RESUMEN
Pasteurella haemolytica-A1 was grown in vitro under iron-rich conditions, iron-depleted conditions, and in vivo within a chamber implanted in the peritoneal cavity of a rabbit to determine if iron regulated outer-membrane proteins were expressed in vivo. The antigenicity of outer membrane (OM) proteins from bacteria grown under these conditions was assessed by immunoblotting with pooled serum from convalescent bovine calves experimentally infected with P. haemolytica-A1 and serum from the implanted rabbit. Pasteurella haemolytica-A1 grown under iron-depleted conditions showed three distinct OM protein bands (71, 77, and 100 kDa) that were present in much lesser amounts when the organism was grown under iron-rich conditions. These same three bands were evident in OM protein preparations from bacteria grown in vivo. Western blotting indicated that these protein bands were recognized immunologically by the convalescent bovine serum and by serum from the implanted rabbit, in cells grown under the iron-depleted conditions and in vivo, but not if the bacteria were grown under the in vitro iron-rich conditions.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Hierro/farmacología , Mannheimia haemolytica/genética , Infecciones por Pasteurella/veterinaria , Animales , Formación de Anticuerpos , Bovinos , Enfermedades de los Bovinos/microbiología , Convalecencia , Cámaras de Difusión de Cultivos , Deficiencias de Hierro , Mannheimia haemolytica/patogenicidad , ConejosRESUMEN
OBJECTIVE: A method that would allow in situ comparison of the degrees of adherence to genital epithelia by the biofilms of the normal flora. SUBJECTS: Four healthy women. SETTING: Departments of Biological Sciences, University of Calgary, and of Medicine, University of British Columbia. METHODS: In situ, scraped specimens were taken from the vagina and ectocervix before and after vigorous vaginal washes, and colony counts of associated bacteria were compared. In vitro, cells from the vulva, vagina and ectocervix were vortexed, centrifuged and sonicated and remaining associated bacteria quantitated by light microscopy. RESULTS: Anaerobic lactobacilli were notably tissue-adherent as colony counts of postwash specimens were comparable to those of their paired prewash specimens, but crucially were higher than those of their paired wash specimens (p less than 0.05, Wilcoxon signed rank test). However, vaginal and ectocervical coagulase-negative staphylococci and ectocervical Lancefield group B streptococci were loosely tissue-adherent, because counts in postwash specimens were lower than those in prewash or wash specimens (p less than 0.05, Wilcoxon signed rank test). In vitro, only vulvar scrapings and vaginal postwash specimens showed a significant decrease in associated bacteria after shear stressing (p less than 0.05, Wilcoxon signed rank test). CONCLUSIONS: The normal flora of the female genitalia features both avidly and loosely tissue-adherent bacterial biofilm populations whose adherence can be influenced partly by their location. Our scraping/washing method can contribute to further characterisation of this phenomenon. The superior adherence of anaerobic lactobacilli may reflect a potential in maintaining or restoring normality.
Asunto(s)
Bacterias/crecimiento & desarrollo , Adhesión Bacteriana/fisiología , Vagina/microbiología , Bacterias/ultraestructura , Técnicas Bacteriológicas , Recuento de Colonia Microbiana , Epitelio/microbiología , Epitelio/ultraestructura , Femenino , Humanos , Lactobacillus/crecimiento & desarrollo , Microscopía de Contraste de Fase , Manejo de Especímenes/métodos , Staphylococcus/crecimiento & desarrollo , Estrés Mecánico , Vagina/ultraestructuraRESUMEN
Strip immunoblotting with specific, hyperimmune antisera and normal human sera (NHS), in conjunction with lectin and avidin ligand blotting and surface iodination, was used to investigate cell envelope components of phenotypes of Staphylococcus aureus NCTC 6571. Cocci were grown with a relatively slow doubling time (48 min) under iron sufficiency (Fe+) and iron insufficiency (Fe-), and with or without a sub-MIC of penicillin G (pen G), to approximate to in vivo conditions. Fe+ phenotypes demonstrated extra bands with prominent antigens at 48, 52 and 54 kD. Iron depletion or pen G simplified profiles, notably in the mid-range. In this particular S. aureus strain, protein A was ascribed a molecular mass of 35.5 kD and its detection was not affected by iron or pen G conditions. NHS reacted poorly with the Fe+ phenotype, supplying indirect evidence that this phenotype may not be common in vivo. Lectin blotting demonstrated the presence of glycosylated residues. Lectin affinities were not affected by the pen G treatment but in the Fe- phenotypes only, a 30 kD fucose-containing structure was seen. Avidin blotting visualised a major 86 kD binding site in all phenotypes which was not detected in immunoblotting. Whole cell radioiodination revealed that five major proteins of 21, 35.5, 48, 52 and 68 kD were surface-associated but their immunoreactivity depended upon the phenotype and source of sera.
