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Cite this article: Bedi B, Maurice NM, Sadikot RT (2017) Pseudomonas aeruginosa Bio Films. JSM Microbiology 5(2): 1040.

*Corresponding authorSadikot RT, Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Atlanta Veterans Affairs Medical Center, Emory University, 1670 Clairmont Rd, Decatur, GA 30033, USA, Email:

Submitted: 12 June 2017

Accepted: 21 June 2017

Published: 30 June 2017

Copyright© 2017 Sadikot et al.

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Keywords•Pseudomonasaeruginosa•Biofilm•Quorum sensing

Review Article

Pseudomonas aeruginosa Bio FilmsBedi B1, Maurice NM1,2, and Sadikot RT1,2*1Atlanta Veterans Affairs Medical Center, USA2Department of Medicine, Emory University, USA

Abstract

Pseudomonas aeruginosa is an important opportunistic pathogen causing a variety of acute infections including nosocomial pneumonias, sepsis, urinary tract infections, keratitis, wound and skin infections. P. aeruginosa continues to be a leading cause of infections in immune compromised host including patients with cystic fibrosis and is among the most virulent of the opportunistic pathogens. The outcome of P. aeruginosa infection depends on the virulence factors displayed by the bacteria as well as the host response. The virulence of P. aeruginosa is multifactorial and includes a variety of cell associated and extracellular proteins. P. aeruginosa has also developed mechanisms to clinically colonize surfaces by co ordinately expressing genes in a density dependent manner by the production of small diffusible molecules called auto inducers or quorum sensing (QS) molecules. Activation of the QS cascade promotes formation of bio films, structured communities that coat mucosal surfaces and invasive devices. These bio films make conditions more favorable for bacterial persistence as embedded bacteria are inherently more difficult to eradicate than those in the planktonic form. Here, we provided an in depth review of development, composition, transcriptional regulation, experimental models and dispersal of P. aeruginosa bio films.

ABBREVIATIONSEPS: Extracellular Polymeric Substances; QS: Quorum

Sensing; AHL: Acylhomoserine Lactone; c-di-GMP: Cyclicdi Guanosine Monophosphate; CF: Cystic Fibrosis; VAP: Ventilator Associated Pneumonia; NET: Neutrophil Extracellular Trap; Polymorphonuclearneutrophil

INTRODUCTIONNaturally occurring microbes exist in two distinct forms,

nomadic (planktonic) or sedentary (sessile). Sessile microbial communities attached to a surface often develop a complex structure called a bio film [1,2]. Bio films can be described as “microbial cities” [3], in which microbial cells adhere to surfaces as well-structured micro colonies surrounded by a complex matrix composed of many extracellular polymeric substances (EPS). In the context of evolution and adaptation, bio films provide homeostasis and stability in the face of fluctuating and harsh environmental conditions including host immune defense [4,5]. When bio films form in living organisms, they often cause serious diseases. Due to the increased resistance of cells associated with bio films.

Pseudomonas aeruginosa is a common opportunistic and nosocomial pathogen and the leading cause of morbidity and mortality in immune compromised as well as cystic fibrosis (CF) patients [6]. P. aeruginosa bio films are associated with the pathogenesis of urinary, ventilator associated pneumonia (VAP),

dialysis catheter infections, bacterial keratitis, otitisexterna and burn wound infections [7]. Due to the many clinical implications, bio film formation by P. aeruginosa is one of the most widely studied bio film models.

BIO FILM MATRIX COMPOSITIONIn most bio films, the microorganism itself accounts for less

than 10% of the dry mass, whereas the matrix can account for more than 90% (8). Bio film structure can be affected by the genetics (active response) as well as environmental (passive response) conditions which are not mutually exclusive [9,10]. The bio film matrix is composed of extracellular material, produced by the pathogen itself, in which the microbial cells are embedded. It comprises of different types of biopolymers- known as extracellular polymeric substances (EPS) [8]. EPS forms the scaffold for the three-dimensional architecture of the bio films as well as glue for adhesiveness. The importance of EPS is demonstrated by the altered morphology of bio films produced by mutants lacking components of EPS [11, 12]. Components of the EPSs primarily include polysaccharides, extracellular DNAs (e DNA), and proteins [13]. Gram negative pathogens such as P. aeruginosa also contain a range of enzymes and DNA [14], which can alter matrix properties, acting as ‘killer vesicles’ of other bio films.

