A chemically defined medium induces resistance to lipopolysaccharide antibody in Haemophilus influenzae type b

Haemophilus influenzae type b was more resistant to killing by lipopolysaccharide (LPS) antibody and complement after growth in defined medium than in conventional broths. Resistance correlated with decreased binding of LPS antibody, as determined by whole-cell enzyme-linked immunosorbent assay. An inhibition radioimmunoassay was used to determine that bacteria grown in defined medium contained about 2.5 times more capsule than bacteria grown in conventional broth. No major differences were noted in the electrophoretic patterns of outer membrane proteins or LPS. The defined medium did not increase the resistance of a capsule-deficient mutant. Resistance and increased encapsulation could be reproduced after growth in conventional broth supplemented with magnesium, glutamic acid, and aspartic acid. Thus, the growth medium may influence the content of capsule on H. influenzae type b, and may in turn, influence the binding and bactericidal activity of LPS antibody to the cells.     

Hydrophobic characterization of thermophilicCampylobacterspecies and adhesion to INT 407 cell membranes and fibronectin

Cell surface hydrophobicity ofCampylobacter jejuni,C. coli,C. lariandC. upsaliensiswas tested by hydrophobic interaction chromatography on octylsepharose CL-4B. The hydrophobicity was influenced by cultivation mode, presence or absence of intact lipopolysaccharide (LPS) and outer membrane protein structures. Species-specific differences of hydrophobic characteristics were not detected. Bacteria grown in fluid medium exhibited a high degree of hydrophobicity. Agar-grown bacteria showed hydrophobic interaction to a significant lower extent. By oxidation of LPS with sodiummeta-periodate the hydrophobicity of agar-grown bacteria was slightly increased. Bacteria pretreated with proteinase K exhibited a marked decrease of hydrophobic interaction, whereas pretreatment with trypsin did not influence the hydrophobic interaction. Live bacteria were allowed to adhere to INT 407 cell membranes. With exception of one aflagellate strain, bacteria grown in fluid medium adhered better to the cellular substrate than agar-grown bacteria. This difference was not found when adhesion to fibronectin was tested. LPS-oxidized bacteria adhered significantly better to both cell membranes and fibronectin, whereas proteinase K treated bacteria exhibited a significant loss of adhesion capacity for both substrates.     

Enhancement of the IgG antibody production to Escherichia coli O antigen by prior exposure to serologically different E. coli bacteria

Exposure of rabbits and mice to serologically different E. coli bacteria enhanced the synthesis of IgG but often suppressed the formation of IgM O antibodies in response to subsequent immunization with E. coli O6 bacteria. The effect differed with the O antigen used. A higher enhancing effect on the IgG antibody synthesis was associated with less suppression of the IgM-antibody formation. The antibody avidity of both immunoglobulin classes was relatively low compared to that in the ordinary immune response to E. coli O6. The findings indicated an enhancing action of endotoxin on the IgG antibody response to the O6 antigen.     

Modulation of Antibody-Mediated Immune Response by Probiotics in Chickens

Probiotic bacteria, including Lactobacillus acidophilus and Bifidobacterium bifidum, have been shown to enhance antibody responses in mammals. The objective of this study was to examine the effects of a probiotic product containing the above bacteria in addition to Streptococcus faecalis on the induction of the chicken antibody response to various antigens, both systemically and in the gut. The birds received probiotics via oral gavage and subsequently were immunized with sheep red blood cells (SRBC) and bovine serum albumin (BSA) to evaluate antibody responses in serum or with tetanus toxoid (TT) to measure the mucosal antibody response in gut contents. Control groups received phosphate-buffered saline. Overall, BSA and SRBC induced a detectable antibody response as early as week 1 postimmunization (p.i.), which lasted until week 3 p.i. Probiotic-treated birds had significantly (P ≤ 0.001) more serum antibody (predominantly immunoglobulin M [IgM]) to SRBC than the birds that were not treated with probiotics. However, treatment with probiotics did not enhance the serum IgM and IgG antibody responses to BSA. Immunization with TT resulted in the presence of specific IgA and IgG antibody responses in the gut. Again, treatment with probiotics did not change the level or duration of the antibody response in the gut. In conclusion, probiotics enhance the systemic antibody response to some antigens in chickens, but it remains to be seen whether probiotics have an effect on the generation of the mucosal antibody response.     