Asunto(s)
Hierro/metabolismo , Penicilina G/farmacología , Staphylococcus aureus/química , Animales , Antígenos Bacterianos/análisis , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Humanos , Sueros Inmunes/inmunología , Immunoblotting , Ligandos , Masculino , Fenotipo , Conejos , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/inmunología , Staphylococcus aureus/ultraestructuraRESUMEN
Chronic osteomyelitis was produced by inoculating Staphylococcus aureus into rat tibia. The infection was characterised grossly by bone deformation and histopathologically by inflammation and the presence of coccal organisms sequestered within the bone tissue. Further observations by scanning electronmicroscopy demonstrated bacteria in microcolonies surrounded by dehydrated amorphous material that was considered to be glycocalyx. Transmission electronmicroscopy, when aided by antibody stabilisation, revealed extensive glycocalyx production within the tibia. These findings indicate that the rat model of chronic S. aureus osteomyelitis mimics the human infection with respect to the sessile mode of growth of bacteria within the bone. Serum antibody levels were assayed by ELISA and immunoblotting procedures. After an initial increase, ELISA titres remained relatively stable, apparently indicating the establishment of chronic osteomyelitis, whereas in immunoblotting an increase in titre over the course of infection was observed. Whole-cell ELISA revealed less subtle differences in antibody titre than did immunoblotting with cell-wall antigen. We found that mid-range antigens, including an antigen implicated as protein A, featured prominently in the immune response in this model of infection.
Asunto(s)
Modelos Animales de Enfermedad , Osteomielitis/patología , Infecciones Estafilocócicas , Staphylococcus aureus/patogenicidad , Animales , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Immunoblotting , Masculino , Microscopía Electrónica , Osteomielitis/inmunología , Osteomielitis/microbiología , Ratas , Ratas EndogámicasRESUMEN
Western blotting of whole-cell preparations of Enterococcus faecalis showed the protein-antigen profiles to be markedly influenced by growth conditions. The E. faecalis-specific antigens of 40 and 37 kDa, which are prominent in endocarditis, were strongly expressed following growth in serum or brain heart infusion, but not after growth in a chemically defined medium. The expression of these antigens in vivo was demonstrated in cells grown as a biofilm on silastic discs in the peritoneum of rabbits. These in vivo culture conditions may be useful in studying the pathogenesis of E. faecalis infections and the effectiveness of antibiotic therapy.
Asunto(s)
Antígenos Bacterianos/biosíntesis , Enterococcus faecalis/inmunología , Animales , Antígenos de Superficie/biosíntesis , Sangre , Western Blotting , Medios de Cultivo , Enterococcus faecalis/crecimiento & desarrollo , Humanos , ConejosRESUMEN
Gardnerella vaginalis has a very thin cell wall with a characteristic gram-negative staining pattern and an apparent lamellar structure when viewed at an oblique angle by electronmicroscopy. Examination at right angles to the cell-wall plane and by freeze-etching showed absence of an outer membrane or any other lamellar structure. Cell-wall extracts made by methods specific for lipopolysaccharide (LPS) gave negative reactions by silver staining and for endotoxin in the limulus amoebocyte lysate assay. 2-Keto-3-deoxy-D-manno-2-octonoic acid (KDO), heptose and hydroxy fatty acids specific for LPS were not detected in the extracts. G. vaginalis cell walls are unequivocally gram-positive in their ultrastructural characteristics and chemical composition.
Asunto(s)
Gardnerella vaginalis/ultraestructura , Haemophilus/ultraestructura , Lipopolisacáridos/análisis , Pared Celular/análisis , Pared Celular/ultraestructura , Grabado por Congelación , Gardnerella vaginalis/análisis , Microscopía Electrónica , Microscopía Electrónica de RastreoRESUMEN
Staphylococcus aureus NCTC 6571 was grown in iron-depleted tryptone soya broth (Fe-TSB) to approximate to in vivo conditions, and in iron-rich TSB (Fe + TSB). Low iron effected a crucial decrease in surface hydrophobicity (SH) and a lack of supernatant Protein A (PrA). Iron availability did not affect PrA detection in immunoblotting and it was identified as a 35.5 kDa antigen in this strain. Fe-phenotypes lacked 34, 48 and 52 kDa antigens. In chemiluminescence, Fe-phenotypes appeared least vulnerable to phagocytosis despite opsonisation.