Biomedical and Biotechnology Research Journal (BBRJ)

: 2018  |  Volume : 2  |  Issue : 4  |  Page : 227--236

Microarchitecture of Pseudomonas aeruginosa biofilms: A biological perspective

Brahmchetna Bedi, Nicholas M Maurice, Ruxana T Sadikot 
 Atlanta Veterans Affairs Medical Center, Decatur; Department of Medicine, Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Emory University, Atlanta, Georgia, USA

Correspondence Address:
Dr. Ruxana T Sadikot
Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Atlanta Veterans Affairs Medical Center, Emory University, 1670 Clairmont Road, Decatur, Georgia 30033


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 immunocompromised host including patients with cystic fibrosis and is among the most virulent of the opportunistic pathogens as listed by the Centers of Disease Control (CDC). P. aeruginosa has also developed mechanisms to colonize surfaces by coordinately expressing genes in a density dependent manner regulated by the production of small diffusible molecules called auto inducers or quorum sensing (QS) molecules. Activation of the QS cascade promotes formation of biofilms which provide an encapsulated communal structure that coats mucosal surfaces and invasive devices. These biofilms make conditions more favorable for bacterial persistence as embedded bacteria are inherently more difficult to eradicate by both antibiotic regimens as well as by innate immune systems as compared with those in the planktonic state. The objective of this report is to provide an overview; (i) propagation of P. aeruginosa biofilms; (ii) components of the biofilm matrix and their transcriptional regulation; (iii) key signaling pathways regulating C-di-GMP dependent biofilm dispersal; (iv) characterization of experimental models of biofilms.

How to cite this article:
Bedi B, Maurice NM, Sadikot RT. Microarchitecture of Pseudomonas aeruginosa biofilms: A biological perspective.Biomed Biotechnol Res J 2018;2:227-236

How to cite this URL:
Bedi B, Maurice NM, Sadikot RT. Microarchitecture of Pseudomonas aeruginosa biofilms: A biological perspective. Biomed Biotechnol Res J [serial online] 2018 [cited 2019 Jan 21 ];2:227-236
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Bacterial communities can exist in two distinct forms, nomadic (planktonic) or sedentary (sessile). Biofilms are comprised sessile microbial communities attached to a surface, where “microbial cities” are formed by well-structured microcolonies adhering to surfaces surrounded by a complex matrix composed of many extracellular polymeric substances (EPS).[1] Biofilms provide a tremendous adaptive advantage to opportunistic pathogens such as Pseudomonas aeruginosa by imparting homeostasis and stability in the face of fluctuating and harsh environmental conditions such as the host environment,[2],[3] as well as the presence of antibiotics. Biofilm formation by P. aeruginosa has serious pathological implications such as urinary, ventilator-associated pneumonia, dialysis catheter infections, bacterial keratitis, otitis externa, and burn wound infections.[4] The role of P. aeruginosa biofilms in cystic fibrosis (CF) pathogenesis is well established.[5] P. aeruginosa is an opportunistic pathogen and a huge threat to public health due to its adaptive qualities and varied antibiotic resistance.[6],[7]

 Biofilm Microarchitecture

Biofilm architecture is in most biofilms influenced by external chemical stimuli and shear forces. These biofilms 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 toward chemical perturbations such as ions, polyelectrolytes, organic molecules, or pH changes.[8] The microorganism itself accounts for <10% of the dry mass, whereas the matrix can account for >90%.[9] Biofilms structure can be affected by the genetics (active response) as well as environmental (passive response) conditions which are not mutually exclusive.[10],[11] The matrix within the biofilm is composed of extracellular material, produced by the pathogen itself, embedding the microbial cells. It comprises different types of biopolymers known as EPS.[9] EPS forms the scaffold for the three-dimensional architecture of the biofilms as well as glue for adhesiveness. Major components of the EPSs primarily include polysaccharides, extracellular DNAs (eDNA), and proteins.[12] Biofilms architecture of P. aeruginosa is specifically sensitive to iron, (which can facilitate its conversion from nonmucoid to mucoid phenotype).[13],[14]

 Major Components


Polysaccharides comprise a major part of the EPS. P. aeruginosa produces at least three distinct exopolysaccharides that contribute to biofilm development and architecture, namely, alginate,[14],[15] pellicle (PeI),[16],[17] and polysaccharide synthesis locus.[18],[19]

Alginate and mucoid phenotype in the cystic fibrosis lung

Alginate is one of the most extensively studied polysaccharides produced by P. aeruginosa. Overexpression of the alginate exopolysaccharide 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.[19],[20],[21] It is a shiny, high-molecular mass, unbranched heteropolymer consisting of 1,4-linked uronic residues of b-D-mannuronate and a-L-guluronate.[9] These components are arranged in homopolymeric blocks of polymannuronate and heteropolymeric sequences with a random distribution of guluronate and partially O-acetylated mannuronate residues.[9] Mucoidy is attained 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.[9],[13] Alginate is not only involved in the establishment of microcolonies at the beginning of biofilm formation. It also imparts mechanical stability to form mature biofilms.[22] A recent study showed that nonmucoid variants of nonCF P. aeruginosa isolates express increased levels of alginate and potentially convert into the mucoid variant under iron-limiting conditions.[13] Mucoid variants are absent among environmental P. aeruginosa isolates, although nonmucoid strains seem to have the genetic makeup necessary for mucoidy. Factors such as hydrogen peroxide or activated polymorphonuclear neutrophils (PMNs) can induce mutations in the mucA gene.[23],[24],[25] Proteomic analysis identified an outer-membrane porin, OprF, the concentration of which is increased 40-fold in anaerobic culture. OprF is detected in the secretions from CF lungs along with circulating antibodies, concurring with P. aeruginosa antibiotic resistance and biofilm formation.[26]

In the nonmucoid not expressing alginate biosynthesis genes, Pel and Psl are involved in the establishment of biofilms.[27],[28] Pel is a glucose-rich polysaccharide, whereas Psl consists of a repeating pentasaccharide containing d-mannose, d-glucose, and l-rhamnose.[28] Pel is essential for the formation of biofilms (called PeIs) at air-liquid interfaces and biofilms that are attached to a surface,[16],[17] and Psl is involved in the adherence to abiotic and biotic surfaces and in the maintenance of biofilm architecture, as well as dispersal. In P. aeruginosa, exopolysaccharides are indispensable for biofilm formation, and mutants that lack the ability to synthesize exopolysaccharides are severely compromised or unable to form mature biofilms.[29],[30],[31] In multi-species biofilms, the presence of a species that produces exopolysaccharides can lead to predomination and integration of other species that do not synthesize matrix polymers.[32]

Extracellular proteins

In P. aeruginosa, the association of extracellular lactonizing lipase (lipA; also, known as lip) with alginate is based on weak-binding forces.[9],[33] Such interactions result in a matrix of exopolysaccharides that are biochemically modified by activated enzymes. This arrangement imparts a physiologic advantage to the microorganisms constituting the biofilm. The enzymatic activity close to the cell(s) and keeps the diffusion distances of enzymatic products short, thereby optimizing their uptake by bacteria.[34]