Effects of serum components on gram-negative bacteria during bactericidal reactions

Biochemical changes which occur in a smooth strain of Escherichia coli were investigated with hyperimmune rabbit serum as a source of antibody and fresh normal guinea pig serum as a source of complement. Ribonucleic acid synthesis, as shown by incorporation of ³H-uridine, was decreased as early as 5 min, and deoxyribonucleic acid synthesis, shown by incorporation of ³H-thymidine, was decreased after 15 min of reaction. Incorporation of glycerol-2-³H into membrane lipid ceased after 25 to 30 min, probably as a result of functional or physical disruption of the membrane, or both. Permeability control (as indicated by loss of ³H-uridine-labeled compounds and by decrease in optical density) and protein synthesis were subsequently shown to be affected after 30 min. The metabolic state of the bacteria was found to be important in determining the outcome of the reaction. This was shown by the influence of the type of medium on the reaction. A complex nutrient medium decreased susceptibility as compared with a simple medium. The energy sources glycerol and acetate also decreased susceptibility. It is postulated that the ability of the cell to prevent or repair damage to the cell membrane may be involved. Therefore, metabolic conditions which allow retention of vital processes associated with the cytoplasmic membrane and cell surface will mitigate the bactericidal effect. Such conditions may occur in vivo during bactericidal reactions.     

Comparison of the activity against Streptococcus mutans of a chlorhexidine-based antiseptic as a suspension or as a biofilm]

When bacteria colonize a surface they form a biofilm whose susceptibility to anti-microbials is different from that of the same bacterial species forming a homogeneous suspension in a liquid. This study investigated colonization of an inert solid phase (Tygon) with Streptococcus mutans ATCC 25,175--one of the strains involved in the initiation of cariogenic dental plaque--in a continuous flow of fresh medium sufficiently diluted so as to preclude growth of suspended bacteria. Only those bacteria which adhered to the solid phase grew, forming a biofilm. The antiseptic activity of Eludril (0.1% chlorhexidine) on this biofilm under dynamic (flowing medium) and static (stagnant medium) conditions was studied by comparison with the same strain in a suspension (in compliance with the AFNOR NF T 72-150 norm) and in a confluent culture on a filtering membrane. The biofilm was less susceptible under dynamic than under static conditions; under both conditions, the biofilm was less susceptible than the suspension. According to this model, the concentration of antiseptic recommended by the manufacturer according to studies using AFNOR norm NF T 72-150 (bacteria in a suspension) may be inadequate for bacteria adhering to tooth surfaces or gingival mucosa.     

Phagocytosis of mastitis isolates of Staphylococcus aureus and expression of type 5 capsular polysaccharide are influenced by growth in the presence of milk.

Phagocytosis by bovine polymorphonuclear granulocytes of seven capsular polysaccharide type 5 Staphylococcus aureus strains isolated from mastitis [corrected] was investigated by means of luminol-dependent chemiluminescence. Bacteria were grown on four different agar media (brain heart infusion, Columbia broth, modified staphylococcus medium 110, and skim milk) and were opsonized by normal bovine serum. When compared to growth on brain heart infusion agar, Columbia agar, and modified staphylococcus medium 110 agar, growth on skim milk agar rendered five of the strains more resistant to opsonization. The other two strains were resistant in all culture media used. Short periods of incubation in milk after growth on brain heart infusion agar did not augment resistance to phagocytosis, indicating that mere adsorption of milk components on bacteria was not responsible. The variability of the chemiluminescence response of polymorphonuclear leukocytes was pronounced among strains with each growth medium except milk. Growth on modified staphylococcus medium 110 and on milk agar favored the masking of teichoic acid, as shown by inagglutinability with rabbit antiserum. Interestingly, agglutination by a monoclonal antibody to capsular polysaccharide type 5 was optimal when bacteria were grown on skim milk agar. This suggests that capsular polysaccharide participated in the masking effect. These findings indicate that masking of the bacterial target of most of the naturally acquired opsonins present in normal bovine serum occurred when bacteria grew in the presence of milk, resulting in an increased resistance to phagocytosis by polymorphonuclear leukocytes.     

