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Objective: To evaluate the genetic diversity of clinical and environmental isolates of Burkholderia pseudomallei (B. pseudomallei) recovered in Cear′a, Brazil, and screen these isolates for the presence of type three secretion system virulence gene. Methods: Nineteen B. pseudomallei isolates (9 from clinical cases and 10 from soils) were analyzed. Random amplified polymorphic DNA was performed with primers OPQ-2, OPQ-4 and OPQ-16 to evaluate the genetic diversity, and type three secretion system gene was detected through polymerase chain reaction. Results: Random amplified polymorphic DNA showed a genetic relatedness of approximately 50% among the tested B. pseudomallei isolates, which were grouped into two clades, of which the biggest ones comprised 18/19 isolates for primer OPQ-2, and 17/19 isolates for primer OPQ-16. Primer OPQ-4 grouped the isolates into three clades comprising 1/19, 3/19 and 15/19 isolates. Additionally, type three secretion system gene was detected in all tested isolates. Conclusions: This is an effort to type B. pseudomallei strains from Cear′a, which is important for better understanding this pathogen, contributing for the epidemiological surveillance of melioidosis in this endemic region.