- Volume 26, Issue 1, 1988
Volume 26, Issue 1, 1988
- Articles
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Adherence of Vero cytotoxin-producing Escherichia coli of serotype O157:H7 to human epithelial cells in tissue culture: role of outer membranes as bacterial adhesins
More LessSUMMARYEscherichia coli of serotype O157: H7 are Vero cytotoxin-producing enteric pathogens that have recently been associated with outbreaks of haemorrhagic colitis, sporadic cases of haemorrhagic colitis and with the haemolytic uraemic syndrome. The organisms demonstrate attaching and effacing binding to the caecum and colon of orally infected gnotobiotic piglets, chickens and infant rabbits. E. coli O157:H7 cells adhere to the surface but do not invade the cytoplasm of human epithelial cell lines in tissue culture. Since outer membranes, lipopolysaccharides and flagella have been identified as bacterial adhesins on other enteric pathogens, we evaluated their roles in the binding of non-fimbriated E. coli O157:H7 to HEp-2 cells. Hyperimmune rabbit antisera were prepared to whole cells, outer membranes and flagella of E. coli O157:H7. The presence of antibody to homologous antigen was confirmed by dot blot immunoassays. Both antisera and purified outer membrane and flagellar antigens were co-incubated with bacteria and HEp-2 cells to quantitate inhibition of bacterial attachment. Adherence of E. coli O157:H7 to tissue culture cells was inhibited by rabbit antisera raised to whole cells (76.0 ± 5.6% inhibition compared with bacterial adherence in the presence of pre-immune rabbit serum) and outer membranes (69.2 ± 3.4% inhibition). In contrast, inhibition of bacterial attachment to tissue-culture cells was significantly less when two antisera to H7 flagella were co-incubated with E. coli O157:H7 and HEp-2 cells (12.4 ± 7.6%; 6.0 ± 3.5% inhibition). Outer-membrane extracts inhibited adherence to E. coli O157:H7 to HEp-2 cells in a concentration dependent manner whereas isolated flagella and lipopolysaccharide antigens did not inhibit bacterial attachment. These data suggest that constituents in the outer membranes of E. coli O157:H7 mediate binding of the organisms to epithelial cells in vitro.
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Adhesion of clinical and environmental Aeromonas isolates to HEp-2 cells
More LessSUMMARYA total of 63 Aeromonas strains isolated from diarrhoeal faeces or water samples were tested for adhesion to HEp-2 cells. An association between diarrhoea and high level adhesion was observed in that 12 of the 34 faecal isolates and none of the 29 environmental isolates yielded > 20 bacteria per HEp-2 cell in the adhesion assay. The proportion of high adherers was significantly greater for A. sobria (57%) than for A. hydrophila isolates (19%). Three of the eight faecal A. caviae isolates were also found to be high adherers. All of the environmental isolates were heavily pilated with pili having a mean diameter of 5 nm and a mean length of 420 nm; these were termed type-S pili. Of the 34 faecal isolates, 32% possessed S pili, and 68% were lightly pilated with up to 15 thin, flexible type-L pili, of mean diameter 2.5 nm and mean length 960 nm. Type-L pilation was associated with a high level of HEp-2 cell adhesion, and was more common in A. sobria and A. caviae than in A. hydrophila isolates. These results suggest that adherence to HEp-2 cells is a useful model for the investigation of Aeromonas enteropathogenicity, and that adhesion may be pilusmediated in this organism.
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Penetration of immunoglobulins through the Klebsiella capsule and their effect on cell-surface hydrophobicity
More LessSUMMARYThe ability of antibodies to cell-surface components of Klebsiella to increase surface hydrophobicity and to gain access to antigens potentially masked by the capsule was investigated. Treatment of capsulate or non-capsulate strains with the respective autologous antiserum resulted in a marked increase in surface hydrophobicity. Antisera raised against a rough non-capsulate (K−O-) strain had little effect on the surface hydrophobicity of either of the capsulate strains K1+O1+ and K2+ O1+, or of the non-capsulate K− O1+ strain. Whereas anti-K−O1+ sera or anti-K2+ sera increased the surface hydrophobicity of the K2+ O1+ strain, only antisera containing anti-K1+ antibodies increased the hydrophobicity of the K1+ O1+ strain. Immunoadsorption of anti-K−O1+ serum by whole capsulate cells revealed that neither the K1 nor the K2 capsular polysaccharide acted as a barrier to anti-O antibodies but that the K1 capsular polysaccharide masked the presence of the immunoglobulin at the cell surface. The Klebsiella capsular polysaccharide does not appear to present a permeability barrier to immunoglobulins although failure to detect outer-membrane proteins in the immune complexes of either of the capsulate strains or of the K−O1+ strain suggests that the O antigen may prevent access of antibodies to these antigens.