The architecture of bio films formed by P. aeruginosa is influenced by many extrinsic factors such as, hydrodynamic

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conditions, nutrient concentrations, specifically iron, (which can facilitate its conversion from non-mucoid to mucoid phenotype) [15], bacterial motility and intracellular communication.

Exo polysaccharides

Polysaccharides comprise a major part of the EPS. P. aeruginosa produces at least three distinct exo polysaccharides that contribute to bio film development and architecture; namely, alginate [16,17], PeI (Pellicle) [18,19] and PsI (polysaccharide synthesis locus) [20, 21].

Alginate

Alginate is one of the most extensively studied polysaccharides produced by P. aeruginosa. Over expression of the alginate exo polysaccharide was first identified as being associated with P. aeruginosa isolates recovered from the lungs of chronically infected CF patients, but rarely from other types of infections [21-23]. It is a shiny, high molecular mass, un branched hetero polymer consisting of 1,4-linked uronic residues of β-D-mannuronate and α-L-guluronate [8]. These components are arranged in homo polymeric blocks of poly mannuronate and hetero polymeric sequences with a random distribution of guluronate and partially O-acetylated mannuronate residues (8). Overproduction of alginate is a characteristic feature the mucoid strain of P. aeruginosa [8,15,21,24]. In CF, such isolates can become mucoid through mutation of one of the genes of the alginate operon (mucA, mucB,mucC,or mucD) which produce factors for sequestering AlgU, required for increased expression of alginate producing genes (8,15). Alginate is not only involved in the establishment of micro colonies at the beginning of bio film formation. It also imparts mechanical stability to form mature bio films [25]. Although alginate is not essential for P. aeruginosa bio film formation, it has a notable effect on bio film architecture when it is present [21].

A recent study showed that mucoid mutants of non-CF P. aeruginosa isolates express increased levels of alginate and potentially convert into the mucoid variant under iron-limiting conditions and not under iron-replete conditions [15].

In the non-mucoid not expressing alginate biosynthesis genes, Pel and Psl are involved in the establishment of bio films [2,26]. Pel is a glucose-rich polysaccharide, whereas Psl consists of a repeating pentasaccharide containing d-mannose, d-glucose and l-rhamnose [2]. Pel is essential for the formation of bio films (called pellicles) at air–liquid interfaces and bio films that are attached to a surface [18,19], and Psl is involved in the adherence to abiotic and biotic surfaces and in the maintenance of bio film architecture, as well as dispersal.

In many bacteria, exo polysaccharides are indispensable for bio film formation, and mutants that lack the ability to synthesize exo polysaccharides are severely compromised or unable to form mature bio films [27-29]. In mixed-species bio films, the presence of a species that produces exo polysaccharides may lead to the integration of other species that do not synthesize matrix polymers [30].

Extracellular proteins

Extracellular enzymes can be efficiently retained in the

bio film matrix by their interaction with polysaccharides. In P. aeruginosa, the association of extracellular lactonizing lipase (lipA; also, known as lip) with alginate is based on weak binding forces [8,31]. Such interactions result in a matrix of exo polysaccharides that are biochemically activated by the attached enzymes. This arrangement retains the enzymatic activity close to the cell(s) and keeps the diffusion distances of enzymatic products short, thereby optimizing their uptake by bacteria [32].

EPS modifying enzymes

These enzymes can potentially degrade EPS components during starvation, targeting EPS made by the bacterium that produces the enzyme or EPS made by other species [33,34]. Exo polysaccharides are degraded mainly by hydrolases and lyases. Furthermore, these enzymes assist in the dispersion of sessile cells from the bio films, which allows new bio films to be formed [29,35].

Structural proteins

The non-enzymatic proteins in the matrix, such as cell surface-associated and carbohydrate-binding proteins (lectins), are involved in the structure and stabilization of the polysaccharide matrix by constituting a link between the bacterial surface and extracellular EPS [2]. Galactose-specific and fucose specific lectin LecA and LecB respectively [36,37] of P. aeruginosa, have been implicated in bio film formation. LecB has a stabilizing effect on in intact bio films of P. aeruginosa. Synthetic high-affinity multivalent ligands targeting LecB induce complete dispersion of established bio films [38]. Proteinaceous appendages such as pili, fimbria, and flagella act as additional supportive structural elements by interacting with other EPS components of the bio film matrix in P. aeruginosa bio films. For example, type IV pili of P. aeruginosa bind DNA and so possibly act as cross-linking structures [39].

