P fimbriae and other adhesins enhance intestinal persistence of Escherichia coli in early infancy.
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Resident and transient Escherichia coli strains were identified in the rectal flora of 22 Pakistani infants followed from birth to 6 months of age. All strains were tested for O-antigen expression, adhesin specificity (P fimbriae, other mannose-resistant adhesins or type 1 fimbriae) and adherence to the colonic cell line HT-29. Resident strains displayed higher mannose-resistant adherence to HT-29 cells, and expressed P fimbriae (P = 0.0036) as well as other mannose-resistant adhesins (P = 0.012) more often than transient strains. In strains acquired during the first month of life, P fimbriae were 12 times more frequent in resident than in transient strains (P = 0.0006). The O-antigen distribution did not differ between resident and transient strains, and none of the resident P-fimbriated strains belonged to previously recognized uropathogenic clones. The results suggest that adhesins mediating adherence to intestinal epithelial cells, especially P fimbriae, enhance the persistence of E. coli in the large intestine of infants. (+info)
Genetic analysis of the Serratia marcescens N28b O4 antigen gene cluster.
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The Serratia marcescens N28b wbbL gene has been shown to complement the rfb-50 mutation of Escherichia coli K-12 derivatives, and a wbbL mutant has been shown to be impaired in O4-antigen biosynthesis (X. Rubires, F. Saigi, N. Pique, N. Climent, S. Merino, S. Alberti, J. M. Tomas, and M. Regue, J. Bacteriol. 179:7581-7586, 1997). We analyzed a recombinant cosmid containing the wbbL gene by subcloning and determination of O-antigen production phenotype in E. coli DH5alpha by sodium dodecyl sulfate-polyacrylamide electrophoresis and Western blot experiments with S. marcescens O4 antiserum. The results obtained showed that a recombinant plasmid (pSUB6) containing about 10 kb of DNA insert was enough to induce O4-antigen biosynthesis. The same results were obtained when an E. coli K-12 strain with a deletion of the wb cluster was used, suggesting that the O4 wb cluster is located in pSUB6. No O4 antigen was produced when plasmid pSUB6 was introduced in a wecA mutant E. coli strain, suggesting that O4-antigen production is wecA dependent. Nucleotide sequence determination of the whole insert in plasmid pSUB6 showed seven open reading frames (ORFs). On the basis of protein similarity analysis of the ORF-encoded proteins and analysis of the S. marcescens N28b wbbA insertion mutant and wzm-wzt deletion mutant, we suggest that the O4 wb cluster codes for two dTDP-rhamnose biosynthetic enzymes (RmlDC), a rhamnosyltransferase (WbbL), a two-component ATP-binding-cassette-type export system (Wzm Wzt), and a putative glycosyltransferase (WbbA). A sequence showing DNA homology to insertion element IS4 was found downstream from the last gene in the cluster (wbbA), suggesting that an IS4-like element could have been involved in the acquisition of the O4 wb cluster. (+info)
Structural analysis of a novel putative capsular polysaccharide from Pseudomonas (Burkholderia) caryophylli strain 2151.
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A novel putative capsular polysaccharide consisting of D-Glcp and D-Fruf in the molar ratio of 1:1 was isolated as minor constituent from the lipopolysaccharide (LPS) fraction of Pseudomonas (Burkholderia) caryophylli. Its structure was determined, using mainly one- and two-dimensional NMR spectroscopy, as: -->6)-alpha-D-Glcp-(1-->1)-beta-D-Fruf-(2-->. (+info)
Structural and serological studies on the O-antigen of Proteus mirabilis O14, a new polysaccharide containing 2-[(R)-1-carboxyethylamino]ethyl phosphate.
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An O-specific polysaccharide was obtained by mild acid degradation of Proteus mirabilis O14 lipopolysaccharide (LPS) and found to contain D-galactose, 2-acetamido-2-deoxy-D-glalactose, phosphate, N-(2-hydroxyethyl)-D-alanine (D-AlaEtn), and O-acetyl groups. Studies of the initial and O-deacetylated polysaccharides using one- and two-dimensional 1H- and 13C-NMR spectroscopy, including COSY, TOCSY, NOESY, H-detected 1H,13C heteronuclear multiple-quantum coherence, and heteronuclear multiple-bond correlation experiments, demonstrated the following structure of the repeating unit: [equation: see text] This is the second bacterial polysaccharide reported to contain alpha-D-Galp6PAlaEtn, whereas the first one was the O-antigen of P. mirabilis EU313 taken erroneously as strain PrK 6/57 from the O3 serogroup [Vinogradov, E. V., Kaca, W., Shashkov, A.S., Krajewska-Pietrasik, D., Rozalski, A., Knirel, Y.A. & Kochetkov, N.K. (1990) Eur. J. Biochem., 188, 645-651]. Anti-(P. mirabilis O14) serum cross-reacted with LPS of P. mirabilis EU313 and vice versa in passive hemolysis and ELISA. Absorption of both O-antisera with the heterologous LPS decreased markedly but did not abolish the reaction with the homologous LPS. These and chemical data indicated that both strains have similar but not identical O-antigens. Therefore, we propose that P. mirabilis EU313 should belong to a new subgroup of the O14 serogroup. (+info)
Structure of the O-specific polysaccharide of a serologically separate strain Proteus penneri 2 from a new proposed serogroup O66.
