Binding studies of a monoclonal antibody specific for 3-deoxy-D-manno-octulosonic acid with a panel of Klebsiella pneumoniae lipopolysaccharides representing all of the O serotypes.

A monoclonal antibody (MAb) raised against Salmonella minnesota R595 and specific for alpha-3-deoxy-D-manno-octulosonic acid (alpha-Kdo) of the inner core was tested for binding to lipopolysaccharides (LPS) of Klebsiella pneumoniae. The MAb was tested in several assay systems (enzyme-linked immunosorbent assay, passive hemolysis, and inhibition of passive hemolysis) with a large panel (n = 23) of K. pneumoniae LPS representing all nine currently known O serotypes. MAb 20 showed reactivity with almost all O serotypes of K. pneumoniae LPS, and this reactivity could be inhibited by synthetic Kdo. This suggests an epitope in the cores of these Klebsiella LPS much like that in the inner core of LPS of S. minnesota. Large differences in reactivity between LPS of different strains belonging to the same O serotype were observed. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LPS followed by immunoblotting, reactivity of MAb 20 was observed only with the fast-moving fraction possibly representing the nonsubstituted core. No binding was seen with the high-molecular-weight fraction that contained core material substituted with several units of O-antigen building blocks. The chemical basis for these differences in reactivity remains to be established. As far as we know, this is the first report containing comprehensive immunochemical data on the LPS core of K. pneumoniae.

Antigenic and immunogenic differences in lipopolysaccharides of Escherichia coli J5 vaccine strains of different origins.

Escherichia coli strain J5 mutants of various origins have often been used as vaccines for induction of cross-reactive, cross-protective antibodies directed against the lipopolysaccharide (LPS) core region. The antigenic composition of LPS from J5 strains of different origin, i.e. strains J5(U), J5(UK), J5(2877) and J5(a), was investigated using monoclonal antibodies (mAbs) reactive only with LPS of a given chemotype, i.e. one specific for the incomplete E. coli core of the Rc chemotype, a second mAb reactive only with the E. coli R3 complete core, and a third specific for the O-antigen of E. coli serovar O111. The LPS of strains J5(U) and J5(a) is almost exclusively composed of LPS of the Rc chemotype, LPS of the J5(UK) strain is composed of Rc LPS and R3 complete core, while LPS of the J5(2877) strain contains Rc, R3 complete core and O-antigen. Growth of the bacteria in medium supplemented with galactose led to increased expression of complete core. The immune responses to the various strains were investigated. Antiserum to the J5 strain expressing the largest amount of R3 core [J5(UK)] had much higher anti-R3 LPS antibody titres compared to antiserum to the other strains. mAb 53, representative of the anti-R3 response to J5 strains containing complete core, bound to those E. coli LPS expressing the R3 core. Thus, the R3 LPS, present in some J5 vaccine strains is at least partially responsible for some of the cross-reactivities exhibited by some anti-J5 antisera.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymyxin B-horseradish peroxidase conjugates as tools in endotoxin research.

The peptide antibiotic Polymyxin B (PMB) binds to bacterial endotoxin (lipopolysaccharide, LPS). We prepared covalent conjugates of PMB and horseradish peroxidase (HRP) by periodation of HRP-linked oligosaccharides followed by direct condensation with PMB. In addition we prepared monoclonal antibodies (Mabs) to PMB. The PMB-HRP conjugates and anti-PMB Mabs were used to study in ELISA the binding of PMB to LPS from Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. In addition, PMB-HRP was used to quantify lipid A in ELISA, and to stain gram-negative bacteria histochemically. For the study of PMB-LPS interaction, PMB-HRP proved to be superior to the anti-PMB Mabs. PMB-HRP conjugates are useful general probes to detect or measure lipid A and LPS of various species using very simple methods and to stain bacteria, and they may obviate the need for many specific antisera. Thus, PMB-HRP conjugates are useful probes for endotoxin research.

Murine ascitic fluids contain varying amounts of an inhibitor that interferes with complement-mediated effector functions of monoclonal antibodies.

The ability of murine monoclonal antibodies (mAbs), directed to the inner core of Gram-negative bacterial lipopolysaccharide (LPS, endotoxin), to enhance complement-mediated killing of bacteria, was investigated. The mAbs were tested as present in ascitic fluid. It was found that ascites contains an factor that inhibited the activity of complement. This effect was evident in assays for complement-mediated lysis of antibody-coated Gram-negative bacteria (bacterial killing) or of opsonised red blood cells. Moreover, the amount of inhibitor was found to vary from one ascites to another and spanned a 60-fold range. Thus, in vitro or in vivo experiments where complement is known to play a determining role may yield incorrect results when ascites is used as a source of antibody; the use of ascites prepared from irrelevant antibody as a negative control does not eliminate this problem.