Exopolysaccharides are degraded mainly by hydrolases and lyases. Furthermore, these enzymes assist in the dispersion of sessile cells from the biofilms, which allows new biofilms to be formed.[31],[35] Other structural proteins such as 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.[28] Galactose-specific and fucose-specific lectin LecA and LecB, respectively[36],[37] of P. aeruginosa, have been implicated in biofilm formation. LecB has a stabilizing effect on in intact biofilms of P. aeruginosa. Other proteinaceous appendages such as pili, fimbriae, and flagella act as additional supportive structural elements by interacting with other EPS components of the biofilm matrix in P. aeruginosa biofilms. Such as, type IV pili of P. aeruginosa bind DNA and so possibly act as cross-linking structures.[38]

Extracellular DNA

eDNA, although initially seen as residual material from lysed cells, has been shown to be in fact an integral part of the biofilm matrix and stationary mode of life.[39],[40] Due to this characteristic, DNase therapy can be used to inhibit biofilm formation.[41],[42],[43] eDNA has multifunctional impacts on the biofilm formation: (i) by aiding in initial adhesion and (ii) by modulating charge and hydrophobic interactions between the bacteria and the surface.[44] The localization of eDNA can vary widely between biofilms. In P. aeruginosa, biofilms eDNA forms a grid-like structure.[45] Notably, high concentrations of eDNA have found in the outer parts of the stalk, thereby forming a border between stalk and cap-forming P. aeruginosa subpopulations.[45] In human sites of infection, such as the CF lung, eDNA detected is almost entirely derived from human PMNs that are recruited heavily to infection sites.[46] Recent studies have shown that PMN-derived genomic DNA can be incorporated into P. aeruginosa biofilms and confer increased bacterial resistance to aminoglycoside treatment.[47] eDNA can limit anti-microbial peptides, membrane damage, and killing.[48],[49] eDNA can also confer antibiotic resistance by acidifying biofilm cultures.[40]

Additional components such as 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.[50] They display surface activity and have been proposed to act in initial microcolony formation, surface-associated bacterial migration, and formation of mushroom-shaped structures, and most importantly playing a part in biofilm dispersion.[51]

 Biofilm Development

Temporal and spatial organization of biofilms

Proteomic studies indicate that there are five stages of P. aeruginosa biofilm formation [Figure 1], the fifth stage being purely dispersal.[52],[53] 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 4 cells aggregate into microcolonies where biofilm cells are dispersed.[53] Studies have also described that LPS can affect surface charge, hydrophobicity as well as dictate alterations in attachments, the transition to sessile growth, and morphology.[54] During biofilm formation, a phenotypic and spatial distribution of cells also takes place. Biofilm 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.[55] The pores or channels in the biofilm structure sustain the supply of nutrients and oxygen for the growth of inhabitant cells. In a mature P. aeruginosa biofilm,[56] cells within the biofilm differentiate into two distinct phenotypes; highly motile cells within the interior of biofilm and nonmotile stationary cells in the outer wall.[57] During the differentiation process, the nonmotile cells form small mushroom “stalks” within the microcolonies, and the motile cells, using the type-IV pili-mediated twitching motility, climb the stalks, aggregating on the top and forming the mushroom cap.[55],[57] The subpopulation of cells constituting the stalk is less metabolically active in comparison to the cells in the cap.[58],[59] The cap-forming and the stalk-forming subpopulations of the mushroom-shaped structures display differential tolerance to antimicrobial compounds such as tobramycin, 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 biofilm [Figure 1].[31],[57] There is a significant difference in the proteomics of planktonic versus biofilm cellular forms of P. aeruginosa, indicating that there are distinct patterns of gene expression occurring in these states.{Figure 1}

 Transcriptional Regulators of Biofilm Formation

Quorum sensing

Quorum sensing (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 “autoinducers.” When the concentration of QS molecules y reaches a threshold, they bind to specific bacterial receptors and direct a coordinated program of gene transcription increasing the bacterial virulence. This mechanism is particularly critical for expression of virulent genes in P. aeruginosa. The two interconnected QS systems of P. aeruginosa utilize acylhomoserine lactone signaling molecules: the Las system and the Rhl system.[60],[61],[62],[63] LasI synthase is responsible for synthesizing N-(3-oxododecanoyl)-L-homoserine lactone, which is recognized by the LasR receptor.[64],[65],[66] In the Rhl system, N-butanoyl homoserine lactone (C4-HSL) is produced by the RhlI synthase which is sensed by the RhlR receptor.[67],[68],[69] The Pseudomonas quinolone signal system mediated by 2-heptyl-3-hydroxy-4-quinolone is considered as a third QS system which closely interacts with the Las and Rhl systems.[70] Recently, a fourth QS system, termed the IQS system, has shown to connect environmental stress cues with the QS networks.[71] The four QS systems are integrated into 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 with nutrient depletion.[60] The most important functional effects of the QS systems is the formation of biofilms which confers advantages to bacterial survival within the host. Las-deficient strains of P. aeruginosa have been shown to form undeveloped biofilms lacking the classical multicellular mushroom-shaped structures. Together, these studies suggest that QS may not be completely necessary for the formation of biofilms; however, they are responsible for the development of mature biofilms. This may reflect the fact that the mushroom-shaped structures are composed of eDNA and rhamnolipids, components which are under the control of QS systems.[41],[45],[72],[73],[74]

Control by sRNA and Two-component systems in Pseudomonas aeruginosa biofilms

P. aeruginosa biofilm is regulated by sRNAs, rsmY, and rsmZ.[75] Three sensor kinases are involved, are involved in this pathway, namely, RetS, LadS, but mainly GacS.[76] GacS phosphorylates GacA,[77] which, in turn, activates the transcription of rsmZ and rsmY. RsmZ and rsmY reduce the activity of effector protein RsmA, being a negative posttranscriptional regulator of the biofilm matrix polysaccharide Psl,[78] and also downregulate another effector, RsmN, controlling the same functions as RsmA.[79] In addition, alternative sigma factor, RpoS expression was also significantly increased in P. aeruginosa biofilms[80] acts as a positive regulator of the expression of the psl gene.[78],[80]

 Biofilm Dispersal

Biologically, biofilm dispersal is a way for bacteria to return to the planktonic mode of life and by which the pathogen sparks new infections between hosts or abiotic surfaces resulting in a new cycle of biofilm development. Biofilm detachment is not defined by a single process and is dependent on the external and internal environmental cues. Detachment can be classified into several different processes: (1) Sloughing, (2) erosion, and (3) seeding dispersal.[53],[56] Sloughing is the process in which biofilm is stripped off as a lump from the surface which causes rapid loss of biofilm mass. In contrast, erosion is a process in which a single cell or small portion of biofilm is gradually and continually detached from outside. While these types of detachment are generally considered shear-dependent and passive processes, seeding dispersal is an active process and refers to the rapid release of single cells or a small portion of cells from the central region of biofilm, leaving hollow cavities.[81] Active dispersal was usually occurs in the final stage of biofilm development in Pseudomonas spp., in which bacterial cells within biofilm actively swim away from the interior of the biofilm leaving central hollows.[58],[82] This “hollowing” phenomena are active processes that are not observed in mutants.

