RESUMEN
An intraperitoneal chamber implant system has been used to investigate the phenotype of Staphylococcus aureus growing in the rat and the effect of the antibiotic flucloxacillin on bacterial growth in vivo. Titanium chambers were implanted in the peritoneum: a period of 3-4 days equilibration allowed diffusion of host proteins into the chamber fluid prior to inoculation with bacteria. S. aureus inoculated into the chamber fluid, grew rapidly over a 72 h period, reaching counts of > 10(9) per ml. Organisms harvested from chambers were analysed by SDS-PAGE and showed significant differences in polypeptide profiles from the same strain grown in nutrient broth in vitro. Analysis of whole cell extracts by Western-blotting revealed that protein A expression was repressed in S. aureus grown in vivo. Following subcutaneous administration, flucloxacillin levels in serum peaked earlier and were higher than those detected in chamber fluid. The inhibitory effect of the antibiotic on the growth of S. aureus in chambers in treated animals could be monitored easily by sequential sampling of the chamber fluid. These results indicate the potential of the chamber implant model for investigation of microbial phenotype in vivo and development of alternative methods for assessment of antimicrobial efficacy in vivo.
Asunto(s)
Cámaras de Difusión de Cultivos , Staphylococcus aureus/crecimiento & desarrollo , Animales , Estudios de Evaluación como Asunto , Femenino , Floxacilina/farmacocinética , Floxacilina/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Microscopía Electrónica , Fenotipo , Ratas , Ratas Wistar , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/ultraestructuraRESUMEN
Staphylococcus epidermidis was grown in vivo in chambers implanted intraperitoneally in rats. The cell wall and cytoplasmic membrane protein profiles of the in vivo-grown organisms were compared with those of S. epidermidis grown in vitro in nutrient broth (NB), in iron-restricted NB, or in pooled human peritoneal dialysate (HPD). Compared with growth in broth and in common with growth in HPD, growth in vivo in chambers resulted in the repression of many S. epidermidis wall proteins, with proteins of 27, 42, 54, and 70 kDa predominating. Growth in vivo also resulted in the induction of two iron-repressible cytoplasmic membrane proteins of 32 and 36 kDa, which were also present in staphylococci grown in HPD and in iron-restricted NB. Immunoblotting experiments revealed that in sera taken 21 days after inoculation of the intraperitoneal chambers, the predominant antibody response to cell envelope proteins was directed against the 32- and 36-kDa iron-repressible membrane proteins.
Asunto(s)
Proteínas Bacterianas/análisis , Proteínas de la Membrana/análisis , Peritoneo/microbiología , Staphylococcus epidermidis/crecimiento & desarrollo , Animales , Pared Celular/química , Citoplasma/química , Ratas , Staphylococcus epidermidis/química , Staphylococcus epidermidis/inmunologíaRESUMEN
An accurate reflection of the pathogenicity of microorganisms and the therapeutic effects of antimicrobial agents on their growth necessitates testing within an in vivo environment. We have developed a novel diffusion chamber, incorporating two 0.22 microns membrane filters, for the growth of in vivo organisms. The chamber, which is implanted intraperitoneally into the rat, has an external sampling portal. This portal allows multiple and sequential sampling of the microbial inoculum without killing the rat, thus significantly reducing the total number of animals used in such studies. In addition, the chamber is superior to other reported implants since it is well tolerated, reusable, easily constructed and can be used within two days of implantation. Staphylococcus epidermidis and a toxic shock syndrome toxin-1 (TSST-1) producing strain of S. aureus have been successfully grown within in vivo chambers, with 10(8)-10(9) organisms per millilitre being recovered within 48 h. Scanning electron microscopy revealed clusters of staphylococci and fibrous material adhering to the inner surface of the filters, with numerous phagocytic cells attached to the outer side. Western immunoblotting indicated that higher levels of TSST-1 were produced by S. aureus grown in vivo as opposed to cells grown in vitro.
Asunto(s)
Antibacterianos/uso terapéutico , Toxinas Bacterianas , Cámaras de Difusión de Cultivos/métodos , Evaluación de Medicamentos/métodos , Superantígenos , Animales , Técnicas Bacteriológicas , Enterotoxinas/análisis , Enterotoxinas/biosíntesis , Femenino , Politetrafluoroetileno , Ratas , Ratas Endogámicas , Choque Séptico/microbiología , Staphylococcus/crecimiento & desarrollo , Staphylococcus aureus/patogenicidad , Staphylococcus epidermidis/patogenicidad , VirulenciaRESUMEN
Measurement of Candida cell wall polysaccharide (mannan) with an enzyme-linked immunosorbent assay (ELISA) was evaluated as an alternative to viable counting for the enumeration of yeast cell numbers in the human vagina. A statistically significant association was found between mannan levels and the number of colony forming units in vaginal washings collected from 40 women infected with Candida albicans (r = 0.81), indicating the accuracy of mannan levels in reflecting yeast cell load in vulvo-vaginal candidosis. Subsequent comparisons revealed a significant association between mannan levels and clinical signs in the vagina, so reflecting the importance of vulvitis and vaginitis as clinical markers for determining the severity of infection. No association was found between yeast load and the clinical symptoms, indicating the high degree of patient subjectivity. Estimation of mannan levels could be developed and used for the rapid laboratory investigation of chronic vulvo-vaginal candidosis.