Résumé
RESUMEN Streptococcus agalactiae (SGB) produce infecciones invasivas en neonatos siendo la transmisión materna la más frecuente. Estudios epidemiológicos utilizan técnicas moleculares que evalúan la diversidad genética, entre ellas la de amplificación aleatoria de ADN polimórfico (RAPD) que resulta ser accesible, sensible y utiliza cebadores arbitrarios para amplificar segmentos polimórficos de ADN mediante PCR. El objetivo fue determinar la relación clonal entre aislamientos de SGB recuperados de madres y sus respectivos recién nacidos. Se estudiaron por RAPD cuatro parejas de aislamientos de SGB obtenidos de hisopados vagino-rectales de madres y de hemocultivos de sus neonatos. Se utilizaron los cebadores OPS11, OPB17 y OPB18 para seleccionar uno con capacidad de discriminar entre cepas no relacionadas genéticamente. Se utilizó la fórmula de Hunter-Gaston que establece el índice de discriminación (D), cuando D>0,90 se considera que los aislamientos pertenecen a clones diferentes. Los perfiles de amplificación para los ocho aislamientos, empleando independientemente cada cebador, permitieron calcular un D=1 para OPS11, y D=0,84 para OPB17 y OPB18. Por lo tanto, OPS11 fue seleccionado para el estudio de la relación clonal de los aislamientos, encontrándose perfiles de amplificación similares por RAPD para cada par de cepas madre-recién nacido. Se observaron diferentes perfiles de amplificación entre los pares de cepas madre-recién nacido, lo que revela la discriminación entre cepas no relacionadas, resultados confirmados por electroforesis en campo pulsante (PFGE). Estos resultados indican transmisión vertical para cada caso estudiado y robustez del cebador OPS11. Se encontraron condiciones apropiadas del ensayo de RAPD, lo que es útil para estudios epidemiológicos.
ABSTRACT Streptococcus agalactiae (GBS) causes invasive infections in newborns, being the most frequent the maternal transmission. Epidemiological studies use molecular techniques that assess genetic diversity, including random amplification of polymorphic DNA (RAPD) that is found to be accessible, sensitive and uses arbitrary primers to amplify polymorphic segments of DNA by PCR. The objective was to determine the clonal relationship between GBS strains recovered from mothers and their respective newborns. Four pairs of GBS isolates obtained from vaginal-rectal swabs of mothers and blood cultures of their newborns were studied with RAPD. Primers OPS11, OPB17 and OPB18 were used to select one with the ability to discriminate between non-genetically related strains. The Hunter-Gaston formula that establishes the discrimination index (D) was used; when D>0.90, it is considered that the isolates belong to different clones. The amplification profiles for the eight isolates, using each primer independently, allowed to calculate a D=1 for OPS11, and D=0.84 for OPB17 and OPB18. Therefore, OPS11 was selected for the study of the clonal relationship of the isolates, and similar amplification profiles were found by RAPD for each mother-newborn pair of GBS isolates. Different amplification profiles were observed between pairs of mother-newborn strains, which reveals the discrimination between unrelated strains, confirmed by pulsating field electrophoresis (PFGE). These results indicated vertical transmission for each studied case and robustness of the OPS11 primer. Appropriate conditions of the RAPD trial were found, which is useful for epidemiological studies.
Résumé
Introduction: Diarrhoea caused by contaminated water is among the most prevalent waterborne diseases in the developing countries like India. In the interest of public health, water supplies should be tested regularly to confirm their freedom from contamination. Objective: The objectives of the study were to screen different water sources for bacterial contamination, to know the antibiotic susceptibility of the common bacterial isolates and typing of the bacterial isolates by random amplification of polymorphic DNA (RAPD) technique. Place and Duration of the Study: Kasturba Medical College Hospital, Microbiology Laboratory, Mangalore, Karnataka, India between August 2007 and August 2009. Methodology: Water samples (n=324) were analyzed by standard microbiological techniques for bacterial contamination. Isolates were identified biochemically and antibiotic susceptibility testing was done by disc diffusion method. Escherichia coli isolates were typed by RAPD technique. Results: Among the water samples tested, 246 were excellent and 78 were contaminated. Contaminated samples showed the growth of commensal bacteria belonging to the family Enterobacteriaceae along with pathogens like Salmonella spp. and Vibrio spp. Many of the isolates were found to be sensitive and a few were found to be resistant to the antibiotics tested. RAPD typing showed genetic similarity and differences among the E. coli isolates from different water sources. Conclusion: Genetic similarity among isolates of E. coli indicates a common ancestral origin or a common source. Bacterial contamination of water samples with pathogens like, Salmonella spp. and Vibrio spp. as well as the faecal coliform is a concern, as water quality is an index of health and well - being of the society. Degree of contamination observed in this study suggests a need to be vigilant to monitor water quality, in order to prevent enteric diseases.