- Volume 38, Issue 2, 1993
Volume 38, Issue 2, 1993
- Articles
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Vaccine efficacies of elastase, exotoxin A, and outer-membrane protein F in preventing chronic pulmonary infection by Pseudomonas aeruginosa in a rat model
More LessSummaryThe protective efficacies of eight vaccine preparations consisting of Pseudomonas aeruginosa outer-membrane protein F, elastase and exotoxin A toxoid, administered either individually or in various combinations, were determined in a rat model of chronic pulmonary infection. Rats were immunised intramuscularly at 2-week intervals (days 0, 14 and 28). On day 42, blood was collected and antisera were obtained from each vaccine group for use in an enzyme-linked immunosorbent assay which determined the titre of IgG antibodies elicited by each vaccine. Also on day 42, rats were challenged by intratracheal inoculation of a clinical isolate of P. aeruginosa encased within agar beads. On day 49, the animals were killed and the lungs were examined macroscopically for the presence of lesions and fixed for histological examination. When compared with control rats immunised with bovine serum albumin, rats immunised with protein F alone as a vaccine received significant protection against the development of severe pulmonary lesions. Elastase used alone as a vaccine provided some protection against severe lung lesions and reduced the incidence of microscopic peribronchial inflammation. However, the combination of protein F plus elastase as a vaccine did not afford protection from severe lesions, and there was an increased incidence of necrotising granulomas in the lungs from this vaccine group. Protection against lung lesions from the three-component vaccine consisting of protein F, elastase and exotoxin A toxoid was similar, to that provided by the protein F vaccine. Neither macroscopic nor histological evidence showed any enhancement of protective efficacy for the three-component vaccine over that of the protein F vaccine. No combinatination of elastase or exotoxin A toxoid with protein F improved the protective efficacy of the protein F vaccine alone.
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Detection of enterovirulent Escherichia coli associated with diarrhoea in Seville, Southern Spain, with nonradioactive DNA probes
SummaryTo assess the role of diarrhoeagenic Escherichia coli in Southern Spain, faecal samples from 135 patients with diarrhoea and 40 healthy subjects from Seville, Andalusia, were investigated. In this prospective study, enterovirulent E. coli were identified by hybridisation with five non-radioactive DNA probes specific for enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC) and verocytotoxin-producing E. coli (VTEC). Probe-positive strains were isolated from four patients (3%) with diarrhoea and from none of the healthy controls. Two patients harboured ETEC and two patients had EPEC probepositive strains in their faeces. No VTEC were isolated during this study. Salmonella spp. were the most frequently identified enteric pathogens, accounting for 10% of the cases, followed by Campylobacter jejuni (3%) and diarrhoeagenic E. coli (3%). This study indicates that enterovirulent E. coli play a modest role in the aetiology of diarrhoea among the indigenous population of Southern Spain.
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Role of fibronectin in staphylococcal colonisation of fibrin thrombi and plastic surfaces
More LessSummaryThe adhesive glycoprotein fibronectin has been proposed as a mediator of adherence of certain gram-positive cocci to host cells and fibrin thrombi. This study compared the role of soluble and immobilised fibronectin in the adherence of coagulasenegative staphylococci (CNS) and Staphylococcus aureus to fibrin thrombi and plastic surfaces. Adherence of S. epidermidis to fibrin thrombi was significantly reduced when fibronectin was removed from the plasma used for thrombus preparation. Adherence was restored through restitution of fibronectin. S. epidermidis also adhered substantially more to plastic surface coated with fibronectin than to non-coated plastic. Increased adherence of CNS to plastic was also observed after coating with the 29-kDa N-terminal fragment of fibronectin. Soluble fibronectin did not affect the adherence of CNS to fibrin thrombi or plastic surfaces. The adherence of S. aureus to fibrin thrombi was significantly increased by the addition of soluble fibronectin, but not by incorporation of fibronectin into the clot. These results indicate that the binding of fibronectin is an important factor in the adherence of staphylococci to fibrin clots and plastic surfaces and, thus, colonisation of these surfaces. However, the two species of staphylococci seem to employ different mechanisms of fibronectin-mediated adherence: S. epidermidis interacts mainly with fibronectin incorporated in fibrin clots or immobilised on implanted synthetic materials, whereas S. aureus adheres to the fibrin matrix through binding of soluble fibronectin present in wound exudates.
