- Volume 28, Issue 1, 1989
Volume 28, Issue 1, 1989
- Articles
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Coaggregation of black-pigmented Bacteroides species with other oral bacteria
More LessSummaryCoaggregation of Bacteroides gingivalis and other black-pigmented bacteroides with several oral bacteria was studied with “reagent” strains specially prepared by methods that have been described previously. B. gingivalis coaggregated with Veillonella, Capnocytophaga and Actinomyces spp., but not with any Streptococcus spp. Coaggregation of B. gingivalis with other bacteria was inhibited and reversed by lactose. Of the asaccharolytic black-pigmented bacteroides, only B. gingivalis demonstrated any coaggregation with other bacteria, whereas within the saccharolytic species, B. loescheii showed a marked ability to coaggregate with several species of oral bacteria. This property of coaggregation by B. gingivalis may be an important factor in the pathogenesis of periodontal infections.
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Cytotoxic activity of crude extracts of Bacteroides gingivalis
More LessSummaryThe cytotoxic activities of culture supernates, crude cell extracts and cell-wall materials of Bacteroides gingivalis were investigated in vitro. Each component was cytotoxic to Vero cells and, to a lesser extent, Wi 38 cells. The cytotoxic agents had similar effects on the cell lines to butyric acid, propionic acid and a partially-purified trypsin-like protein extracted from a clinical isolate of B. gingivalis; the effects were eliminated by heat. Cytotoxic materials obtained from young cultures were more susceptible to heat than those from older cultures. The heat-labile substance inside and outside the bacterial cell in young cultures of B. gingivalis may contribute to its overall cytotoxic activity.
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Aggregation by fragilis and non-fragilis Bacteroides strains in vitro
More LessSummaryBacteroides fragilis is associated with the formation of intra-abdominal abscesses, whereas other Bacteroides species are rarely involved. Since bacterial clumping may contribute to the survival of bacteria in the face of host defence mechanisms, the hypothesis has been put forward that differences in aggregation between fragilis and non-fragilis strains of Bacteroides may account for their differences in survival in vivo. All seven B. fragilis strains tested formed aggregates within 4 h, but strains not associated with intra-abdominal sepsis—B. vulgatus, B. thetaiotaomicron and B. distasonis—did not form aggregates in vitro. Aggregation occurred at 37°C, but not at 4°C or 2°C. Treatment with pronase partially inhibited aggregation. Periodate treatment killed the cells and caused them to form clumps which were distinguishable from the control aggregates. Heat-killed B. fragilis cells formed similar distinct clumps, but cells killed by glutaraldehyde and formaldehyde did so to a lesser degree. No inhibition was found upon addition of carbohydrates, ethylenediaminetetraacetic acid or after treatment with trypsin. These results demonstrate that aggregate formation occurs with B. fragilis strains alone, and that surface proteins probably mediate this interaction.
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A new methicillin-and gentamicin-resistant Staphylococcus aureus in Dublin: Molecular genetic analysis
SummaryIn June 1985 two new strains of methicillin- and gentamicin-resistant Staphylococcus aureus (MGRSA) were isolated in a Dublin hospital. Of these, one strain spread rapidly, affecting a total of 65 patients during the following 18 months, and subsequently spread to a second Dublin hospital. Detailed laboratory studies, including plasmid screening, plasmid restriction enzyme digest pattern analysis, hybridisation analysis, location of resistance determinants, and bacteriophage typing with a set of experimental S. aureus typing phages, demonstrated that the “new” MGRSA organisms, termed Phenotype III Dublin isolates, were completely distinct from, and unrelated to, the MGRSA strains responsible for serious nosocomial infections in Dublin hospitals during the decade before June 1985. These Phenotype III isolates were very similar to MGRSA organisms isolated in a Baghdad hospital during 1984. Data from plasmid curing, plasmid transfer and hybridisation experiments indicated that 20% of the Phenotype-III isolates expressed chromo-somally encoded, high level resistance to ethidium bromide (MIC 120 μg/ml), and that this was possibly due to chromosomal integration of a penicillinase-like plasmid.
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Characterisation of methicillin-resistant isolates of Staphylococcus aureus by analysis of whole-cell and exported proteins
More LessSummaryThirty-four isolates of methicillin-resistant Staphylococcus aureus (MRSA) from patients in Glasgow Royal Infirmary were studied. Whole-cell protein profiles obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were compared with banding patterns produced by immunoblots of exported proteins. Human plasma was used as a source of staphylococcal antibodies for the immunoblots. SDS-PAGE of whole-cell extracts did not usefully distinguish different isolates of MRSA. Reproducible banding patterns were obtained by immunoblots of exported proteins. Analyses of immunoblots by use of the Dice coefficient demonstrated that isolates of MRSA could be divided into two main groups. Immunoblots of exported proteins provided a rapid, reproducible and sensitive method for characterisation of MRSA.
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Immunobiological relationships among new cholera toxins produced by CT gene-negative strains of Vibrio choleraeO1
More LessSummaryThe enterotoxic activities of partially purified new cholera toxins (NCT) prepared from the CT gene-negative strains of Vibrio cholerae serotype O1 isolated from cases of diarrhoea in man and from diverse environmental sources were assayed in the rabbit ileal-loop model and the toxin unit was calculated in μUg of protein content. The secretory activities of one unit of homologous and heterologous enterotoxins were completely neutralised by 2.5 units of crude antiserum raised against one NCT preparation. In immunodiffusion tests, the NCT preparations from all strains tested gave precipitin bands against one antiserum showing reaction of complete identity. Thus, the present study clearly demonstrated that NCTs elaborated by CT gene-negative strains of V. cholerae O1 are immunobiologically similar.
