RESUMEN
Clostridium perfringens is one of the etiologic agents of gas gangrene that can occur when a wound is contaminated with soil. Type A C. perfringens can cause foodborne and nonfoodborne gastrointestinal illnesses due to an enterotoxin (CPE) produced by some strains during sporulation. We developed a quantitative real-time PCR assay based on fluorescence resonance energy transfer hybridization chemistry that targets the C. perfringens-specific phospholipase C (plc) gene and the enterotoxigenic gene (cpe) with the LightCycler and the Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.). The assay can detect as few as 20 copies of target sequences per PCR. The total assay time, from extraction to PCR analysis, is 90 min. This assay is rapid, sensitive, and specific and will allow direct detection of C. perfringens in water, food, and stool samples. It should prove helpful in investigating foodborne illnesses due to C. perfringens and can be used as a tool to ensure the safety of food and water supplies.
Asunto(s)
Clostridium perfringens/aislamiento & purificación , ADN Bacteriano/análisis , Microbiología de Alimentos , Reacción en Cadena de la Polimerasa/métodos , Microbiología del Agua , Secuencia de Bases , Clostridium perfringens/clasificación , Enterotoxinas/análisis , Enterotoxinas/genética , Fluorescencia , Contaminación de Alimentos/análisis , Datos de Secuencia Molecular , Filogenia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Esporas Bacterianas , Factores de TiempoRESUMEN
Streptococcus agalactiae (Group B Streptococcus: GBS) is the major causative agent of neonatal sepsis. Neonates at risk for GBS infections are empirically administered broad-spectrum antibiotics for at least 48 h pending blood culture results. A rapid assay to expedite detection of GBS would facilitate initiation of specific antibiotic therapy. Conversely, expeditious proof of absence of infection will avoid unnecessary antibiotic use. Using the LightCycler, we evaluated a hybridization probe polymerase chain reaction (PCR) assay to detect GBS-specific cfb gene target DNA sequence in blood specimens. Both sensitivity and specificity of the real-time PCR assay was 100%. The assay demonstrated 100% specificity when tested against 26 non-GBS bacteria. This method is capable of detecting as few as approximately 100 copies or 10 pg of GBS genomic DNA. This real-time PCR method is rapid, sensitive, and specific for the detection of GBS in neonatal blood samples and holds great promise in its utility in the diagnostic laboratory.
Asunto(s)
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones Estreptocócicas/sangre , Streptococcus agalactiae/aislamiento & purificación , Secuencia de Bases , Southern Blotting , ADN Bacteriano/análisis , Electroforesis en Gel de Agar , Femenino , Humanos , Recién Nacido , Masculino , Datos de Secuencia Molecular , Sensibilidad y Especificidad , Infecciones Estreptocócicas/diagnóstico , Factores de TiempoRESUMEN
TolC is the outer-membrane component of several multidrug resistance (MDR) efflux pumps and plays an important role in the survival and virulence of many gram-negative bacterial animal pathogens. We have identified and characterized the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16. The gene was found to encode a 51-kDa protein with 70% identity to its Escherichia coli homologue. The E. chrysanthemi gene was able to functionally complement the E. coli tolC gene with respect to its role in MDR efflux pumps. A tolC mutant of E. chrysanthemi was found to be extremely sensitive to antimicrobial agents, including several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective in the efflux of berberine, a model antimicrobial plant chemical. These results suggest that by conferring resistance to the antimicrobial compounds produced by plants, the E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue.