Direct observations have revealed that the bacteria that cause device-related and other chronic diseases grow in matrix-enclosed biofilms, adherent to the surfaces of biomaterials and tissues. In this biofilm mode-of-growth, the organisms are virtually impervious to antibiotics, and to the antibodies and phagocytes that constitute the defense systems of virtually all mammals. Within the biofilm community the cells communicate by means of chemical and (possibly) electrical signals, so that these sessile communities can coordinate their responses to host countermeasures, and persist for months or even for years. Biofilm communities can withstand the attacks of antibacterial agents (e.g. antibiotics) that would readily kill planktonic cells of the same strain, and many medical specialties surgically remove the biofilms and their living or inert substrata, as their rational basis of anti-biofilm therapy. In general, biofilms cause damage to the affected tissues by their persistence. When host defenses and antibiotic therapy fail to resolve chronic infections, the inflammation that they stimulate becomes the predominant factor in damage to affected tissues.
Because of recent advances in Biofilm Microbiology, the clinical pendulum is swinging away form frontal attacks with antibiotics, towards the use of immune modulation to minimize the effects of inflammation on the host tissues. These same data have stimulated interest in the use of signals to minimize biofilm formation, and even to stimulate the dissolution of existing biofilms by promoting detachment. The notion of using physical forces (e.g. DC fields, and ultrasonic waves) to disrupt the internal communications within biofilms is also gaining traction, and a more complete understanding of the structure and function of whole microbial communities will engender new and practical technologies for biofilm control.
There has been an explosion in research directed at understanding the mechanisms of how bacteria communicate and cooperate to perform a variety of multicellular behaviors, including biofilm formation. Not until very recently have microbiologists also begun to investigate these behaviors from the perspective of social evolution. Our goal is to integrate mechanistic and evolutionary approaches to investigate communication, also termed quorum sensing (QS), and cooperation in the model bacterium and opportunistic pathogen Pseudomonas aeruginosa. P. aeruginosa communicates via diffusible acyl-homoserine lactone signals to coordinate the expression of hundreds of genes, many of which encode extracellular virulence factors. On a mechanistic level, we have utilized a variety of different approaches, including transcriptomics, ChIP-chip, and mutagenesis, to identify directly and indirectly regulated genes, and to characterize additional regulators of the QS system. With respect to sociobiology, we have utilized in vitro evolution and analysis of natural P. aeruginosa populations to gain insight into the propensity of cheating in bacterial populations, which is a threat common to social systems across all domains of life. We identified variants that ceased production of shared extracellular factors and took advantage of their production by the group. The existence of these cheaters demonstrates the sociality of microbes, and provides a compelling resolution to the long-standing paradox in P. aeruginosa pathogenesis that although QS is required for infection in animal models, QS-deficient variants are commonly associated with infections. In addition to cheating, our evolution-in-a-test-tube experiment also revealed a mechanism of cheater control. Before cheating became detrimental to the population, a novel type of cooperator with superior fitness had evolved from a cheating ancestor. Experiments are underway to define the underlying mechanism. As an extension of our own work, an attempt will be made to compare and contrast current mechanistic and sociobiological views on biofilm formation. A combination of both perspectives appears necessary to build a complete model of biofilm formation and guide appropriate treatment strategies.
Pathogen populations produce persisters, specialized survivor cells that are dormant and highly tolerant to all known antibiotics. Molecular mechanisms of persister formation will be discussed, as well as their role in disease, such as biofilm infections of catheters, cystic fibrosis, and oropharyngeal candidiasis. Approaches to eradicating persisters will be discussed as well.
The important human pathogen Pseudomonas aeruginosa has been linked to numerous biofilm-related chronic infections Biofilms are complex communities of microorganisms encased in a matrix and attached to surfaces. It is well recognized that biofilm cells differ from their free swimming counterparts with respect to gene expression, protein production, and resistance to antibiotics and the human immune system. However, little is known about the underlying regulatory events that lead to the formation of biofilms, the primary cause of many chronic and persistent human infections. By mapping the phosphoproteome over the course of P. aeruginosa biofilm development, demonstrated that biofilm formation following the transition to the surface attached lifestyle is regulated by three previously undescribed two-component systems: BfiSR (PA4196-4197) harboring an RpoD-like domain, an OmpR-like BfmSR (PA4101-4102), and MifSR (PA5511-5512) belonging to the family of NtrC-like transcriptional regulators. we identified three novel two-component regulatory systems that were required for the development and maturation of P. aeruginosa biofilms. These two-component systems become sequentially phosphorylated during biofilm formation. Inactivation of bfiS, bfmR, and mifR arrested biofilm formation at the transition to the irreversible attachment, maturation-1 and -2 stages, respectively, as indicated by analyses of biofilm architecture, and protein and phosphoprotein patterns. Moreover, discontinuation of bfiS, bfmR, and mifR expression in established biofilms resulted in the collapse of biofilms to an earlier developmental stage indicating a requirement for these regulatory systems for the development and maintenance of normal biofilm architecture. Interestingly, inactivation did not affect planktonic growth, motility, polysaccharide production, or initial attachment. Further, we demonstrate the interdependency of this two-component systems network with GacS (PA0928), which was found to play a dual role in biofilm formation. This work describes a novel signal transduction network regulating committed biofilm developmental steps following attachment, in which phosphorelays and two sigma factor-dependent response regulators appear to be key components of the regulatory machinery that coordinates gene expression during P. aeruginosa biofilm development in response to environmental cues.
Driven by recent advances in noninvasive microscopy, staining techniques, and genetic probes, there has been enormous increase in our understanding of biofilms. Along with this increase in understanding, has been increasing interest in mathematical models of biofilms to get at important mechanisms. Most recent modeling in the field has been directed towards understanding the mechanisms underlying the remarkable spatial structure of biofilms which has become evident through the use of modern imaging techniques. Most of these models are so complex that they can be investigated only using sophisticated numerical simulations.
On the other hand, there are relatively few simple, conceptual biofilm models which are amenable to mathematical analysis yet which yield significant and useful results. Here, we speak of models which do not attempt to provide much detail on the spatial structure of biofilms but which provide information on conditions suitable for biofilm formation and maintenance and which model the formation of biofilms directly, starting from an inoculum of planktonic bacteria. Freter et al. formulated a mathematical model to understand the phenomena of colonization resistance in the mammalian gut (stability of resident microflora to colonization). Essentially, their model can be viewed as a crude biofilm model. In contrast to state of the art biofilm models, the Freter model completely ignores the three-dimensional spatial structure of the biofilm. Yet it can give useful results. The model and its implications will be surveyed.