Analysis of electrophoretically heterogeneous lipopolysaccharides of Escherichia coli by immunoblotting

Karch, Helge

doi:10.1016/0378-1097(84)90007-7

Cited by 10 publications

(11 citation statements)

References 8 publications

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“…However, when C. jejuni LPS was analyzed by SDS-PAGE and visualized by the immunoblotting method, the LPSs of some strains appeared to have a long 0-specific side chain forming high-molecular-weight ladderlike bands which could not be visualized by silver staining (38). The immunoblotting method has been found to be superior to traditional silver staining for visualization of LPS profiles on SDS-PAGE gel (12,15,24,31,38).…”

Section: Resultsmentioning

confidence: 99%

Electrophoretic and chemical characterization of lipopolysaccharides of Vibrio parahaemolyticus

Han

Chai

1992

J Bacteriol

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Lipopolysaccharides (LPSs) isolated from three Kanagawa-positive and three negative strains of Vibrio parahaemolyticus were characterized by using electrophoretic, immunochemical, and chemical methods. The results of this study indicated that the LPSs of all six strains of V. parahaemolyticus examined did not have an 0-specific side chain. These V. parahaemolyticus LPSs appeared to have molecular weights similar to that of the rough-type (Ra) LPS of Salmonella typhimurium TV-119 and might just contain lipid A and a core region. However, the microheterogeneity of V. parahaemolyticus LPS observed was greater than that of S. typhimurium LPS. The profile of V. parahaemolyticus LPS consisted of closely spaced triplet or quadruplet bands, but that of S. typhimurium consisted of doublet bands. Slower-moving bands appeared on sodium dodecyl sulfatepolyacrylamide gel electrophoresis gels only when large amounts of V. parahaemolyticus LPS were loaded. These bands were proven to be the aggregates of the fastest-moving low-molecular-weight bands by re-electrophoresis. The banding pattern of V. parahaemolyticus LPSs produced on nitrocellulose membranes by immunoblotting indicated that the V. parahaemolyticus LPSs did not have an 0-specific side chain. The low ratio of total carbohydrate to lipid A of V. parahaemolyticus LPSs also suggested that they were like rough-type LPS. The mobility and profile of V. parahaemolyticus LPS on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and its chemical composition were closely related to the serotype of a specific strain but not with the Kanagawa phenomenon.Vibrio parahaemolyticus is a gram-negative pathogen to humans and other creatures (47). Since it is an inhabitant of estuarine and coastal waters of the ocean (28), fresh seafood is often contaminated with this pathogen (41). Most gastroenteritis outbreaks resulting from seafood consumption are caused by V. parahaemolyticus (22). As seafood consumption is increasing in this country, it is becoming more and more important to understand this pathogen and its virulence factors. However, the virulence factors of pathogenic V. parahaemolyticus are still unknown and not all V. parahaemolyticus strains are pathogenic (23). Most (96.5%) of the pathogenic strains isolated from patients are able to lyse erythrocytes on blood agar medium containing 7% NaCl (44), i.e., they are Kanagawa positive (K+), but 99% of the strains isolated from natural environments do not have such hemolytic activity, i.e., they are Kanagawa negative (K-), and are usually nonpathogenic (23). Although the thermostable direct hemolysin which accounts for K+ activity has fluid-accumulating activity in the rabbit ileal loop (46), identification of thermostable direct hemolysin as the virulence factor is still unclear owing to its relatively low activity (21 Extraction of LPS. LPS was extracted from lyophilized cells by using the phenol-water method (45). This method had been demonstrated to be better than the phenol-chloroform-petroleum ether method (13) fo...

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Section: Resultsmentioning

confidence: 99%

Electrophoretic and chemical characterization of lipopolysaccharides of Vibrio parahaemolyticus

Han

Chai

1992

J Bacteriol

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“…A 20-,ug portion of lipopolysaccharide of the respective strain was applied to the gel; conditions corresponded to those maintained for the outer membrane analysis. Gels were developed by the silver-staining procedure (16 Table 2). Variants selected in the presence of imipenem (Imir) exhibited resistance exclusively to imipenem (Table 2).…”

Section: * Corresponding Authormentioning

confidence: 99%

Imipenem resistance in Pseudomonas aeruginosa resulting from diminished expression of an outer membrane protein

