Screening And Mechanism Study Of Chinese Herbal Extracts Against Pseudomonas Aeruginosa Biofilms

It becomes clear in the last two decades that bacterial biofilms play an important role the persistence of bacterial infections. Bacterial biofilms are formed when unicellular organisms come together to form a community that is attached to a solid surface and encased in an exopolysaccharide matrix. Biofilms can be made up of single or multiple bacterial species. It has been reported that bacteria living in biofilm exhibit dramatically increased resistance to antibiotics.. In fact, when cells exist in a biofilm, they can become10-1000times more resistant to antimicrobial agents than planktonic cells. There is no doubt that biofilms have enormous implications in the development and treatment of bacterial diseases..Pseudomonas aeruginosa is an important human pathogen which often forms biofilms. Many studies have been done on inhibition of P.a biofilm by antibiotics. In this study we aim to screen for inhibitory compounds against P. aeruginosa biofilms from draditional Chinese medicine; and to investigate the mechanism of inhibition observed. The connection between biofilm and quorum sensing was analyzed as well. The ultimate goal is to develop novel compounds from Chinese herbal medicine for treatment of P. aeruginosa biofilm.To construct a biofilm gene reporter system, we selected12gene clusters that are associated with biofilm synthesis and regulation. The promoter ergions of these genes were by amplified by polymerase chain reaction (PCR), and inserted in the upstream of the promoterless luciferase gene cluster (luxCDABE). After transfering to P. aeruginosa the promoter-reporter were used to monitor changes in gene expression by measuireing light production. Such a system was then used to screen for inhibitors targeting biofilm related genes.A compound library consisting of water and ethanol extracts from60Chinese herbs was screened using the above system. Samples affecting biofilm-releated gene expression were selected for further investigation. A total of20herbal extract showed an effect on biofilm related genes. Their effect on biofilm formation was then tested by muti-well plate biofilm assays.the resultsconfirmed the effect of12extracts from following Chinese herbs in the order of inhibition strenghth; dark plum fruit, radixsanguisorbae, honeysuckle flower, baical skullcap root, common threewingnut root, China ixeris, agrimony, areca seed, pomegranate rind, java brucea fruit, andrographis paniculata, and ash bark. No growth inhibition was observed with these extracts at the test concentrations.It has been reported that the formation and maintenance of biofilm are regulated by the quorum sensing systems. We also studied the impact of sub-inhibitory concentrations of herbal drugs on QS systems and its regulated products. They include the production of pyocyanin, elastase, rhamnolipid, and alginate. The results indicate that6herbs had inhibitory effect on alginate production. In summary, the results obtained in this study provides a basis for further exploration of Chinese herbs for the novel compounds against P. aeruginosa biofilms.