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Longitudinal study of brucellosis in mice by immunoassay of lipopolysaccharide-related antigens in blood and urine
More LessSUMMARYImmunoassays based on latex agglutination or enzyme labelling (ELISA) were devised for the detection of lipopolysaccharide (LPS) of Brucella abortus, or its degradation products, in biological fluids of infected mice. The agglutination of latex was measured by counting of the remaining non-agglutinated particles in an automated immunoassay analyser. LPS was assayed by agglutination with antibodycoated latex and by competitive inhibition of agglutination of LPS-coated latex by anti-LPS antiserum. The inhibition system was more sensitive for the detection of degradation products of LPS. Correlation between ELISA and agglutination inhibition immunoassay was excellent (r = 0.96). Degradation of LPS occurred during storage, particularly when the samples contained specific antibodies. It could be prevented by removing cells immediately after collecting blood samples and by heating or alkaline denaturation of plasma.
CBA/H mice were infected with various doses [65-(65 × 106) cfu] of B. abortus biovar 3 cells and the course of infection followed by immunoassay of LPS-related antigens in serum and urine, and by titration of specific antibodies and non-specific circulating immune complexes. The concentration of LPS degradation products, assayed by the agglutination inhibition assay, was related to the severity of the infection, which was assessed by viable counts of B. abortus in the spleen. A close correlation was observed between the values of antigenaemia, the number of cfu (r = 0.97), and the inoculum size (r = 0.99 at day 28).
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Identification of leptospiral flagellar antigens by gel electrophoresis and immunoblotting
More LessSUMMARYFlagella extracted from five serovars, representative of the pathogenic and saprophytic species of the Leptospiraceae, were morphologically similar. Analysis of Leptospira interrogans flagellar preparations by polyacrylamide gel electrophoresis revealed three common major bands in the (30-40) × 103-mol. wt region, and serovar-specific bands in the lower region of the gels. Although some differences were observed, flagella extracted from L. biflexa serovar patoc and Leptonema illini revealed similar electrophoretic profiles to those seen in L. interrogans flagella. Immunoblot analysis showed that while flagellar components in the (20−30) × 103-mol. wt region were recognised only by homologous rabbit antisera, a major protein doublet of (33−34) × 103 or (35−36) × 103-mol. wt, depending on the species, was also demonstrated by heterologous antisera. The serovar-specific bands in the (20−30) × 103-mol. wt region were composed of lipopolysaccharide (LPS). These results show that leptospiral flagella are immunogenic and contain antigens which are conserved among the different genera of the family Leptospiraceae.
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Role of murine macrophages and complement in experimental campylobacter infection
More LessSUMMARYThe roles of macrophages and the complement system as potential host defence mechanisms in mice against campylobacter infection were studied in vivo, by depleting the murine serum-complement or the phagocytic cells. Macrophage-depletion was performed by intraperitoneal (i.p.) injection of silica dust, Liquoid or dextran sulphate. During 5 days after infection, such mice showed a significant increase in mortality, compared with controls. In contrast, mice that were previously decomplemented by i.p. injection of Cobra Venom Factor showed no significant increase in mortality. The results with combined macrophage depletion and decomplementation did not differ from those with macrophage depletion alone. These experiments suggest that macrophages seem to be more important than complement in the defence of mice against experimental campylobacter infection.
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Bacteriuria and bacteraemia in patients with long-term indwelling catheters-a domiciliary study
More LessSUMMARYMen with indwelling catheters and men and women with suprapubic catheters were studied in their homes. Urine and blood were cultured and body temperature recorded after every catheter change. Nearly all patients had infected urine after 4 weeks of catheterisation, and all had bacteriuria after longer periods, usually with a mixture of organisms. Culture on selective media revealed a wider range of organisms than was detected on routine C.L.E.D. and blood agar with antibiotic sensitivity disks, but routine culture gave adequate information for clinical purposes. Bacteraemia was demonstrated after 20 of 197 changes of urethral catheter and after one of 19 changes of suprapubic catheter; but no patient had pyrexia or other symptoms. However, two had rigors on other occasions. When assessing “risk factors” for blood-stream infection in catheterised patients, it is important to record the total incidence of bacteraemia, asymptomatic as well as symptomatic.
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Trimethoprim resistance determinants encoding a dihydrofolate reductase in clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci
More LessSUMMARYThe molecular and biochemical basis of resistance to high concentrations (MIC ± 1000 mg/L) of trimethoprim (Tpr(h)) was examined in Australian isolates of Staphylococcus aureus and coagulase-negative staphylococci. The Tpr(h) determinant (dfrA) was located within a 2.75-Kb Bg/II fragment on the S. aureus aminoglycoside-resistance plasmids pSK1 and pSK16 as judged by comparative restriction mapping with two naturally-occurring variants, pSK9 and pSK14, which did not encode trimethoprim resistance. This was confirmed in DNA-DNA hybridisation experiments in which a 0.9-Kb sequence of pSK1 DNA was used as a specific probe for the Tpr (h) gene. Plasmid DNA from three strains of coagulase-negative staphylococci, and the chromosomal material of one other isolate, were found to share homology with the probe DNA. Dihydrofolate reductases produced by these strains were virtually identical to the type S1 enzyme encoded by the S. aureus plasmid pSK1. Interspecies transfer may have been responsible for the conservation of Tpr(h) gene sequences among staphylococci.
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