immunogenic in humans, to varying degrees depending on the strain, and that TBP2s (like the equivalent proteins of Haemophilus influenzae type b) are among the most important iron-regulated outer membrane antigens expressed during infection. Other immunogenic outer membrane proteins (some iron-regulated) are also expressed during infection; in a previous study in mouse, three of these proteins (with M(r) of 50, 70 and 77 kDa) did not induce an immune response. Our cross-reactivity data provide some support for Robki et al.'s two-group classification of N. meningitidis strains, and provide evidence against the possibility that the antigenic domains shared by the TBP2s of all N. meningitidis strains induce immune responses in vivo.

Reliability of laboratory models in the analysis of TBP2 and other meningococcal antigens.

The lack of experimental models suitable for the study of meningococcal pathogenicity led us to investigate if those actually in use (culture in iron-restricted media and animal models) provide results comparable with the responses observed in vivo during infection. In this work we studied three invasive strains cultured both in laboratory media and in human plasma, analysing the immune responses elicited in mice against membrane antigens and comparing them with those seen using homologous human convalescent sera. Outer membrane protein profiles observed after culture in plasma were different and more complex than those obtained after growth in laboratory media. Analogous differences were observed in the antigenic profiles, detecting some antigens recognized by human, but not mouse sera, and vice versa. However, the response to one of the major iron-regulated outer membrane antigens, the transferrin binding protein 2 (TBP2), was unaffected by the culture medium or the model, human or mouse, used for the analysis. In conclusion, we have found that results of antigenic analysis change depending on the culture conditions and animal models used. For the meningococcal antigen TBP2, growth in iron-restricted laboratory media and a mouse model provide results which correlate well with those observed using convalescent human serum from individuals recovered from infections. We suggest that careful analysis and evaluation of experimental results and their comparison with in vivo elicited immune responses are essential in order to get accurate extrapolations for experimental vaccine designs.

Bactericidal activity of antibodies elicited against the Neisseria meningitidis 37-kDa ferric binding protein (FbpA) with different adjuvants.

The 37-kDa ferric binding protein, FbpA, from three Neisseria meningitidis strains was purified to homogeneity with iron-affinity chromatography and used for immunisation of mice employing four different adjuvants: aluminium hydroxide, Freund's, the saponin Quil-A, and a Ribi adjuvant system (RAS). Controls immunised without adjuvant were also included. All sera obtained were monospecific for the meningococcal FbpA, with antibody titres higher when RAS and Quil-A were used (256), PBS resulting in titres similar to those of Freund's (64), and, surprisingly, with no antibodies elicited when aluminium hydroxide, the only approved adjuvant for use in humans, was used. All anti-FbpA sera bound to intact meningococcal cells, showing a complete cross-reactivity, but the bactericidal activity of anti-FbpA antibodies, demonstrated for the first time in this work, was low (32% of killing with the homologous strain), and the analysis of immunoglobulin isotypes showed that the non-bactericidal IgG1 was predominant. The results confirm that the FbpA is surface-exposed, antigenic, and able to elicit bactericidal antibodies, although, in the conditions and with the adjuvants tested, killing efficacy was low and cross-killing was very variable, not supporting the inclusion of this protein in vaccine formulations. Nevertheless, given the high conservation of the FbpA in the genus Neisseria, its surface exposure and its antigenicity, studies on immunisation with peptides corresponding to the exposed epitopes and/or new adjuvant systems could improve the bactericidal response to this protein, making it suitable for vaccine development.

Analysis of Neisseria lactamica antigens putatively implicated in acquisition of natural immunity to Neisseria meningitidis.