Extracellular DNA (e DNA)

Extracellular DNA, although initially seen as residual material from lysed cells, has been shown to be in fact an integral part of the bio film matrix and stationary mode of life [40,41]. e DNA is a major matrix component in P. aeruginosa bio films, in which it functions as an intercellular connector. Due to this characteristic, DNA ase inhibits the formation of bio films in P. aeruginosa [42-44]. e DNA has multiple functions in bio film formation and stability: by aiding in initial adhesion, by modulating charge and hydro phobic interactions between the bacteria and the surface [45]. The localization of e DNA can vary widely between bio films. In P. aeruginosa bio films e DNA forms a grid-like structure [46].

The e DNA is involved in the structural maturation of P. aeruginosa bio films by localizing in a time-dependent manner in the stalks of mushroom-shaped micro colonies. Notably, high concentrations of e DNA have found in the outer parts of the stalk, there by forming a border between stalk and cap-forming P. aeruginosa subpopulations [46]. In human sites of infection, such as the CF lung, e DNA detected is almost entirely derived from human polymorpho nuclear neutrophils (PMNs) that are recruited heavily to infection sites [47]. Recent studies have shown that PMN-derived genomic DNA can be incorporated into P. aeruginosa bio films and confer increased bacterial resistance

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to amino glycoside treatment [48]. eDNA can limit anti-microbial peptides (AMPs), membrane damage and killing [49,50]. Extracellular DNA can also confer antibiotic resistance acidifying bio film cultures [41].

Surfactants, lipids, and water

Surfactants and lipids comprise the hydrophobic properties of the EPS. Rhamnolipids, can act as surfactants, and have been found in the EPS matrix of P. aeruginosa [51]. They display surface activity and have been proposed to act in initial micro colony formation, surface-associated bacterial migration and formation of mushroom-shaped structures, and most importantly playing a part in bio film dispersion [52]. Water is the largest component of the matrix, and the EPS provides a highly-hydrated environment. It has the ability to buffer the bio film against external environmental fluctuations pertaining to water availability. P. aeruginosa matrices are EPS are hygroscopic and seem to retain water entropically rather than through specific water binding mechanisms [8].

Mechanical properties of bio films

The viscoelastic properties of P. aeruginosa bio films are influenced by external chemical stimuli and shear forces. These bio films display remarkable viscoelastic behavior, which allows them to fully regain their initial stiffness after being exposed to external forces. Specifically, this recovery process is highly robust towards chemical perturbations such as ions, poly electrolytes, organic molecules, or pH changes. Trivalent ions such as Fe (III) and Al3+, but not divalent ions, significantly enhance the bio film elasticity and also alter their texture. In contrast, citric acid as a potent fluidization agent for pre-formed bio films [53].

P. aeruginosa mucoid phenotype in the CF lung

CF is an autosomal recessive disease that is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that renders impairment of mucociliary clearance [54]. P. aeruginosa grows in bio films within the CF lung [16,55,56]. A salient feature of P. aeruginosa colonization of the CF lung is the selection of mucoid variants of P. aeruginosa, which are characterized by overproduction of alginate [16,17]. Initial isolates of P.aeruginosa from the CF lungare non mucoid. However, chronicity of infections coincides with host immune evasion and prevalence of [57] mucoid isolates. Mucoid variants are absent among environmental P. aeruginosa isolates, although non-mucoid strains seem to have the genetic makeup necessary for mucoidy. Factors such as hydrogen peroxide or activated PMNs can induce mutations in the mucA gene [58-60].

The increased viscosity of CF airway mucous acts as a matrix scaffold which decreases clearance [61], worsened by thick mucus plaques and depleted O2 in the respiratory epithelium, leading to the colonization by P. aeruginosa that further exacerbates the pathology of CF pneumonia and sustenance of the mucoid phenotype. Anaerobic growth might be an important feature of P. aeruginosa growing in bio films in CF patients leading to the build-up of toxic nitrogen metabolites [62]. Proteomic analysis identified an outer-membrane porin, OprF, the concentration of which increased 40-fold in anaerobic culture. Detection of OprF in the secretions from CF lungs along with circulating

antibodies against OprF were found in chronically infected CF patients, concurring with P. aeruginosa antibiotic resistance and bio film formation [63]. Alterations in salt concentrations as well as iron depletion can facilitate the enhancement of the mucoid phenotype [15,64].

