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PubMed1976

Molecular order of carbohydrate components in cell walls of bacteria, fungi and algae according to the topo-optical reaction of the vicinal OH groups.

Selective topo-optical staining of vicinal OH groups with aldehyde-bisulphite-toluidine blue (ABT) has been used for studying the molecular structural order of polysaccharide components in microbial cell walls and capsules. (i) By intensive metachromatic staining in the light microscope, the method demonstrates carbohydrates, containing free vicinal OH or acylaminohydroxyl groups. (ii) The birefringence induced by oriented toluidine blue binding of the ABT reaction, provides information about the linear order of the reacting carbohydrate chains, a possibility not offered by other ultrastructural methods. Furthermore the different optical character of birefringence induced by ABT is indicative of the presence of (a) tangentially oriented polysaccharide chains in the cell wall of the molds and bacterial capsules; or (b) radially oriented OH groups suggesting helical glycan chains of peptidoglucomannane and mucopeptide in the yeasts and bacterial cell walls.

Bacteria↗
PubMed2004

Revising the role of the pneumococcal vex-vncRS locus in vancomycin tolerance.

Vancomycin is used increasingly to treat invasive infections caused by multidrug-resistant Streptococcus pneumoniae. Although no vancomycin-resistant strains have been isolated to date, tolerant strains that fail to die rapidly and that cause relapsing disease have been described. The vex123-pep27-vncRS locus, consisting of an ABC transporter, a presumed signaling peptide, and a two-component system, respectively, has been implicated in vancomycin tolerance. Recent findings, however, challenged this model. The data presented here indicate that erythromycin in the growth medium induces a vancomycin-tolerant phenotype and that loss of function of Pep27 or VncRS does not alter autolysis. However, a role for the ABC transporter encoded by the vex123 genes in tolerance was confirmed. A vex3 mutant was considerably more tolerant to vancomycin treatment than the wild-type strain T4, and the strength of the phenotype depended on the orientation of the resistance cassette used to construct the mutant. Microarray results suggested a number of genes that might be involved in tolerance in the vex3 mutant. Although the exact function and regulation of the vex123-pep27-vncRS locus remains to be determined, several factors influence the autolysis behavior of S. pneumoniae, including the bacterial capsule, erythromycin, and the lytA and vex3 gene products.

ATP-Binding Cassette Transporters↗
PubMed1981

Attachment of bacteria to intestinal epithelial cells in diarrhea caused by Escherichia coli strain RDEC-1 in the rabbit: stages and role of capsule.

RDEC-1 is a strain of Escherichia coli that, in rabbits, attaches to intestinal mucosal epithelial cells bereft of microvillar borders and causes diarrhea by an unknown mechanism. The stages of attachment of RDEC-1 bacteria to mucosal epithelial cells were examined using high-voltage electron microscopy of thick (0.5-micrometers) sections of ileum and cecum of rabbits with diarrhea. The tissues were stained with ruthenium red or antisera to strain RDEC-1 OK antigens. Micrographs, including stereopairs, demonstrated several stages of bacterial attachment. Bacteria were attached to the glycocalyxes of epithelial cell microvilli and to pedestal-like extrusions of the surfaces of epithelial cells lacking microvilli. Structures consistent with bacterial pili were rarely visualized. Attachment to microvilli appeared to be a result of the interaction of polysaccharides of the microvillar glycocalyx and the K antigen of the bacterial capsule.

Animals↗
PubMed1991

Differential handling of bacterial antigens in macrophages infected with Mycobacterium leprae as studied by immunogold labeling of ultrathin sections.