Internal and external environmental cues regulating biofilm dispersal

Internal environmental stimuli or host factors such as the presence of antibiotics, EPS degradation enzymes, and the addition of rhamnolipids that directly impact biofilm stability such as motility, EPS formation, rhamnolipids, cell death, and lysis directly modulate biofilm dispersal. This phenomenon is especially noted in CF. Even minor changes in environmental cues such as nutrients, oxygen, nitric oxide (NO), and chelating chemicals[82] can impact biofilm dispersal. It is well studied that a shift in carbon sources such as glutamate and glucose can cause drastic reductions in biofilm mass by the dispersal process, by altering the levels of cyclic dimeric guanosine monophosphate (c-di-GMP).[35] Oxygen levels can alter the shape of the biofilm, by creating an oxygen gradient from the outer to the inner core of the biofilm. The inner core tends to be anaerobic with low metabolic activity and high antibiotic tolerance.[35],[82] In P. aeruginosa, biofilms, NO is produced by anaerobic respiration, which in turn induced dispersal.[83] More recently, prodrugs releasing NO after an activation step in the bacterium, such as diethylamine (DEA) NONOate-cephalosporin prodrug (DEACP), were also shown to promote dispersal,[84],[85] other studies have shown that dispersal was induced with low, sublethal concentrations (25–500 nM) of the NO donor sodium nitroprusside,[83] by decreasing c-di-GMP levels.

Cyclic dimeric guanosine monophosphate and transcriptional regulators of dispersal

c-di-GMP is recognized as an intracellular signaling molecule, essentially a second messenger coordinating the “lifestyle transition” from motility to sessility and vice versa (i.e., dispersion).[86],[87] The inverse correlation between c-di-GMP concentration in the cell and biofilm formation or between low c-di-GMP levels and motility has been widely studied across different bacterial species.[88] P. aeruginosa biofilms are estimated to contain on average 75–110 pmol of c-di-GMP per mg of total cell extract, whereas planktonic cells contain < 30 pmol/mg.[89] It is proposed that the bacterial cells use c-di-GMP as a checkpoint to proceed through the distinct stages of biofilm development until they fully commit to the biofilm lifestyle, although they may still retain the option to revert the decision at any time.[90],[91] The levels of c-di-GMP in the cell are modified by the rate of its synthesis and degradation. The molecule is synthesized from two molecules of GTP by enzymes called diguanylate cyclases (DGCs) and is degraded into 5′-phosphoguanylyl-(3′-5′)-guanosine (pGpG) and/or GMP by phosphodiesterases (PDEs). The genome of P. aeruginosa PAO1 encodes 41 c-di-GMP proteins predicted to participate in c-di-GMP metabolism, while P. aeruginosa PA14 has 40 such proteins.[92] Most of these proteins are also linked to various sensory input domains on their N-terminus, CheY-like receiver domain, conserved receiver domain of response regulator proteins (REC), sensory input domain Per-Arnt-Sim (PAS), sensory input domain in cGMP regulated phosphodiesterases, adenlyl cyclases, and transcription factor FhlA,[93] presumably transducing environmental stimuli to cellular response (s). Therefore, c-di-GMP has been implicated in numerous cellular functions including regulation of cell cycle, differentiation, biofilm formation and dispersion, motility, and virulence.[87],[94],[95],[96]

Using bioinformatics, biochemical, and structural approaches, the catalytic domains of DGCs and PDEs have been identified and characterized: the former carrying a GGDEF active site motif, and the latter carrying either EAL or HD-GYP domains.[97],[98] Moreover, GGDEF and EAL domains can both be present in the same protein. One such example in P. aeruginosa is the GGDEF and the EAL domains of MucR. These domains are activated differently, such that in planktonic cells, MucR functions as a DGC and as a positive regulator of alginate biosynthesis, whereas in biofilms, it functions as a PDE and is a positive regulator of biofilm dispersal induced by NO or glutamate.[99] The P. aeruginosa genome encodes one of the highest numbers of DGCs and PDEs: 18 GGDEF, 5 EAL, 16 GGDEF/EAL, and 3 HD-GYP predicted proteins.

In P. aeruginosa, the first biochemical characterization of a DGC stems from the work on WspR, which contains a REC-GGDEF domain organization. The DGC was named after its regulatory role on the P. aeruginosa wrinkly spreader phenotype that is correlated with a thick biofilm due to increased production of exopolysaccharides.[100] The control of WspR activity occurs by three different routes that are proposed to occur subsequentially. First, on sensing growth on the surface, the Wsp signal transduction complex phosphorylates WspR and triggers c-di-GMP synthesis.[100],[101] In turn, the WspR phosphorylation triggers subcellular WspR oligomerization and cluster formation, which further increases the DGC activity.[101] PDEs consist of EAL or HD-GYP domain proteins. In P. aeruginosa, the CheY-EAL domain protein RocR was identified as a response regulator in the RocSAR signaling system.[102] This system is composed of a membrane sensor RocS1 and two response regulators, RocA1 and RocR. RocR activity is triggered by phosphorylation at the CheY domain, and the protein competes with RocA1 for the phosphoryl transfer from the RocS1 sensor.

In P. aeruginosa, two of the three HD-GYP proteins (PA4108, PA4781, and PA2572) were shown to have a PDE activity in vivo and in vitro. In addition, the 3′-5′exoribonuclease Orn has been identified in P. aeruginosa as primarily responsible for the pGpG cleavage into two GMP molecules.[103],[104]

Besides WspR and RocR, described previously, other DGCs and PDEs have been reported as key players in P. aeruginosa biofilm formation. At least five DGCs have been described to specifically control the transition from planktonic to surface-associated growth: WspR, SadC, RoeA, and SiaD.[105],[106] GcbA and NicD DGCs or the DipA (Pch), RbdA, BifA, and NbdA PDEs have been linked to biofilm dispersal.[89],[107],[108],[109],[110] BdlA acts as a sensor of intracellular c-di-GMP levels and modulates its downregulation to enable dispersion. Native BdlA is inactive and is activated by proteolytic cleavage and phosphorylation.[111],[112]

The cleaved fragments interact with each other and associate with DipA and other PDEs to make a multi-protein complex and degrade c-di-GMP.