BIO FILM DEVELOPMENT

Temporal and spatial organization of bio films

Proteomic studies indicate that bio film formation in P. aeruginosa proceeds as a regulated developmental sequence, with five stages, with the fifth stage being purely dispersal [65,66]. First and second stages include initial attachment to surfaces-identified by a loose or transient association with the surface using van der waals forces, followed by robust adhesion. During stages 3 and 4cells aggregate into micro colonies where bio film cells are dispersed [66]. Studies have also described the role of LPS mediated bio film stability [67]. LPS has the ability to affect surface charge, hydrophobicity as well as dictate alterations in attachments, transition to sessile growth and morphology [67].

Bio film structures can be flat or mushroom-shaped with a highly porous EPS matrix, depending on the nutrient source, which seems to influence the interactions between localized clonal growth and the subsequent rearrangement of cells through Type IV pilus-mediated gliding motility in response to the nutritional cues [68]. The pores or channels in the bio film structure sustain the supply of nutrients and oxygen for the growth of inhabitant cells. The fully structured bio films can be maintained for a long period, with eventual dispersion [1]. There is a significant difference in the proteomics of planktonic versus bio film cellular forms of P. aeruginosa. These results demonstrate that there is distinct genomics associated with the physiology of bio film cells, and this developmental stage [66].

During bio film formation, a phenotypic and spatial distribution of cells also takes place. In a mature P. aeruginosa bio film [69], cells within the bio film differentiate into two distinct phenotypes; highly motile cells in the interior of bio film and non-motile stationary cells in the outer wall [70]. During the differentiation process, the non-motile cells form small mushroom “stalks” within the micro colonies, and the motile cells, using the type-IV pili mediated twitching motility, climb the stalks, aggregating on the top and forming the mushroom cap [68,70]. The subpopulation of cells constituting the stalk is less metabolically active in comparison to the cells in the cap [71,72]. The cap-forming and the stalk-forming subpopulations of the mushroom-shaped structures display differential tolerance to antimicrobial compounds such as to bramycin, which preferentially kills bacteria in the cap portion, this spatial differentiation creates central hollows by causing the evacuation of motile cells in the center of the bio film [29,70]. Since these hollow cavities devoid of bio film matrix are important for dispersal [73]. The process of central hollowing is a motility-based evacuation phenomenon and is a distinct process from ordinary detachment and is called ‘seeding dispersal’ or dispersal (or dispersion) [66,74].

Mucoid Pseudomonas aeruginosa bio film architecture

Bio films formed by (alginate-overproducing) strains of P.

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aeruginosa are lumpy and uneven [8] where as spontaneous non-mucoid revert ant strains of P. aeruginosa form smoother and flatter bio films. The role of calcium appears to be important in bio film morphology as well, with the presence of Ca2+ stabilizing the cross bridges between alginate molecules and allowing a thicker and more stable bio film to be formed [8,75]. The presence of O-acetyl groups in alginate in mucoid bio film architecture is important. A mutant P. aeruginosa strain lacking in its ability to acetyl at alginate loses it mucoid phenotype [8].

Transcriptional regulators of bio film formation

Biofilm formation are highly controlled processes regulated at the genetic level and by environmental signals. In several gram-negative species such as P. aeruginosa, current literature points to quorum sensing (QS), bis-(3’-5’) cyclic diguanosine monophosphate (c-di-GMP), and small RNAs (s RNAs) as the main regulators of bacterial bio films [76,77].