Mycobacterium leprae were purified from the livers of experimentally infected armadillos, and the purity of the bacterial preparation was established by electron microscopy, immunoelectrophoresis of purified bacilli with rabbit serum raised against liver tissues from a noninfected armadillo, and gas chromatography. Such purified and intact bacilli were fixed and embedded by a gelatin-Lowicryl method for electron microscopy which preserved the mycobacterial antigens. Ultrathin sections were labeled with antisera raised in rabbits against the total antigens of the following species of mycobacteria: M. leprae, M. bovis BCG, M. avium, and a rapid-growing, nonpathogenic species, M. fallax. Bacteria were also labeled using serum raised against 2,3-diacyl-trehalose-2'-sulfate (sulfolipid-IV or SLIV) isolated and purified from M. tuberculosis. The immunolabeling was visualized under the electron microscope (EM) by using a secondary probe (goat-antirabbit IgG, H+L, coupled to 5 nm gold particles; GAR-5). EM results showed that M. leprae bacilli were highly labeled with all of the antisera used except SLIV, which was present only in discrete amounts. All of the antisera used labeled the bacterial "capsule," showing that this structure was not an artifact since it contained mycobacterial antigens. In parallel experiments, the murine J-774 macrophage cell line was infected with purified M. leprae, and fixed for EM at various time intervals for 1 week. Although the phagocytized bacteria did not multiply during the 1-week experiment, macrophages were unable to lyse them. Immunogold labeling of bacterial antigens in ultrathin sections of infected macrophages helped us to conclude: a) bacterial death and/or lysis is not a prerequisite for processing of antigens by infected macrophages; b) there was conclusive evidence for a differential antigen handling, i.e., some antigens were rapidly released (within 2 days, mostly capsular antigens) inside infected macrophages and transported to the macrophage surface, whereas others (the majority of them located in the cell-wall skeleton and in deeper bacterial structures) remained unreleased even after 4 to 7 days of infection; c) although relatively fewer epitopes reacting with anti-SLIV antibodies were found, they were rapidly released (within 2 days) inside macrophages, and exocytized to the macrophage surface. These novel findings are discussed in relation to leprosy and the current knowledge about the processing of bacterial antigens.

Animals↗
PubMed1994

Transposon mutants of Staphylococcus epidermidis deficient in elaboration of capsular polysaccharide/adhesin and slime are avirulent in a rabbit model of endocarditis.

Virulence comparisons were made in a rabbit model of endocarditis between wild-type and transposon mutants of Staphylococcus epidermidis deficient in elaboration of the capsular polysaccharide/adhesin (PS/A) and slime. The parental phenotype grew from 36 (61%) of 59 cultures of blood. The PS/A-negative phenotype grew in 1 (1%) of 98 cultures of blood (P < .001). No animals infected with PS/A-negative strains developed endocarditis compared with 75% of rabbits infected with PS/A-positive strains. PS/A-producing strains survived better than did PS/A-deficient strains in intact, absorbed rabbit or human serum plus human leukocytes. There was also greater deposition of C3 onto the PS/A-deficient strains than with the PS/A-producing isogenic strains. PS/A functions as an antiphagocytic bacterial capsule preventing C3 deposition and phagocytosis; loss of this structure increases the strain's susceptibility to opsonic killing and decreases its virulence.

Analysis of Variance↗
PubMed1991

Complement deposition by antibodies to Pseudomonas aeruginosa mucoid exopolysaccharide (MEP) and by non-MEP specific opsonins.

The failure of cystic fibrosis patients to limit chronic infection due to mucoid Pseudomonas aeruginosa might be due to ineffective opsonins produced against this bacterium. Nonopsonizing antibody to the bacterial capsule, mucoid exopolysaccharide (MEP), appears at elevated titers during chronic colonization of cystic fibrosis patients, as do opsonins not specific for MEP. Nonopsonic antibodies to MEP occur naturally in most adults and can be induced in animals by immunization. A limited number of humans produce MEP-specific opsonic antibodies after immunization. The purpose of this study was to compare the activation and deposition of C components onto the bacterial surface in the presence of these different antibodies. Opsonic killing uses the classical C pathway. MEP-specific opsonic and nonopsonic antibodies bound to whole bacteria and activated C to a comparable degree, but opsonic antibody deposited 3 to 40 times more C3 onto bacteria, mostly as C3bi, compared to nonopsonic antibody. In addition, two to three times as much nonopsonic mAb as opsonic mAb (both IgG2b) bound to the bacteria at comparable input concentrations, indicating the difference in C deposition was not due to differences in antibody binding. Non-MEP-specific opsonins also bound C3 to the bacteria, but only a mean of 27 +/- 14% was ester linked, compared with 81 +/- 11% of C3 deposited by MEP-specific opsonins. Immunoprecipitation experiments indicated that two-thirds of the C3 bound in the presence of MEP-specific opsonins was linked to MEP, whereas non-MEP-specific opsonins obtained from infected patients deposited the C3 onto LPS and other unidentified Ag. These data show that MEP-specific opsonins function by depositing C3 onto the outer bacterial surface that differentiates them from non-MEP-specific opsonins produced in response to chronic infection.

Animals↗
PubMed1994

[Detection of virulence factors using DNA probes in uropathogenic strains of Escherichia coli].