Transferrins and heme-compounds as iron sources for pathogenic bacteria.

The low concentration of free iron in body fluids creates bacteriostatic conditions for many microorganisms and is therefore an important defense factor of the body against invading bacteria. Pathogenic bacteria have developed several mechanisms for acquiring iron from the host. Siderophore-mediated iron uptake involves the synthesis of low molecular weight iron chelators called siderophores which compete with the host iron-binding glycoproteins lactoferrin (LF) and transferrin (TF) for iron. Other ways to induce iron uptake, without the mediation of siderophores, are the possession of outer membrane protein receptors that actually recognize the complex of TF or LF with iron, resulting in the internalization of this metal, and the use of heme-compounds released into the circulation after lysis of erythrocytes. In this review, the nonsiderophore-mediated iron-uptake systems used by certain pathogenic bacteria are emphasized. The possible contribution of these iron-uptake systems to the virulence of pathogens is also discussed.

Human immune response to an iron-repressible outer membrane protein of Bacteroides fragilis.

Under conditions of iron starvation, Bacteroides fragilis expresses various iron-repressible outer membrane proteins (IROMPs). A 44-kDa protein appears to be one of the major outer membrane proteins (OMPs) in B. fragilis under iron stress and plays a role in heme uptake by this bacterium. To determine whether the 44-kDa IROMP of B. fragilis is expressed in vivo and whether this protein is immunogenic, we used Western immunoblotting to examine serum samples from patients with an infection caused by Bacteroides species. All the serum samples from patients and from normal controls showed reactivity with several proteins of B. fragilis. Only serum samples from patients infected with B. fragilis showed immunoreactivity with the 44-kDa protein. We also used a rat infection model to study the immune response against this protein during the process of an intra-abdominal infection in these animals. During the first 8 days of infection a gradual increase of antibodies to the 44-kDa protein in the rat was detected. These results suggest that the 44-kDa IROMP is expressed in vivo, since it induces an antibody response in patients and animals. We also analyzed 85 strains of the B. fragilis group for the presence of proteins antigenically related to the B. fragilis 44-kDa protein. The data indicate that this protein was conserved in B. fragilis strains and was absent in the other bacterial strains tested.

Iron-regulated outer membrane protein of Bacteroides fragilis involved in heme uptake.

Under iron starvation, Bacteroides fragilis expresses various iron-regulated outer membrane proteins. In this study, a deferrated minimal medium was used in growth experiments, and the role of one of these iron-regulated outer membrane proteins (a 44-kDa protein) in an iron uptake mechanism which acquires iron from heme compounds was elucidated. When a specific 44-kDa protein antiserum was used in a medium with heme as the only iron source, growth inhibition was observed. These results demonstrate that the 44-kDa outer membrane protein plays an important role in the uptake of heme in B. fragilis.

Purification and partial characterization of an iron regulated outer membrane protein of B. fragilis under non-denaturing conditions.

The CHAPS-PAGE gelsystem we applied gave a good separation of the proteins of Bacteroides fragilis under non-denaturing conditions. We succeeded with preparative CHAPS-PAGE in purifying an iron regulated outer membrane protein (a 44 kDa polypeptide on SDS-PAGE) of B. fragilis. This integral membrane protein proved to be a lipopolysaccharide binding protein with an isoelectric point of approximately pH 5.5. This method of purifying membrane proteins could be an important step in research into the function of membrane proteins.

Preservatives in insulin preparations impair leukocyte function. In vitro study.

m-Cresol and methyl p-hydroxybenzoate are preservatives in insulin preparations. As previously reported, in diabetic patients on continuous subcutaneous insulin infusion, users of insulin-containing m-cresol had significantly more inflamed infusion sites than users of insulin with methyl p-hydroxybenzoate. This study assessed the influence of insulin with and without these preservatives on leukocyte function. Leukocyte function was investigated in a killing experiment, expressed as the percentage of bacteria killed after 60 min incubation of bacteria (Staphylococcus aureus), polymorphonuclear leukocytes, serum, and insulin preparations. Because preservative is retained by the infusion device, insulin with preservative was tested before and after 1 and 4 days perfusion with a PVC pump catheter. After perfusion, the amount of preservative was reduced (percentage of original concentration after 1 and 4 days 8 and 30% m-cresol and 42 and 72% methyl p-hydroxybenzoate, respectively). The killing percentage in insulin with m-cresol reduced compared with insulin without preservative (mean +/- SE 95.4 +/- 0.8%) and the control without insulin (95.8 +/- 0.8%), both before and after 1 and 4 days perfusion (74.8 +/- 0.7, 80.2 +/- 2.8, and 80.6 +/- 1.6%, respectively; P less than 0.01). The same occurred in insulin with methyl p-hydroxybenzoate (85.0 +/- 0.9% before and 88.4 +/- 0.9 and 86.2 +/- 0.8% after 1 and 4 days perfusion; P less than 0.05). All insulin preparations with m-cresol caused lower killing percentages than corresponding insulin preparations with methyl p-hydroxybenzoate (P less than 0.05). These results demonstrate that both preservatives impaired leukocyte function, but m-cresol was the most noxious in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)

Further characterization of monoclonal antibodies to lipopolysaccharide of Salmonella Minnesota strain R595.