In the CF, lungs increased levels of c-di-GMP and exopolysaccharides are observed in small colony variants (SCV). SCVs develop as a consequence of persistence and accumulation of phenotypic heterogeneity in P. aeruginosa.[113],[114] These are slow growing and antibiotic resistant.[115]

In vitro and in vivo systems for determination/quantitation of biofilm dispersal

Cells dispersed from biofilms 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.[116] The rationale for evaluating biofilm 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 biofilm, 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 biofilms 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.[116]

 Multi Species Biofilm Formation

Multispecies interactions have also been shown to promote biofilm formation. Clinical associations have been described in patients who are co-infected with respiratory syncytial virus (RSV) infections and P. aeruginosa. RSV lung infections may facilitate the conversion of opportunistic P. aeruginosa infection into a chronic colonization state.[117] A recent study by Hendricks et al. has shown that RSV infection enhances biofilm formation by P. aeruginosa. Mechanistically, this was shown to be related to modulation of nutritional immunity, including iron homeostasis involving transferrin.,[118] Mixed-species biofilms also demonstrate the occurrence of cooperative behavior in biofilms formed by P. aeruginosa, P. protegens and Klebsiella pneuomoniae.[119] This scenario can partly explain the high tolerance of these communities to toxin accumulation, particularly of their planktonic counterparts.[119] P. aeruginosa can also associate with bacteria such as Staphylococcus epidermis[4] and Staphylococcus aureus[120] to cause severe chronic infections in burn wounds. In CF patients and CF mouse models, the coinfection 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 can inhibit P. aeruginosa virulence, signaling, and biofilm formation.[121],[122] The fatty acid cis-2-decenoic acid, produced by P. aeruginosa, can induce dispersal in other bacterial species, including B. subtilis and S. aureus.[123] P. aeruginosa rhamnolipids when added exogenously can disperse biofilms produced by Bordetella bronchiseptica and Salmonella Typhimurium.[124],[125]

 Overview of Models Used for Characterization of Pseudomonas Aeruginosa biofilm In Vitro and In Vivo

In vitro systems

There are several approaches used to study biofilms. Microtiter plate assay is one of the simplest methods of studying surface-attached biofilms. In this assay, the adherence of P. aeruginosa can be easily monitored in a 96 well plate, with crystal violet staining.[126] Although crude, this is an effective way to study the effects of prospective anti-biofilm agents. However, one of the limitations is that it is not very reproducible, and does not differentiate between live and dead cells as well as the nonadherent or planktonic counterparts.[126],[127] Biofilm formation in co-culture models of P. aeruginosa with host cells such as epithelial cells and neutrophils can also be studied.[128] Other quantitative methods such as confocal microscopy by employing P. aeruginosa GFP strains, by itself or in co-culture models can be performed using biofilms grown on glass coated with collagen. The images can be obtained by using software such as image J or comstat.[118],[129],[130] Centers for Disease Control (CDC) reactor, tests biofilm formation on discs spinning in the media. Some of the other methodologies that are used include continuous flow cell system which was first described by Sternberg et al.[131] This technique is used to study biofilm formation particularly in a setting where carbon is available continuously and produces highly structured biofilms. To study biofilms in medical devices, a drip slow reactor is used. All these techniques allow for assessing colony forming units, microscopy and structural characterization.[132]

For detecting biofilms of multispecies such as P. aeruginosa and S. aureus laser ablation electrospray ionization mass spectrometry can be employed.[120] EPS constituting biofilms can be visualized in situ by confocal laser scanning microscopy (CLSM) in combination with fluorescent dyes, such as fluorescently labeled lectins and antibodies. Specific multi labeling with both lectins and antibodies has been reported.[9] Raman microscopy and microspectroscopy are upcoming approaches that are used in combination with CLSM-based lectin-binding analysis.[133] This combination gives a more in-depth insight into EPS composition. Local enzymatic activity in biofilms can be assessed by microscopic visualization of fluorogenic substrates. Examples include phosphatase activity using the water-soluble substrate ELF-97 phosphate. eDNA can be detected with dye-specific for nucleic acid.[9]

In vivo systems

Nonmammalian hosts such as Zebrafish, Drosophila, melanogaster, Caenorhabditis elegans,[134] and mammalian hosts such as guinea pigs[135] and minks[136] have also been used to assess virulence of P. aeruginosa. Rodent models using rats and mice are most commonly employed to study acute and chronic infections by P. aeruginosa. Introduction of planktonic free-living P. aeruginosa cells into organs such as lungs, skin, or eyes are commonly used. For an acute model of lung infection, P. aeruginosa is either administered intratracheally or via aerosol challenge, or intranasally[137],[138] or intranasal application. Acute P. aeruginosa infection in a burn wound model can be achieved by challenging the mice topically with P. aeruginosa, underneath the burn wound.[134]

Chronic lung infections, mimicking CF type lung infections by P. aeruginosa are attained by embedding its cells in agar, agarose, or seaweed alginate. Alternatively introducing small plastic tube with bacterial inoculum can result in a chronic focal infection. The first model of chronic bronchopulmonary infection was performed e in rats using agar beads by Cash et al. in 1979.[139] The embedding of P. aeruginosa in beads causes the beads to be physically retained in the airways. This creates an environment that mimics biofilm formation and micro aerobiosis and can recapitulate the conditions of a CF lung. Given its simplicity, efficacy and low cost, this model is still frequently employed to study the impact of chronic P. aeruginosa infections. Minor variations of this established model are also widely used.[134],[137],[138] In this model, the P. aeruginosa infection load is evaluated by colony counts or other in situ visualization methods described above.

In vitro simulation of biofilm pathology

The on-going challenge is 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 like that of expectorated sputum in patients with CF, known as synthetic CF sputum medium (SCFM).[132] This medium approximates P. aeruginosa gene expression, and virulence factors like that observed in expectorated sputum in CF patients. 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.[132] To delineate host inflammatory and immune response semisolid collagen matrix in which P. aeruginosa and S. aureus can both form aggregates can be employed. This approach mimics wound bed or the mucopurulent pus of the CF lung. These model systems could be very useful in determining influences of individual bacterial factors.[140] Artificial biofilms that impersonate important aspects of the real biofilms can be used to study these complex systems. Agarose is used as a hydrogel matrix to simulate extracellular polymeric matrix in which microorganisms such as P. aeruginosa are colonized.[140] Furthermore, this model can also be used to mimic and compare biofilms formed by nonmucoid versus mucoid strains of P. aeruginosa and can help test the efficacy of antibiotics.

 Host Response

The host response against P. aeruginosa infections is complex and involves the coordinated activity of a variety of cell types including structural and immune cells. A clear understanding of the in vivo host response to P. aeruginosa biofilms is made more challenging by the difficulty in modeling biofilms 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 response to P. aeruginosa biofilm infections.[141],[142],[143] We have recently in depth reviewed the host response to P. aeruginosa biofilms and therapeutic approaches that can modulate biofilm formation.[7]

 Summary and Conclusion

The International health organizations such as the CDC and the World Health Organization have highlighted P. aeruginosa as a major health threat and strongly urged the research and development of alternate and new antimicrobial therapies due to the high rates of antibiotic resistance. We have barely scratched the surface to begin to understand both the immensely complex bacterial factors that influence biofilm development, maturation, and dispersal, as well as the host factors that influence the immune response to biofilm infections. However, a more thorough understanding of biofilm biology and host immune response to biofilms is urgently needed given the recalcitrance of P. aeruginosa infections.

