- Volume 23, Issue 2, 1987
Volume 23, Issue 2, 1987
- Short Article
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Variations in the virulence, for pregnant guinea pigs, of campylobacters isolated from man
More LessSummaryPregnant guinea pigs were used to compare the virulence of four human isolates of Campylobacter fetus ss. fetus and four of C. jejuni on the basis of their ability to cause abortion and bacteraemia. Of the four strains of C. fetus ss. fetus two produced abortion readily after intramuscular injection. The four C. jejuni isolates were, however, of comparatively low virulence and no differences between them were demonstrated. Some of the isolates differed in their ability to survive in vitro in human and guinea-pig serum. It is suggested that Campylobacters vary in their virulence for man and that this may influence the outcome of infections. Guinea pigs may prove useful in studying the pathogenesis of systemic Campylobacter infections.
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- Article
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Western blot analysis of staphylococcal antibodies present in human sera during health and disease
More LessSummaryIgG antibodies directed against Staphylococcus aureus were examined by Western blotting in sera from 15 healthy individuals isolated for a year in Antarctica. Sera reacted with many staphylococcal antigens in whole-cell extracts and individuals showed unique and unchanging blot profiles. The IgG and IgM profiles of patients with deep-seated staphylococcal infections were also examined by Western blotting. Anti-staphylococcal IgM antibodies that reacted with an antigen of apparent molecular mass 31 × 103 were present in all patients with staphylococcal disease, and were absent from, or detected in much smaller amounts in, control sera.
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Plasmid-mediated resistance to gentamicin in Staphylococcus aureus: the involvement of a transposon
More LessSummaryResistance to gentamicin, tobramycin and kanamycin (GmrTmrKmr) in strains of Staphylococcus aureus isolated from clinical sources in Australia is mediated by a 4.7 kb transposable element, designated Tn4001. A 2.5 kb HindIII fragment which maps symmetrically within Tn4001, and encompasses the aminoglycosideresistance coding region, has been shown to hybridise with fragments of identical size in HindIII digests of three different GmrTmrKmr plasmids, two of which were self-transmissible, from strains of S. aureus isolated in the USA. Examination by electronmicroscopy of self-annealed molecules of the North American GmrTmrKmr plasmids revealed the presence of stem and loop structures similar to those produced by Tn4001, but with shorter inverted repeats. These results suggest that GmrTmrKmr in strains of S. aureus isolated in the USA is, or once was, transposable, and that transposable elements analogous to Tn4001 may be found in isolates of GmrTmrKmr S. aureus worldwide.
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Antibiotic resistances and plasmids in Staphylococcus aureus from Italian hospitals
More LessSummaryA total of 473 Staphylococcus aureus isolates from six Italian hospitals was examined for susceptibility to several antimicrobial agents and for plasmid content. Methicillin-resistant S. aureus (MRSA) were characterised by a plasmid of mol. wt (106) 18–22 or 25 that carried the determinants for penicillinase production, resistance to cadmium ions and resistance to tetracycline. MRSA isolates usually harboured other smaller plasmids of mol. wt (106) 2.8, 2.6 and 1.65 that encoded resistance to tetracycline, chloramphenicol and erythromycin, respectively, and cryptic plasmids of mol. wt (106) c. 2 and 1 were found frequently. Methicillin-sensitive S. aureus (MSSA) that produced penicillinase often carried plasmids of mol. wt (106) 11 or 13. No particular difference was found in plasmid patterns of strains from the various sources. Analysis of plasmids by EcoRI digestion showed that plasmids of similar mol. wt and phenotypic characteristics may have different restriction patterns, but often share one or more fragments in common.
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Bacteriophages associated with multiresistant Staphylococcus aureus in Australia
More LessSummaryNineteen multiresistant strains of Staphylococcus aureus from Australian hospitals were examined for lysogenic bacteriophage. Thirteen strains contained prophage inducible with mitomycin C. Three of these lysed completely on induction producing a phage referred to as type 1; this phage plated on S. aureus propagating strains 6, 53 and 77, which are hosts for phages of serogroup-lysogroup A III, B III and F III respectively. Type-1 phage did not plate on other propagating strains representative of the other serogroup-lysogroup combinations in the International Typing Set for S. aureus. Ten strains of S. aureus lysed incompletely when treated with mitomycin C, yielding phage type 2, that plated only on propagating strain 6. The virions of phage types 1 and 2 had isometric heads and flexible tails, and the genome consisted of c. 40 kilobases of double stranded DNA. The DNA from the two phage types was different, as shown by endonuclease digestion and by hybridisation to reference phage DNAs. The remaining six S. aureus strains contained no phage inducible with either mitomycin C or ultraviolet irradiation. However, all contained type 2 DNA, as shown by Southern blotting, present presumably in a defective prophage state. Moreover, the three strains yielding type-1 phage on induction also contained type-2 DNA. Thus, type-2 DNA was found in all 19 strains of multiresistant S. aureus from geographically diverse Australian hospitals.