Quorum sensing (QS)

QS is a mechanism employed by many bacteria to coordinate gene transcription and group activity in response to population density. In QS, bacteria constitutively express signaling molecules, termed QS molecules or “auto inducers”. As the bacteria replicate, the concentration of QS molecules eventually reaches a threshold upon which they will bind to specific bacterial receptors and direct a coordinated program of gene transcription. In P. aeruginosa, QS plays an important role in the regulation of microbial virulence. There are two primary interconnected QS systems that utilize acylhomoserine lactone (AHL) signaling molecules: the Las system and the Rhl system (78-81). In the Las system, the QS molecule, N-(3-oxododecanoyl)-L-homo serine lactone (3-oxo-C12-HSL), is synthesized by the LasI synthase and recognized by the LasR receptor [82-84]. In the Rhl system, N-butanoyl homo serine lactone (C4-HSL) is produced by the RhlI synthase and sensed by the RhlR receptor [85-87]. A third QS system that does not rely on AHL signaling has also been characterized. The Pseudomonas quinolone signal (Pqs) system, which is mediated by the molecule, 2-heptyl-3-hydroxy-4-quinolone, closely interacts with the Las and Rhl systems [88]. Recently, a fourth QS system, termed the IQS system, has also been shown to connect environmental stress cues with the QS network [89]. The four QS systems are integrated in a multi-layered hierarchical fashion with the Las system acting as the predominant controller, but with built-in redundancies and plasticity if either the Las system becomes inactive or under conditions of nutrient depletion [78].

Intact QS systems have been implicated as a requirement for bio film formation. Transcriptomic analysis revealed that QS systems regulate the gene expression observed in P. aeruginosa bio films [90]. Additionally, Davies et al., demonstrated that Las-deficient strains of P. aeruginosa formed undeveloped bio films lacking the classical multi cellular mushroom-shaped structures. Additionally, unlike the wild-type P. aeruginosa bio films, the Las-deficient bio films were susceptible to the detergent, SDS [91]. Furthermore, bio films treated with quorum-sensing inhibitors demonstrated a disrupted architecture and loss of bacterial biomass [92]. Similar results have also been seen in bacterial strains with disrupted Rhl and PQS systems [93,94]. However, other investigators have demonstrated that QS is

not always required for bio film formation in in vitro models [95,96]. The involvement in QS signaling in bio film formation appears to depend on the specific in vitro conditions of the bio film assay used. Taken together, these studies suggest that QS is not completely necessary for the formation of bio films, but only responsible for the development of the mushroom cap portion of mature bio films. This may reflect the fact that the mushroom-shaped structures are composed of extracellular DNA and rhamnolipids, components which are under the control of QS systems [42,46,93,97,98]. This disruption of mature bio film maturation could explain why bio films of QS-deficient P. aeruginosa are less stable than QS-intact P. aeruginosa bio films [91,99].

Control by s RNA

P. aeruginosa bio film is regulated by s RNAs, rsmY and rsmZ [76]. Three sensor kinases are involved in this pathway, namely, RetS, LadS, butmainly GacS [100]. GacS phosphorylates GacA [101], which, in turn, activates the transcription of rsmZ and rsmY. RsmZ and rsmY reduce the activity of effector protein RsmA, being a negative post-transcriptional regulator of the bio film matrix polysaccharide Psl [102], and also downregulate another effector, RsmN, controlling the same functions as RsmA [103]. Additionally, alternative sigma factor, Rpo Sexpression was also significantly increased in P. aeruginosa bio films [104] acts as a positive regulator of the expression of the psl gene [102,104].

BIO FILM DISPERSALP. aeruginosa displays swarming dispersal behavior which has

been best described in the context of non-mucoid P. aeruginosa bio films. After initial bio film growth, the micro colonies differentiate to form an outer wall of stationary bacteria and an inner ‘liquefied’ region of motile cells of planktonic phenotype. These motile cells can then ‘swim’ out of the micro colony, leaving a hollow mound [66,105].

Environmental factors regulating bio film dispersal

Environmental stimuli or host factors can affect all aspects of bio film dispersal, such as motility, EPS formation, rhamnolipids, cell death and lysis. The main players involved in this process include, nutrients, oxygen, nitric oxide (NO), and chelating chemicals [1]. A shift in carbon sources such as glutamate and glucose, can cause drastic reductions in bio film mass by the dispersal process, by altering the levels of c-di-GMP [35]. Oxygen levels can alter the shape of the bio film, by creating an oxygen gradient from the outer to the inner core of the bio film. The inner core tends to be anaerobic with low metabolic activity and high antibiotic tolerance [1,35]. In P. aeruginosa, biofilms, NO is produced by anaerobic respiration, which in turn induced dispersal [106]. The factors regulating these processes are discussed further.

Transcriptional regulation of bio film dispersal

It is unclear as to the involvement of the quorum sensing signaling pathway in regard to the relationship to dispersal. Experimentally, P. aeruginosa incapable of synthesizing AHL suggest that QS is not involved in seeding dispersal [107,108].