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Serum and tissue protein binding and cell surface properties of Staphylococcus lugdunensis
More LessSummaryEleven strains of Staphylococcus lugdunensis from different clinical sources were investigated for their ability to bind125I-labelled collagen (Cn) type I and IV, fibronectin (Fn), vitronectin (Vn), laminin (Lm), fibrinogen (Fg), thrombospondin, plasminogen (glu- and lys-form) and human IgG. All the strains bound these proteins, although a higher degree of binding was obtained for Cn types I and IV and IgG with mean values of 36%, 32% and 26% binding, respectively. In tests with proteins immobilised on latex beads in a particle agglutination assay, eight of the 11 strains bound Cn type I and seven bound Fg, whereas no strain bound immobilised IgG. Binding to immobilised Cn-I, Fg, Lm and Vn was abolished when the bacterial cells were treated with proteases or heat, indicating cell-surface receptors with protein characteristics. Cell-surface extracts of S. lugdunensis 2342 were able to totally inhibit binding of the homologous strain and S. aureus Cowan 1 to latex-immobilised proteins Cn-1, Lm, Vn, Fn and Fg. The binding of125I-labelled Cn IV by S. lugdunensis 2342, was heat sensitive, whereas the binding to S. aureus Cowan 1 was heat resistant. The strains gave negative results in tests for the presence of protein A with a S. aureus protein A gene probe and with sensitised red blood cells. No production of heat-stable nuclease (TNase) could be detected by monoclonal antibodies against TNase or by the polymerase chain reaction with an oligonucleotide sequence from S. aureus TNase as primer. When the cell surface characters of the S. lugdunensis strains were studied, five were found to be hydrophobic and negatively charged, four hydrophilic and positively charged and two hydrophobic with positive net charge.
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In-vitro and in-vivo characterisation of resistance to colonisation with Clostridium difficile
H. E. Larson and A. WelchSummaryIn hamsters, resistance to colonisation by Clostridium difficile appears to be mediated by micro-organisms that are present in the gut in relatively low concentrations. Small amounts of normal caecal contents inhibited the growth of C. difficile when added to cultures in vitro or given to animals which had been treated with clindamycin. Filtrates of caecal contents, frozen and thawed contents and contents diluted to 0.1% wet weight lost their inhibitory properties. However, caecal contents retained their protective capacity after culture for 7 days in vitro. Antibiotic treatment altered resistance to colonisation by only a few species of clostridia. Faeces of animals treated with ampicillin but not clindamycin recovered colonisation resistance after incubation at 37°C in vitro. Since human faeces could also restore colonisation resistance to hamsters, the hamster model may be useful for the study of resistance to colonisation by C. difficile in man.
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Characterisation of Clostridium difficile strains by polymerase chain reaction with toxin A- and B-specific primers
More LessSummaryA total of 218 Clostridium difficile strains was examined for production of toxin A by ELISA, production of toxin B by a cytotoxin assay and the presence of toxin A and B geneassociated sequences by the polymerase chain reaction (PCR). After saturation amplification with toxin B-specific primers, the characteristic amplification product (591 bp) was detected in all 184 toxigenic strains examined. PCR with toxin A-specific primers gave positive results with all but one of the toxigenic strains. By contrast, PCR with toxin A- and toxin B-specific primers yielded negative results with all 34 non-toxigenic strains tested. This suggests that PCR detection of either the toxin A or B gene is a good indication of toxin production. PCR did not require DNA extraction or hybridisation and was convenient, sensitive and rapid. Toxigenic C. difficile could be detected in mixed cultures, suggesting a role for PCR in the identification of toxigenic C. difficile in primary culture.