Financial support and sponsorship

This study was financially supported by the Department of Veterans Affairs, Emory University Department of Medicine, Emory University, Atlanta, GA, USA.

Conflicts of interest

There are no conflicts of interest.


1Flemming HC, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. Biofilms: An emergent form of bacterial life. Nat Rev Microbiol 2016;14:563-75.
2Hancock RE, Speert DP. Antibiotic resistance in Pseudomonas aeruginosa: Mechanisms and impact on treatment. Drug Resist Updat 2000;3:247-55.
3Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, et al. Bacterial biofilms in nature and disease. Annu Rev Microbiol 1987;41:435-64.
4Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. Science 1999;284:1318-22.
5Ciofu O, Tolker-Nielsen T, Jensen PØ, Wang H, Høiby N. Antimicrobial resistance, respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients. Adv Drug Deliv Rev 2015;85:7-23.
6Sadikot RT, Blackwell TS, Christman JW, Prince AS. Pathogen-host interactions in Pseudomonas aeruginosa pneumonia. Am J Respir Crit Care Med 2005;171:1209-23.
7Maurice NM, Bedi B, Sadikot RT. Pseudomonas aeruginosa biofilms: Host response and clinical implications in lung infections. Am J Respir Cell Mol Biol 2018;58:428-39.
8Lieleg O, Caldara M, Baumgärtel R, Ribbeck K. Mechanical robustness of Pseudomonas aeruginosa biofilms. Soft Matter 2011;7:3307-14.
9Flemming HC, Wingender J. The biofilm matrix. Nat Rev Microbiol 2010;8:623-33.
10Hall-Stoodley L, Stoodley P. Developmental regulation of microbial biofilms. Curr Opin Biotechnol 2002;13:228-33.
11Kjelleberg S, Molin S. Is there a role for quorum sensing signals in bacterial biofilms? Curr Opin Microbiol 2002;5:254-8.
12Wei Q, Ma LZ. Biofilm matrix and its regulation in Pseudomonas aeruginosa. Int J Mol Sci 2013;14:20983-1005.
13Wiens JR, Vasil AI, Schurr MJ, Vasil ML. Iron-regulated expression of alginate production, mucoid phenotype, and biofilm formation by Pseudomonas aeruginosa. MBio 2014;5:e01010-13.
14Koch C, Høiby N. Pathogenesis of cystic fibrosis. Lancet 1993;341:1065-9.
15Govan JR, Deretic V. Microbial pathogenesis in cystic fibrosis: Mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 1996;60:539-74.
16Yang L, Hu Y, Liu Y, Zhang J, Ulstrup J, Molin S, et al. Distinct roles of extracellular polymeric substances in Pseudomonas aeruginosa biofilm development. Environ Microbiol 2011;13:1705-17.
17Li Z, Chen JH, Hao Y, Nair SK. Structures of the PelD cyclic diguanylate effector involved in pellicle formation in Pseudomonas aeruginosa PAO1. J Biol Chem 2012;287:30191-204.
18Jackson KD, Starkey M, Kremer S, Parsek MR, Wozniak DJ. Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation. J Bacteriol 2004;186:4466-75.
19Wozniak DJ, Wyckoff TJ, Starkey M, Keyser R, Azadi P, O'Toole GA, et al. Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms. Proc Natl Acad Sci U S A 2003;100:7907-12.
20Lyczak JB, Cannon CL, Pier GB. Lung infections associated with cystic fibrosis. Clin Microbiol Rev 2002;15:194-222.
21Murray TS, Egan M, Kazmierczak BI. Pseudomonas aeruginosa chronic colonization in cystic fibrosis patients. Curr Opin Pediatr 2007;19:83-8.
22Orgad O, Oren Y, Walker SL, Herzberg M. The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment. Biofouling 2011;27:787-98.
23Mathee K, Ciofu O, Sternberg C, Lindum PW, Campbell JI, Jensen P, et al. Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: A mechanism for virulence activation in the cystic fibrosis lung. Microbiology 1999;145 (Pt 6):1349-57.
24DeVries CA, Ohman DE. Mucoid-to-nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT, encoding a putative alternate sigma factor, and shows evidence for autoregulation. J Bacteriol 1994;176:6677-87.
25Mathee K, McPherson CJ, Ohman DE. Posttranslational control of the algT (algU)-encoded sigma22 for expression of the alginate regulon in Pseudomonas aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN). J Bacteriol 1997;179:3711-20.
26Drenkard E, Ausubel FM. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 2002;416:740-3.
27Ryder C, Byrd M, Wozniak DJ. Role of polysaccharides in Pseudomonas aeruginosa biofilm development. Curr Opin Microbiol 2007;10:644-8.
28Mann EE, Wozniak DJ. Pseudomonas biofilm matrix composition and niche biology. FEMS Microbiol Rev 2012;36:893-916.
29Watnick PI, Kolter R. Steps in the development of a Vibrio cholerae El tor biofilm. Mol Microbiol 1999;34:586-95.
30Danese PN, Pratt LA, Kolter R. Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. J Bacteriol 2000;182:3593-6.
31Ma L, Conover M, Lu H, Parsek MR, Bayles K, Wozniak DJ, et al. Assembly and development of the Pseudomonas aeruginosa biofilm matrix. PLoS Pathog 2009;5:e1000354.
32Sutherland IW. The biofilm matrix – an immobilized but dynamic microbial environment. Trends Microbiol 2001;9:222-7.
33Mayer C, Moritz R, Kirschner C, Borchard W, Maibaum R, Wingender J, et al. The role of intermolecular interactions: Studies on model systems for bacterial biofilms. Int J Biol Macromol 1999;26:3-16.
34Skillman LC, Sutherland IW, Jones MV. The role of exopolysaccharides in dual species biofilm development. J Appl Microbiol 1998;85 Suppl 1:13S-8S.
35Sauer K, Cullen MC, Rickard AH, Zeef LA, Davies DG, Gilbert P. Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J Bacteriol 2004;186:7312-26.
36Tielker D, Hacker S, Loris R, Strathmann M, Wingender J, Wilhelm S, et al. Pseudomonas aeruginosa lectin lecB is located in the outer membrane and is involved in biofilm formation. Microbiology 2005;151:1313-23.
37Diggle SP, Stacey RE, Dodd C, Cámara M, Williams P, Winzer K, et al. The galactophilic lectin, lecA, contributes to biofilm development in Pseudomonas aeruginosa. Environ Microbiol 2006;8:1095-104.
38van Schaik EJ, Giltner CL, Audette GF, Keizer DW, Bautista DL, Slupsky CM, et al. DNA binding: A novel function of Pseudomonas aeruginosa type IV pili. J Bacteriol 2005;187:1455-64.
39Montanaro L, Poggi A, Visai L, Ravaioli S, Campoccia D, Speziale P, et al. Extracellular DNA in biofilms. Int J Artif Organs 2011;34:824-31.
