Controlling Bacterial Biofilms (2009)

Films you don't want to see: The medical community faces a tremendous challenge in overcoming diseases stemming from the formation and persistence of bacterial biofilms. A concise review of current approaches employed to control bacterial biofilms is discussed. The ubiquitous nature of bacteria in the environment, and the role they play in infectious disease has been one of the most extensively researched areas in biomedical science. It has led to tremendous scientific breakthroughs aimed at eradicating a myriad of diseases and improving the overall quality of life. However, within the past 20–30 years, there has been an increased understanding that bacterial biofilms are a major factor in the morbidity and mortality of most infectious diseases.1–3 This is significant because bacterial biofilms are resistant to common therapeutic approaches that would eliminate their free-floating (planktonic) counterparts. Biofilms are described as surface-associated communities of microorganisms encased in a protective extracellular matrix.4 Approximately 80 % of the world's microbial biomass resides in the biofilm state, and the National Institutes of Health (NIH) estimates that upwards of 75 % of microbial infections that occur in the human body are underpinned by the formation and persistence of biofilms.5 Common diseases associated with the formation of biofilms include lung infections of individuals who suffer from cystic fibrosis (CF), burn wound infections, otitis media, bacterial endocarditis, and tooth decay (Table 1).5, 6 Additionally, the infection of indwelling medical devices (IMDs), such as catheters and heart stents, is also of major concern.3, 7, 8 Biofilms formed on IMDs provide a continual source of infection, thereby often necessitating their complete removal from the patient. Biofilm communities of bacteria differ from the traditional thinking of planktonic bacteria as they are complex, dynamic entities that undoubtedly represent a remarkable evolutionary attempt by the bacteria to survive against a plethora of environmental pressures.9 Organism Biofilm-associated disease Pseudomonas aeruginosa cystic fibrosis lung infection Burkholderia cepacia cystic fibrosis lung infection Acinetobacter baumannii burn wound, trauma infection Helicobacter pylori gastrointestinal infection Escherichia coli urinary, catheter infection Haemophilus influenzae otitis media Bordetella pertussis respiratory infection Legionella pneumophila legionnaires' disease Staphylococcus auerus burn wound, catheter, trauma infection Staphylococcus epidermidis sepsis, catheter infection Streptococcus mutans dental plaques, gingivitis vancomycin-resistant enterococci (VRE) nosocomial infections One of the most significant explanations for interest in biofilm research is the continued emergence and threat of antibiotic-resistant bacteria.10, 11 As the pool of available antibiotic treatments that have not selected for resistance continually diminishes, new avenues for the exploration of suitable therapies to provide adequate health care for the general populous are needed. In the United States alone, antibiotic-resistant infections have been approximated to cost upwards of $20 billion per year.10 Biofilms exacerbate this problem because they are known to be insensitive to the antibiotics and microbicides that would eliminate the bacterium in its planktonic state.12 By understanding the biofilm development model, it becomes clear how biofilms perpetuate many of the problems faced by the medical community in treating bacterial-based infectious diseases. Therefore, this review seeks to highlight how bacteria form biofilms and some of the obstacles faced by the medical field resulting from their development. This will then lead to an overview of a few of the current approaches and research therapies aimed at targeting these communities of bacteria. The purpose of this report is not to be a comprehensive review, but instead to provide the reader with a suitable framework for introduction to the topic of biofilms. Whereas many bacteria need to meet certain requirements to initiate the formation of a biofilm, it is generally thought that most biofilms grow in a cyclic nature and share many similarities to one another.9 The formation of biofilms has been extensively studied in a number of bacteria including, but not limited to, Pseudomonas aeruginosa, Escherichia coli, and Vibrio cholerae.13, 14 Much of what is known about biofilm inception originates from studies documenting the phenomenon of quorum sensing (QS). QS is the term employed to describe how bacteria communicate with one another in an effort to make a coordinated attempt at altering gene expression within the population.15–17 In Gram-negative bacteria, small molecules such as acyl homoserine lactones (AHLs) have been identified as the major players in this communication system (Scheme 1).17 Even across multiple bacterial