Molecular typing of Burkholderia contaminas by PCR fingerprinting in comparison with Fourier transform infrared-spectroscopy-based phenotyping.

PABLO MARTINA, GONZALO SEQUEIRA, CONSTANZA MANNINO, ALEJANDRA BOSCH, ANTONIO LAGARES, AND OSVALDO YANTORNO*

Hotel Portal del Lago, Villa Carlos Paz, Cordoba

Workshop; II Workshop in Current Topics in Pseudomonas and Burkholderia Research; 2009

SAMIGE

The Burkholderia cepacia complex (BCC) is a closely related group of Gram-negative bacteria found in many niches of both natural and clinical environments. Members of the BCC are symbionts of plant rhizospheres, contaminants of pharmaceutical and industrial products, inhabitants of soil and surface waters, and opportunistic pathogens, capable of causing disease in plants, invertebrates, animals, and humans. They can be particularly devastating, highly virulent, cystic fibrosis (CF) pathogens that are also able to cause nosocomial infections. BCC taxonomy has undergone considerable changes over the last years, and is known to include at least 15 distinct species. The identification and discrimination of BCC at the species level need multiple diagnostic tests due to misidentification that can easily occur.A relatively new technique that is fast becoming the "gold standard" of bacterial typing methods is multilocus sequence typing which represents the sequencing of 7 different genes. However, this technique is expensive, laborious, time consuming, and considered unattractive for routine application. In this work we report the use of a combination between the PCR-RFLP patterns of recA and gyrB genes applying HaeIII digestion enzyme as a tool for rapid discrimination and identification of BCC isolates. Fifty-seven isolates belonging to different environments and hospitals previously identified by biochemical method and recA gene sequencing (as gold standard identification method) were used. PCR-RFLP analyse were performed by amplification of recA and gyrB genes, subsequent digestion with HaeIII enzyme and the program NEBcutter V2.0 was applied. The recA-RFLP patterns obtained were the ones described previously for all the species, which do not allow the discrimination among B. cenocepacia , B. stabilis, and B. contaminas. Nevertheless, when these patterns were combined with the ones obtained by gyrB-RFLP treated with HaeIII restrictive enzyme, the BCC species of incidence in our country could be discriminated. In addition, gyrB-RFLP HaeIII, which formal patterns for the different species is described here for the first time, revealed an important diversity among B. contaminans isolates, the species of major incidence in Argentina, In conclusion, the strategy of combining these two RFLP patterns proved to be adequate, rapid and simple for the proper discrimination among the most closely related BCC species.