Effect of Antiflagellar Human Monoclonal Antibody on Gut-Derived Pseudomonas aeruginosa Sepsis in Mice

We evaluated the effect of antiflagellar human monoclonal antibody on gut-derived Pseudomonas aeruginosa sepsis. Mice were given a suspension of P. aeruginosa SP10052 in their drinking water and were simultaneously treated with ampicillin (200 mg/kg of body weight) to disrupt the normal bacterial flora. Cyclophosphamide was then administered to induce leukopenia and translocation of the P. aeruginosa that had colonized the gastrointestinal tract, thereby producing gut-derived generalized sepsis. In this model, intraperitoneal injection of 100 μg of antiflagellar human monoclonal antibody (SC-1225) per mouse for 5 consecutive days significantly (P < 0.01) increased the survival rate compared with that for mice treated with bovine serum albumin (BSA). Treatment with SC-1225 significantly reduced the average number of viable bacteria in portal blood, liver, and heart blood compared with the average number after treatment with BSA. Furthermore, the presence in serum of the inflammatory cytokines tumor necrosis factor alpha and interleukin 6 were evaluated as markers of severity of infection, and the results showed that the levels of these cytokines in mice treated with SC-1225 were significantly decreased in comparison with those in BSA-treated control mice. Although there was no significant difference in the number of bacteria that colonized the intestine, SC-1225 treatment significantly increased bacterial opsonophagocytosis by cultured peritoneal macrophages from mice with or without cyclophosphamide pretreatment. Our results indicate that antiflagellar human monoclonal antibody SC-1225 protects mice against gut-derived sepsis caused by P. aeruginosa and suggest that such an effect is due to its opsonophagocytic activity and the reduced motility of the translocated bacteria once the bacteria move from the intestine into the bloodstream.     

Adjuvant Activity of Naturally Occurring Monophosphoryl Lipopolysaccharide Preparations from Mucosa-Associated Bacteria

Natural heterogeneity in the structure of the lipid A portion of lipopolysaccharide (LPS) produces differential effects on the innate immune response. Gram-negative bacterial species produce LPS structures that differ from the classic endotoxic LPS structures. These differences include hypoacylation and hypophosphorylation of the diglucosamine backbone, both differences known to decrease LPS toxicity. The effect of decreased toxicity on the adjuvant properties of many of these LPS structures has not been fully explored. Here we demonstrate that two naturally produced forms of monophosphorylated LPS, from the mucosa-associated bacteria Bacteroides thetaiotaomicron and Prevotella intermedia, function as immunological adjuvants for antigen-specific immune responses. Each form of mucosal LPS increased vaccination-initiated antigen-specific antibody titers in both quantity and quality when given simultaneously with vaccine antigen preparations. Interestingly, adjuvant effects on initial T cell clonal expansion were selective for CD4 T cells. No significant increase in CD8 T cell expansion was detected. MyD88/Toll-like receptor 4 (TLR4) and TRIF/TLR4 signaling pathways showed equally decreased signaling with the LPS forms studied here as with endotoxic LPS or detoxified monophosphorylated lipid A (MPLA). Natural monophosphorylated LPS from mucosa-associated bacteria functions as a weak but effective adjuvant for specific immune responses, with preferential effects on antibody and CD4 T cell responses over CD8 T cell responses.     