We have shown here that despite the use of monoclonal antibodies with well-defined epitope-specificities, and despite testing them in the most simple animal model available (i.e., mixing of homologous LPS with Mab prior to injection), we are not yet able to explain why some of the antibodies were effective and others not. For some of the clones (e.g., clone 20), an even better definition of binding sites is currently taking place in an attempt to obtain this understanding. We also do not yet understand why clone 20 was not effective in the mucin model, while using much lower amounts of injected antibody, and much higher challenge doses, this Mab was effective against E. coli in the gentamicin-treated mouse model. Very clear is, however, that in order to be protective in the latter model, Mabs are not required to be specific for lipid A. In the future it will be essential to develop procedures which measure specific interaction between smooth LPS/bacteria and antibodies to the LPS core region. In addition, it will be of great help when the chemical structure of non-substituted, rough-form LPS, as occurring in smooth LPS preparations, would be defined. This applies also to O-substituted core molecules.

Epidemiology of the Bacteroides fragilis group in the colonic flora in 10 patients with colonic cancer.

We report the relative frequencies of members of the Bacteroides fragilis group in the faeces, in colon lavage fluid obtained pre-operatively, and in colonic tissue specimens obtained at operation from 10 patients with colonic cancer. B. vulgatus was the most and B. fragilis and B. ovatus were the least frequently isolated Bacteroides spp. in the faeces of the 10 subjects. B. uniformis and B. thetaiotaomicron ranked second and third in the faeces. The relative frequencies of all species except B. fragilis were lower in the lavage fluid and in cultures of mucosa. The relative frequency of B. fragilis increased from 4% in faeces to 39% in the final lavage fluid and to 42% in the colonic mucosa culture. Our results suggest that B. fragilis has a more intimate association with the gut mucosa than other members of the B. fragilis group, which might be one explanation for the high incidence of this species in gut-associated intra-abdominal infections.

Prevention of lethal endotoxemia in actinomycin D-sensitized mice by incubation of Salmonella minnesota R595 lipopolysaccharide with monoclonal antibodies to R595.

Murine monoclonal antibodies reacting with lipopolysaccharide (LPS) of Salmonella minnesota strain R595 (Re chemotype) were prepared, and tested for their ability to protect actinomycin D-sensitized mice against lethal endotoxemia. Protection was found with some antibodies up to a 90-fold increase in LD50, whereas others exhibited no protection. The various protective antibodies did not all bind to the same epitope. The same applied for non-protective clones. Protective and non-protective clones could not be discriminated by ELISA. One protective monoclonal antibody (clone 20) was specific for ketodeoxyoctonate, a structural element common to various LPS. These findings show that the involvement of lipid A in the binding site of monoclonal antibodies is no prerequisite for protection.

Comparison of monoclonal antibodies to the deeper core region of gram-negative lipopolysaccharide by means of polymyxin B inhibition studies.

Previously we have described a panel of 12 monoclonal antibodies (Mabs) directed to lipopolysaccharide (LPS) of the Salmonella minnesota Re mutant R595. Six of them had been found to decrease mortality of LPS for actinomycin D-sensitized mice. The other six clones were not effective. It is known and we have confirmed that polymyxin B (PMB) also neutralizes LPS endotoxicity. We now tested the hypothesis that protective clones bound near or at the PMB binding site, by an in vitro assay where PMB and Mab competed for binding to R595 LPS. Our results show that this hypothesis must be rejected and that the LPS epitopes recognized by protective clones are interspersed by those recognized by non-protective ones. We could, however, demonstrate that this sort of inhibition assays are of value in estimating the localization on the core of the binding sites of various Mabs.

Production and characterisation of mouse monoclonal antibodies reacting with the lipopolysaccharide core region of gram-negative bacilli.

Monoclonal antibodies to the lipopolysaccharide (LPS) core region were produced by immunising mice with Escherichia coli strain J5 (chemotype Rc). One of these bound to the deepest part of the core, i.e., Lipid A, and reacted with other heat-killed but not live gram-negative bacilli, including E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Eight other monoclonal antibodies, binding to the terminal glucose residue of Rc LPS, reacted with live cells of E. coli strains only. Thus, the O antigen does not necessarily render the core inaccessible to antibody. However, despite binding to live bacteria, these monoclonal antibodies neither enhanced phagocytic killing, nor protected mice from dying from gram-negative infection or endotoxaemia. It is concluded that antibodies reacting with the most immunodominant parts of the J5 core are not protective.