BACKGROUND: There are several bacterial determinants that contribute to the onset of urinary tract infection by E. coli. The present study focuses on some of the virulence factors considered to be most important, as P fimbriae, the siderophore aerobactin and bacterial capsule, which were studied among 123 uropathogenic E. coli strains isolated from outpatients from the Basque Community. METHODS: Virulence factors were detected using Molecular Biology techniques, namely DNA hybridization to specific probes prepared in our laboratory. RESULTS: When probe pap2, specific for fimbrial adherence was used, 36.5% of the strains showed positive hybridization, and 66 and 73% of the strains hybridized to probes for aerobactin and common capsule region, respectively. CONCLUSIONS: We believe that this technology provides a very useful tool for rapid and easy screening of strains harbouring different virulence factors. Nevertheless, the fact that these methods detect genetic determinants that are not always being expressed must be borne in mind.

Bacterial Adhesion↗
PubMed2005

Molecular basis for improved anthrax vaccines.

The current vaccine for anthrax has been licensed since 1970 and was developed based on the outcome of human trials conducted in the 1950s. This vaccine, known as anthrax vaccine adsorbed (AVA), consists of a culture filtrate from an attenuated strain of Bacillus anthracis adsorbed to aluminum salts as an adjuvant. This vaccine is considered safe and effective, but is difficult to produce and is associated with complaints about reactogenicity among users of the vaccine. Much of the work in the past decade on generating a second generation vaccine is based on the observation that antibodies to protective antigen (PA) are crucial in the protection against exposure to virulent anthrax spores. Antibodies to PA are thought to prevent binding to its cellular receptor and subsequent binding of lethal factor (LF) and edema factor (EF), which are required events for the action of the two toxins: lethal toxin (LeTx) and edema toxin (EdTx). The bacterial capsule as well as the two toxins are virulence factors of B. anthracis. The levels of antibodies to PA must exceed a certain minimal threshold in order to induce and maintain protective immunity. Immunity can be generated by vaccination with purified PA, as well as spores and DNA plasmids that express PA. Although antibodies to PA address the toxemia component of anthrax disease, antibodies to additional virulence factors, including the capsule or somatic antigens in the spore, may be critical in development of complete, sterilizing immunity to anthrax exposure. The next generation anthrax vaccines will be derived from the thorough understanding of the interaction of virulence factors with human and animal hosts and the role the immune response plays in providing protective immunity.

Animals↗
PubMed1997

Effect of bile on Vibrio parahaemolyticus.

Many enteric pathogens are thought to enter a viable but nonculturable state when deprived of nutrients. Virulent strains of the enteric pathogen Vibrio parahaemolyticus are rarely isolated from their low-nutrient aquatic environments, possibly due to their nonculturability. Host factors such as bile may trigger release from dormancy and increase virulence in these strains. In this study, the addition of bile or the bile acid deoxycholic acid to estuarine water-cultured bacteria led to an increase in the direct viable count and colony counts among the virulent strains. This effect was not demonstrated in the nonvirulent strains, and it was reversed by extraction of bile acids with cholestyramine. Bile-treated V. parahaemolyticus had lower levels of intracellular calcium than untreated cells, and this effect coincided with an increase in the number of metabolically active cells. Chelation of intracellular calcium with BAPTA/AM (R. Y. Tsien, Biochemistry 19:2396-2402, 1980) produced similar results. Addition of bile to V. parahaemolyticus cultures in laboratory medium enhanced factors associated with virulence such as Congo red binding, bacterial capsule size, and adherence to epithelial cells. These results suggest that a bile acid-containing environment such as that found in the human host favors growth of virulent strains of V. parahaemolyticus and that bile acids enhance the expression of virulence factors. These effects seem to be mediated by a decrease in intracellular calcium.

Bacterial Adhesion↗
PubMed1984

A plasmid-encoded outer membrane protein, TraT, enhances resistance of Escherichia coli to phagocytosis.

The presence of the outer membrane protein TraT, encoded by plasmid R6-5, reduces the sensitivity of Escherichia coli cells to phagocytosis by macrophages. This effect is independent of the bacterial capsule and is more evident in the presence of adsorbed normal human serum. The property of inhibiting phagocytosis is specifically abolished by anti-TraT protein antiserum and anti-TraT immunoglobulin G but not by Fab fragments. These results indicate that the TraT protein is a passive inhibitor of phagocytosis. Inhibition of phagocytosis is produced because the TraT protein antagonizes opsonization by complement, such that C3 deposition is reduced and altered in distribution.

Antibodies, Bacterial↗
PubMed1998

Enhancement of T cell-independent immune responses in vivo by CD40 antibodies.