40Wilton M, Charron-Mazenod L, Moore R, Lewenza S. Extracellular DNA acidifies biofilms and induces aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2016;60:544-53.
41Yang L, Barken KB, Skindersoe ME, Christensen AB, Givskov M, Tolker-Nielsen T. Effects of iron on DNA release and biofilm development by Pseudomonas aeruginosa. Microbiology 2007;153:1318-28.
42Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS. Extracellular DNA required for bacterial biofilm formation. Science 2002;295:1487.
43Vilain S, Pretorius JM, Theron J, Brözel VS. DNA as an adhesin: Bacillus cereus requires extracellular DNA to form biofilms. Appl Environ Microbiol 2009;75:2861-8.
44Das T, Sharma PK, Busscher HJ, van der Mei HC, Krom BP. Role of extracellular DNA in initial bacterial adhesion and surface aggregation. Appl Environ Microbiol 2010;76:3405-8.
45Allesen-Holm M, Barken KB, Yang L, Klausen M, Webb JS, Kjelleberg S, et al. A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol Microbiol 2006;59:1114-28.
46Lethem MI, James SL, Marriott C, Burke JF. The origin of DNA associated with mucus glycoproteins in cystic fibrosis sputum. Eur Respir J 1990;3:19-23.
47Chiang WC, Nilsson M, Jensen PØ, Høiby N, Nielsen TE, Givskov M, et al. Extracellular DNA shields against aminoglycosides in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 2013;57:2352-61.
48Mulcahy H, Charron-Mazenod L, Lewenza S. Extracellular DNA chelates cations and induces antibiotic resistance in Pseudomonas aeruginosa biofilms. PLoS Pathog 2008;4:e1000213.
49Johnson L, Mulcahy H, Kanevets U, Shi Y, Lewenza S. Surface-localized spermidine protects the Pseudomonas aeruginosa outer membrane from antibiotic treatment and oxidative stress. J Bacteriol 2012;194:813-26.
50Davey ME, Caiazza NC, O'Toole GA. Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. J Bacteriol 2003;185:1027-36.
51Boles BR, Thoendel M, Singh PK. Self-generated diversity produces “insurance effects” in biofilm communities. Proc Natl Acad Sci U S A 2004;101:16630-5.
52Stoodley P, Sauer K, Davies DG, Costerton JW. Biofilms as complex differentiated communities. Annu Rev Microbiol 2002;56:187-209.
53Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG. Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol 2002;184:1140-54.
54Rocchetta HL, Burrows LL, Lam JS. Genetics of O-antigen biosynthesis in Pseudomonas aeruginosa. Microbiol Mol Biol Rev 1999;63:523-53.
55Klausen M, Heydorn A, Ragas P, Lambertsen L, Aaes-Jørgensen A, Molin S, et al. Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol 2003;48:1511-24.
56Purevdorj-Gage B, Costerton WJ, Stoodley P. Phenotypic differentiation and seeding dispersal in non-mucoid and mucoid Pseudomonas aeruginosa biofilms. Microbiology 2005;151:1569-76.
57Klausen M, Aaes-Jørgensen A, Molin S, Tolker-Nielsen T. Involvement of bacterial migration in the development of complex multicellular structures in Pseudomonas aeruginosa biofilms. Mol Microbiol 2003;50:61-8.
58Harmsen M, Yang L, Pamp SJ, Tolker-Nielsen T. An update on Pseudomonas aeruginosa biofilm formation, tolerance, and dispersal. FEMS Immunol Med Microbiol 2010;59:253-68.
59van Gestel J, Vlamakis H, Kolter R. Division of labor in biofilms: The ecology of cell differentiation. Microbiol Spectr 2015;3:MB-0002-2014.
60Lee J, Zhang L. The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell 2015;6:26-41.
61Rasamiravaka T, El Jaziri M. Quorum-sensing mechanisms and bacterial response to antibiotics in P. Aeruginosa. Curr Microbiol 2016;73:747-53.
62Bjarnsholt T, Tolker-Nielsen T, Høiby N, Givskov M. Interference of Pseudomonas aeruginosa signalling and biofilm formation for infection control. Expert Rev Mol Med 2010;12:e11.
63Pesci EC, Pearson JP, Seed PC, Iglewski BH. Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J Bacteriol 1997;179:3127-32.
64Gambello MJ, Iglewski BH. Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression. J Bacteriol 1991;173:3000-9.
65Jones S, Yu B, Bainton NJ, Birdsall M, Bycroft BW, Chhabra SR, et al. The lux autoinducer regulates the production of exoenzyme virulence determinants in erwinia carotovora and Pseudomonas aeruginosa. EMBO J 1993;12:2477-82.
66Pearson JP, Gray KM, Passador L, Tucker KD, Eberhard A, Iglewski BH, et al. Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci U S A 1994;91:197-201.
67Ochsner UA, Reiser J. Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 1995;92:6424-8.
68Brint JM, Ohman DE. Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of rhlR-rhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive luxR-luxI family. J Bacteriol 1995;177:7155-63.
69Pearson JP, Passador L, Iglewski BH, Greenberg EP. A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 1995;92:1490-4.
70Pesci EC, Milbank JB, Pearson JP, McKnight S, Kende AS, Greenberg EP, et al. Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 1999;96:11229-34.
71Lee J, Wu J, Deng Y, Wang J, Wang C, Wang J, et al. A cell-cell communication signal integrates quorum sensing and stress response. Nat Chem Biol 2013;9:339-43.
72Yang L, Nilsson M, Gjermansen M, Givskov M, Tolker-Nielsen T. Pyoverdine and PQS mediated subpopulation interactions involved in Pseudomonas aeruginosa biofilm formation. Mol Microbiol 2009;74:1380-92.
73Pamp SJ, Tolker-Nielsen T. Multiple roles of biosurfactants in structural biofilm development by Pseudomonas aeruginosa. J Bacteriol 2007;189:2531-9.
74Barken KB, Pamp SJ, Yang L, Gjermansen M, Bertrand JJ, Klausen M, et al. Roles of type IV pili, flagellum-mediated motility and extracellular DNA in the formation of mature multicellular structures in Pseudomonas aeruginosa biofilms. Environ Microbiol 2008;10:2331-43.
75Wolska KI, Grudniak AM, Rudnicka Z, Markowska K. Genetic control of bacterial biofilms. J Appl Genet 2016;57:225-38.
76Ventre I, Goodman AL, Vallet-Gely I, Vasseur P, Soscia C, Molin S, et al. Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes. Proc Natl Acad Sci U S A 2006;103:171-6.