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O-specific polysaccharide chain of Proteus penneri strain 2 lipopolysaccharide was studied by full and partial acid hydrolysis, Smith degradation, methylation analysis, and NMR spectroscopy, including two-dimensional rotating-frame NOE spectroscopy (ROESY) and 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments. Together with D-glucose and 2-acetamido-2-deoxy-D-glucose, the polysaccharide was found to contain two rarely occurring sugars, 6-deoxy-L-talose (L-6dTal) and 2,3-diacetamido-2,3,6-trideoxy-L-mannose (L-RhaNAc3NAc), and the following structure of a non-stoichiometrically O-acetylated tetrasaccharide repeating unit was established: [equation: see text] The O-specific polysaccharide studied has a unique composition and structure and, accordingly, P. penneri 2 is serologically separate among Proteus strains. Therefore, we propose for P. penneri 2 a new Proteus O-serogroup O66 where this strain is at present the single representative. (+info)
Protein conjugates of synthetic saccharides elicit higher levels of serum IgG lipopolysaccharide antibodies in mice than do those of the O-specific polysaccharide from Shigella dysenteriae type 1.
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Our development of vaccines to prevent shigellosis is based on the hypothesis that a critical (protective) level of serum IgG to the O-specific polysaccharide (O-SP) domain of Shigella lipopolysaccharide (LPS) confers immunity. The O-SP is a hapten and must be conjugated to a protein to induce serum antibodies. The O-SP of Shigella dysenteriae type 1 (approximately 27 tetrasaccharide repeat units), prepared by acid hydrolysis of the LPS, was bound to human serum albumin (HSA) by multiple point attachment (O-SP-HSA): The molar ratio of HSA to O-SP was 1.0. Synthetic saccharides, composed of one or multiples of the O-SP tetrasaccharide, equipped with a spacer at their reducing end, were bound to HSA by a single point attachment: The average molar ratios of the saccharides to HSA ranged from 4 to 24. Serum IgG anti-LPS, elicited in mice by O-SP-HSA or synthetic tetra-, octa-, dodeca-, and hexadecasaccharide fragments, was measured by ELISA. Outbred 6-week-old female mice were injected s.c. three times at biweekly intervals with 2.5 micrograms of saccharide as a conjugate and were bled 7 days after the second and third injections. Excepting the tetramer, conjugates of the octamer, dodecamer and hexadecamer elicited IgG LPS antibodies after the second injection, a statistically significant rise (booster) after the third injection, and higher levels than those vaccinated with O-SP-HSA (P = 0.0001). The highest geometric mean levels of IgG anti-LPS were elicited by the hexadecamer with 9 chains or 9 moles of saccharide/HSA (15.5 ELISA units) followed by the octamer with 20 chains (11.1 ELISA units) and the dodecamer with 10 chains (9.52 ELISA units). Clinical evaluation of these synthetic saccharides bound to a medically useful carrier is planned. (+info)
Identification of Acinetobacter baumannii strains with monoclonal antibodies against the O antigens of their lipopolysaccharides.
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Despite the emergence of Acinetobacter baumannii strains as nosocomial pathogens, simple methods for their phenotypic identification are still unavailable. Murine monoclonal antibodies specific for the O-polysaccharide moiety of the lipopolysaccharide (LPS) of two A. baumannii strains were obtained after immunization with heat-killed bacteria. The monoclonal antibodies were characterized by enzyme immunoassay and by Western and dot blot analyses and were investigated for their potential use for the identification of A. baumannii strains. The antibodies reacted with 46 of the 80 A. baumannii clinical isolates that were investigated, and reactivity was observed with 11 of 14 strains which were isolated during outbreaks in different northwestern European cities; no reactivity was observed with Acinetobacter strains of other genomic species, including the closely related genomic species 1 (Acinetobacter calcoaceticus), 3, and 13 sensu Tjernberg and Ursing, or with other gram-negative bacterial strains. The results show that O-antigen-specific monoclonal antibodies such as the ones described are convenient reagents which can be used to identify Acinetobacter strains in clinical and research laboratories. (+info)
Safety and immunogenicity of Shigella sonnei and Shigella flexneri 2a O-specific polysaccharide conjugates in children.
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O-specific polysaccharide conjugates of shigellae were safe and immunogenic in young adults, and a Shigella sonnei conjugate conferred protection [1-3]. Shigellosis is primarily a disease of children; therefore, the safety and immunogenicity of S. sonnei and Shigella flexneri 2a conjugates were studied in 4- to 7-year-old children. Local and systemic reactions were minimal. The first injection of both conjugates elicited significant rises in geometric mean levels of serum IgG only to the homologous lipopolysaccharide (LPS) (S. sonnei, 0.32-8.25 ELISA units [EU]; S. flexneri 2a, 1.15-20.5 EU; P<.0001). Revaccination at 6 weeks induced a booster response to S. flexneri 2a LPS (20.5-30.5 EU, P=.003). Six months later, the geometric mean levels of IgG anti-LPS for both groups were higher than the prevaccination levels (P<.0001). Similar, but lesser, rises were observed for IgM and IgA anti-LPS. The investigational Shigella conjugates were safe and immunogenic in children and merit evaluation of their efficacy. (+info)