Respiratory infections from bacteria that develop within hospital drinking water systems are not uncommon, such as Legionella pneumophila and Mycobacterium avium complex pathogens. Colonization success by any pathogen results from an array of complex interactions between biotic and abiotic factors. Low level seeding of drinking water distribution systems may occur periodically, most likely due to pathogens being associated with particulate matter breaking through water treatment, some of which may be within amoebae cysts. However, pathogen numbers only reach an important threshold once replication occurs, most of which is thought to occur within pipe biofilms (mostly within premise plumbing). For example, occurrence of L. pneumophila in hospital plumbing systems and outbreaks of legionellosis within hospitals around the world support its ubiquitous occurrence, no matter how good municipal drinking water treatment may be. The outstanding unknown is how much sporadic legionellosis and other biofilm-related disease occurs from non-institutional drinking water exposures? Hence, mathematical models are being developed to aid in identifying research needs to better estimate community disease risks from aerosolized drinking waters. Key parameters seem to be the density of pathogens within amoeba/biofilms were they replicate, and the proportion that may be released from the biofilm in respiratory-sized (< 10 Ám) aerosols. Shower heads would appear to be important sites for pathogen development and release, but upstream plumbing could be equally important, depending on the residual disinfectant type and concentration in the drinking water.
It is well known that a biofilm lifestyle provides many layers of protection. Many of the protective mechanisms, such as delayed penetration, are transient. The persister hypothesis is a mechanism of more permanent tolerance; however, there are many questions that remain to be answered. We will discuss how mathematical modeling can be used to explore different hypothesis and behaviors.
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.
Cis-2-decenoic acid has been shown to act as a cell-to-cell signaling molecule responsible for inducing biofilm dispersion in Gram-negative bacteria, Gram-positive bacteria and fungi. Recently, we have shown that this signaling system is responsible for a number of effects that together lead to the transition from a sessile mode of existence to an active disseminating lifestyle. Induction of dispersion of P. aeruginosa biofilms was demonstrated to result in the release of degradative enzymes involved in hydrolysis of protein, nucleic acids and polysaccharides. Dispersed bacteria were observed to become motile and alter expression of virulence determinants, including elevated expression of pyochelin and exotoxin T. In addition, cis-2-decenoic acid led to increased dissemination of infection in a lettuce virulence model. The altered phenotype induced by cis-2-decenoic acid has also been demonstrated to result in enhanced susceptibility of bacteria to antimicrobial agents. Against P. aeruginosa, in the presence of cis-2-decenoic acid, triclosan showed an average 3.4 Log increase in cidal activity, tobramycin and ciprofloxacin showed an average 1.8 Log increase, and polymyxin B showed a 0.9 Log increase compared to the cidal activity of the antimicrobial agents alone. It has also been determined that cis-2-decenoic acid was able to resuscitate dormant bacteria and that it enhanced the recovery of viable-non-culturable bacteria on solid medium, yielding higher cell numbers and shorter lag periods, compared to bacteria cultured on medium not containing cis-2-decenoic acid. Taken together, our results demonstrate that cis-2-decenoic acid acts to induce bacteria to transition from a biofilm phenotype typically associated with chronic infections (in which bacteria show a reduction in metabolic activity, growth and motility, and an enhanced resistance to antimicrobial agents), to bacteria with a phenotype more typically associated with planktonic growth or acute phase infections (as characterized by enhanced growth, activity and susceptibility to antimicrobial agents). These observations suggest that treatment with cis-2-decenoic acid alone or in combination with antibacterial agents, should have a significant enhancing effect on the killing of bacteria associated with chronic infections.
Many bacteria use the size and density of their colonies to regulate the production of a large variety of substances. This phenomenon is called quorum sensing. We present a review of mathematical models of quorum sensing and their use in biofilm Modeling.
Quorum sensing (in the strict sense) is a cell signaling mechanism used by bacteria to coordinate gene expression according to the size of the population. Cells constantly produce quorum sensing molecules, so called autoinducers. These accumulate in the environment and when the concentration passes a certain threshold, the bacteria undergo up-regulation. The autoinducer molecules are subjected to mass transfer, which, in non-homogeneous environments, leads to concentration gradients. In this case the autoinducer concentration contains also spatial information (diffusion sensing). Recently both concepts have been unified under the umbrella efficiency sensing. We present a mathematical model for this process for patchy biofilm systems, in which the quorum sensing mechanism enable inter-colony communication. We discuss the well-posedness of the model and use computer simulations to illustrate the effect of autoinducer production on neigboring colonies.