Characterization of a Monoclonal Antibody That Binds to an Epitope on Soluble Bacterial Peptidoglycan Fragments

We employed an inhibition-type enzyme-linked immunosorbent assay (ELISA) to characterize a murine immunoglobulin M monoclonal antibody (MAb) that bound soluble macromolecular peptidoglycan (PG). With this ELISA, the MAb was capable of detecting soluble PG concentrations of less than 10 ng/ml. Enzymatic digestion of PG reduced binding by more than 100-fold, implying that the epitope recognized by this antibody depended on repeating subunits within the glycan backbone. Additionally, the MAb bound to epitopes on both O-acetylated and non-O-acetylated PG fragments from gram-negative bacteria, as well as PG fragments from Staphylococcus aureus and PG fragments released into the medium by a number of gram-positive and gram-negative bacteria.     

Antibody response to Escherichia coli O antigens and influence of the protein moiety of the endotoxin

Exposure of mice to different serotypes of E. coli bacteria, either O4 or O6, resulted in an enhanced indirect IgG-PFC response to the alternate bacteria. This effect seemed to be mediated by a protein connected with the endotoxin structure. This protein moiety had some weak adjuvant activities and increased the antibody response against sheep red blood cell about two-fold. This effect was not likely to be due to any contamination with the B-cell mitogen lipid A, a constituent of the endotoxin from which the protein was isolated.

In addition, experiments were performed in which irradiated spleen cells (0–400 R) from mice injected with E. coli O4 bacteria were transferred to irradiated (800 R) recipients together with E. coli O6 bacteria. Decreasing numbers of antibody forming cells with increasing irradiation dose were found. The parallel experiment employing E. coli O6 bacteria for both primary and secondary antigen injections revealed an increased immune response for an irradiation dose of 50 R, showing that suppressor cells are more irradiation sensitive than the other cells involved in this immune response, but that the effect of such cells is possibly overcome by the influence of the protein residue isolated from endotoxin.

A secondary response to E. coli O6 bacteria was also noted in agreement with previous results. It was found that this immune response could be reduced drastically by injecting primed thymocytes, simultaneously with the second injection.

     

Production of common antigen by enteric bacteria grown in a synthetic culture medium

Enterobacteriaceae share a common antigen (CA). The present investigation was carried out to determine whether this antigen is produced by representative strains of Escherichia, Salmonella, and Serratia grown in a completely synthetic medium. For comparative purposes, antigen production by the same strains grown in infusion broth was determined. CA, as assayed by indirect hemagglutination and immunogenicity studies in the rabbit, was produced by the microorganisms cultured in both media. The amount of CA produced by the strains varied according to the size of microbial population and to the length of culture. With the exception of E. coli O14, the strains studied, on day 7, produced 1.4 to 8 times more CA in infusion broth than in the synthetic medium; with E. coli O14, the ratio of CA in the respective media was 96:1. E. coli O14, but not E. coli O111 or S. typhimurium, when grown in the synthetic medium, engendered CA antibodies upon intravenous injection into rabbits. Ethanol extraction of the latter two strains yielded an immunogenic ethanol-soluble antigen preparation. The ethanol-soluble fraction of E. coli O111 also induced a secondary response in animals primed with E. coli O111 or S. typhimurium cultures grown in the synthetic medium. It is concluded that CA produced in a completely synthetic culture medium has the same attributes as CA produced in infusion broth.     

EFFECT OF STRESS ON PRODUCTION OF HEAT LABILE ENTEROTOXIN BY ESCHERICHIA COLI

Enterotoxigenic Escherichia coli (ETEC) is an important pathogen responsible for secretory diarrhoea. The production of heat labile enterotoxin (LT), by ETEC, is largely responsible for the pathogenesis of diarrhoea. In the present study we investigated the effect of stress factors such as temperature, pH, osmotic stress and nutritional limitation on the production of LT by ETEC using in-house GMI-ELISA. Four strains of E. coli consisting, one standard strain MTCC 723 and three clinical isolates were used in the study. Maximum amount of LT (OD 3.285) was produced at 37°C followed by 40°C (OD 3.305). Growth of E. coli in medium with pH 8.6 resulted in maximum amount of LT production (OD 3.489). LT was not detectable when bacteria were grown in medium with pH ≤7.2 and ≥9.2. Sodium chloride concentration of 0.2 M stimulated maximum amount of LT production. Maximum amount of LT was produced when the bacteria were grown in medium containing 2.5g/l of glucose. All the stress factors had a significant effect on the LT production by E. coli, though quantitative differences in the various strains were observed.     