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Stability of cefdinir (CI-983, FK482) to extendedspectrum plasmid-mediated β-lactamases
More LessSummaryFourteen plasmid-encoded extended-spectrum β-lactamases were purified from Escherichia coli transconjugants of original clinical isolates. The Vmax, Km and Vmax/Km were each determined for ampicillin, carbenicillin, cephaloridine, cephalexin, cefuroxime, cefuroxime, cefixime, cefdinir, ceftazidime and cefotaxime as substrates with eight of these enzymes and with the narrow-spectrum β-lactamase, TEM-1. The relative rates of hydrolysis of ampicillin, cephaloridine, cephalexin, cefuroxime, cefixime, cefdinir, ceftazidime and cefotaxime were also determined for the remaining six enzymes. Cefdinir had Vmax/Km or relative rates of hydrolysis values either equal to or lower than ampicillin, cephaloridine, cephalexin and cefotaxime for all the enzymes tested. Overall, cefdinir was more stable to the 15 β-lactamases tested than either cefuroxime or cefixime; however, ceftazidime was more stable than cefdinir to hydrolysis by eight of the enzymes tested.
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Immunological analysis of the plasmid-encoded proteins from the highly pathogenic Yersinia enterocolitica serogroup 08 and the less pathogenic serogroup 03
More LessSummaryThe plasmid-encoded proteins of the pathogenic Yersinia enterocolitica serogroups O3 and O8 were analysed with respect to their immunological relationship. Common epitopes on yersinia outer-membrane proteins (YOPs) and released proteins (RPs) were recognised by orally-induced antisera against living bacteria and by monospecific antisera induced against single RPs of Y. enterocolitica serogroup O8. A major difference between the pathogenic serogroups O3 and O8 was YOP42, which was detected only in the outer membrane of the highly pathogenic O8+ bacteria. The YOP42 may be responsible for the greater virulence of serogroup O8 bacteria.
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Genetic characterisation of isolates of Listeria monocytogenes from man, animals and food
More LessSummaryMultilocus enzyme electrophoresis was used to assess genetic relationships between 95 isolates of Listeria monocytogenes, most of which were isolated in Australia and New Zealand from man, animals and food. The isolates were separable into two major genetic divisions; the majority of those from human patients and animals were in division I, and the majority from those foods that were not specifically associated with human listeriosis were in division II. Isolates in division I were virulent, whereas many isolates from food were probably less virulent and did not pose a large threat to human health. However, isolates from certain foods, particularly paté, were indistinguishable from those causing disease in man, and the consumption of these products represented a clear risk factor for infection. Isolates from infected human patients in Australia and New Zealand belonged to the same clone of serotype 4b that has been responsible for major epidemics in the northern hemisphere. However, a separate clone of serotype 1/2b strains, present in both Australia and New Zealand, was responsible for two major outbreaks that occurred in Western Australia in 1978–80 and 1990–91.
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Evolutionary origin and radiation of the avian-adapted non-motile salmonellae
SummaryMultilocus enzyme electrophoresis was employed to estimate chromosomal genotypic diversity and relationships among 131 isolates of the non-motile Salmonella biotypes Gallinarum and Pullorum (serotype 1, 9, 12:–:–) that cause fowl typhoid and pullorum disease, respectively. Thirteen electrophoretic types (ETs), marking clones, were distinguished, and construction of a neighbour-joining phylogenetic tree revealed three lineages: one consisted of five ETs of Gallinarum, a second included seven ETs of Pullorum, and a third was represented by a single ET (Ga/Pu 1) that is intermediate between those of the other two lineages in both multilocus enzyme genotype and biochemical properties. Enzyme genotype analysis and comparative nucleotide sequencing of the phase 1 flagellin gene (fliC), the hook-associated protein 1 gene (flgK), and the 6-phosphogluconate dehydrogenase gene (gnd) identified serotype Enteritidis (1, 9,12:g, m:–) as a close relative of the non-motile salmonellae. In most strains of biotype Gallinarum, the fliC gene is complete, intact and identical in sequence to that of Enteritidis, but isolates of three ETs had a stop codon at position 495. The fliC sequences of the ETs of Pullorum differed from that of Enteritidis in having non-synonymous changes in either two or three codons and a synonymous change in one codon. The sharing of distinctive alleles at three metabolic enzyme loci and a stop codon in flgK indicates that the non-motile salmonellae are monophyletic and that their most recent common ancestor was non-motile. Since diverging from that ancestor, the Pullorum lineage has evolved more rapidly than the Gallinarum and Ga/Pu 1 lineages.