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c-di-GMP and transcriptional regulators of dispersal

c-di-GMP is extensively implicated for its involvement in regulating the formation and dispersal of bio films. The intracellular level of c-di-GMP determines the transition between planktonic and bio film phenotypes. An increase in intracellular c-di-GMP level facilitates bio film formation, whereas a decrease facilitates the planktonic mode of life [1,109]. c-di-GMP is synthesized from two GTPs by di guanylate cyclase (DGC, proteins with GGDEF domains) activity, and degraded into pGpG and finally into two GMPs by phosphor di esterase (PDE, proteins with EAL or GYP domains) activity. The level of c-di-GMP is balanced between these two reciprocal enzymatic activities [109].

Transcriptional regulators of P. aeruginosa dispersion include: bio film dispersion locus A(BdlA) and other factors with which it associates such as [110], Nutrient induced cyclase D (NicD), Dispersion-induced phosphor di esterase A (DipA), and GcbA, in a complex functional combination [1].

NicD possesses DGC enzymatic activity and is required for dispersion induced in the presence of carbon sources including glutamate, succinate and glucose. In the presence of glutamate, NicD is dephosphorylated on its cyto plasmic domain to activate its DGC activity, it causes temporary increases in c-di-GMP preceding its reduction. This process leads to the phosphorylation and cleavage of BdlA [111]. BdlA: Activation of BdlA enables bio film dispersion. Native BdlA is inactive and is activated by proteolytic cleavage and phosphorylation. Dephosphorylated NicD (in the presence of glutamate) phosphorylates BdlA and induces its proteolytic cleavage. The cleaved fragments interact with each other and associate with DipA to make a multi-protein complex. DipA has PDE activity that is enhanced by the activated BdlA in the multi-proteincomplex, this complex formation leads to ultimate reduction in the intracellular c-di-GMP level, resulting in the bio film dispersal [111-113]. The dipA mutant is dispersion- deficient in response to glutamate, NO, ammonium chloride, and mercury chloride. GcbAhas DGC activity, contributes to the bio film-specific posttranslational modifications of BdlA and is required for the dispersion by increasing nutrients, heavy metals, and NO [114]. Although both GcbA and BdlA have the capacity to enhance dispersal to the same environmental cues, they oppositely adjust the intracellular c-di-GMP level.

RbdA has PDE activity and regulates dispersion in response to oxygen levels [115]. It reduces c-di-GMP by directly interacting with BdlA levels in oxygen depleted conditions. RbdA can facilitate the bio film dispersal by controlling the production of rhamnolipids, as well as down regulating the production of exo polysaccharides [115].

MucR and NbdA are c-di-GMP-modulating enzymes responsible for the NO effects on bio film dispersion studied in P. aeruginosa [116]. MucR acts as a PDE under planktonic growth conditions and acts as a DGC in bio films. NbdA has only PDE activity that decreases c-di-GMP level in response to NO in bio film growth [117].

In P. aeruginosa, the fatty acid cis-2-decenoic acid induces a cascade of events that result in degradation of the bio film matrix and bio film dispersal [118,119]. Exogenous cis-2-decenoic acid has been shown to induce bio film dispersal in P. aeruginosa and other phylogenetically diverse bacteria.

MIXED SPECIES BIO FILMS Clinical associations have been described in patients with

respiratory syncytial virus (RSV) infections and P. aeruginosa co-infections. RSV lung infections often facilitate conversion of opportunistic P. aeruginosa infection into a chronic colonization state [120]. A recent study by Hendricks et al has shown that RSV infection enhances biofilm formation by P. aeruginosa by modulating nutritional immunity, including iron homeostasis involving transferring [121]. Study models of mixed-species bio films clearly demonstrate the occurrence of cooperative behavior in bio films formed by P. aeruginosa, P. protegens and Klebsiella pneuomoniae [122]. Stress tolerance in this environment is present equally in all the community members. This scenario can partly explain the high tolerance of these communities to toxin accumulation, significantly more than their planktonic counterparts [122]. P. aeruginosa can associate with skin bacteria like Staphylococcus epidermis [7] and S. aureus [123] to cause severe chronic infections in burn wounds. In CF patients and CF mouse models, the co infection of common pathogens such as Burkholderia cenocepacia and P. aeruginosa leads to the formation of a mutualistic relationship, where P. aeruginosa persists and B. cenocepacia alters the inflammatory response. Virulence factors of B. cenocepacia inhibit P. aeruginosa virulence, signaling, and bio film formation [124,125].