Recombinant GroEL enhances protective antigen-mediated protection against Bacillus anthracis spore challenge

The fatal inhalation infection caused by Bacillus anthracis results from a complex pathogenic cycle involving release of toxins by bacteria that germinate from spores. Currently available vaccines against anthrax consist of protective antigen (PA), one of the anthrax toxin components. However, these PA-based vaccines are only partially protective against spore challenge in mice. This shows that exclusive elicitation of high anti-PA titer does not directly correlate with protection. Here, we demonstrate that inclusion of GroEL of B. anthracis with PA elicits enhanced protection against anthrax spore challenge in mice. GroEL was included as it has been reported to be present both on the exosporium and in the secretome in addition to the cell surface of B. anthracis. It has also been found protective against other pathogens. In the present study, immunization with GroEL alone was also potent enough to induce high humoral and cell-mediated response and significantly prolonged the mean time to death in spore-challenged mice. As a surface antigen, opsonization of spores with anti-GroEL IgG showed increased uptake of treated spores and therefore accelerated rate of spore destruction by phagocytic cells leading to the protection of mice. We found that GroEL was able to enhance nitric oxide release from lymphocytes and also reduce bacterial load from the organs, probably through the activation of macrophages and over-expression of certain innate immunity receptors. Therefore, the present study emphasizes that GroEL is an effective immunomodulator against B. anthracis infection.     

Effects of in vitro and in vivo growth conditions on expression of type 8 capsular polysaccharide by Staphylococcus aureus.

Type 8 capsular polysaccharide (CP8) is widely prevalent among clinical isolates of Staphylococcus aureus, but the role that the capsule plays in the pathogenesis of staphylococcal infections is unclear. This study was performed to identify growth conditions that would optimize the production of CP8 and to determine whether enhanced CP8 expression would influence staphylococcal virulence. S. aureus Becker grown in a chemically defined broth medium with < 1 microM ferric nitrate produced up to eightfold more CP8 per milligram of biomass than did bacteria cultivated in the same medium containing 20 microM ferric nitrate. The bacteria produced > 350-fold more cell-associated CP8 per milligram of biomass when grown on the surface of Columbia agar than when grown in Columbia broth. Most of the CP8 produced by broth-grown cells was secreted into the culture medium. S. aureus cultivated on the surface of nitrocellulose membranes floating on Columbia broth produced levels of CP8 similar to those produced by cells grown on Columbia agar. Similarly, bacteria harvested from endocardial vegetations of rabbits infected with S. aureus produced high levels of CP8. These results indicate that staphylococci grown on surfaces, both in vitro and in vivo, produce larger quantities of cell-associated CP8 than those grown in liquid cultures. However, no differences were observed in the 50% lethal dose for mice of strain Becker grown on solid medium (high levels of capsule expression) or in liquid medium (low levels of capsule expression).     

Adaptation of Francisella tularensis to the mammalian environment is governed by cues which can be mimicked in vitro

The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and freshwater amoeba. It was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod- or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacterium's infection-derived phenotype. We compared Francisella LVS grown in brain heart infusion broth (BHI; a standard microbiological medium rarely used in Francisella research) to that grown in Mueller-Hinton broth (MHB; the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHI- and macrophage-grown F. tularensis cells showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supraphysiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that by extracellular bacteria; BHI-grown bacteria mirrored the latter, while MHB-grown bacteria resembled neither. Naïve macrophages responding to BHI- and macrophage-grown bacteria produced markedly lower levels of proinflammatory mediators than those in cells exposed to MHB-grown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella organisms that have emerged from macrophages.     

Streptococcus uberis resists the bactericidal action of bovine neutrophils despite the presence of bound immunoglobulin.