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Identification of Bordetella pertussis in nasopharyngeal swabs by PCR amplification of a region of the adenylate cyclase gene
More LessSummaryThe polymerase chain reaction (PCR) was used to amplify a 522-bp region of the adenylate cyclase toxin (cyaA) gene of Bordetella pertussis. As few as 100cfu from a suspension of B. pertussis could be detected by this procedure when the amplified PCR product was detected by ethidium bromide staining of agarose gels. However, simulated clinical specimens, prepared from swabs impregnated with known numbers of B. pertussis cells, only yielded a positive reaction with ⩾104cfu. Hybridisation of a Southern blot of the PCR products from the swab samples with a cya-specific probe gave a positive reaction with as few as 8 cfu, but the hybridisation signal was uniformly weak with fewer than 104cfu. Nevertheless, three of 13 nasopharyngeal swabs, taken from suspected clinically defined cases of whooping cough and stored frozen for up to 18 months, gave a positive PCR reaction.
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Campylobacter jejuni adapts to aerobic metabolism in the environment
More LessSummaryCampylobacter jejuni, when left on blood agar for prolonged periods, was found to survive better in air than under micro-aerobic conditions. After a period of 2–3 days in air, all strains of C. jejuni examined grew freely in air on subculture, and could be further subcultured apparently indefinitely in air. This adaptation to aerobic metabolism was accompanied by a change in colony morphology and some changes in outer-membrane protein patterns, but no change in serotyping reactions. The ability to colonise mice was unaltered as was the helical morphology of growing cells. The important survival phase of C. jejuni, when outside the animal gut, involves not only a change to coccal morphology but also fundamental changes in the metabolism of the organism. These changes are likely to be relevant to techniques required for culturing C. jejuni from foods and environmental sources.
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Phagocytosis of bacterial strains isolated from acute dentoalveolar abscess
More LessSummaryThe phagocytosis by human polymorphonuclear leucocytes of 37 bacterial strains identified as Streptococcus milleri (10 strains), strictly anaerobic gram-positive cocci (10) Prevotella intermedia (6), Pr. oralis (5) and Fusobacterium nucleatum (6) was investigated in vitro. The ingestion of S. milleri and strictly anaerobic gram-positive cocci was significantly greater (p<0.001) than that of strains of Prevotella spp. and F. nucleatum. The degree of uptake of capsulate and non-capsulate strains did not differ.
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- Editorial
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- Books Received
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Progress in Medical Virology, volume 38
More LessEdited by J. L. MELNICK. 1991. ISBN 3-8055-5229-7 S. Karger AG, Basel. Pp. 208. £84.80.
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Developments in Biological Standardization, volume 73. Pertussis: Evaluation and Research on Acellular Pertussis Vaccines
More LessEdited by Y. SATO, H. SATO, M. TIRU and F. BROWN. 1991. ISBN 3-8055-5457-5. S. Karger AB, Basel. Pp.278, £104.40.
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Quality Control. Principles and Practice in the Microbiology Laboratory
More LessEdited by J. J. S. SNELL, 1. D. FARRELL and C. ROBERTS. 1991. ISBN 0 901 144 31 2. Public Health Laboratory Service. Pp. 172. A5 Paperback £12.50.
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Current Topics in Clinical Virology
More LessEdited by P. MORGAN-CAPNER. 1991. ISBN 0 901 14430 4. Cambridge University Press. Pp. 304. £24.95.
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