Phenotypic characterization of three clinical isolates of Burkholderia pseudomallei in Ceará, Brazil

Abstract

Melioidosis is a serious human disease caused by Burkholderia pseudomallei. Isolation of B. pseudomallei from human samples had not been reported in Brazil until 2003(Miralles et al. 2004, Braga & Almeida 2005. The purpose of this study was to compare the characteristics of these Brazilian isolates of the species with others reported in the literature, and to alert microbiologists in the region to these characteristics in order to enable them to recognize subsequent cases.In this study three isolates of B. pseudomallei were analyzed. These were isolated from clinical samples from a small cluster of cases that occurred in Tejuçuoca, Ceará, Brazil, involving three patients from the same family, aged 10-15 years old. Two strains of B. pseudomallei were initially isolated on MacConkey agar from the spleen, liver, and lung and one strain from blood culture in BHI broth. All procedures involving viable cells were performed in a category 3 containment laboratory.Strains were identified phenotipically according to the criteria described for gram-negative nonfermentative bacilli and by characteristics on Ashdown selective medium (Ashdown 1979, Gilligan & Whittier 1999, Koneman et al. 2001. The identification of B. pseudomallei was confirmed by biochemical tests using the API 20NE system (bioMérieux) (Dance et al. 1989).The bacteria were short, motile bipolar gram-negative bacilli. All three isolates showed characteristic bipolar staining, probably because of intracellular deposits of β-hydroxy butyric acid (Inglis et al. 2001, Sprague & Neubauer 2004. A thick and dry pellicle with a matt surface was seen on BHI broth cultures. This pellicle forms at the broth-air interface, possibly because of positive aerotaxis of B. pseudomallei, and reflects a type of a multicellular organization that resembles a biofilm.On MacConkey agar after 24 h of incubation the colonies were pink-transparent, small, smooth, with sheen and convex. After seven days of culture, isolates 1 and 3 showed larges, circulars, viscous, with sheen on surface and entire borders colonies. Isolate 2 also produced circulars, with surface sheen and pink colonies, however showed consistence dry and wrinkled.Ashdown's selective agar is commonly used to culture the organism from a mixed flora. All the B. pseudomallei isolates had characteristic colonial morphology on Ashdown agar. The colonies were smooth, moist, purple in color, dome shaped, about 0.8 to 1 mm in diameter, on plates after 24 h of incubation at 37ºC. After seven days of incubation isolate 2 grew as purple colonies, wrinkled, 5 to 6 mm in diameter, which had centrally umbonated, with radiating ridges at the periphery and a characteristic earthy odor; this showed a typical colonial morphology (Ashdown 1979). Isolates 1 and 3 included both smooth and mucoid colonies. The isolation of mucoid variant strains from clinical specimens is relatively uncommon (Inglis et al. 2001). Howard and Inglis (2003) have suggested that this may reflect inhibitory effect of crystal violet in Ashdown's agar. However, th...