- Volume 36, Issue 3, 1992
Volume 36, Issue 3, 1992
- Article
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Serological and biological characteristics of “Streptococcus milleri” isolates from systemic purulent infections
More LessSummaryNinety-one Streptococcus milleri strains isolated from various systemic purulent lesions of 68 patients were examined by physiological and serological tests. Most strains formed a smooth colony (66 strains), did not form spontaneous aggregation of cells in BHI broth culture (79), were non-β-haemolytic (α-35 or non-41), and belonged to biotype Ia (49) or Ib (34) and to API taxa S. milleri I (41) or II (38). Almost all of the β-haemolytic strains as well as two-fifths of the non-β-haemolytic belonged to API taxon I; strains of API taxa II and III were non-β-haemolytic and non-haemolytic, respectively. Two-fifths (38) of the isolates belonged to one of eight serotypes, a—g and k, and more than half (47) to Lancefield groups A, C, F or G, the most frequent being type b (19) and group F (33). Fifteen strains carried simultaneously type a/group A, b/C, c/C, e/G, f/F or k/G antigens. Nineteen were neither typable nor groupable. All the 38 serotypable isolates were non-β-haemolytic and not members of API taxon III, and were serologically and physiologically similar to oral S. milleri. The isolates from various infected sites—sputum, thorax, abdomen, urogenitalia, skin, eye and dental—exhibited distinct combinations of biological and serological properties. These results suggest that serotyping, haemolytic properties and API taxon, and their combinations, would be useful methods to trace oral S. milleri in systemic infections.
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A numerical taxonomic study of the “Streptococcus milleri” group based upon conventional phenotypic tests and pyrolysis mass spectrometry
SummaryClinical strains presumptively identified as Streptococcus milleri (60), and blind coded collection strains (21) were characterised in conventional tests and pyrolysis mass spectrometry. Comparison of the clusters found by these two approaches revealed five clearly distinct centres of variation. Three corresponded to the DNA homology groups suggested by Whiley and Hardie (1989) as representing the species S. anginosus, S. intermedius and S. constellatus; a fourth comprised three Lancefield group C β-haemolytic strains; the fifth may represent a biotype of S. anginosus. The characteristics of the latter group are described.
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Localisation of the mitogenic epitope of staphylococcal enterotoxin B
More LessSummaryLimited digestion of staphylococcal enterotoxin B (SEB) with trypsin resulted in the generation of a 12-Kda amino-terminal fragment and a 17-Kda carboxy-terminal fragment which were isolated by preparative iso-electric focusing. The carboxy-terminal fragment exhibited significant mitogenic activity for murine splenocytes, whereas the isolated amino-terminal fragment possessed little detectable mitogenic activity. Monoclonal antibodies (MAbs) specific for the carboxy-terminal fragment neutralised most of the mitogenic activity of both the intact toxin and the carboxy-terminal fragment. MAbs specific for the amino-terminal fragment had no detectable neutralising activity. These results support the hypothesis that the epitope(s) responsible for mitogenic activity is located in the carboxy-terminal region of SEB.
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Comparison of enterotoxins and haemolysins produced by methicillin-resistant (MRSA) and sensitive (MSSA) Staphylococcus aureus
More LessSummaryA collection of 201 isolates of Staphylococcus aureus was examined: 152 methicillin-sensitive S. aureus (MSSA) comprised 48 blood culture isolates (BC) and 58 isolates from routine diagnostic specimens (RD) from Glasgow Royal Infirmary (GRI), and 46 strains from nasal swabs of patients attending a general practitioner (GP); 49 isolates were of methicillin-resistant S. aureus (MRSA) from GRI. We have previously shown that the MRSA could be divided into two sub-groups on the basis of sensitivity or resistance to aminoglycoside antibiotics. Production of enterotoxins A, B, C and D, and α-, β-, γ- and δ-haemolysins was detected by reverse passive latex agglutination (RPLA) and agar overlay methods respectively: 60% of BC MSSA and a similar proportion of MSSA from other sources produced enterotoxin; 87% of aminoglycoside-sensitive MRSA produced enterotoxin (89% of these produced enterotoxin A alone) whereas only 27% of aminoglycoside-resistant MRSA were enterotoxin-positive, significantly less than either MSSA or aminoglycoside-sensitive MRSA. The proportion of haemolysin-producing isolates did not differ amongst the isolates of MSSA and MRSA; there was no difference in the distributions of haemolysins between aminoglycoside-sensitive and -resistant strains of MRSA. GP MSSA had higher and lower numbers of γ- and δ-haemolysin producers respectively than other S. aureus isolates. α-Haemolysin producers were commoner amongst MRSA isolates, which were also more likely than MSSA isolates to produce several haemolysins. Differences in enterotoxin production between aminoglycoside-sensitive and -resistant MRSA isolates reflect subgroups previously defined by biotype, phage type, immunoblot and restriction enzyme fragmentation pattern data, and provide further evidence for the existence of two major MRSA clones in GRI.