Biofilms are colonies of microbial cells encased in a self-produced organic polymeric matrix and represent a common mode of microbial growth. Microbes growing as biofilm are highly resistant to commonly used antimicrobial drugs. Recently, microbial biofilms have gained prominence because of the increase in infections related to indwelling medical devices (IMD). Candida albicans, the pathogenic fungus which is a major cause of morbidity and mortality in blood stream infections, is the most common fungal pathogen isolated from patients with IMD-associated infections. Biofilm formation by Candida species is believed to contribute to invasiveness of these fungal species. We discuss experimental methods used to study fungal biofilms as well as the biology of biofilm formation by clinically relevant Candida species. This lecture will take us in a journey from basic science to translational research.
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 human oral biofilms known as supra- and subgingival dental plaque have been studied intensively from a bacteriological standpoint for nearly 50 years, and a wealth of taxonomic and physiological information is available on the hundreds of bacteria found in the oral cavity. The ecology of plaque biofilm development is better known than for most other natural (i.e., multispecies) biofilm systems. One driving force hypothesized to be important for assemblage of microbial communities in plaque is the trait known as coaggregation: cell-cell recognition and binding that brings different bacteria into direct contact. Coaggregation interactions have partner specificity that results from adhesin-receptor recognition, a specificity we hypothesize to have arisen through co-evolution of metabolic processes. However, coaggregation traits are known primarily through in vitro studies of culture-collection bacterial isolates - little data exist for clinical isolates. Is coaggregation truly important in nature? If so, can coaggregation be used as a scaffold for modeling bacterial interaction in dental plaque? Models of dental plaque exist that address topics such as diffusion and mass transport. Is a model built on cooperative bacterial physiology possible? Data on bacterial interactions in in vitro systems exist, and it may be possible to translate these data from the laboratory to the tooth surface. While factors such as host response and community complexity are major obstacles to predictive modeling of oral disease, simple plaque biofilm models are likely to be broadly applicable to other natural microbial systems.
P. aeruginosa produces at least 3 types of exopolysaccaride (EPS): alginate, Pel and Psl. The Pel polysaccharide is a glucose-rich polymer and contributes to the formation of surface-associated biofilm. It has been reported that increasing the level of cyclic diguanylate (c-di-GMP) also results in the increased transcription of pel expression. This is due to derepression of pel expression by the c-di-GMP binding regulator, FleQ. In this work, we focused on regulation of pel expression and its relationship to c-di-GMP in biofilm communities. To measure the pel transcription in a biofilm, we used a tube biofilm culturing system. P. aeruginosa PAO1 was injected into the tube and incubated for 30 min. Control cells were incubated statically. Biofilms were grown at room temperature at 50 ml/h flow rate for 48 hours. Total RNA was isolated from biofilm cells and quantitative RT PCR was performed.
In a tube biofilm, pelA transcription increased about 8 times higher than that of control after only one hour post attachment and after 12 hours it was 20 times higher. This induction appeared to be Psl-specific, psl transcription was not induced under these conditions. It was shown that the transcription of PA4625 was elevated under high c-di-GMP conditions. In this tube biofilm, the transcription of PA4625 was increased 10 times higher, too. With the fleQ mutant, pelA transcription was still high and it was not induced by surface attachment. From these results, we hypothesized that c-di-GMP is elevated by initial attachment in biofilms. To test this, c-di-GMP levels of attached and planktonic cells were measured by HPLC/MS/MS system. The c-di-GMP levels of attached cells were found to be about 3 times higher than that of planktonic cells. Our data suggested that surface attachment induced the accumulation of c-di-GMP in the cell through FleQ dissociation from the pel promoter region, activating pel transcription and biofilm formation.
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.
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.
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.
Vibrio cholerae causes the disease cholera and is a natural inhabitant of aquatic environments. V. cholerae's ability to form biofilms, matrix-enclosed, surface-associated communities, is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. I will discuss production of biofilm matrix components and regulation of biofilm formation in V. cholerae.