strains, slight structural modifications in the aliphatic tails of these molecules are often times the only differences documented (1–4). AHLs and AI-2 utilized by various Gram-negative bacteria for QS. In Gram-positive bacteria, autoinducing peptides (AIPs), which bind to receptor histidine kinases embedded within the cellular membranes are commonly used for communication.18 The only definitive quorum-sensing mechanism identified to date shared by both Gram-negative and Gram-positive bacteria involves the production and use of autoinducer-2 (AI-2, 6 and 7) as effective signaling molecules. AI-2 molecules are derived from the common synthetic precursor (S)-4,5-dihydroxy-2,3-pentanedione (DPD) 5, the synthase enzyme of which has been identified in over 55 species of genetically diverse bacteria (Scheme 1).19, 20 Biofilm formation and the production of other virulence factors are known to be heavily influenced by QS pathways. Numerous studies have shown that the inhibition of certain QS pathways can lead to the formation of biofilms exhibiting distinct morphological differences in biomass structure.1, 9, 13, 21 This, coupled to the effect that the local environment can play in shaping biofilm development, explains how biofilms range from flat to highly organized structures. Throughout the biofilm formation process, bacteria are observed to exhibit distinct phenotypic differences from their planktonic counterparts during each subsequent developmental stage.1, 4, 9 Planktonic bacteria first localize on a substrate suitable for attachment to begin the growth cycle (Scheme 2). This attachment is initially characterized as reversible, yet it becomes irreversible once the bacteria begin to secrete an exopolymeric substance (EPS). The EPS is one of the most significant attributes of bacterial biofilms. Composed of a variety of biomolecules originating from both the bacteria and the surrounding environment, it serves as a barrier to defend the gathering microorganisms from microbicides and to provide an enclosed space for the biofilm to mature.22 Early maturation of the biofilm is often observed by the structure becoming three dimensional in space. As development continues, the morphology and topography of the biofilm becomes very distinct. Unique pillar shapes protrude from the biomass that allow for maximization of nutrient adsorption and waste disposal. Additionally, the formation of cavities or hollow channels throughout the biofilm becomes evident. These structural features provide the biofilm with the transport system necessary to funnel water and planktonic bacteria throughout the community and are thought to arise from the production of rhamnolipids by the bacteria. Biofilm growth cycle. A) Planktonic bacteria B) reversibly attach to a surface suitable for growth. C) Bacteria begin secretion of the EPS and attachment becomes irreversible. D) The maturing biofilm begins to take on a 3-dimensional shape. E) The biofilm fully matures, and complex architecture is observed. F) Bacteria disperse from the biofilm to reinitiate biofilm colonization of a distal surface. Rhamnolipids are biomolecules composed of glycolipids containing one or more rhamnose sugars, which have been observed to play a crucial role in the development and maintenance of P. aeruginosa biofilms.23 Studies have documented that these molecules are responsible for the shaping of the maturing biofilm by presumably altering cell-to-cell and cell-to-surface interactions; this ultimately leads to structural changes in the biofilm matrix.24 Mutant biofilms deficient in rhamnolipid production lack a distinct three-dimensional topography; this thus results in a biofilm composed of an uniformly thick layer of cells, whereas those that overexpress the genes encoding for rhamnolipid production have been observed to hyper-detach from surfaces.25 The last stage in the developmental cycle is the detachment of bacteria from the biofilm. These cells shed from the biofilm (which have been documented to be phenotypically similar to their planktonic brethren), move on to further colonize distal sites, and repeat the development cycle. This is consistent with reports of dispersed bacteria reverting back to an antibiotic-susceptible form when treated with antimicrobials.26 The exact mechanism of how or why dispersion occurs is still under investigation, yet most likely it can be attributed to a number of factors including various environmental pressures such as starvation conditions or overcrowding. As discussed in the introduction, antibiotic resistance is the preeminent obstacle faced by the medical community in regard to biofilm-driven infections. Approximately 70 % of bacteria frequently associated with nosocomial infections are resistant to at least one of the drugs commonly used to treat them.27 Many of these resistance mechanisms are tied to the morphology of the biofilm, which provides a unique shelter for the bacteria to thrive (Figure 