Büscher¹,

Cullmann²,

Dick³

et al. 1987

Antimicrob Agents Chemother

117

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The mechanism of Pseudomonas aeruginosa resistance to imipenem in five imipenem-susceptible clinical isolates and in their resistant counterparts was investigated. The frequency for selecting imipenem-resistant variants ranged from 2.7 x 10-5 to 2.1 x 10-8 and was comparable to those for other ,-lactams.Cross-resistance between imipenem and other 13-lactam compounds was not observed. In all imipenemresistant variants, induction of chromosomal j3-lactamase by imipenem was markedly diminished compared with that in the susceptible parent strain. This was not the case for other inducers such as ampicillin or cefoxitin, suggesting an impaired uptake of imipenem as an explanation for resistance. Analysis of the outer membrane proteins revealed a marked decrease of either a 46-or a 45-kilodalton protein. The lipopolysaccharide of the outer membrane in the imipenem-resistant variants was not altered.Within the last 2 years, several reports have emphasized the development of resistance to imipenem in clinical Pseudomonas aeruginosa isolates (1,8,10,19,22,24). Emergence of resistance was observed especially in patients suffering from either cystic fibrosis or nosocomial lower respiratory tract infections (1,19,22). Consequently, development of cross-resistance between imipenem and other ,B-lactam compounds must be evaluated.Enzymatic inactivation of imipenem has been reported thus far only for Pseudomonas maltophilia (21). Since resistance to newer cephalosporins and broad-spectrum penicillins is often chromosomally mediated, we studied five imipenem-susceptible clinical isolates of P. aeruginosa and their derived imipenem-resistant counterparts for the production of imipenem-inactivating enzymes and for outer membrane alterations. As it was not clear whether mutation or selection of a resistant subpopulation occurred, the term "variants" will be used for the resistant counterparts.MATERIALS AND METHODS Strains. The P. aeruginosa strains used were recent clinical isolates from our laboratory and were identified by standard procedures (12). Strain FRA was kindly supplied by G. Seibert, Frankfurt, Federal Republic of Germany. None of the strains exhibited resistance to antipseudomonal P-lactam compounds.MICs. MICs were determined in Mueller-Hinton broth (E. Merck AG, Darmstadt, Federal Republic of Germany) by twofold serial dilutions representing a final inoculum of 5 x 105 CFU/ml (microdilution procedure).Selection of resistant variants. Resistant variants were selected by plating 0.05 ml of an overnight culture in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) onto Mueller-Hinton agar plates (Merck) containing the antibiotic in twofold serial dilution steps above the MIC. The frequency of resistant variants was expressed as the ratio of CFU grown in the presence of the antibiotic to the CFU of the control (grown without an antibiotic). All assays were carried out in triplicate. * Corresponding author.Kinetics of j3-lactamase production. ,B-Lactamase production was studied during exponential growth. Ove...

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“…2(b). Such differences in the detection of LPS sidechains between silver-staining and Western blotting have been described previously in the LPS of E. coli (Karch et al, 1984), Coxiella burnetii (Hackstadt et al, 1985) and Campylobacter jejuni (Preston & Penner, 1987). The differences observed between the LPS of the biotype A and T isolates provide further evidence that these two groups should be regarded as different species (Biberstein & Francis, 1968;Bisgaard & Mutters, 1986).…”

Section: Discussionmentioning

confidence: 52%

“…Characterization of the LPS profiles of large numbers of isolates of a given bacterial species is able to identify strain differences and subtypes within that species and such analyses have proved extremely useful in epidemiological and virulence studies (Inzana, 1983;Tsai et al, 1983;Lugtenberg et al, 1984;Rimler, 1990). Variation in LPS structure can be further analysed by examining antigenic differences by Western blotting (Karch et al, 1984;Sturm et al, 1984;Manning et al, 1986;Preston & Penner, 1987;Tsai et al, 1987).…”

Section: Introductionmentioning

confidence: 99%

Lipopolysaccharide heterogeneity in Pasteurella haemolytica isolates from cattle and sheep

Ali

Davies

Parton

et al. 1992

Journal of General Microbiology

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Analysis of electrophoretically heterogeneous lipopolysaccharides of Escherichia coli by immunoblotting

Cited by 10 publications

References 8 publications

Electrophoretic and chemical characterization of lipopolysaccharides of Vibrio parahaemolyticus

Electrophoretic and chemical characterization of lipopolysaccharides of Vibrio parahaemolyticus

Imipenem resistance in Pseudomonas aeruginosa resulting from diminished expression of an outer membrane protein

Lipopolysaccharide heterogeneity in Pasteurella haemolytica isolates from cattle and sheep

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