This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.In spite of The US National Institutes of Healths recognition that: biofilms are medically important, accounting for over 80 percent of microbial infections in the body , an in-depth understanding of biofilm and strategies to control its formation, remain lacking. Adaptation of bacterially-derived, biofilm-degrading enzymes offers a novel option to biofilm control. Bacitracin, a peptide antibiotic derived from the biofilm-forming Bacillus licheniformis, inhibits the cell wall formation of Gram-positive bacteria. In humans, the use of this antibiotic is limited by its toxicity. In animals, however, bacitracin is widely used as a growth-promoting food additive and thought to contribute to the development of antibiotic resistance. We have found commercial bacitracin, including bacitracin formulated for human use by injection, to be contaminated with significant quantities of bacterially-derived protease, DNAse, RNAse, lipase, phosphatase and cellulose/ beta glucanase among others. Our data show that these enzymes are exceptionally active, resilient, and show biofilm-degrading capabilities. Our research is aiming to improve to our understanding of the degradative enzymes found in antibiotic bacitracin and to characterize their impact on biomedically-relevant biofilms. Our recent progress includes biochemical characterization, about 100-fold purification of the protease and the DNAse, and separation of the contaminating enzymes. Peptide sequence fragments, obtained by mass spectrometry of a 27KDa protein contaminant, are homologous to subtilisin Carlsberg, an alkaline serine protease secrete by Bacillus licheniformis. Currently, we are growing S. epidermis and P. fluorescens biofilms, defining the biofilm structure as a function of time, and, using SEM and a variety of biochemical approaches, to characterize the biofilm-inhibitory or mass-reducing effect on biofilm formation. We have additionally found chitosan, and a cationic detergent, to have a potent inhibitory effect on biofilm accumulation. Based on these finding, our current studies focus on the development of novel materials, including nanoparticles, which may mitigate biofilm formation or facilitate its removal.
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