Streptococcus uberis 0140J was more resistant to the bactericidal action of bovine neutrophils after growth in chemically defined medium (CDM) supplemented with casein hydrolysate than when grown in CDM alone. Neither adult bovine serum obtained prior to vaccination nor hyperimmune serum raised against this bacterium was capable of acting as an effective opsonin towards S. uberis grown in the presence of casein hydrolysate. There was no detectable difference in the ability of bacteria grown in either CDM or CDM supplemented with casein hydrolysate to bind immunoglobulin G1 (IgG1), IgG2, or IgM from either hyperimmune serum or preparations of immunoglobulin from the same serum. Bacteria of both the phagocytosis-resistant and phagocytosis-sensitive phenotypes presented the same amount of IgG2 Fc terminus on their surfaces. It is concluded that the inducible resistance of S. uberis to bactericidal action of bovine neutrophils is not mediated by inhibition of antibody binding.     

A periodate-sensitive anti-phagocytic surface structure, induced by growth in milk whey, on Staphylococcus aureus isolated from bovine mastitis.

The phagocytic and chemiluminescent activity of purified bovine neutrophils in response to two Staphylococcus aureus strains isolated from mastitic bovine milk and grown in milk whey was studied. The activity was significantly reduced compared with the response elicited by the same strains grown in tryptic soy broth (TSB). A mild periodate treatment of the milk whey-grown strains resulted in a significant increase of both chemiluminescence and phagocytosis, whereas trypsin, subtilisin or papain treatment had no effect. The decreased binding of complement factor C3 to milk-whey-grown bacteria was restored to the level of TSB-grown homologous organisms by periodate treatment. Moreover, this treatment, but not treatment with trypsin, increased the surface hydrophobicity of milk-whey-grown bacteria. The chemiluminescent activity was as high towards heat-killed as towards live bacteria. Also, incubation of heat-killed TSB-grown bacteria in milk whey did not alter the chemiluminescent response, indicating that the reduced neutrophil activity towards milk-whey-grown bacteria was not due to binding of milk components to the microorganisms. These results strongly suggest that bovine mastitis S. aureus strains grown in milk whey produce an anti-phagocytic surface structure. This structure is heat- and protease-resistant and renders the bacterial surface hydrophilic. The anti-phagocytic material is altered or, more likely, released from the bacterial surface on periodate treatment and is probably of carbohydrate nature.     

Artificial Salmonella Vaccines: Salmonella typhimurium O-Antigen-Specific Oligosaccharide-Protein Conjugates Elicit Protective Antibodies in Rabbits and Mice

Several saccharides representative of the O-antigenic polysaccharide chain of Salmonella typhimurium (O antigens 4 and 12) were used as haptenic groups covalently linked to bovine serum albumin. The disaccharide abequose 1 → 3 D-mannose was synthesized, and the [Formula: see text] tetra-, octa- and dodecasaccharides were isolated after cleavage of isolated S. typhimurium O-polysaccharide chains by using bacteriophage endo-glycosidases. Rabbits immunized with the saccharide-protein conjugates suspended in Freund complete adjuvant readily responded with O4 antibody titers as high, or almost as high, as those elicited by heat-killed bacteria. The octa- and dodecasaccharide conjugates also gave rise to O12 antibody titers. The antibody response in mice differed in two ways from that seen in rabbits: mice did not respond with measurable antibody production against the disaccharide haptens, and the highest S. typhimurium lipopolysaccharide antibody response elicited by the saccharide haptens was still approximately 50-fold lower than that elicited by heat-killed bacteria. The latter difference may be a consequence of the fact that the mouse preferentially produces antibodies against the galactose residue which is terminal in the hapten but not in the native O-antigenic polysaccharide chain. Antibodies elicited in rabbits against all saccharide-protein conjugates protected passively transferred mice against intraperitoneal challenge with 100 times the 50% lethal dose of S. typhimurium SH 2201 (O4, 12) but not against challenge with S. enteritidis SH 2204 (O9, 12). The antibodies elicited by the saccharide-protein conjugates protected as well as antibodies elicited by heat-killed bacteria.