- Volume 36, Issue 6, 1992
Volume 36, Issue 6, 1992
- Article
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Candida albicans HSP 90: link between protective and auto immunity
More LessSummaryHeat shock proteins (HSP) are thought to play a role in the aetiology of autoimmune diseases, but are also common targets for the immune response to many infections. Patients recovering from systemic candidosis produce antibodies to Candida albicans HSP 90, both to species-specific epitopes and, more commonly, to epitopes shared with human HSP 90. One such autoreactive antibody was protective in a mouse model of systemic candidosis.
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Rapid identification of Legionella species from a single colony by gas-liquid chromatography with trimethylsulphonium hydroxide for transesterification
More LessSummaryTransesterification of bacterial fatty acids to methyl esters with trimethylsulphonium hydroxide (TMSH) was compared with a conventional method for the identification of Legionella species by capillary gas chromatography. There was an extensive coincidence in the gas chromatographic profiles of bacterial fatty acid methyl esters (FAMEs) obtained by the two methods. However, the TMSH procedure needs less initial material and is much more simple and rapid. The chromatographic pattern of FAMEs obtained from a single colony is sufficient for the identification of the genus Legionella, and L. pneumophila can be clearly distinguished from other Legionella species.
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Dissemination and proliferation of Salmonella typhimurium in genetically resistant and susceptible mice
More LessSummaryGenetically resistant A/J and CBA mice were inoculated intraperitoneally with either 103 or 104 organisms of a virulent strain of Salmonella typhimurium; susceptible C57BL/6J and BALB/c mice were inoculated with either 102 or 103 organisms. Except with the smaller dose in resistant mice, fatal infection ensued. Bacteraemia occurred within 1 h after inoculation, except that it was not detectable during the first 6 h in the susceptible mice inoculated with 102 organisms. From day 2, the circulating bacterial population continued to increase in all infected mice, except that it remained under control in the resistant mice inoculated with the lower dose (103 organisms). The pathogen proliferated logarithmically in the liver from day 2, and a bacterial count of c. 108 cfu/g of tissue was reached when the animals died at 5–7 days; again, the resistant mice inoculated with 103 organisms were an exception in which the hepatic bacterial population was kept under control and the mice survived.
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Application of biochemical fingerprinting to the investigation of clonal groups of Salmonella of serotype Havana
More LessSummaryA computerised typing method based on biochemical fingerprinting was used to investigate biochemical phenotypes (BPTs) among 70 strains of Salmonella of serotype Havana isolated from human cases of gastroenteritis in Iran and other parts of the world. A total of 16 BPTs comprising five common and 11 single phenotypes was identified. The most frequently found BPT contained 24 isolates from Iran and nine from other countries. Three common BPTs with two, seven and 15 isolates were found among Iranian strains only and one common BPT with two isolates was found among non-Iranian strains only. Antibiotic-resistance patterns and virulence properties of strains from these common BPTs suggested that they might be unique clones. Forty-two Iranian isolates shared multi-resistance to between three and seven antibiotics. In contrast, none of the isolates from other countries was resistant to antibiotics. Furthermore, 43 Iranian isolates showed mannose-resistant adhesion to HeLa cells and 24 of them possessed an aerobactin-mediated iron-uptake system, whereas none of the isolates from other countries possessed any of these virulence properties. These findings suggest that four unique clones of Salmonella Havana with different BPTs and virulence properties are common in Iran; two particular clones were responsible for a majority of Havana infections there. However, the most prevalent BPT found among Iranian strains was also common in strains from other countries. It is concluded that biochemical fingerprinting, as used in this study, is a reliable method for identifying clonal groups of Havana strains. The method is reproducible, easy to perform and can be used alone, or in combination with other typing methods, in epidemiological studies of serotype Havana.
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In-vitro and in-vivo characteristics of TnphoA mutant strains of Salmonella serotype Gallinarum not invasive for tissue culture cells
More LessSummaryInsertion mutants of a strain of Salmonella serotype Gallinarum, the cause of fowl typhoid, were produced with transposon TnphoA. Eight mutants were identified as being less invasive in cell culture than the parent strain. Neither the parent strain nor the mutants showed mannose-sensitive haemagglutination of various red blood cell species. Although two mutants gave mannose-resistant (MR) haemagglutination of red cells of different animal species these MR activities could not be correlated with other characteristics in vitro or in vivo. The mutant strains were divisible into three classes by their patterns of invasiveness and adhesiveness in vitro and by changes in membrane proteins. Strains of classes 1 and 2 had single transposon insertions, detected by Southern hybridisation, of 8•9 and 2.4 kb EcoRV-digested chromosomal-fragments, respectively, and were slightly less invasive than the parent strain. They were no less virulent for chickens by the oral route than a mutant strain shown to be as invasive in vitro as the parent strain. The class-2 mutant was also less adhesive than the other strains. The single mutant strain of class 3 which contained insertions in several chromosomal fragments, was non-invasive in Vero cells (
10 1.0 cfu/ml recovered compared with log10 2.88 for the parent strain) and showed reduced virulence by both oral and intramuscular routes. The mutant strains of all classes were taken up equally rapidly from the blood by the spleen. Intramuscular immunisation of chickens with the class-3 mutant strain gave complete protection against oral challenge 3 weeks later with 1000 oral LD50 doses of the virulent parent strain.
