Influence of the culture medium in the evaluation of biofilm inhibitory concentrations for isolates of Burkholderia cenocepacia

Bacteria belonging to the Burkholderia cepacia complex (Bcc) principally cause chronic pulmonary infections in cystic fibrosis (CF) patients. They are intrinsically resistant to many antibiotics, so therapeutic options are often limited. Moreover, their ability to form biofilms (BF) can retard contact with some antimicrobials, making these infections difficult to eradicate even by drugs that display a Minimal Inhibitory Concentration (MIC) lower than the breakpoint. But the MIC is evaluated on the planktonic forms whereas the higher resistance of the sessile forms (BIC: Biofilm Inhibitory Concentration) towards some antibiotics commonly used in the clinical management of lung infections has been widely described (REF). One variable experimental condition in the evaluation of the BIC is the choice of the medium. Data available till now have been obtained using different media, as no guidelines have been established yet. However, preliminary results suggest that the composition of the BF matrix (protein/EPS ratio) varies depending on the medium used to culture bacteria (Cescutti, personal communication). We evaluated the antibiotic susceptibility of a B. cenocepacia clinical isolate, named BTS2, which has stably colonized a patient for ten years, is a good biofilm producer in the sample taken in 2000 (BTS2-00) while shows a significant reduction in this capacity 9 years later (BTS2-09). We compared the susceptibility of its sessile forms grown inside the biofilm synthesized in three different media: Muller Hinton Broth (MHB), commonly used for antimicrobial susceptibility tests; Yeast Extract Medium (YEM), where Bcc produce large amount of the EPS cepacian (REF); and Synthetic Cystic Fibrosis Medium (SCFM), a synthetic medium that mimics the nutritional composition of CF sputum (REF). A modification of the Calgary Device method was used (REF). Bacteria were initially grown as biofilm using one of the media listed above. Then, the incubation with the antibiotic was always carried on in MHB. Some of the tested drugs showed different activity levels on the BF depending on the culture medium used, but the most important difference was observed after incubation with aztreonam, whose BICs in different media diverged more than 50x even in absence of significant differences of the BF production. So, as it seems difficult to ascribe only to the thickness of the BF matrix the protection against the drug, we are planning to analyse its ability to spread into the different BF matrices following the course of a labelled molecule by confocal microscopy analysis.