Biologically-Inspired Strategies for Combating Bacterial Biofilms

Abstract

Infections caused by bacterial biofilms are a significant global health problem, causing considerable patient morbidity and mortality and contributing to the economic burden of infectious disease. This review describes diverse strategies to combat bacterial biofilms, focusing firstly on small molecule interference with bacterial communication and signaling pathways, including quorum sensing and two-component signal transduction systems. Secondly we discuss enzymatic approaches to the degradation of extracellular matrix components to effect biofilm dispersal. Both these approaches are based upon non-microbicidal mechanisms of action, and thereby do not place a direct evolutionary pressure on the bacteria to develop resistance. Such approaches have the potential to, in combination with conventional antibiotics, play an important role in the eradication of biofilm based bacterial infections.

Introduction

In the last twenty years, bacterial infections have reemerged as a major health threat. Hospital-acquired infections are now responsible for more deaths annually in the United States than emphysema, AIDS, Parkinson’s disease, and homicide combined [1] and cost the U.S. health care system over $20 billion annually [2]. An estimated 80% of bacterial infections in humans are caused by biofilms, according to the National Institutes of Health [3], leading the Centers for Disease Control to declare biofilms among the most pressing clinical impediments of the century [4]. Despite the increased virulence of biofilms and their obvious threat to human health, there are no clinically available drugs to inhibit or disperse biofilms in vivo [**5].

Biofilms are formed by multiple bacterial cells attached to a surface that arrange themselves into a complex tertiary structure encased in an extracellular matrix comprised of carbohydrates, proteins, and other macromolecules [6, 7]. Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii and other medically relevant bacterial strains colonize clinical surfaces and medical devices via biofilms and resist common eradication methods including desiccation, antibiotic treatment, and nutrient deprivation [8]. Bacteria associated with a biofilm are up to 1,000 times more resistant to antibiotic therapies in comparison to their planktonic counterparts and are insensitive to the host immune response, allowing them to persist and promote continued infection despite aggressive antibiotic therapy [8, 9].[8, 9]. Of particular concern are biofilms that form on indwelling medical devices (IMDs), creating a continuous source of infection that often necessitates removal of the device [7].

Biofilm formation is a complex process involving multiple bacterial signaling systems including quorum sensing, nutrient and chemical signal response, and extracellular matrix formation (Figure 1). As such, very few chemical scaffolds have been identified that can inhibit or disperse bacterial biofilms. Numerous approaches have been investigated to both inhibit and disperse bacterial biofilms[**10]. This review describes approaches that involve inhibition of intercellular communication and signaling pathways with small molecules, in addition to approaches that center on degrading the integrity of the extracellular matrix. We provide an overview of important quorum sensing pathways and two component systems involved in biofilm formation and the effects of their inhibition by novel antibiofilm compounds. We also describe methods for disrupting the extracellular matrix required for the formation of robust biofilms. Finally, we conclude with future perspectives for the discovery and development of biofilm inhibitors as important and necessary therapeutic agents. This is not meant to be an exhaustive review of every anti-biofilm approach, which be beyond the scope of this document, and instead provides the reader with an overview of several of the most important anti-biofilm strategies, giving select examples in each case.

Figure 1.

Stages of the Biofilm Lifecycle. In stage 1, planktonic bacteria initiate attachment to an abiotic surface, which becomes irreversible in stage 2. Stages 3 and 4 feature biofilm maturation and growth of the three dimensional community. Dispersion occurs in stage 5 and releases planktonic bacteria from the biofilm to colonize additional sites.

Disruption of Intercellular Communication and Signaling Pathways

Quorum Sensing

Quorum sensing (QS) describes the intercellular communication required for bacterial communities to act in coordinated ways to alter gene expression based on population density [11]. QS can be reduced to interplay between two proteins; the first produces a signaling molecule known as an autoinducer (AI), and a second protein that responds to the AI. Autoinducers encompass several classes of structurally related molecules including acyl homoserine lactones (AHLs), autoinducing peptides (AIPs) and autoinducer-2 (AI-2) [12].