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The susceptibility to lysozyme of β-lactamase-producing and non-producing derivatives of Staphylococcus aureus strain 1030
More LessSummaryThe killing of Staphylococcus aureus strain 1030 and derived variants of it by lysozyme increased with increased lysozyme concentrations or decreased concentrations of sodium chloride. β-Lactamase-producing and non-producing derivatives of strain 1030 were constructed. The former were less susceptible to lysozyme. Induction of β-lactamase synthesis with 2-2carboxyphenyl-benzoyl-6-penicillanic acid increased the resistance of producer strains to lysozyme. These results are discussed in relation to the spread of β-lactamase-producing strains of S. aureus.
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Extracellular and membrane-bound β lactamase of Staphylococcus aureus: their importance for the expression of penicillin resistance
W. Bruns and H. KeppelerSummaryThe synthesis and excretion of β lactamase by several strains of Staphylococcus aureus from different clinical sources and the ability of both the extracellular and membrane-bound enzyme to mediate penicillin resistance was studied. When β-lactamase production was maximally induced with penicillin G or ampicillin, about 50% of the β lactamase was excreted from the cells, the amount of extracellular enzyme correlating well with the degree of resistance established by an in-vitro test model. From penicillin-binding experiments it became apparent, however, that the membrane-bound β lactamase can also constitute a barrier, strong enough on its own to prevent penicillins from reaching their target. This could be of clinical relevance if, under certain conditions in vivo, the extracellular β lactamase is insufficient for full protection of the staphylococcal cells.
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Penicillin-binding proteins in Streptococcus faecalis and S. faecium
More LessSummaryPenicillin-binding proteins (PBPs) of Streptococcus faecalis NCTC 775, S. faecium NCTC 7171 and an isolate of S. faecium (strain 37) highly resistant to β-lactam antibiotics were visualised by autoradiography. Five PBPs were detected in S. faecalis NCTC 775 and six in S. faecium NCTC 7171. Additional PBPs could not be found in the resistant isolate of S. faecium.
The PBP affinities of β lactams were compared with MIC values. The affinities of PBPs 3 and 4 of S. faecalis NCTC 775 for penicillin G, ampicillin, cefathiamidine, cephaloridine and cephazolin were related to the sensitivity of the strain to these antibiotics as were the affinities of PBPs 4 and 5 in each S. faecium strain for the β lactams. It is postulated that PBPs 3 and 4 of S. faecalis NCTC 775 and PBPs 4 and 5 of S. faecium are the relevant target enzymes of the test antibiotics. PBPs 4 and 5 of the highly β-lactam-resistant S. faecium strain 37 showed proportionally low affinities for the five β lactams compared to that of the less resistant strain S. faecium NCTC 7171. Decreased affinities of PBPs 4 and 5 may account for the resistance in S. faecium strain 37 to β lactams. The affinities for PBP 1, 2 and 5 in S. faecalis NCTC 775 and PBPs 1, 2, 3 and 6 in S. faecium were not related to the antibiotic sensitivities.
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The effect of antibiotics on the cell morphology of Legionella pneumophila
More LessSummaryLegionella pneumophila, in Buffered Yeast Extract broth, was treated for 5 h at 37°C with rosaramicin, erythromycin, cefotaxime, dibekacin, penicillin, methicillin, cefoxitin, cephalothin, ticarcillin, carbenicillin or polymyxin B at near-MIC levels and above. Electronmicroscopy demonstrated morphological changes to the bacteria in some, but not all, of the antibiotic-treated suspensions. Penicillin, at 1000 µ/ml (40 × MIC) but not less, produced smooth bubble-like structures on cell surfaces; methicillin produced rough bubble-like structures at 100 µ/ml (MIC) but not at 1000 µ/ml. In each case, these structures resembled spheroplasts. Polymyxin B induced small-bleb formation on the bacterial cell surfaces at all concentrations tested (MIC-10 × MIC). The other eight antibiotics did not induce any morphological changes at any concentration tested.
