Biofilms are surface-attached groups of microbial cells encased in an extracellular matrix that are significantly less susceptible to antimicrobial agents than non-adherent, planktonic cells. Biofilm-based infections are, as a result, extremely difficult to cure. A wide range of molecular mechanisms contribute to the high degree of recalcitrance that is characteristic of biofilm communities. These mechanisms include, among others, interaction of antimicrobials with biofilm matrix components, reduced growth rates and the various actions of specific genetic determinants of antibiotic resistance and tolerance. Alone, each of these mechanisms only partially accounts for the increased antimicrobial recalcitrance observed in biofilms. Acting in concert, however, these defences help to ensure the survival of biofilm cells in the face of even the most aggressive antimicrobial treatment regimens. This review summarises both historical and recent scientific data in support of the known biofilm resistance and tolerance mechanisms. Additionally, suggestions for future work in the field are provided.

Biofilms are surface-attached microbial communities with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts. One of the best-known of these biofilm-specific properties is the development of antibiotic resistance that can be up to 1,000-fold greater than planktonic cells. We report a genetic determinant of this high-level resistance in the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa. We have identified a mutant of P. aeruginosa that, while still capable of forming biofilms with the characteristic P. aeruginosa architecture, does not develop high-level biofilm-specific resistance to three different classes of antibiotics. The locus identified in our screen, ndvB, is required for the synthesis of periplasmic glucans. Our discovery that these periplasmic glucans interact physically with tobramycin suggests that these glucose polymers may prevent antibiotics from reaching their sites of action by sequestering these antimicrobial agents in the periplasm. Our results indicate that biofilms themselves are not simply a diffusion barrier to these antibiotics, but rather that bacteria within these microbial communities employ distinct mechanisms to resist the action of antimicrobial agents.

In living organisms, biofilms are defined as complex communities of bacteria residing within an exopolysaccharide matrix that adheres to a surface. In the clinic, they are typically the cause of chronic, nosocomial, and medical device-related infections. Due to the antibiotic-resistant nature of biofilms, the use of antibiotics alone is ineffective for treating biofilm-related infections. In this review, we present a brief overview of concepts of bacterial biofilm formation, and current state-of-the-art therapeutic approaches for preventing and treating biofilms. Also, we have reviewed the prevalence of such infections on medical devices and discussed the future challenges that need to be overcome in order to successfully treat biofilms using the novel technologies being developed.

Bacterial biofilms are the basis of many persistent diseases. The persistence of these infections is primarily attributed to the increased antibiotic resistance exhibited by the cells within the biofilms. This resistance is multifactorial; there are multiple mechanisms of resistance that act together in order to provide an increased overall level of resistance to the biofilm. These mechanisms are based on the function of wild-type genes and are not the result of mutations. This article reviews the known mechanisms of resistance, including the ability of the biofilm matrix to prevent antibiotics from reaching the cells and the function of individual genes that are preferentially expressed in biofilms. Evidence suggests that these mechanisms have been developed as a general stress response of biofilms that enables the cells in the biofilm to respond to all of the changes in the environment that they may encounter.

Bacteria growing in biofilms are more resistant to antibiotics than their planktonic counterparts. How this transition occurs is unclear, but it is likely there are multiple mechanisms of resistance that act together in order to provide an increased overall level of resistance to the biofilm. We have identified a novel efflux pump in Pseudomonas aeruginosa that is important for biofilm-specific resistance to a subset of antibiotics. Complete deletion of the genes encoding this pump, PA1874 to PA1877 (PA1874-1877) genes, in an P. aeruginosa PA14 background results in an increase in sensitivity to tobramycin, gentamicin, and ciprofloxacin, specifically when this mutant strain is growing in a biofilm. This efflux pump is more highly expressed in biofilm cells than in planktonic cells, providing an explanation for why these genes are important for biofilm but not planktonic resistance to antibiotics. Furthermore, expression of these genes in planktonic cells increases their resistance to antibiotics. We have previously shown that ndvB is important for biofilm-specific resistance (T. F. Mah, B. Pitts, B. Pellock, G. C. Walker, P. S. Stewart, and G. A. O'Toole, Nature 426:306-310, 2003). Our discovery that combining the ndvB mutation with the PA1874-1877 gene deletion results in a mutant strain that is more sensitive to antibiotics than either single mutant strain suggests that ndvB and PA1874-1877 contribute to two different mechanisms of biofilm-specific resistance to antibiotics.

The stable association of the N gene transcriptional antiterminator protein of bacteriophage A with transcribing RNA polymerase requires a nut site (boxA+ boxB) in the nascent transcript and the Escherichia coli factors NusA, NusB, NusG, and ribosomal protein S10. We have used electrophoretic mobility shift assays to analyze the assembly of N protein, the E. coli factors, and RNA polymerase onto the nut site RNA in the absence of a DNA template. We show that N binds boxB RNA and that subsequent association of NusA with the N-nut site complex is facilitated by both boxA and boxB. In the presence of N, NusA, and RNA polymerase the nut site assembles ribonucleoprotein complexes containing NusB, NusG, and S10. The effects on assembly of mutations in boxA, boxB, NusA, and RNA polymerase define multiple weak protein-protein and protein-RNA interactions (e.g., NusB with NusG; NusA with boxB; NusA, NusB, and NusG with boxA) that contribute to the overall stability of the complex. Interaction of each component of the complex with two or more other components can explain the many observed cooperative binding associations in the DNA-independent assembly of a stable antitermination complex on RNA polymerase.

Biofilm formation in living organisms is associated to tissue and implant infections, and it has also been linked to the contribution of antibiotic resistance. Thus, understanding biofilm development and being able to mimic such processes is vital for the successful development of antibiofilm treatments and therapies. Several decades of research have contributed to building the foundation for developing in vitro and in vivo biofilm models. However, no such thing as an ''all fit'' in vitro or in vivo biofilm models is currently available. In this review, in addition to presenting an updated overview of biofilm formation, we critically revise recent approaches for the improvement of in vitro and in vivo biofilm models.

SummaryThe association of the essential Escherichia coli protein NusA with RNA polymerase increases pausing and the ef®ciency of termination at intrinsic terminators. NusA is also part of the phage l N protein-modi®ed antitermination complex that functions to prevent transcriptional termination. We have investigated the structure of NusA using various deletion fragments of NusA in a variety of in vitro assays. Sequence and structural alignments have suggested that NusA has both S1 and KH homology regions that are thought to bind RNA. We show here that the portion of NusA containing the S1 and KH homology regions is important for NusA to enhance both termination and antitermination. There are two RNA polymerase-binding regions in NusA, one in the amino-terminal 137 amino acids and the other in the carboxy-terminal 264 amino acids; only the aminoterminal RNA polymerase-binding region provides a functional contact that enhances termination at an intrinsic terminator or antitermination by N. The carboxy-terminal region of NusA is also required for interaction with N and is important for the formation of an N±NusA±nut site or N±NusA±RNA polymerase±nut site complex; the instability of complexes lacking this carboxy-terminal region of NusA that binds N and RNA polymerase can be compensated for by the presence of the additional E. coli elongation factors, NusB, NusG and ribosomal protein S10.