The fatty acid cis-2-decenoic acid, produced by P. aeruginosa, can induce dispersal in other bacterial species, including B. subtilis and S. aureus [119]. Exogenously added P. aeruginosa rhamnolipids can disperse bio films produced by Bordetella bronchiseptica and Salmonella enteric serovar typhimurium [26,127].

Components of the P. aeruginosa Las and RhlQS systems act in sync to disperse E. coli bio films. Rhamnolipids complement Las signaling by inducing selective permeability of the E. coli membrane to lipo philic AHLs suggesting that rhamnolipids play a critical role in P. aeruginosa, E. coli interspecies signaling [128].

OVERVIEW OF MODELS USED FOR CHARACTERI-ZATION OF P. AERUGINOSA BIO FILMS IN VITRO AND IN VIVO

Visualization and quantitation of bio films in vitro

One of the simplest methods of studying surface-attached bio films is the micro titer plate assay. In this assay, the adherence of P. aeruginosa can be easily monitored in a 96 well plate, with crystal violet (CV) staining [129]. This is a crude but effective way to study prospective anti-bio film agents. However, it is not very reproducible, which does not differentiate between live and dead cells as well as the non-adherent or planktonic counter-parts [29,130]. Bio film formation in co-culture models of P. aeruginosa with host cells such as neutron phils and epithelial cells can also be studied [131].

Confocal microscopy with P. aeruginosa GFP strains or live dead stains, by itself or in a co-culture models can be performed using bio films grown on glass coated with collagen, using softwares such as image J or comstat [121, 132,133].

Other methodologies include Centers for Disease Control

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(CDC) reactor, which tests bio film formation on discs spinning in the media. Continuous flow cell system first described by Strenberg et al., [134] is used to study bio film formation in a setting where carbon is available in a continuously supply. This method produces highly structured bio films. A drip slow reactor is used for studying bio films on surface placed at an angle, such as in medical devices. All these methods allow for assessing colony forming units (CFUs), microscopy and structural characterizations [135].

Bio films formed by commonly associated bacterial species, such as P. aeruginosa and S. aureus can be characterized and visualized by laser ablation electrospray ionization mass spectrometry (LAESI-MS) [123]. EPS constituting bio films can be visualized in situ by confocal laser scanning microscopy (CLSM) in combination with different fluorescent dyes [8], such as fluorescently labeled lectins and antibodies. Exo polysaccharides can then be visualized according to their interaction with specific target sugars. Specific multi labeling with both lectins and antibodies has been reported [8]. Another upcoming approach is the use of CLSM-based lectin-binding analysis in combination with Raman microscopy and microspectroscopy [136]. This combination gives a more in-depth insight into EPS composition. Local enzymatic activity in bio films can be achieved by microscopic visualization of fluorogenic substrates. Such as phosphatase activity using the water-soluble substrate ELF-97phosphate. EDNA can be detected with dye specific for nucleic acid [8].

In vivo models of bio film and chronic infection

Non-mammalian hosts such as Zebrafish, Drosophila melanogaster, Caenorhabditis elegans [137], and mammalian hosts such as guinea pigs [138] and minks [139] have been used to assess virulence of P. aeruginosa. Based on reagent availability, cost, ease of use as well as pertinence to the human host, most common animal models currently used to study acute and chronic infections by P. aeruginosa are rodents, namely rats and mice models.

Introduction of free-living and mobile P. aeruginosa cells into the lungs provides an acute model of lung infection. P. aeruginosa is either administered via aerosol challenge, inter-tracheal (IT) installation, [140,141] or intra nasal application. Rapid clearance or acute sepsis and death is associated with this model [137]. Acute infections in a burn wound model are achieved by challenging the mice ectopically with P. aeruginosa, underneath the burn wound [137].

