Pseudomonas aeruginosa is both a model biofilm-forming organism and an opportunistic pathogen responsible for chronic lung infections in cystic fibrosis (CF) patients and infections in burn patients, among other maladies. Here we describe the development of an efficient high-throughput screen to identify small-molecule modulators of biofilm formation. This screen has been run with 66,095 compounds to identify those that prevent biofilm formation without affecting planktonic bacterial growth. The screen is a luminescence-based attachment assay that has been validated with several strains of P. aeruginosa and compared to a wellestablished but low-throughput crystal violet staining biofilm assay. P. aeruginosa strain PAO1 was selected for use in the screen both because it forms robust biofilms and because genetic information and tools are available for the organism. The attachment-inhibited mutant, strain PAO1 ⌬fliC, was used as a screening-positive control. We have also developed and validated a complementary biofilm detachment assay that can be used as an alternative primary screen or secondary screen for the attachment screening-positive compounds. We have determined the potencies of 61 compounds against biofilm attachment and have identified 30 compounds that fall into different structural classes as biofilm attachment inhibitors with 50% effective concentrations of less than 20 M. These small-molecule inhibitors could lead to the identification of their relevant biofilm targets or potential therapeutics for P. aeruginosa infections.Establishment of a bacterial infection in the form of a biofilm, a complex, three-dimensional, attached bacterial community, can have devastating consequences for patient morbidity and mortality. Individual cells within a biofilm are slowly growing and are embedded in an exopolymeric substance. These biofilm cells are relatively insensitive to many environmental stresses, including antibiotics and host immune responses (6). Because of the biofilm cells' intrinsic resistance to antibiotics, the infections that they cause persist, and eradication of these biofilm-related infections is a challenge (7). A new strategy for combating biofilms and persistent infections is desperately needed.Biofilm infections cause, contribute to, or complicate several conditions, including endocarditis, burns, periodontal disease, ear infections, chronic urinary tract infections, and pneumonia in patients with cystic fibrosis (CF) (7,29). Devices such as catheters (9) and ventilators (2) that are associated with longer hospital stays and prosthetic and implanted devices such as artificial heart valves, joints, and stents (11) provide surfaces for bacterial attachment, resulting in high rates of morbidity and mortality from nosocomial infections (18,20). In the United States, these infections are estimated to result in a 20% rate of mortality and to have an annual cost of $1 billion (18), so improvements in the prevention and treatment of biofilmrelated persistent infections represent a significant therape...

Phages have recently been implicated as important in biofilm development, although the mechanisms whereby phages impact biofilms remain unclear. One defective lambdoid phage carried by Escherichia coli K-12 is DLP12. Among the genes found in DLP12 are essD, ybcS and rzpD/rzoD, which are homologues of the Lambda phage genes encoding cell-lysis proteins (S, R and Rz/Rz 1 ). The role that these DLP12 lysis genes play in biofilm formation was examined in deletion mutants of E. coli PHL628, a curli-overproducing, biofilm-forming K-12 derivative. Strains lacking essD, ybcS and rzpD/rzoD were unable to form wild-type biofilms. While all mutants were compromised in attachment to abiotic surfaces and aggregated less well than the wild-type, the effect of the essD knockout on biofilm formation was less dramatic than that of deleting ybcS or rzpD/rzoD. These results were consistent with electron micrographs of the mutants, which showed a decreased number of curli fibres on cell surfaces. Also consistent with this finding, we observed that expression from the promoter of csgB, which encodes the curli subunits, was downregulated in the mutants. As curli production is transcriptionally downregulated in response to cell wall stress, we challenged the mutants with SDS and found them to be more sensitive to the detergent than the wild-type. We also examined the release of 14 C-labelled peptidoglycan from the mutants and found that they did not lose labelled peptidoglycan to the same extent as the wild-type. Given that curli production is known to be suppressed by N-acetylglucosamine 6-phosphate (NAG-6P), a metabolite produced during peptidoglycan recycling, we deleted nagK, the N-acetylglucosamine kinase gene, from the lysis mutants and found that this restored curli production. This suggested that deletion of the lysis genes affected cell wall status, which was transduced to the curli operon by NAG-6P via an as yet unknown mechanism. These observations provide evidence that the S, R and Rz/Rz 1 gene homologues encoded by DLP12 are not merely genetic junk, but rather play an important, though undefined, role in cell wall maintenance.

Escherichia coli strain PHL628 was subjected to saturating Tn5 transposon mutagenesis and then grown under competitive planktonic or biofilm conditions. The locations of transposon insertions from the remaining cells were then mapped on a gene array. The results from the array mapping indicated that 4?5 % of the E. coli genome was important under these conditions. Specifically, 114 genes were identified as important for the biofilm lifestyle, whereas 80 genes were important for the planktonic lifestyle. Four broad functional categories were identified as biofilm-important. These included genes encoding cell structures, small-molecule transport, energy metabolism and regulatory functions. For one of these genes, arcA, an insertion mutant was generated and its biofilm-related phenotype was examined. Results from both the transposon array and insertion mutagenesis indicated that arcA, which is known to be a negative response regulator of genes in aerobic pathways, was important for competitiveness in E. coli PHL628 biofilms. This work also demonstrated that ligation-mediated PCR, coupled with array-based transposon mapping, was an effective tool for identifying a large variety of candidate genes that are important for biofilm fitness.

The biofilm-specific gene expression of Escherichia coli PHL628 was compared with that from exponentially growing planktonic cells using macroarray technology. In duplicate experiments, both biofilm and planktonic cells were grown in separate continually stirred tank reactors at 57% of the maximal planktonic growth rate. When transcriptional results from planktonic cultures were compared with that of biofilm grown cells, c. 4.5% of the genome showed a significant change in expression. The results presented here point to an extremely heterogeneous biofilm wherein specific gene induction was consistent with the response of biofilm cells to gradients in electron acceptors, nutrients, carbon source and a variety of stresses. A mutant in one of the genes, gspM (pshM), that was induced in biofilms was constructed and was shown to be compromised for its ability to form mature biofilms. This analysis provides additional insight into the genes induced during biofilm development, the gradients they respond to, the contribution of one gene to biofilm development, and a comparison of this with other transcriptional profiles from E. coli biofilms.

These results call into question the long-term utility of triclosan incorporation into ABS plastic and highlight the need for proof of efficacy regarding the antimicrobial properties of such materials.