In this report we describe a potentially powerful method for vaccinating infants against encapsulated bacterial pathogens such as Haemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitidis. High levels of antibody directed against the polysaccharides of the bacterial capsule are normally protective. Unfortunately, the capsular polysaccharides are T cell-independent antigens (TI); lacking T-cell help, they induce only weak, predominantly IgM antibody responses, with infants responding especially poorly. T-cell help, given to B cells during responses to protein antigens, causes stronger antibody responses and isotype switching to the IgG isotypes. T-cell help is mainly mediated through ligation of the B-cell surface antigen, CD40, by its cognate T-cell ligand, CD154. Here we show that administering anti-CD40 monoclonal antibody to mice, along with pneumococcal polysaccharide, provides a substitute for T-cell help and results in the generation of strong, isotype-switched antibody responses, which are protective. The work points the way toward a possible effective and inexpensive means of protecting susceptible groups against important bacterial pathogens.

Animals↗
PubMed2004

Corticosteroids and peritonsillar abscess formation in infectious mononucleosis.

Peritonsillar abscess formation is an uncommon complication of infectious mononucleosis (IM). Early case reports implicated corticosteroids in the development of such abscesses, however, subsequent studies suggested that these drugs do not promote the formation of abscesses at several sites outside the central nervous system. It has recently been demonstrated that zwitterionic polysaccharides, in bacterial capsules, form complexes with CD4(+) T lymphocytes leading to abscess formation. A patient is presented who developed peritonsillar abscess a few days after initiation of corticosteroid therapy for IM; the medical literature was reviewed in respect of this subject. It appears that the occurrence of these abscesses in IM is not strongly linked to corticosteroid treatment. The authors, therefore, recommend that steroids should not be withheld from patients with severe IM on the basis that they may precipitate the development of peritonsillar abscess.

Administration, Oral↗
PubMed1996

In situ localization of Streptococcus pyogenes during acute tonsillitis: an immunocytochemical study with gold markers.

Epithelial cells were harvested from the surface of the palatine tonsils of seven patients with current acute tonsillitis, proven culture-positive for Streptococcus pyogenes. The epithelial cells harboured attached bacteria, which expressed positive affinity to gold-labelled antiserum to S. pyogenes. The gold particles adhered selectively to the bacterial capsules. The microorganisms were held in place by projections protruding from the epithelial cells, which were in close contact with the pili of the bacteria. In some areas, positive immunogold-labelled bacteria intermingled with bacteria lacking such labelling. None of the culture-negative controls harboured epithelial cells with positive immunogold-labelled bacteria. Orally administered phenoxymethylpenicillin caused a significant reduction in both culture-positive S. pyogenes and bacteria displaying positive coating with specific gold-labelled antiserum to S. pyogenes.

Acute Disease↗
PubMed1991

Antigenic homology among gram-negative organisms isolated from cattle with clinical mastitis.

This study examined the degree of serologic homology among mastitis pathogens. Antibodies were raised against the Rc mutant, Escherichia coli O111:B4 (strain J5) and affinity purified against lipopolysaccharide derived from the Ra mutant, Salmonella typhimurium TV119. These antibodies reacted with a battery of unrelated Gram-negative bacteria in whole cell ELISA. Bacteria with strong cross-reactions included a heterologous, smooth E. coli, Salmonella dublin, S. typhimurium, Salmonella newport, and Pseudomonas aeruginosa. Recognition of Klebsiella pneumoniae and Bordetella bronchisepticum was observed, but reactions were weaker than with the other isolates. The reduced recognition of these isolates probably reflects a masking effect of the bacterial capsule and variations in lipopolysaccharide structure. The polyclonal antibody did not recognize a Gram-positive isolate, Staphylococcus aureus. These immunoglobulins were then tested using whole cell ELISA against a panel of bacteria recovered from the mammary glands of cattle with clinical mastitis. Marked reactivity was noted against a variety of Gram-negative pathogens. Gram-positive isolates had lower recognition by Gram-negative core antigen specific immunoglobulin. The results suggest immunization with rough mutant bacteria may have broad application in the prevention of coliform mastitis.

Animals↗
PubMed1998

[Efficacy of pneumococcal vaccination].

POLYVALENT VACCINE: Pneumococcal vaccination is based on the antigenic properties of polysides carried on the bacterial capsule which induce production of bactericidal serotype-specific antibodies. The 23 serotypes contained in the current vaccine cover 85 to 90% of all strains observed in pneumococcal infections in Europe. PROVEN EFFICACY: Prospective and retrospective studies have demonstrated that pneumococcal vaccination can prevent 60 to 80% of invasive pneumococcal infections in immunocompetent elderly subjects and/or persons in high-risk groups with underlying disorders. TARGET POPULATION: Immunocompetent persons in the 60-65 year and older age group comprise the target population for pneumococcal vaccination. Incidence and mortality of pneumococcal infections is high in this group. Simple vaccination strategies should be implemented.

