PHENOTYPIC CHANGES OF CLINICAL ISOLATES OF BURKHOLDERIA CONTAMINANS ASSOCIATED TO LONG-TERM CYSTIC FIBROSIS LUNG INFECTION

CLAUDIA PRIETO, PABLO MARTINA, KATRIN HENTSCHEL, AILÉN NATALE, ALEJANDRA BOSCH AND OSVALDO YANTORNO

Mar del Plata

Congreso; VIII Congreso Argentino de Microbiología General; 2012

Soceidad Argentina de Microbiología General

Burkholderia cepacia complex (Bcc) comprises Gram-negative bacteria which are opportunistic pathogens that cause infections in the airways of cystic fibrosis (CF) patients. We and other have previously reported that B. contaminans (Bc) is the species of major incidence among Bcc bacteria that infect CF patients in our country. In addition, a number of studies have been carried out to elucidate the mechanisms used by Bcc bacteria for their persistence in the host lungs with the aim to identify vulnerabilities and potential targets for future antimicrobial agents. Colonization and persistence of Burkholderia species in the lungs have been associated with their capacity of biofilm formation. Besides, long-term Bcc lung infection gave rise to genotypic and phenotypic variation among isolates, contributing to their fitness while maintaining their ability for survival in this human niche. Here we studied two B. contaminans isolates sequentially recovered from sputum samples of the same CF patient. One isolate was recovered when the infection was originally detected (Bc 64) and the other after 3 years of infection (Bc 187). Growth rate of planktonic cells, colony morphology, production of haemolysin and protease and antibiotic resistance for planktonic (MIC-P) and sessile (MIC-B) cells were determined for each isolate. Biofilm studies were carried out using multiwell plates, column bioreactors and continuous flow chambers. Biofilm formation was monitored by crystal violet stain (CV), and Fourier transform infrared spectroscopy (FT-IR). The architecture of biofilms was analyzed by the application of staining methods with fluorescent microscopy (FM) and confocal laser scanning microscopy (CLSM). To test genetic variability both isolates were firstly identified by traditional techniques and sequencing of recA gene. Then, they were characterized by PCR- recA RFLP using HaeIII as restriction enzyme and by fingerprinting of DNA using BOX-PCR. Both isolates showed similar pattern in all these studies, so they could come from a similar clon. The isolate recovered from the first patient’s infection showed in LB solid medium: yellow colonies with higher polysaccharide content, while the Bc 187 isolate showed white colonies of lower size. The haemolysin production was higher for the chronic isolate but the production of proteases was similar in both cases. In LB liquid cultures both isolates showed similar specific growth rate and yield but the Bc 187 isolate grown in aggregates. For biofilm cultures the Bc 64 isolate showed higher growth rate adhered to surfaces than the Bc 187 isolate. The structure of mature biofilm was flat for Bc 64 isolate. In contrast, the isolate retrieved from the long-term infection showed a defined cloud structure. Both isolates showed a similar behavior in MIC-P and MIC-B studies, displaying a significant higher antibiotic resistant when they growth as biofilm than in planktonic mode. Overall, adaptation during Bcc chronic lung infections showed phenotypic variation that could contribute to increase the capacity of these bacteria for survival and persistence in their host.