77Goodman AL, Merighi M, Hyodo M, Ventre I, Filloux A, Lory S, et al. Direct interaction between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial pathogen. Genes Dev 2009;23:249-59.
78Irie Y, Starkey M, Edwards AN, Wozniak DJ, Romeo T, Parsek MR. Pseudomonas aeruginosa biofilm matrix polysaccharide psl is regulated transcriptionally by rpoS and post-transcriptionally by rsmA. Mol Microbiol 2010;78:158-72.
79Marden JN, Diaz MR, Walton WG, Gode CJ, Betts L, Urbanowski ML, et al. An unusual csrA family member operates in series with rsmA to amplify posttranscriptional responses in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 2013;110:15055-60.
80Waite RD, Paccanaro A, Papakonstantinopoulou A, Hurst JM, Saqi M, Littler E, et al. Clustering of Pseudomonas aeruginosa transcriptomes from planktonic cultures, developing and mature biofilms reveals distinct expression profiles. BMC Genomics 2006;7:162.
81Boles BR, Thoendel M, Singh PK. Rhamnolipids mediate detachment of Pseudomonas aeruginosa from biofilms. Mol Microbiol 2005;57:1210-23.
82Kim SK, Lee JH. Biofilm dispersion in Pseudomonas aeruginosa. J Microbiol 2016;54:71-85.
83Barraud N, Hassett DJ, Hwang SH, Rice SA, Kjelleberg S, Webb JS, et al. Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa. J Bacteriol 2006;188:7344-53.
84Cutruzzolà F, Frankenberg-Dinkel N. Origin and impact of nitric oxide in Pseudomonas aeruginosa biofilms. J Bacteriol 2016;198:55-65.
85Barraud N, Kardak BG, Yepuri NR, Howlin RP, Webb JS, Faust SN, et al. Cephalosporin-3'-diazeniumdiolates: Targeted NO-donor prodrugs for dispersing bacterial biofilms. Angew Chem Int Ed Engl 2012;51:9057-60.
86Ha DG, O'Toole GA. c-di-GMP and its Effects on Biofilm Formation and Dispersion: a Pseudomonas Aeruginosa Review. Microbiol Spectr 2015;3:MB-0003-2014.
87Valentini M, Filloux A. Biofilms and cyclic di-GMP (c-di-GMP) signaling: Lessons from Pseudomonas aeruginosa and other bacteria. J Biol Chem 2016;291:12547-55.
88Simm R, Morr M, Kader A, Nimtz M, Römling U. GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol Microbiol 2004;53:1123-34.
89Basu Roy A, Sauer K. Diguanylate cyclase nicD-based signalling mechanism of nutrient-induced dispersion by Pseudomonas aeruginosa. Mol Microbiol 2014;94:771-93.
90Römling U, Galperin MY, Gomelsky M. Cyclic di-GMP: The first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 2013;77:1-52.
91Hengge R. Principles of c-di-GMP signalling in bacteria. Nat Rev Microbiol 2009;7:263-73.
92Boles BR, McCarter LL. Vibrio parahaemolyticus scrABC, a novel operon affecting swarming and capsular polysaccharide regulation. J Bacteriol 2002;184:5946-54.
93Seshasayee AS, Fraser GM, Luscombe NM. Comparative genomics of cyclic-di-GMP signalling in bacteria: Post-translational regulation and catalytic activity. Nucleic Acids Res 2010;38:5970-81.
94Chen MW, Kotaka M, Vonrhein C, Bricogne G, Rao F, Chuah ML, et al. Structural insights into the regulatory mechanism of the response regulator rocR from Pseudomonas aeruginosa in cyclic di-GMP signaling. J Bacteriol 2012;194:4837-46.
95Paul R, Weiser S, Amiot NC, Chan C, Schirmer T, Giese B, et al. Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain. Genes Dev 2004;18:715-27.
96Hickman JW, Tifrea DF, Harwood CS. A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels. Proc Natl Acad Sci U S A 2005;102:14422-7.
97Ryjenkov DA, Tarutina M, Moskvin OV, Gomelsky M. Cyclic diguanylate is a ubiquitous signaling molecule in bacteria: Insights into biochemistry of the GGDEF protein domain. J Bacteriol 2005;187:1792-8.
98Schmidt AJ, Ryjenkov DA, Gomelsky M. The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase: Enzymatically active and inactive EAL domains. J Bacteriol 2005;187:4774-81.
99Hay ID, Remminghorst U, Rehm BH. MucR, a novel membrane-associated regulator of alginate biosynthesis in Pseudomonas aeruginosa. Appl Environ Microbiol 2009;75:1110-20.
100Güvener ZT, Harwood CS. Subcellular location characteristics of the Pseudomonas aeruginosa GGDEF protein, wspR, indicate that it produces cyclic-di-GMP in response to growth on surfaces. Mol Microbiol 2007;66:1459-73.
101Huangyutitham V, Güvener ZT, Harwood CS. Subcellular clustering of the phosphorylated wspR response regulator protein stimulates its diguanylate cyclase activity. MBio 2013;4:e00242-13.
102Kulasekara HD, Ventre I, Kulasekara BR, Lazdunski A, Filloux A, Lory S, et al. A novel two-component system controls the expression of Pseudomonas aeruginosa fimbrial cup genes. Mol Microbiol 2005;55:368-80.
103Orr MW, Donaldson GP, Severin GB, Wang J, Sintim HO, Waters CM, et al. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover. Proc Natl Acad Sci U S A 2015;112:E5048-57.
104Cohen D, Mechold U, Nevenzal H, Yarmiyhu Y, Randall TE, Bay DC, et al. Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 2015;112:11359-64.
105Klebensberger J, Birkenmaier A, Geffers R, Kjelleberg S, Philipp B. SiaA and siaD are essential for inducing autoaggregation as a specific response to detergent stress in Pseudomonas aeruginosa. Environ Microbiol 2009;11:3073-86.
106Malone JG, Jaeger T, Spangler C, Ritz D, Spang A, Arrieumerlou C, et al. YfiBNR mediates cyclic di-GMP dependent small colony variant formation and persistence in Pseudomonas aeruginosa. PLoS Pathog 2010;6:e1000804.
107Petrova OE, Cherny KE, Sauer K. The Pseudomonas aeruginosa diguanylate cyclase gcbA, a homolog of P. Fluorescens gcbA, promotes initial attachment to surfaces, but not biofilm formation, via regulation of motility. J Bacteriol 2014;196:2827-41.
108Roy AB, Petrova OE, Sauer K. The phosphodiesterase dipA (PA5017) is essential for Pseudomonas aeruginosa biofilm dispersion. J Bacteriol 2012;194:2904-15.
109Li Y, Heine S, Entian M, Sauer K, Frankenberg-Dinkel N. NO-induced biofilm dispersion in Pseudomonas aeruginosa is mediated by an MHYT domain-coupled phosphodiesterase. J Bacteriol 2013;195:3531-42.
110Kuchma SL, Brothers KM, Merritt JH, Liberati NT, Ausubel FM, O'Toole GA, et al. BifA, a cyclic-di-GMP phosphodiesterase, inversely regulates biofilm formation and swarming motility by Pseudomonas aeruginosa PA14. J Bacteriol 2007;189:8165-78.