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Comparison of rapid methods for detection of heat-labile (LT) and heat-stable (ST) enterotoxin in Escherichia coli
SummaryOxoid VET-RPLA, ST-EIA and Pharmacia Phadebact ETEC-LT enterotoxin tests were compared to find a simple but reliable method for detecting enterotoxigenic Escherichia coli (ETEC) in Hungary. In the Oxoid tests, all six reference LT-or ST-producing strains, except one ST-producer, gave positive results. Of 11 reference porcine enterotoxigenic strains, all four LT-producers gave positive reactions for LT but three of 10 ST-producers gave negative reactions for ST. Thirteen of 50 strains from culture collections of H. Steinrück (Germany) were LT+ and nine of 33 were ST+. When 31 isolates were tested simultaneously with the Oxoid and the Pharmacia LT tests, 12 strains were LT+ by the Oxoid LT test but by the Phadebact LT test only seven of these strains were LT+ and, of the remainder, three gave uncertain results and two gave negative results. Of 69 porcine strains, seven were LT+ and three ST+. Of 901 human strains isolated in Hungary, 10 were LT+ and one of 24 tested was ST+. In two cases, ETEC strains were isolated from contacts of travellers returning from Mongolia and Bangladesh. Results of comparative studies with reference strains corresponded well to those of the classical toxin detection tests. The Oxoid test was rapid, sensitive, specific and easy to perform and is recommended for use in screening ETEC isolates.
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Adhesion of Shigella dysenteriae type 1 and Shigella flexneri to guinea-pig colonic epithelial cells in vitro
More LessSummaryAdhesion of bacteria to guinea-pig colonic epithelial cells in vitro was inhibited by fucose with all the four strains tested (two of Shigella dysenteriae type 1 and two of S. flexneri). N-acetyl neuraminic acid and N-acetyl mannosamine also caused inhibition, suggesting a multiplicity of receptors on the epithelial cell. Congo-red binding of the strains correlated with their adhesive ability, whereas haemagglutination of rabbit erythrocytes by the bacteria did not.
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Mechanism of persistent infection associated with peritoneal implants
More LessSummaryThe ability of rabbits to clear an intraperitoneal injection of Pseudomonas aeruginosa in the presence or absence of a surgically implanted peritoneal device was investigated. Sham-operated rabbits without an implant eliminated a P. aeruginosa challenge of 5 × 106 cfu/ml; lavage fluid and peritoneal tissues became culture-negative within 96 h. However, peritonitis developed in rabbits that were given the same number of bacteria in the presence of an implant; high bacterial counts were recovered from the lavage fluid and the device itself. Scanning and transmission electronmicroscopy revealed bacterial biofilms on the surface of the device. Insertion of pre-colonised devices demonstrated a rapid multiplication of sessile organisms within the resulting bacterial biofilm. Counts reached a plateau of about 1 × 107 cfu/cm2 of Silastic by day 16 and fluctuated around this level until the end of the study. Pre-immunisation with formalin-killed whole cells of P. aeruginosa did not reduce this bacterial growth despite high levels of specific IgG. The results confirm the failure of peritoneal defences to clear an infection in the presence of an implant following either challenge with planktonic bacteria or insertion of a pre-colonised device, and demonstrate the rapid development of bacterial biofilms on the surface of the implant which appear to protect the bacteria from host defences, even when primed by pre-immunisation.
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Ribotyping of coagulase-negative staphylococci
More LessSummaryThe discriminative capacity of ribotyping was initially assessed without knowledge of results obtained for the same isolates by use of more established typing methods. Forty-eight isolates of coagulase-negative staphylococci (CNS) from peritoneal fluids were studied. They were collected prospectively during 31 consecutive episodes of infection associated with peritoneal dialysis in 17 patients. DNA was digested by the restriction endonucleases EcoRI or HindIII and ribotyped by means of a biotinylated cDNA probe to 16S + 23S staphylococcal ribosomal RNA gene sequences. These methods in combination produced a total of 27 types which compared well with numbers of groups distinguished by other typing methods: limited biotype–antibiotic resistogram (ARB; 28), antibiotic resistogram alone (25), API-Staph (12), phage typing (9) and plasmid analysis (22). Ribotyping was highly reproducible and typed all isolates, including those that were not phage-typable (35) or did not contain plasmids (4). When used in a hierarchical manner with ARB, ribotyping results produced 13 additional types in comparison with the other three methods. When used hierarchically with all other typing systems, a further five types were found among isolates from two patients. However, some of the differences observed as a result of ribotyping could have been due to subtle changes produced by mutation, lysogenisation or gene transposition. Since the method requires additional time, expense and technical expertise, it is likely to be useful only when answers to specific epidemiological problems are required or as an initial screen before using other methods of genetic analysis.