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Use of contour-clamped homogeneous electric field (CHEF) electrophoresis to type methicillin-resistant Staphylococcus aureus
M.-Q. Wei, Fu Wang and W. B. GrubbSummaryStrains of methicillin-resistant Staphylococcus aureus (MRSA) from Australia and the UK were compared by digesting their chromosomal DNA with the low-frequency-cutting restriction enzyme SmaI and separating the restriction fragment length polymorphisms (RFLPs) by contour-clamped homogeneous electric field (CHEF) electrophoresis. The numbers of restriction fragments produced were in the range 14–17 and the sizes of the bands were 7–700 kb. Generally, the results confirmed previous conclusions based on antimicrobial resistance and plasmid profiles. The earlier MRSA isolates were different from more recent isolates, and the epidemic MRSA from eastern Australia (EA MRSA) was the same as the epidemic MRSA (EMRSA) found in London hospitals. However, contrary to previous results, the EA MRSA did not constitute a homogeneous group. The results showed that comparison of RFLPs by CHEF electrophoresis is a useful technique for studying the epidemiology of MRSA.
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Identification of a human lactoferrin-binding protein in Staphylococcus aureus
More LessSummaryHuman lactoferrin (HLf) is an iron-binding protein with antimicrobial activity that is present in high concentrations in milk and various exocrine secretions. HLf is also an acute-phase protein secreted by polymorphonuclear leucocytes, and its binding to a large number of clinical isolates of Staphylococcus aureus has been described recently from our laboratory. We have now characterised the HLf-staphylococcal interaction in S. aureus strain MAS-89. The binding of 125I-HLf to strain MAS-89 reached saturation in <90 min and was maximal between pH 4 and 9. Unlabelled HLf displaced 125I-HLf binding. Various plasma and subepithelial matrix proteins, such as IgG, fibrinogen, fibronectin, collagen and laminin, which are known to interact specifically with S. aureus, did not interfere with HLf binding. A Scatchard plot was non-linear; this implied a low affinity (1‡55 × 107 L/mol) and a high affinity (2‡70 × 108 L/mol) binding mechanism. We estimated that there were c. 5700 HLf binding sites/cell. The staphylococcal HLf-binding protein (HLf-BP) was partially susceptible to proteolytic enzymes or periodate treatment and was resistant to glycosidases. An active HLf-BP with an apparent Mr of c. 450 Kda was isolated from strain MAS-89 cell lysate by ion-exchange chromatography on Q-sepharose. In SDS-PAGE, the reduced HLf-BP was resolved into two components of 67 and 62 Kda. The two components demonstrated a positive reaction with HLf-HRPO in a Western blot. These data establish that there is a specific receptor for HLf in S. aureus.
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Isolation of a novel siderophore from Pseudomonas cepacia
More LessSummaryA novel iron-binding compound was identified in ethyl acetate extracts of the supernates from Pseudomonas cepacia cultures. This compound, named azurechelin, was produced by 88% of P. cepacia strains isolated from the respiratory tract. Production of azurechelin was regulated by the iron concentration in the culture medium. Azurechelin enhanced the growth of P. cepacia in a medium containing transferrin 200 mg/L. Azurechelin released iron from transferrin in an equilibrium dialysis assay, suggesting that it could compete with transferrin for iron. Azurechelin could also stimulate iron uptake by P. cepacia. This siderophore appeared to have a novel structure with neither the typical characteristics of catechol nor of hydroxamate compounds.
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A non-haemagglutinating form of Clostridium difficile toxin A
More LessSummaryAnalysis of crude culture filtrate of Clostridium difficile by Mono Q-anion exchange fast protein liquid chromatography (FPLC) demonstrated that toxin A had distinct peaks of activity for cytotoxicity and haemagglutination, as also did highly purified toxin A obtained by thyroglobulin affinity chromatography (TG) followed by two sequential anion-exchange chromatographic steps with Q-Sepharose FF and Mono Q. From TG unbound fractions a highly cytotoxic but weakly haemagglutinating variant (toxin A′) of toxin A was obtained by Q-Sepharose FF and Mono Q chromatography. Analysis of toxins A and A’ from cultures of C. difficile in a chemically defined medium, and of toxin A dialysed against brain heart infusion broth, indicated that A’ was not merely toxin A coupled to a component of the growth medium. Polyacrylamide gel electrophoresis under non-denaturing conditions showed that toxins A and A’ had the same Mr. Immunoblotting with mouse monospecific A antitoxin showed that five bands larger than the major 240-Kda band were more strongly developed in toxin A than in A’ in denaturing but non-reducing conditions, and in reducing conditions eight bands (38–175 Kda) were seen in toxin A but not A′. Immunoblotting with a monoclonal antibody (PCG-4) showed that, in both reducing and non-reducing conditions, two bands of 160 and 155 Kda were more prominent in toxins A and A’ respectively, and four bands (195, 180, 175 and 125 Kda) were detected only in toxin A′.