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The use of phage-sensitivity patterns for tracing heat labile toxin-positive (LT+) Escherichia coli
More LessSummaryThe heat-labile toxin (LT) positive Escherichia coli colonies from 785 stool specimens obtained during a cholera vaccine trial were examined for their phage-sensitivity pattern to 31 E. coli phages. These specimens originated from 105 index cases and their contacts. Isolates with common phage-sensitivity patterns were grouped and were studied further both serologically and for their plasmid profile. The largest group (42 isolates) belonged to serogroup O78 and the second largest group (19 isolates) belonged to serogroup O6. There were 23 index cases which had E. coli with the same phage-sensitivity pattern as some of their contact strains. The identity of isolates from 16 index cases with strains from their respective contacts could be verified serologically. For the remaining seven index cases and their contacts, the isolates did not agglutinate with available antisera. However, subsequent studies demonstrated that, when the phage-sensitivity pattern among the strains matched, the plasmid profiles of these strains also matched. This further indicates the ability of phage-sensitivity patterns to serve as markers in tracing strains.
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An endocytic process in HEp-2 cells induced by enteropathogenic Escherichia coli
More LessSummaryInfection of HEp-2 cells by enteropathogenic Escherichia coli (EPEC) was examined by transmission and scanning electronmicroscopy. EPEC strains of serogroups O111:K58 and O55:K59 recently isolated from human patients did not exhibit enterotoxic activity, as judged by the Vero-cell and suckling-mouse assays, or invasive ability as judged by the Sereny test. These strains attached to and penetrated HEp-2 cells. Transmission electronmicroscopy showed bacteria in close contact with cell membranes 15 min after infection; later, intense swelling and budding of membranes and penetration of EPEC into the cell cytoplasm occurred. Intracellular bacteria were enclosed in membrane-bound vacuoles in the cell cytoplasm underlying localised adherence sites observed by light microscopy. Scanning electronmicroscopy showed morphologically altered membranes only at the sites of bacterial attachment. Bacteria inactivated by ultraviolet light were not internalised and cytochalasin B (≥ 10 mg/L) markedly inhibited uptake. These observations suggest that penetration of EPEC into HEp-2 cells occurs by an endocytic process in metabolically active bacteria.
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Invasion of Vero cells by Salmonella species
More LessSummaryThe invasiveness of Salmonella strains for Vero cells was studied by quantitative bacteriology; the technique was more sensitive than phase contrast microscopy. All of 59 Salmonella strains, of 19 different serotypes, were more invasive than Escherichia coli K12. Three strains of Shigella were as invasive as most of the Salmonella strains whereas 29 strains of E. coli, two of Proteus, three of Klebsiella and one of Serratia were much less invasive. Two Citrobacter strains exhibited intermediate invasiveness. Eleven Salmonella strains were also shown to be invasive in HeLa, int 407, bovine kidney, chick kidney and chick embryonic fibroblast cells. The difference between invasive and non-invasive organisms was apparent irrespective of the numbers of bacteria in contact with Vero cells or the duration of bacteria-cell contact. There was little intracellular multiplication of S. typhimurium in Vero cells. Unlike the situation with Shigella, incubation of Salmonella or Salmonella-cell mixtures at 41°C, 22°C or 0°C had little effect on invasiveness. Non-viable Salmonella organisms were non-invasive. Incubation of Vero cells with cholera toxin, dinitrophenol, iodoacetic acid, cytochalasin B or d-mannose did not substantially reduce invasiveness. Virulence-associated plasmids were not essential to invasion by S. typhimurium, S. gallinarum or S. pullorum. Neither somatic antigens nor mannose-sensitive haemagglutinins were essential to the invasiveness of an S. infantis strain, but an additional factor, eliminated by N-methyl, N-nitro, N-nitrosoguanidine mutagenesis did contribute to invasiveness.
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Evaluation of a latex agglutination test for Rickettsia conorii antibodies in seropositive patients
More LessSummaryA latex agglutination test for antibodies to Rickettsia conorii was compared with micro-immunofluorescence (the reference test for total antibodies); 179 sera were from 115 confirmed cases of Mediterranean Spotted Fever, and 101 were from pregnant women (control group) who had no detectable antibodies by the reference method. The micro-immunofluorescence test for specific IgM antibodies was used to clarify some discordant results. The agreement obtained between latex-R. conorii and micro-immunofluorescence was 95%. Sensitivity and specificity were 96% and 93% respectively. When micro-immunofluorescence results for specific IgM antibodies were included, these figures rose to 96 and 99%, and agreement was almost 97%. Latex agglutination is a simpler and more rapid technique than micro-immunofluorescence and is suitable for the screening as well as for the titration of R. conorii antibodies.
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- Technical Report
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Rapid identification of Vibrio cholerae serotype O1 from primary isolation plates by a coagglutination test
More LessSummaryA coagglutination test was developed for identifying suspected colonies of Vibrio cholerae serotype O1 directly from primary isolation plates. Visible agglutination occurs when V. cholerae O1 antibody attached to cell-wall protein A of Staphylococcus aureus reacts with its homologous antigen. From 314 faecal samples from clinically suspected cases of cholera, 210 colonies from thiosulphate citrate bile salts sucrose (TCBS) agar and 222 colonies from taurocholate tellurite gelatin (TTG) agar were tested as suspect V. cholerae. In each case 204 isolates were identified as V. cholerae O1 by conventional methods and also gave positive results for V. cholerae O1 in the coagglutination test; with one partial exception, no other colonies tested gave positive results. The coagglutination test is simple and inexpensive and provides a result 24 h earlier than conventional methods.
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