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Relationship between lipopolysaccharide composition and virulence of Haemophilus ducreyi
More LessSummaryThe relationship between lipopolysaccharide (LPS) composition and virulence of Haemophilus ducreyi strains was investigated. Glycoses identified in LPS by gas-liquid chromatography were glucose, galactose, and their amino derivatives glucosamine and galactosamine. Fucose was found in trace amounts but mannose and rhamnose, characteristic of the O-side chain of LPS in many species, were not detected. Qualitatively, the LPS composition of the eight strains examined was similar and differences were mainly quantitative. The total glycose: KDO ratio of the LPS of virulent strains exceeded that of avirulent strains. All strains had similar fatty-acid composition but lacked lauric acid. SDS-polyacrylamide gel electrophoresis of the LPS of virulent and avirulent strains also revealed differences in their electrophoretic mobilities. The LPS profiles of avirulent strains were similar, but differed from those of virulent strains. These profiles lacked high mol. wt bands representing O-side chain repeating units. Thus, differences in the electrophoretic mobilities of the LPS of virulent and avirulent strains may reflect differences in the amount of carbohydrates associated with the core polysaccharide.
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A monoclonal antibody directed against a serotype-specific, outer-membrane protein of Haemophilus influenzae type b
More LessSummaryMonoclonal antibodies (Mabs) were produced against outer-membrane proteins (OMPs) of Haemophilus influenzae type b. The clones were screened by ELISA with outer-membrane preparations of H. influenzae type b and untypable strains as coating antigens. Antibodies directed against the proteins of mol. wt (103) 43, 37 and 13 were identified by immunoblotting of SDS-PAGE patterns of OMPs. Proteolytic enzyme treatments of the OMPs resulted in reduction of Mab reactivity as measured by ELISA. Furthermore, the absence of reactivity of Mab Hb-2 with a preparation of lipopolysaccharide confirmed the protein nature of its corresponding epitope. Binding assays with live bacteria showed that Hb-2 reacted with a cell surface-exposed antigenic determinant. Mab Hb-2 was bactericidal in vitro in the presence of complement. The characterisation of Hb-2 (IgG2a) by Western immunoblotting analysis revealed that it was directed against the 37 × 103-mol. wt OMP. In a dot-enzyme immunoassay, Hb-2 reacted specifically with 326 strains of H. influenzae type b. It did not cross-react with the other serotypes or untypable strains of H. influenzae or with other bacterial species. This is the first report of a monoclonal antibody identifying a serotype—specific surface-exposed OMP of H. influenzae type b.
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Purification of cytotoxic enterotoxin of Aeromonas sobria by use of monoclonal antibodies
More LessSummaryCytotoxic enterotoxin of Aeromonas sobria was purified by affinity chromatography with monoclonal antibodies. The purified enterotoxin gave a single protein band in polyacrylamide gradient gel electrophoresis and its mol. wt estimated by this technique was 63000; it had a pI of 6.2. The purified enterotoxin caused fluid accumulation in rat ileal loops and in infant mice, was cytotoxic to cultured cells, was haemolytic to human erythrocytes, and was lethal to mice after intravenous injection. The relative concentrations of enterotoxic, cytotoxic and haemolytic activities were approximately the same in a culture filtrate and in purified, electrophoretically homogeneous enterotoxin. The three activities were also inactivated to the same extent after incubation for 10 min at 56°C. There was no immunological cross-reactivity with cholera toxin (CT) nor did antiserum to CT neutralise the biological effects of the toxin.
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Aeromonas cytotonic enterotoxin cross reactive with cholera toxin
More LessSummaryIsolation by affinity chromatography from crude culture filtrate of Aeromonas sobria of protein that cross reacted with cholera toxin (CT) revealed a toxin that produced fluid accumulation in rat ileal loops and in infant mice and caused rounding of Y1 adrenal cells. All these activities were neutralised by antiserum to CT. There was no haemolytic or cytotoxic activity associated with this CT-cross reactive cytotonic enterotoxin. CT-cross reactive material detected in enzyme linked immunosorbent assay (ELISA) was produced by 25% of Aeromonas isolates from faeces of children with or without diarrhoea—26% of A. sobria, 20.0% of A. hydrophila and 24% of A. caviae tested gave positive ELISA results. Most strains that produced this cytotonic enterotoxin but no cytotoxic enterotoxin were isolated from children without diarrhoea. Toxin preparations from Aeromonas spp. that completely inhibited adenosine-5′-diphosphate-induced platelet aggregation, an effect related to elevation of intracellular cAMP, were, with one exception, cross reactive with CT in ELISA.
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