Outer membrane proteins of Bacteroides fragilis and Bacteroides vulgatus in relation to iron uptake and virulence.

A virulent strain B. fragilis BE1 and an avirulent strain B. vulgatus BE20 were grown in a culture medium with and without the addition of a synthetic chelator (Bipyridyl) to induce iron limitation. Cells grew more slowly under iron stress, although the growth rate of the B. vulgatus strain was more affected under these conditions than the strain of B. fragilis. The outer membrane protein profile of these strains was studied in relation to the iron concentration in the growth medium by means of SDS-polyacrylamide gel electrophoresis. Four proteins, with the apparent molecular weights of 89, 49, 44 and 23.5 kDa, were consistently present in the outer membrane of B. fragilis BE1 grown under iron restricted conditions. In B. vulgatus BE20 cells a 44 and a 23.5 kDa protein were absent and only the expression of an 89 kDa protein was clearly seen under these conditions. The iron regulated proteins, particularly the 44 kDa protein, could be involved to an iron uptake mechanism in B. fragilis. So the presence of these proteins might play an important role in the virulence of this anaerobic bacterium.

Comparison of biochemical and serological typing results and antimicrobial susceptibility patterns in the epidemiological investigation of Klebsiella spp.

An analysis of the serological and biochemical typing results of 925 clinical isolates of klebsiella revealed that biotyping and serotyping of klebsiella could replace each other for epidemiological purposes. The combination of both typing methods provided even more epidemiological information in analysing clusters of particular serotypes and biotypes in time. Clustering serotypes, mainly of neonatal origin, were nearly uniformly more resistant to the antibiotics in common use than other serotypes. Biotyping as well as serotyping of klebsiella isolates recovered from environmental surveys in the neonatal ward showed that epidemic and non-epidemic klebsiella isolates could occasionally be cultured from the environment and from the staff.

Influence of rosmarinic acid on opsonization and intracellular killing of Escherichia coli and Staphylococcus aureus by porcine and human polymorphonuclear leucocytes.

The influence of rosmarinic acid on the function of porcine and human polymorphonuclear leucocytes was tested. Rosmarinic acid inhibited the chemiluminescence of PMNL, induced by preopsonized Zymosan or phorbol myristate acetate. The killing of Escherichia coli was inhibited by rosmarinic acid at a concentration of 2 mM, but not that of Staphylococcus aureus. The inhibition of the killing was due to an impaired opsonization, caused by an adverse influence of rosmarinic acid on complement activation. Direct effects of rosmarinic acid on the killing mechanisms of PMNL were not observed.

Effect of Bacteroides fragilis cellular components on chemotactic activity of polymorphonuclear leukocytes towards Escherichia coli.

Chemotaxis of polymorphonuclear leukocytes (PMNL) in response to cell components of Bacteroides fragilis alone or in combination with Escherichia coli was evaluated. E. coli produced much more powerful chemotactic factors than B. fragilis. The culture filtrate (CF), outer membrane (OM) preparation, and lipopolysaccharide (LPS) of B. fragilis slightly stimulated chemotactic activity of PMNL. The culture filtrate and OM preparation were capable of inhibiting the chemotaxis of PMNL in response to the chemotactic factors of E. coli but LPS of B. fragilis was not able to do so. Reduction by B. fragilis of PMNL chemotaxis in response to E. coli was not specific for B. fragilis but also occurred in the presence of facultative bacteria. In parallel with chemotaxis, lysozyme release, but not beta-glucuronidase release, by PMNL was significantly stimulated by E. coli but not by B. fragilis.

Characterization of fimbriae from Bacteroides fragilis.

Fimbriae derived from Bacteroides fragilis strain BE1 (BE1 fimbriae) appeared to be composed of subunits with a molecular weight of 40,000. Under the electron microscope the fimbriae could be visualized as straight filaments with a diameter of 4 nm. It appeared that production of the BE1 fimbriae is repressed under conditions of iron limitation, and at a growth temperature of 20 degrees C. Antibodies raised against the 40,000 dalton polypeptide, purified by means of preparative SDS-polyacrylamide gelelectrophoresis, recognized the native fimbriae, as was shown by immunogold labelling of intact bacterial cells, and by immunoprecipitation. Immunoblot experiments showed that other strains of B fragilis tested produced polypeptides, ranging in molecular weight from 40,000 to 42,000, that are antigenically related to the BE1 fimbrial subunit. No haemagglutination activity could be associated with the BE1 fimbriae.