Over 70 species of Gram-negative bacteria use AHLs for intraspecies communication, with specificity imparted by variation in the oxidation state and length of the acyl side chain [12]. AHLs can freely diffuse through the bacterial membrane such that AHL concentration correlates to bacterial concentration and enables population-mediated control of gene expression. Ultimately, this gene expression results in various phenotypes including the production of virulence factors and biofilm formation [13]. As a result of their extensive study over the last three decades, AHLs have provided a scaffold for many potential biofilm inhibitors [14, 15]. The Blackwell group has reported the synthesis and activity of several unnatural AHLs, two of which (1 and 2) (Figure 2) significantly reduced biofilm formation in P. aeruginosa PA01 at 50 μM [16]. Spring et al. also investigated analogs of P. aeruginosa AHLs by replacing the lactone functionality with an N-acylated cyclopentylamide. One compound, known as C10-CPA (3), was able to abrogate biofilm formation in P. aeruginosa PA01:GFP after seven days under flow conditions at a concentration of 250 μM [17]. Using the crystal structure of the P. aeruginosa transcriptional regulator LasR [12, 18], Bottomley and coworkers designed and synthesized covalent LasR inhibitors that inhibited quorum sensing and, in the case of the lead compound, 4, could inhibit wild type PA01 biofilm formation by close to 50% at 50 μM.

Figure 2.

Quorum sensing inhibitors.

In Gram-positive bacteria, the predominant molecules used for QS are autoinducing peptides (AIPs). Many AIPs contain hydrophobic domains crucial for activity, which are hypothesized to help promote hydrophobic interactions that lead to receptor activation [19]. In S. aureus, threshold levels of AIP bind AgrC, resulting in expression of RNA-III, a small noncoding RNA that ultimately downregulates genes controlling adhesins required for biofilm formation [20]. Phosphorylation of RNA-III activating protein (RAP) activates target of RNA-III activating peptide (TRAP), thereby increasing cell adhesion and biofilm formation [21]. Conversely, RNA-III inhibiting peptide 5 (RIP), prevents phosphorylation of TRAP and thus reduces biofilm formation and cell adhesion. RIP has been evaluated extensively and has been shown to prevent infections, including those by antibiotic resistant strains, in several animal models [22] without any signs of toxicity or induction of RIP resistance [23]. Other analogs of S. aureus AIP-1 have been shown to be potent agonists of AgrC-1 QS [24] or promoters of S. aureus biofilm formation [25]. Recently, Blackwell and colleagues reported a novel class of AIP-III mimetics designed to inhibit AgrC receptors in S. aureus [26]. Although their effects on biofilm formation were not reported, the lead compounds were shown to inhibit all four AgrC receptors and block hemolysis, which is under the control of QS, at picomolar levels. Given the role of AgrC in biofilm formation, it would be very interesting to evaluate the effects of these inhibitors on S. aureus biofilm formation and maintenance.

An alternative approach to disrupting AHL based QS is to exploit the lability of the AHL scaffold and degrading AHLs enzymatically, thereby inhibiting QS. This phenomenon is known as quorum quenching, and unsurprisingly, given the role of AHL based QS in bacterial virulence, is utilized by numerous organisms, both bacterial and eukaryotic.[27] This approach to inhibiting quorum sensing was first exploited by Dong et al. in which a gene encoding a lactonase from Bacillus sp. was shown to inhibit AHL activity by hydrolyzing the lactone bond of several AHLs from P. aeruginosa and the plant pathogens Erwinia carotovora and Agrobacterium tumefaciens.[28, 29] Applying this approach as an anti-biofilm strategy, the hydrolase BpiB05, derived from the soil metagenome, was shown to affect biofilm formation, in addition to motility and pyocyanin synthesis, in P. aeruginosa. When transformed with the bpiB05 gene, P. aeruginosa PAO1 formed poorly developed biofilms that did not progress beyond initial surface attachment.[30] This work gives some indication the potential of the quorum quenching approach as an anti-biofilm strategy, though much more investigation is required.

Both Gram-positive and Gram-negative bacteria utilize autoinducer-2 (AI-2) in quorum sensing mechanisms, thus providing a basis for a universal language across bacterial species [31]. Nucleoside analogs (6-9) have been shown to interfere with AI-2 mediated quorum sensing and, in some cases, affect biofilm formation [32, 33]. Recently, Bentley and coworkers have described novel AI-2 analogs that were capable of inhibiting maturation of E. coli biofilms in vitro and that could, when combined with antibiotics near MIC levels, could almost completely clear pre-formed E. coli biofilms in a microfluidic device [*34].

