Effects of extracts from Bulgarian medicinal plants on biofilm formation by UPEC clinical isolates
S. Stoitsova1, B. Mustafa1, J. Staneva2, M. Marhova3, A. Vacheva1, S. Kostadinova3, M. Todorova2, R. Ivanova1
1The Stephan Angeloff Institute of Microbiology, BAS; 2Institute of Organic Chemistry with Centre of Phytochemistry, BAS;3Plovdiv University ‘P. Hilendarski’
Bacteria grown as biofilm cause serious trouble in medical practice as a source of both contamination of indwelling medicinal devices, and nosocomial infections. Due to more rapid development of antibiotic resistance, if compared with planktonic microorganisms, novel anti-biofilm strategies address the search of substances that may suppress biofilm growth without killing the microorganisms themselves. This report presents the results of a primary screening test for plant substances corresponding to these requirements that may be applicable as lubricants of urinary catheters. In an initial series of experiments, 28 strains of clinically isolated uropathogenic E. coli (UPEC) have been tested for biofilm formation. Three of them form sufficient biofilm and were chosen to further check anti-biofilm activities. Antibiotic resistance of the chosen strains was determined by the disk-diffusion assay. The effects of 14 extracts in different organic solvents from 4 medicinal plants were tested. The dried extracts were dissolved as stocks in ethanol. Disk diffusion assay with different amounts of the extracts showed no antibacterial activity against the selected strains. Extracts had no significant influence on growth curves as well. Biofilm growth was examined by the crystal violet assay after growth for 24 h in M63 medium alone or supplemented with 10 μg/ml of the dried extracts. The strains were initially cultivated on nutrient agar and three separate colonies of each strain were selected and further used. The experiments were performed on 96-well microtitre plates. The wells were filled with 150 μl of the supplemented media, and 10 μl of overnight TSB bacterial cultures were inoculated. Each variant was repeated in 6 wells per colony. After removal of plankton, the wells were washed, colored with crystal violet, solubilized and analysed on ELISA reader. Biofilm suppression was registered with most of the extracts. However the effect of some of them showed variability between the UPEC strains and sometimes even - between the subcultures from single colonies of a given strain. Several of the extracts showed consistency in biofilm inhibitory effects, with lowest degree of inhibition against strain PU-19 which is also multidrug resistant. Purified substances from these extracts shall be further tested.
Acknowledgements: This study was funded by the National Science Fund of Bulgaria, Contract VU-L-321/07. The support of A. Vacheva by BG051PO001-3.3.04/32 HR Project should also be acknowledged.