Chronic lung infections by P. aeruginosa are attained by embedding its cells in agar, agarose, or seaweed alginate. The embedding of P. aeruginosa in beads is essential for the initiation and establishment of chronic infection, where beads are physically retained in the airways. This creates an environment that mimics bio film formation and micro aero biosis pertaining to the CF lung. The first model of chronic broncho pulmonary infection was done in rats using agar beads by Cash et al in 1979 [142]. Given its simplicity, efficacy and low cost, this model is still frequently used today. Current models of chronic infections currently employed are in fact modifications of this established model. P. aeruginosa beads are then introduced into the mouse IT

[137,140] or directly into the trachea though the mouth [141]. P. aeruginosa infection load is evaluated by colony counts or other in situ visualization methods described above.

Determination/quantitation of bio film dispersal

Cells dispersed from bio films represent a distinct stage in the transition from the sessile to planktonic forms. However, these dispersed cells from both their sessile and planktonic counterparts, as is reflected in the transcriptome and c-di-GMP levels [143]. The rationale for evaluating bio film dispersal is to (i) to explore new dispersal methodologies for the treatment of chronic infections, (ii) to evaluate the metabolic phenotype or other biomarkers of dispersed cells, such as c-di-GMP levels, and (iii) to evaluate and control the spread of infection. In vitro dispersion is measured by first growing the planktonic cells into a bio film, followed by dispersion. Final evaluation of dispersed cells is performed by colony counts and HPLC analysis of c-di-GMP levels.

In vivo measurement of dispersal is performed by growing P. aeruginosa bio films on implants, followed by insertion of the implant into the peritoneal cavity of the animal. Dispersal agents can then be tested on the animal model, with subsequent measurements of c-di-GMP and colony counts [143].

Simulation of bio film pathology

The question still remains of how to re-create or simulate in vivo conditions in vitro. Examples of this include creating a system based on the nutritional and constitutional cues similar to expectorated CF sputum, known as synthetic CF sputum medium (SCFM) [135]. This medium approximates P. aeruginosa gene expression and virulence factors to that observed in expectorated CF sputum. This medium can also be used to test the efficacy of antibiotics. SCFM can be considered as an infection medium, which could then be differently supplemented further to better understand host factors contributing to infection ex vivo [135]. A semisolid collagen matrix in which P. aeruginosa and S. aureus can both form aggregates, mimicking a wound bed or the muco purulent pus of the CF lung, can be used to delineate host inflammatory factors involved in exacerbating both infection models.

Since bio films are complex systems, model systems could be very useful in determining influences of individual factors [144]. Artificial bio films that impersonate important aspects of the real bio films, and can be used to study defined variations in parameters. Agarose is used as a hydro gel matrix to simulate extracellular polymeric matrix. Microorganisms such as P. aeruginosa are colonized in this type of matrix [144]. Furthermore, this model can be used to mimic and compare bio films formed by non-mucoid versus mucoid strains of P. aeruginosa to test the efficacy of antibiotics and in lieu of host factors.

HOST RESPONSE AGAINST P. AERUGINOSA BIO FILMS

The host response against P. aeruginosa infections is complex and involves the coordinated activity of a variety of cell types comprising both the innate and adaptive immune systems. A clear understanding of the in vivo host response to P. aeruginosa

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bio films is made more challenging by the difficulty in modeling bio films in human disease. In fact, the majority of research investigating the host response to P. aeruginosa has utilized bacterial infection models with the bacteria in the planktonic state. However, a growing body of literature supports a role for both the innate and adaptive immune systems in the response to P. aeruginosa bio film infections [145-147]. Discussion of how the host cells respond to bio film formation is beyond the scope of this review.

SUMMARY AND CONCLUSIONOver the past few decades, there has been a vast growth in

the knowledge of P. aeruginosa bio films and their importance in causing human disease. Both the CDC and the World Health Organization have highlighted the health threat of antibiotic-resistant P. aeruginosa and urged the development new antimicrobial therapies for P. aeruginosa given the high rates of antibiotic resistance. Furthermore, given the tolerance and recalcitrance of P. aeruginosa in many important clinical bio film infections, urgent novel anti-bio film treatments are needed. We have begun to better understand both the bacterial factors that influence bio film development, maturation, and dispersal, and also the host factors that influence the immune response to bio film infections. However, a more thorough understanding of bio film biology and host immune response to bio films is urgently needed given the recalcitrance of bio film infections to host defenses and antimicrobial therapy. Novel therapeutic strategies are currently in development and these will hopefully prove successful as adjunctive treatments for P. aeruginosa bio film infections.

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