RESUMO
INTRODUCTION: Group A streptococci (GAS) is responsible of several human diseases ranging from mild infection to severe invasive toxin-mediated disease and post-infectious sequelae. Accordingly, a GAS surveillance program based on molecular techniques is advisable for its epidemiological control. Pulsed-field gel electrophoresis (PFGE) is the gold standard for GAS molecular subtyping, but a major disadvantage is the length of the procedure, which takes 1-3 days of work, minimum. The aim of this study was to develop a rapid and cost-effective procedure for PFGE subtyping of GAS isolates. METHODOLOGY: Different incubation times of GAS, immobilized in agarose miniplugs, in solutions containing lysozyme and/or mutanolysine followed by solutions with urea instead of proteinase K, were assayed. DNA was restricted with SmaI and the fingerprints were obtained in clamped homogeneous electric field (CHEF) chambers and minichambers. The modified procedure was used to subtype 22 GAS isolates. RESULTS: Intact DNA molecules of GAS immobilized in agarose miniplugs were prepared incubating the cells, in situ, with a solution containing lysozyme for 4 hours, followed by the incubation in a non-enzymatic solution with urea for 2 hours. SmaIDNA macrorestriction fragments were well resolved in 5 hours and 14 minutes by electrophoresis in a CHEF minichamber at 10 V/cm. This procedure for GAS DNA preparation was useful for fingerprinting GAS strains in the format of CHEF Mapper (BioRad). CONCLUSIONS: The procedure took 13 hours for GAS strains subtyping. Both sample preparation and electrophoresis in CHEF minichamber represent an economic alternative for performing massive epidemiological studies of this human pathogen.
Assuntos
DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado/economia , Eletroforese em Gel de Campo Pulsado/métodos , Ácidos Nucleicos Imobilizados/genética , Tipagem Molecular/economia , Tipagem Molecular/métodos , Streptococcus pyogenes/classificação , Custos e Análise de Custo , DNA Bacteriano/isolamento & purificação , Humanos , Ácidos Nucleicos Imobilizados/isolamento & purificação , Epidemiologia Molecular/métodos , Streptococcus pyogenes/genética , Fatores de TempoRESUMO
Las células mononucleares fagocíticas obtenidas de la cavidad peritoneal de la rata, cambian las magnitudes de los potenciales de transmembranas de acuerdo con el estímulo fagocítico y en relación con el receptor estimulado. La incubación de estas células en medios iónicos, donde indistintamente se les bajó las concentraciones extracelulares de Na+, K+, Ca++ y Mg++ mostraron despolarizaciones de la membrana antes de inducir la fagocitosis que estimularon los receptores específicos Fc con latex cubierto con inmunoglobulinas IgG en los medios bajos Na+, Ca+, y Mg++, no se modificaron para los medios de K+. El estímulo fagocítico; provocó hiperpolarizaciones de la membrana; para todos los cambios iónicos pero fueron de menor magnitud para los decrementos de Na+, Ca+, y Mg++, respecto a lo registrado en las soluciones tyrode normales. Los tiempos en que ocurren estos cambios de PT también se modifican. Estos resultados muestran la importancia de las concentraciones iónicas extracelulares en la capacidad fagocítica de los macrófagos