Screening for genes involved inKlebsiella pneumoniaebiofilm formation using a fosmid library

Abstract

Klebsiella pneumoniae is a well-known opportunistic pathogen, often causing catheter-associated urinary tract infections. Biofilm formation on the catheter surfaces is an important step in the development of these infections. To identify the genes involved in the ability of K. pneumoniae to form a biofilm on abiotic surfaces, a novel strategy was used. A clone library was constructed by cloning the entire K. pneumoniae genome of the clinical isolate C3091 into a fosmid vector and the clone library was expressed in Escherichia coli. A total of 1152 clones were screened for enhanced biofilm formation compared with the E. coli parent strain using a biofilm microtiter plate assay. Nine clones with significantly enhanced biofilm formation were identified, subjected to random Tn5 transposon mutagenesis, screened for biofilm deficiency and the biofilm-promoting genes identified. Five of the clones contained the type 3 fimbriae gene cluster, a well-known K. pneumoniae virulence factor and biofilm promoter. Thus, the effectiveness of our approach was confirmed. Furthermore, genes encoding cell surface proteins and proteins involved in metabolism, none of them previously associated with biofilm formation in K. pneumoniae, were identified by our screening method. In conclusion, the use of fosmid libraries is an effective high throughput screening method to identify the genes involved in biofilm formation.