111Petrova OE, Sauer K. Dispersion by Pseudomonas aeruginosa requires an unusual posttranslational modification of bdlA. Proc Natl Acad Sci U S A 2012;109:16690-5.
112Li Y, Petrova OE, Su S, Lau GW, Panmanee W, Na R, et al. BdlA, dipA and induced dispersion contribute to acute virulence and chronic persistence of Pseudomonas aeruginosa. PLoS Pathog 2014;10:e1004168.
113Häussler S, Tümmler B, Weissbrodt H, Rohde M, Steinmetz I. Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis. Clin Infect Dis 1999;29:621-5.
114Malone JG. Role of small colony variants in persistence of Pseudomonas aeruginosa infections in cystic fibrosis lungs. Infect Drug Resist 2015;8:237-47.
115Smith EE, Buckley DG, Wu Z, Saenphimmachak C, Hoffman LR, D'sArgenio DA, et al. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci U S A 2006;103:8487-92.
116Chua SL, Hultqvist LD, Yuan M, Rybtke M, Nielsen TE, Givskov M, et al. In vitro and in vivo generation and characterization of Pseudomonas aeruginosa biofilm-dispersed cells via c-di-GMP manipulation. Nat Protoc 2015;10:1165-80.
117Petersen NT, Høiby N, Mordhorst CH, Lind K, Flensborg EW, Bruun B, et al. Respiratory infections in cystic fibrosis patients caused by virus, chlamydia and mycoplasma – Possible synergism with Pseudomonas aeruginosa. Acta Paediatr Scand 1981;70:623-8.
118Hendricks MR, Lashua LP, Fischer DK, Flitter BA, Eichinger KM, Durbin JE, et al. Respiratory syncytial virus infection enhances Pseudomonas aeruginosa biofilm growth through dysregulation of nutritional immunity. Proc Natl Acad Sci U S A 2016;113:1642-7.
119Lee KW, Periasamy S, Mukherjee M, Xie C, Kjelleberg S, Rice SA, et al. Biofilm development and enhanced stress resistance of a model, mixed-species community biofilm. ISME J 2014;8:894-907.
120Dean SN, Walsh C, Goodman H, van Hoek ML. Analysis of mixed biofilm (Staphylococcus aureus and Pseudomonas aeruginosa) by laser ablation electrospray ionization mass spectrometry. Biofouling 2015;31:151-61.
121Deng Y, Boon C, Chen S, Lim A, Zhang LH. Cis-2-dodecenoic acid signal modulates virulence of Pseudomonas aeruginosa through interference with quorum sensing systems and T3SS. BMC Microbiol 2013;13:231.
122Bragonzi A, Farulla I, Paroni M, Twomey KB, Pirone L, Lorè NI, et al. Modelling co-infection of the cystic fibrosis lung by Pseudomonas aeruginosa and Burkholderia cenocepacia reveals influences on biofilm formation and host response. PLoS One 2012;7:e52330.
123Davies DG, Marques CN. A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. J Bacteriol 2009;191:1393-403.
124Mireles JR 2nd, Toguchi A, Harshey RM. Salmonella enterica serovar typhimurium swarming mutants with altered biofilm-forming abilities: Surfactin inhibits biofilm formation. J Bacteriol 2001;183:5848-54.
125Irie T, Watarai S, Iwasaki T, Kodama H. Protection against experimental Aeromonas salmonicida infection in carp by oral immunisation with bacterial antigen entrapped liposomes. Fish Shellfish Immunol 2005;18:235-42.
126O'Toole GA. Microtiter dish biofilm formation assay. J Vis Exp 2011. pii: 2437.
127Walker TS, Tomlin KL, Worthen GS, Poch KR, Lieber JG, Saavedra MT, et al. Enhanced Pseudomonas aeruginosa biofilm development mediated by human neutrophils. Infect Immun 2005;73:3693-701.
128Moreau-Marquis S, Redelman CV, Stanton BA, Anderson GG. Co-culture models of Pseudomonas aeruginosa biofilms grown on live human airway cells. J Vis Exp 2010. pii: 2186.
129Bedi B, Maurice NM, Ciavatta VT, Lynn KS, Yuan Z, Molina SA, et al. Peroxisome proliferator-activated receptor-γ agonists attenuate biofilm formation by Pseudomonas aeruginosa. FASEB J 2017;31:3608-21.
130Jurcisek JA, Dickson AC, Bruggeman ME, Bakaletz LO.In vitro biofilm formation in an 8-well chamber slide. J Vis Exp 2011. pii: 2481.
131Sternberg C, Christensen BB, Johansen T, Toftgaard Nielsen A, Andersen JB, Givskov M, et al. Distribution of bacterial growth activity in flow-chamber biofilms. Appl Environ Microbiol 1999;65:4108-17.
132Roberts AE, Kragh KN, Bjarnsholt T, Diggle SP. The limitations of in vitro experimentation in understanding biofilms and chronic infection. J Mol Biol 2015;427:3646-61.
133Sandt C, Smith Palmer T, Pink J, Pink D. Quantification of local water and biomass in wild type PA01 biofilms by confocal raman microspectroscopy. J Microbiol Methods 2008;75:148-52.
134Filloux A, Ramos JL. Preface. Pseudomonas methods and protocols. Methods Mol Biol 2014;1149:v.
135Blackwood LL, Pennington JE. Influence of mucoid coating on clearance of Pseudomonas aeruginosa from lungs. Infect Immun 1981;32:443-8.
136Kirkeby S, Wimmerová M, Moe D, Hansen AK. The mink as an animal model for Pseudomonas aeruginosa adhesion: Binding of the bacterial lectins (PA-IL and PA-IIL) to neoglycoproteins and to sections of pancreas and lung tissues from healthy mink. Microbes Infect 2007;9:566-73.
137Facchini M, De Fino I, Riva C, Bragonzi A. Long term chronic Pseudomonas aeruginosa airway infection in mice. J Vis Exp 2014(85).
138Guilbault C, Martin P, Houle D, Boghdady ML, Guiot MC, Marion D, et al. Cystic fibrosis lung disease following infection with Pseudomonas aeruginosa in cftr knockout mice using novel non-invasive direct pulmonary infection technique. Lab Anim 2005;39:336-52.
139Cash HA, Woods DE, McCullough B, Johanson WG Jr., Bass JA. A rat model of chronic respiratory infection with Pseudomonas aeruginosa. Am Rev Respir Dis 1979;119:453-9.
140Strathmann M, Griebe T, Flemming HC. Artificial biofilm model – A useful tool for biofilm research. Appl Microbiol Biotechnol 2000;54:231-7.
141Jensen PØ, Givskov M, Bjarnsholt T, Moser C. The immune system vs. Pseudomonas aeruginosa biofilms. FEMS Immunol Med Microbiol 2010;59:292-305.
142Watters C, Fleming D, Bishop D, Rumbaugh KP. Host responses to biofilm. Prog Mol Biol Transl Sci 2016;142:193-239.
143Moser C, Pedersen HT, Lerche CJ, Kolpen M, Line L, Thomsen K, et al. Biofilms and host response-helpful or harmful. APMIS 2017;125:320-38.