In addition to AI-2, bis-(3′5′)-cyclic di-guanylic acid (c-di-GMP) is believed to be a ubiquitous second messenger signal molecule. c-di-GMP regulates exopolysaccharide synthesis, and thereby influences exopolysaccharide-dependent biofilm formation, in proteobacteria including Vibrio cholera, P. aeruginosa, and E. coli [35], and has been implicated in biofilm dispersion in P. aeruginosa where elevated c-di-GMP levels inhibited effective surface detachment [36]. Treatment of S. aureus with exogenous c-di-GMP inhibited in vitro biofilm formation and adherence to HeLa cells [37]. While many analogs to c-di-GMP have been synthesized and evaluated [37-40], cell permeability and inferior receptor binding and affinity as compared to c-di-GMP have hampered their utility [41].

Two Component Systems

In addition to quorum sensing, bacteria use other pathways to recognize and respond to various external signals and stimuli. In many instances, the signal receptions and responses are mediated by two-component regulatory systems (TCS), which couple a membrane sensor kinase (histidine kinase, HK) to a response regulator (RR) [42, 43]. TCS have been extensively reviewed, with several recent articles highlighting the role of TCS in both virulence and antibiotic resistance [5, 44-46]. In a prototypical TCS, the HK receives extracellular signals and phosphorylates the N-terminal domain of the RR on a highly conserved aspartate residue [43, 47-49]. The phosphorylated RR typically undergoes a conformational change and dimerization leading to DNA binding and activation or repression of gene transcription via the C-terminal domain [47-50]. In this way, the bacteria can produce appropriate gene products based on extracellular signals. TCS are not found in vertebrates, thus representing a novel target for therapeutics [45]. Furthermore, there are very few essential TCSs, such that targeting them could disrupt the bacteria’s ability to respond appropriately to external stimuli without accompanying bactericidal effects, thus side-stepping direct evolutionary pressure for resistance to the TCS inhibitor [45].

Genetic screens of bacterial mutants incapable of producing biofilms has led to an extensive list of TCS involved in biofilm formation and maintenance [5]. However, despite this vast availability of protein targets, few compounds are known to target TCS responsible for biofilm formation and maintenance [5]. Walkmycin C 10 (Figure 3) is a member of the walkmycin family of natural products produced by Streptomyces sp. strain MK632-100F11 [51, 52]. Originally identified as an inhibitor of autophosphorylation of YycG in S. aureus and Bacillus subtilis, it was later found to interact with the conserved catalytic domain of the histidine kinase and has activity against several HKs [51]. In S. mutans, walkmycin C inhibits VicK, a non-essential orthologue of YycG involved in cellular growth, surface adhesion, sucrose-dependent biofilm formation, and competence development, with an IC₅₀ of 2.87 μM (2.53 μg/mL). Walkmycin C also inhibits CiaH, an HK with roles in sucrose-dependent biofilm formation, competence development, and stress tolerance, with an IC₅₀ of 4.87 μM (4.29 μg/mL) [51, 53]. At levels below the minimum inhibitory concentration (MIC), walkmycin C reduces biofilm mass, induces abnormal biofilm formation, and represses acid tolerance and competence in S. aureus [51].

Figure 3.

Two-component system inhibitors.

The 2-aminoimidazole (2-AI) class of small molecules, derived from marine sponge alkaloids, has shown broad spectrum biofilm inhibition and dispersion activity [10, 54-59]. Compound 11, representing the reverse amide class of 2-AIs, has been shown to inhibit and disperse biofilms in both P. aeruginosa and A. baumannii [60]. Recently, a biotinylated analogue of this compound was employed as a chemical probe to identify the A. baumannii response regulator BfmR as the molecular target of this class of compounds [**61]. BfmR belongs to the OmpR family of response regulators and has been implicated in biofilm formation [42]. A bfmR mutant showed significantly lowered ability to form biofilms and altered planktonic cellular morphology,[42, 62] thus, BfmR represents an attractive target for anti-biofilm compounds. Using a BfmR homology model, docking studies suggested that compound 11 targets the interface between the N and C-terminal domains of BfmR. The docking studies were validated with pull-down assays using truncated protein containing either the N- or C-domains only, showing that both domains make contact with the compound. Further studies indicated that 11 interacts with several other response regulators, but displays no binding to any of the tested non-response regulator proteins [**61].