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Detection by ELISA of low numbers of Shiga-like toxin-producing Escherichia coli in mixed cultures after growth in the presence of mitomycin C
More LessSummaryTechniques currently available to detect Shiga-like toxin (SLT)-producing Escherichia coli lack sensitivity or require specialised equipment and facilities, and in some cases detect only strains belonging to serotype O157. We have used an ELISA technique, capable of detecting both SLTI and SLTII with crude P1 glycoprotein from hydatid cysts, in combination with enhancement of toxin production by culture with mitomycin C. Supernates of Tryptone Soya Broth cultures containing mitomycin C 200 ng/ml were tested for SLTII. For SLTI, cell lysates pre-treated with polymyxin B were tested. In tests with E. coli O157:H7 in mixed culture with E. coli strain C600 alone, or with E. coli C600, Proteus mirabilis and Enterococcus faecalis, SLTI could be detected when the proportion of toxigenic organisms represented 1% of the mixture, and SLTII when the proportion was 0‡025%. When faecal samples with added E. coli O157:H7 were examined in this system, SLTII-producing strains were detected when they comprised < 0‡1% of the coliform population. This technique is a sensitive and specific assay for detecting low numbers of SLT-producing organisms in mixed culture such as occurs in cases of haemolytic uraemic syndrome and haemorrhagic colitis.
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Host factors versus virulence-associated bacterial characteristics in neonatal and infantile bacteraemia and meningitis caused by Escherichia coli
SummaryPossession of P fimbriae, virulence-associated O and K antigens, haemolysin and aerobactin production, and susceptibility to 10 antimicrobial agents were studied in 63 Escherichia coli strains isolated from blood or CSF of infants who were grouped according to their clinical characteristics. These isolates were compared with 35 faecal E. coli strains from healthy infants. Individual virulence factors showed a relatively weak association with invasive infection except for P fimbriae in urosepsis and aerobactin production in meningitis. Combinations of factors were generally more predictive for defining virulent clones, particularly in infants defined as being at normal risk of developing septicaemia. Thus, 62% of isolates from such infants had characteristics typical of previously described uropathogenic or meningitis-associated clones of E. coli, compared with 32% of the isolates from high-risk infants (i.e., those defined as being at high risk of developing septicaemia) and only 9% of the faecal isolates (p<0‡001 and <0‡05, respectively). Overall, 45% of the episodes of invasive infection were caused by such clones, whereas risk factors (conditions considered to be associated with increased risk of invasive infection) were present in 59% of the infected infants (39% in meningitis and urosepsis, 78% in cryptogenic septicaemia and untreated bacteraemia). The results indicated that bacterial factors played a significant causative role in neonatal meningitis and urosepsis, particularly in normal-risk infants, whereas predisposing host factors contributed greatly to cryptogenic septicaemia and untreated bacteraemia.
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Identification of an endoflagellar associated protein in Borrelia burgdorferi
More LessSummaryDNA of Borrelia burgdorferi was cleaved by the endonuclease EcoRI and ligated with the bacteriophage expression vector λ gt11. After infection of the Escherichia coli strain Y1089, the plaques of recombinant phages were screened with a B. burgdorferi antiserum (human) for fusion proteins containing borrelia antigens. A positive clone produced a hybrid protein (p200) of c. 200 Kda. The corresponding native borrelia protein (p97) was identified as having an Mr of 97 Kda. To localise protein p97 in the B. burgdorferi cell, immunoelectronmicroscopy and a Western blot of isolated flagella were used. Antibodies directed against proteins p200 and p97 recognised epitopes associated with the flagella.
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Variation of the O antigen of Campylobacter jejuni in vivo
More LessSummaryDuring the course of a clinical study on patients with campylobacteriosis, three consecutive isolates of Campylobacter jejuni from the same patient were sent for O-serotyping. Marked differences in the specificities of the O antigens of the isolates were observed between the first and third isolates when a passive haemagglutination assay system developed for serotyping C. jejuni was used. Differences in specificity were also demonstrated by immunoblots of lipopolysaccharides (LPS) from proteinase K-digested whole cells. The three isolates could not be distinguished either by restriction endonuclease analysis of chromosomal DNA, by gel electrophoresis of whole-cell proteins or by silver-stained LPS gels, thus providing evidence that they were of the same strain and that antigenic variation had occurred in vivo.
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