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
). 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
). 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 biofilm-related persistent infections represent a significant therapeutic opportunity.
In an attempt to identify therapeutic agents for and the therapeutic targets of biofilm-forming opportunistic pathogens, much research has focused on Pseudomonas aeruginosa
. In addition to genes for biofilm formation, the P. aeruginosa
genome contains genes for several drug efflux pumps, including mexAB-oprM
, and mexXY
, that contribute to the organism's ability to resist antibiotics (27
) and its ability to create an infection in host tissues, such as the lungs of CF patients (12
). P. aeruginosa
colonizes the lungs of approximately 21% of CF patients within the first year of life, and by the age of 26 years, nearly 80% of CF patients are colonized. The irreversible damage caused by the recurrent P. aeruginosa
lung infections is a serious problem facing most CF patients, and P. aeruginosa
contributes to the death of 90% of CF patients (14
). Recent advances in antimicrobial therapy and the discovery and use of drugs with antipseudomonal activities, including ceftazidime, aztreonam, ciprofloxacin, and imipenem, have decreased the incidence of P. aeruginosa
). Despite the advances in antibiotics, the incidence of P. aeruginosa
bacteremia compared to that of infections caused by other gram-negative bacteria has not drastically declined in the past 20 years (26
In addition to the medical reasons given above, P. aeruginosa
is also an excellent gram-negative bacterial model for the study of the biology of biofilms because of the genetic and physiological information available. In particular, molecular tools which facilitate genetic manipulation have already been developed for P. aeruginos
a, particularly strain PAO1. Systematic resources are also available, including microarrays and the P. aeruginosa
strain PAO1 genome (36
). Several libraries of P. aeruginosa
mutants have been created, and the organisms have been examined for their biofilm-forming phenotype. Mutations in motility, notably, flagellar motility, decreased the amount of initial biofilm attachment (28
); and a mutant deficient in flagellum synthesis and initial biofilm attachment (PAO1 ΔfliC
) provided an ideal screening-positive phenotype for this work.
A potential strategy for the prevention and treatment of P. aeruginosa
biofilm infections would be the use of small molecules to inhibit biofilm development and/or promote biofilm dispersal without the use of a lethal selection pressure. The cells dispersed from a biofilm would be more susceptible to conventional antibiotics and the immune system (8
). The halogenated furanones illustrate the potential of small molecules to disrupt bacterial chemical signaling and biofilm formation by some bacteria, although not P. aeruginosa
). These molecules structurally resemble bacterial acyl-homoserine lactone quorum-sensing molecules (17
) and effectively interfere with the reception of the signal, the subsequent gene expression, and the swarming phenotype (24
). High-throughput screening (HTS) could be used to identify other compounds effective against P. aeruginosa
biofilm development. Ultimately, such compounds could be developed either as small-molecule tools that could be used to study biofilm formation or as therapeutic agents for the prevention and treatment of biofilm infections.
An HTS method requires an appropriate assay. A crystal violet (CV) staining assay had been developed and widely adopted as a means of examining biofilm development on synthetic surfaces (28
). CV is a common and inexpensive bacterial cell membrane stain that has been used to quantify biofilm attachment. Although the CV method has been successfully used to screen for attachment and pellicle mutants, it has shortcomings in terms of its dynamic range, the amount of time required per plate, and its reproducibility; and these shortcomings preclude its use as a robust HTS method. An improved assay is needed to screen large, chemically diverse small-molecule libraries in an HTS format.
This report describes the development of luminescence-based biofilm screens for both attachment and detachment and the validation and implementation of the attachment screen to identify small molecules that disrupt biofilm development by P. aeruginosa in an HTS format. The results from the established CV method and the new luminescence-based method are included for comparison; and the development, validation, and use of this protocol to screen 66,095 compounds for their specific antibiofilm activities are described.