Non-Small Molecule Approaches

Enzymatic Degradation of Matrix Components

The biofilm matrix typically accounts for over 90% of the dry mass of a biofilm and consists predominantly of biopolymers produced by the bacteria themselves, these biopolymers are known as extracellular polymeric substances (EPS) and, as mentioned above, include polysaccharides, proteins, lipids and nucleic acids. The matrix forms the basis of the three-dimensional structure of the biofilm and immobilizes the cells, keeping them in close proximity to each other and allowing for cell-cell communication. The matrix contains the contents of lysed bacterial cells, serving as a nutrient source and acting as a reservoir of genes for horizontal gene transfer [*63] (Figure 4). The matrix also contributes to the tolerance of bacterial cells to antibiotics, acting as a diffusion barrier to prevent access to the cells [64]. One approach to the eradication of preformed biofilms is therefore to destroy the integrity of the biofilm matrix, typically by enzymatic degradation of components of the EPS, leading to subsequent detachment of cells from the biofilm. This mechanism of biofilm dispersal is an innate phenomenon employed by several diverse bacterial species. Bacteria secrete enzymes such as glycosidases, proteases, and DNases that degrade various components of the EPS [65]. Examples of endogenously produced matrix degrading enzymes include the DNase thermonuclease, which is produced by S. aureus, the glycoside hydrolase dispersin B, which is produced by Aggregatibacter actinomycetemcomitans, and alginate lyase, which is produced by P. aeruginosa. These enzymes, and many others, are used by the bacteria to initiate active dispersion of the biofilm, which then allows for the release of cells into the surrounding environment, contributing to bacterial survival and disease transmission [65]. Several of these matrix-degrading enzymes have been investigated as potential therapeutic agents.

Figure 4.

Schematic of the various matrix-degrading enzymes used to initiate biofilm inhibition and dispersal.

Dispersin B has been shown to inhibit the formation of biofilms by several medically relevant bacterial species including S. aureus [66], Staphylococcus epidermidis [66], E. coli [67], and Yersinia pestis [67], in addition to dispersing preformed S. epidermidis [66, 68] and E. coli [67] biofilms and sensitizing S. epidermidis biofilm cells to the action of antimicrobials [66, 69]. Dispersin B has also demonstrated activity in vivo, lowering the rate of catheter colonization by S. aureus in combination with triclosan in a rabbit model of infection [70]. Alginate lyase degrades a polysaccharide known as alginate and has been shown to enhance the microbicidal activity of aminoglycosides against P. aeruginosa biofilms in vitro [71, 72], and has also demonstrated in vivo efficacy, enhancing the clearance of mucoid P. aeruginosa when coadministered with amikacin in a rabbit model of endocarditis [73].

Extracellular DNA is an important component of the biofilm matrix [74] and the use of nucleases as an anti-biofilm strategy has been explored against a number of bacterial strains. Biofilms formed in the presence of DNase exhibit reduced biomass resulting from a reduced number and size of microcolonies within the biofilm, bacterial and decreased antibiotic tolerance [75]. The fact that degradation of extracellular DNA in the biofilm matrix by DNase has been shown to result in a biofilm that displays decreased tolerance to environmental factors makes the use of DNase an attractive anti-biofilm strategy [75]. An extracellular DNase (NucB), produced by Bacillus licheniformis induces rapid biofilm dispersal activity against preformed biofilm of several species of both Gram-positive and Gram-negative bacteria including B. subtilis, E. coli, and Micrococcus luteus [73]. S. aureus also produces a nuclease, known as Nuc, when the sigma factor B (sigB) gene is absent that has been shown to inhibit biofilm formation [76], while a nuc mutant was shown to form a thicker biofilm that contained increased levels of extracellular DNA [77]. Recombinant human DNase I (rhDNase) has been shown to inhibit biofilm formation by both S. aureus and S. epidermidis, disperse preformed S. aureus biofilms, and increase the susceptibility of S. aureus biofilm cells to killing by chlorhexidine gluconate and povidone iodine. rhDNase displayed activity in vivo, increasing the survival of S. aureus-infected Caenorhabditis elegans in when administered in combination with tobramycin [**78]. rhDNase has also been shown to enhance the microbicidal activity of aminoglycosides against P. aeruginosa biofilms in vitro [72], and to effect a significant loss of cells and biomass from biofilms of several strains of Streptococcus pneumoniae [79]. rhDNase I (also known as dornase alfa and marketed as Pulmozyme by Genentech) is used in the clinic for the treatment of pulmonary disease in cystic fibrosis (CF) patients [80], in which biofilm mediated P. aeruginosa infections are a major contributing factor to lung tissue damage [81]. Administration of Pulmozyme has been shown to lead to reduced demand for antibiotics and improved lung function in CF patients [82].