1). Exchange of genetic material within biofilms through the conjugation process has been observed to occur at rates up to 1000-fold higher than those found in planktonic populations.3, 28, 29 Hypermutation, in which bacteria mutate at higher rates to evolve under stress, also occurs more frequently within biofilms.30, 31 Therefore, development of resistance mechanisms such as specialized efflux pumps or upregulation of porin proteins on the cell surface can quickly be selected for and propagated throughout the community. Conceptual diagram of biofilm resistance mechanisms to antimicrobial therapy. Biofilms are fluid, dynamic structures that can greatly vary in local composition.32 The spider-web-like nature of the biofilm EPS matrix can trap certain classes of microbicides before they can elicit their biological effects. Evidence for this stems from reports of differential penetration distances into biofilm matrices exhibited by a range of antibiotics including tobramycin and ciprofloxacin.33 Many antimicrobials fail to completely penetrate the core of the biofilm by becoming trapped in the EPS or by coming into contact with resistant cells, which block further penetration.32 Biofilms are composed of a heterogeneous population of bacterial cells that exhibit different metabolic properties. Cells that are embedded deep within the biofilm might grow at a slower rate because of lack of nutrients and oxygen in comparison with those located near the surface of the community.34 Because of this, these cells often times display increased insensitivity to antimicrobial compounds due to their reduced metabolic activity. Perhaps one of the most fundamental problems with the treatment of biofilm-associated infections is the high rate of reoccurring infection, which often times results in a more aggressive disease state (Scheme 3). It is now believed that this problem is attributed to the existence of persistor cells.35, 36 These cells are found in both planktonic and biofilm populations and are defined as cells that are inherently resistant to the actions of antibiotics or microbicides.37 It has been suggested that these cells are not mutants or cells that reside in a “special” state of growth or development, but instead might in fact be cells with a disabled programmed death mechanism.36 The progeny of persistor cells can exhibit increased resistance to the particular antimicrobial while simultaneously repopulating the community. In the treatment of infectious diseases underpinned by biofilms, this phenomenon is observed as initial treatment therapies aimed at controlling the infection appear to be successful with dissipation of disease symptoms within the patient. However, when persistor cells repopulate the community, treatment methods are less efficacious with each subsequent infection. Furthermore, persistor cells are not eradicated by the host immune response because they are protected by the biofilm matrix. Persistor cells in the biofilm model. As previously discussed, the implementation of functional QS systems in bacteria is essential for their survival. Work has demonstrated that by inhibiting QS in biofilm-forming bacteria, certain degrees of susceptibility to biocides can be obtained.21, 38 In particular, the use of detergents such as SDS has been documented to disrupt mutant lasI P. aeruginosa biofilms.21 Most of these studies have involved manipulation of the production and use of acyl homoserine lactones (AHLs). From a small-molecule development approach, a considerable amount of effort has been put into the design of potential biofilm controls based on this molecular scaffold (Scheme 4).39, 40 Most derivatives of these analogues are unique in that they elicit their activity in a nonmicrobicidal manner, thus possibly mitigating the development of resistance to the ligand. Various modifications to natural AHLs have resulted in the discovery of analogues that possess the ability to inhibit biofilm formation in P. aeruginosa and a number of QS systems in a multitude of bacteria (8–11).41–45 A few problems associated with the development of AHLs as small-molecule controls of biofilms include their chemical reactivity (prone to hydrolysis) and immunomodulatory properties.46–48 Attempts at overcoming these hurdles include the work of some research groups aimed at tuning the chemical reactivity of AHLs through modification of the lactone head in hopes of obtaining more robust compounds.49–52 In recently related developments, the exogenous addition of the fatty acid messenger cis-2-decenoic acid 12 has also been reported to induce the dispersion of biofilms in a cross-kingdom manner.53 By dispersing established biofilms from bacteria and fungi such as E. coli, S. auerus, and C. albicans, the authors demonstrate that the broad-spectrum activity might result from a evolutionary point that is similarly shared between these organisms. 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