Sera from healthy human volunteers, patients convalescent from meningococcal meningitis, and mice immunized with outer membrane proteins from Neisseria meningitidis and Neisseria lactamica strains were used to analyze and identify antigens cross-reactive to both neisserial species. All classes of meningococcal proteins except class 1 (PorA) and class 5 cross-reacted with N. lactamica proteins and two other proteins of 65 and 55 kDa (an iron-regulated protein). Results obtained with the mouse sera demonstrate that cross-reactive antibodies can be elicited by either N. meningitidis or N. lactamica. These results support the suggestion that N. lactamica contributes to the development of natural immunity against N. meningitidis during the first years of life. The use of vaccines containing proteins other than PorA could interfere in colonization of mucosal surfaces by N. lactamica, hampering the natural mechanisms of immunity acquisition in humans. Only convalescent sera reacted with the 55 and 65 kDa proteins, which suggests that they might be relevant for pathogenicity.

Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species.

Two mouse sera against outer membrane proteins from a pathogenic Neisseria meningitidis strain and a commensal N. lactamica strain and two human sera from patients recovering from meningococcal meningitis were used to identify antigens common to pathogenic and commensal Neisseria species. Two major antigens of 55 kDa and 32 kDa, present in all N. meningitidis and N. lactamica strains tested, were demonstrable with all the sera used; the 55-kDa protein was iron-regulated. Demonstration of other common antigens was dependent on the serum used: a 65-kDa antigen was visualised with the human and the mouse anti-N. lactamica sera; a 37-kDa antigen identified as the meningococcal ferric binding protein (FbpA) was only detected with the mouse sera, and two antigens of 83 kDa and 15 kDa were only shown with the mouse anti-N. meningitidis serum. The results demonstrate the existence of several outer membrane antigens common to N. lactamica and N. meningitidis strains, in agreement with the hypothesis that natural immunity against meningitis is partially acquired through colonisation by commensal species, and open new perspectives for the design of vaccine formulations and the development of strategies for vaccination against meningitis.

Role of R-plasmids in adherence and hydrophobicity in Escherichia coli.

Twenty-five R-plasmids from different incompatibility groups were conjugatively transferred to the laboratory strain Escherichia coli JE2571 and the variations in surface hydrophobicity and adherence to human buccal epithelial cells were investigated. It was found that some R-plasmids produce significant variations in adherence and/or hydrophobicity but that these variations show no quantitative or qualitative correlation.

Failure of phenotypic characteristics to distinguish between carrier and invasive isolates of Staphylococcus epidermidis.

Thirty carrier and 29 invasive Staphylococcus epidermidis isolates were analysed for production of slime, extracellular enzymes and antibiotic resistance. Evaluation of slime production was by two methods which gave differing results, but both showed no difference between the two groups of isolates. Exoenzyme production by the two groups of isolates was also similar, and the only differences were shown in resistance to some antibiotics; carrier isolates were more resistant to novobiocin and cephalothin, but resistance to chloramphenicol, tunicamycin, teicoplanin, penicillin, ampicillin and tetracycline was similar in both groups. Cluster analysis based on exoenzyme production showed no consistent difference between invasive and carrier isolates, with the lowest similarity coefficient over 88% (except for one isolate). Consequently, none of the characteristics studied was useful in differentiating potentially invasive isolates.

Phage typing and phage induction in carrier and invasive Staphylococcus epidermidis isolates.

Fifty-nine Staphylococcus epidermidis isolates (30 carrier and 29 invasive) were phage typed using the phage set developed by Pulverer. Poor results were obtained as only 24% of the invasive and 27% of the carrier isolates were typable, with no differences between these percentages (P greater than 0.05). Isolation of lysogenic phages present in the isolates was attempted by three different methods, induction being satisfactory only by overnight incubation with 0.1 mg l-1 mitomycin C and detection with 2,3,5-triphenyl-tetrazolium chloride. All isolates produced phages, and the lytic activity of these phages was expressed on more than 50% of the isolates in all cases (except one phage which lysed 10% of the carrier isolates and 13.8% of the invasive isolates). The percentages of isolates lysed in the two groups (carrier and invasive) were statistically different (P less than 0.05) only for phages derived from four isolates.