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An ultrastructural study of Helicobacter mustelae and evidence of a specific association with gastric mucosa
Jani O’Rourke, A. Lee and J. G. FoxSummaryThe ultrastructure of Helicobacter mustelae, a natural inhabitant of the ferret stomach, has been studied and compared with the human gastroduodenal pathogen H. pylori. H. mustelae is a short, slightly curved rod, 2 μm × 0.5 μm, with four or more sheathed flagella. The most common flagellar configuration is a single flagellum at one terminus, bipolar arrangement at the other end and a lateral flagellum. Dense inclusion bodies were observed below the flagellar insertion sites. It is suggested that this configuration is a specialised adaptation to motility in a viscous environment. On examination of the ferret gastric mucosa, similarities to H. pylori were observed such as adherence to gastric tissue and the formation of adhesion pedestals. However, unlike H. pylori, significant numbers of bacteria were intracellular. Furthermore, a much greater proportion of H. mustelae were attached to the mucosa with few bacteria lying free in the mucus, as is seen with H. pylori. It is concluded that the ferret is an important model for the study of adherence of bacteria to gastric mucosa and their possible role in peptic ulceration.
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Virulence and adhesive properties of serotypes A and B of Candida albicans isolated from paediatric burn patients
More LessSummaryThe virulence and adhesive properties of 50 isolates of Candida albicans serotypes A and B collected over 6 years from 48 paediatric burn patients were examined to provide more detailed information about candidal pathogenesis in burn patients and to examine the relevance of the commonly used epithelial cell adhesion assay for determining fungal virulence. The isolates represented a fair distribution of serotypes (29 isolates were serotype A and 21 isolates were serotype B) and a total of 28 serotype-biotype combinations were found; 32% of the serotype-biotype combinations appeared only once, while 44% of the isolates showed similar biotype tests for two of three digits. Adhesion of the isolates to plastic and to buccal epithelial cells (BECs) was examined and compared after growth in a chemically defined medium. There were significant differences in the adhesion of individual isolates to plastic or BECs, but no correlation was found between biotype and adhesiveness. Serotype B isolates were found to be more adhesive to BECs (p<0.05) but not to plastic. There was no apparent correlation between candidal adhesiveness and site of isolation from these patients (autografts, blood, faeces, throat swabs, tracheal aspirates, wounds and intravenous catheters), although isolates from catheters were generally less adhesive to epithelial cells. Virulence in a systemic infection mouse model revealed that there were significant differences in virulence between isolates, but no correlation was found between virulence and the biotype, serotype or site of isolation. Similarly, no correlation was found between virulence and adhesiveness or cell-surface hydrophobicity. These results suggest that, although variations in adhesiveness between isolates of C. albicans can be detected in an epithelial cell adhesion assay, such tests may not be useful as reliable predictors of virulence. These data also suggest that candidal pathogenesis is the result of a multifactorial process whereby, in some instances, adhesion may play only a transient role in infection.
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Quantitative analysis of immunoglobulin G subclass responses to Pseudomonas aeruginosa antigens in cystic fibrosis
More LessSummaryThe four subclasses of IgG have different structures, functions and implications in the antibody response. IgG subclass reactions to individual Pseudomonas aeruginosa structural antigens in 22 adolescents and young adults with cystic fibrosis (CF) were studied qualitatively and quantitatively by densitometric analysis of Western blot assays. These patients had been infected by P. aeruginosa for 7 years or longer and were divided into two groups according to their pulmonary status: Group 1 comprised 11 patients with relatively good pulmonary status; Group 2 consisted of 11 patients with poor pulmonary status. There was a relative decrease of IgG1 and a relative increase of IgG2 and, especially, of IgG3 and IgG4 antibodies against P. aeruginosa antigens in the CF patients. Comparison of the two CF patient groups showed a significant increase in the proportion of IgG3 in the Group 2 patients. This could be a potential cause or effect in the deterioration of their pulmonary function. Densitometric analysis of Western blots revealed more than 24 P. aeruginosa antigens and indicated those that were the targets of the isotype antibody response(s) that were apparently most harmful. Thus, there was a significant increase of IgG2 or IgG3 reactivity (or of both) against proteins F, H (H1 and H2), and I in the Group 2 patients. One other striking observation of this study was the high reactivity of IgG4 antibodies to protein H. IgG4 was the major antibody to this protein in seven of the 11 Group 1 patients compared to two of the 11 in Group 2. We hypothesise that IgG4 antibodies may antagonise IgG2 antibodies, helping to preserve stable pulmonary function.
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