Adult↗
PubMed1989

Definition of a bacterial virulence factor: sialylation of the group B streptococcal capsule.

Sialylation of bacterial capsules has been proposed as an important virulence factor for several species of encapsulated pathogens, including group B Streptococcus. We have constructed a transposon mutant strain of type III group B Streptococcus that expresses a capsular polysaccharide differing from the wild type only in that the mutant strain's capsule lacks sialic acid. The mutant polysaccharide is antigenically identical to the capsular polysaccharide of type 14 Streptococcus pneumoniae, as predicted by the structures of the type III group B Streptococcus and S. pneumoniae polysaccharides. Loss of capsular sialic acid was associated with loss of virulence in the mutant strain in a neonatal rat model of lethal group B Streptococcus infection. These studies demonstrate directly that capsular sialic acid is a critical virulence determinant for type III group B Streptococcus and support the general hypothesis that surface sialylation aids pathogenic microorganisms in evading host defenses.

Bacterial Toxins↗
PubMed1992

[Immunology and diagnostic test results in Lyme borreliosis].

Borrelia burgdorferi (B. burgdorferi), the etiologic agent of Lyme borreliosis, shows both a variety of outer surface proteins with molecular weights between 16 and 100 kiloDalton (kD) and a 41 kD flagellar protein, which induce the immunologic response. Lipopolysaccharides, another constituent of the bacterial capsule, are responsible for the inflammatory reaction, constitutional symptoms and for the Jarisch-Herxheimer reaction. First a vigorous T-cell immune response develops, followed later by a more slowly evolving humoral B-cell immune response. The delayed onset of the humoral immune response may explain why antibodies against B. burgdorferi could not be detected early in the course of the disease. But the antibody titers increase with duration of the illness. The humoral response shows the usual pattern of IgM appearing first, followed by IgG and IgA. The IgM titer normalizes after recovery while the IgG titer could persist over years or decades. It is hardly possible to detect B. burgdorferi microscopically or by cultivation from blood, joint or cerebrospinal fluid. For routine diagnosis the fluorescent antibody staining and the ELISA methods are available which detect IgM or IgG antibodies against B. burgdorferi. But the sensitivity and specificity of these tests are still unsatisfactory. Other methods such as the ELISA-capture method, complement binding reaction, passive hemagglutination or the polymerase chain reaction are not yet established for routine purposes. Western blot analysis did not yield an essential diagnostic advantage but may be helpful in long term observation or in special cases. The measurement of the cellular immune response by T-cell proliferation tests remains controversial. First of all Lyme borreliosis has to be diagnosed by clinical findings and by elimination through differential diagnosis. An elevated antibody titer or a positive T-cell proliferation test may confirm the diagnosis but cannot prove it. Without consistent clinical findings they are of no practical significance. Some general guidelines for interpretation of laboratory results are given.

Antibodies, Bacterial↗
PubMed1986

Haemophilus influenzae b polysaccharide vaccine. Physician acceptance and use of a new vaccine.

The introduction of Haemophilus Influenzae type b (Hib) polysaccharide vaccine in 1985 provided an opportunity to study the extent to which physicians have adopted the use of the new vaccine in their routine practice behavior, the factors that predict Hib vaccine use by physicians, and how physicians have chosen to deal with differing recommendations for its administration. We surveyed all physicians providing primary care to children in New Mexico six to eight months after vaccine licensure to assess their knowledge of existing recommendations and their current behavior regarding use of the Hib vaccine. Of the 369 primary care physicians who responded, 100% of pediatricians, 98% of family practitioners, and 91% of general practitioners were aware that a vaccine against invasive Hib disease had been licensed. Sixty-three percent of physicians surveyed were currently using the vaccine. Vaccine usage varied significantly by specialty, with 86% of pediatricians reporting use compared with 61% of family practitioners and 31% of general practitioners. Physicians were significantly more likely to use Hib vaccine if they were young and if they worked in the private sector rather than the public sector. The three sets of recommendations for Hib vaccine use had been widely read; 85% of respondents had read at least one set. Practitioners confronted with differing recommendations had elected to give the vaccine permissively within existing guidelines. Our findings suggest that practitioners have adapted quickly to the introduction of Hib vaccine but that nonuse of the vaccine in the public sector remains an obstacle to full implementation of this prevention strategy.

Attitude of Health Personnel↗