Other enzymatic anti-biofilm approaches include the use of proteases to modulate biofilms by degradation of the protein component of the biofilm matrix. It is known that endogenous proteases play a role in biofilm dispersal [83] and it has also been shown that exogenously added proteases can exhibit dispersal activity against established biofilms. For example, the serine protease Esp, which is produced by S. epidermidis, inhibits S. aureus biofilm formation and eradicates preformed biofilms of this bacterium. Esp has also been shown to enhance the susceptibility of S. aureus biofilms to the antimicrobial peptide human beta-defensin 2 (hBD2), while the activity of Exp in vivo has been demonstrated by the ability of both Esp secreting S. epidermidis and purified Esp to eliminate human nasal colonization by S. aureus [84]. The elastase LasB and proteinase K have also demonstrated anti-biofilm activity against S. aureus, while the supernatant of LasB producing P. aeruginosa was shown to induce the expression of several endogenous protease genes. Proteinase K was been shown to increase proteolytic activity, which is believed to be the mechanism by which these enzymes effect S. aureus biofilm dispersal [85]. Finally, the metalloprotease serratopeptidase (SPEP) is produced by Serratia marcescens and is widely used as an anti-inflammatory therapeutic. SPEP has been shown inhibit biofilm formation and enhance the activity of ofloxacin against biofilms of both P. aeruginosa and S. epidermidis [86] and to inhibit biofilm formation by Listeria monocytogenes [87].

Antibodies as an Anti Biofilm Strategy

A recently reported non-small molecule approach to the discovery of anti-biofilm agents is the use of antibodies for the eradiation of bacterial biofilms. Monoclonal antibodies (mAbs) that bind the P. aeruginosa Psl, a ubiquitous cell surface anchored exopolysaccharide that plays a role in the formation and maintenance of biofilms by acting as a scaffold for other biofilm initiating components [88, **89], were identified from a screen of an M13 phage-based human antibody library. Lead mAbs were shown to inhibit host cell attachment by P. aeruginosa and impart significant protection in multiple animal models of P. aeruginosa infection including a mouse acute lethal pneumonia model and a thermal injury model [**89]. Antibodies to the partially de-N-acetylated form of the Staphylococcal surface polymer poly-N-acetylglucosamine (PNAG), which promotes biofilm formation, increased killing of S. aureus by human neutrophils, while passive immunization of mice with anti-dPNAG-DT rabbit sera resulted in increased clearance of S. aureus from the blood compared to mice treated with normal rabbit sera [90].

Conclusion and Future Perspectives

Bacterial biofilms are a major threat to human health as they are inherently resistant to clearance by both the host immune system and antibiotics. This review highlights recent strategies to combat biofilms both through small molecules and protein-based methods focusing on strategies that do not rely on toxic mechanisms to inhibit or disperse biofilms. As the scientific community learns more about bacterial biofilms and searches for methods to combat them, we must be mindful of the lessons learned from antibiotic development over the last century. Finding treatments that can alter the phenotype of the bacteria without inducing and selecting for genetic modifications that can lead to resistance is key in winning the battle against these pathogens. Antibiofilm strategies that focus on interrupting complex regulatory systems involved in biofilm formation and maintenance without killing the bacteria should disrupt the biofilm without also selecting for a resistant population. Such strategies have the potential, when paired with conventional antibiotics, to prevent or destroy biofilms, thereby greatly impacting human health and medicine. Furthermore, the identification of treatments that can disrupt bacterial regulation and communication may have great implications as antibiotic adjuvants as well as antibiofilm therapies.

Biologically-Inspired Strategies for Combating Bacterial Biofilms Highlights.

• Biofilms are inherently resistant to antibiotics and are a major health threat

• Quorum sensing antagonists inhibit biofilm formation

• Inhibition of two-component systems disrupt biofilm formation

• Approaches to target the extracellular matrix to disrupt biofilms are discussed