|Year : 2018 | Volume
| 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
|Date of Submission||17-Aug-2018|
|Date of Decision||25-Aug-2018|
|Date of Acceptance||16-Nov-2018|
|Date of Web Publication||11-Dec-2018|
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
Source of Support: None, Conflict of Interest: None
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.
Keywords: Biofilm, dispersal, Pseudomonas aeruginosa, quorum sensing
|How to cite this article:|
Bedi B, Maurice NM, Sadikot RT. Microarchitecture of Pseudomonas aeruginosa biofilms: A biological perspective. Biomed Biotechnol Res J 2018;2:227-36
|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 2020 Aug 3];2:227-36. Available from: http://www.bmbtrj.org/text.asp?2018/2/4/227/247248
| Introduction|| |
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). 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,, 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. The role of P. aeruginosa biofilms in cystic fibrosis (CF) pathogenesis is well established. P. aeruginosa is an opportunistic pathogen and a huge threat to public health due to its adaptive qualities and varied antibiotic resistance.,
| 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. The microorganism itself accounts for <10% of the dry mass, whereas the matrix can account for >90%. Biofilms structure can be affected by the genetics (active response) as well as environmental (passive response) conditions which are not mutually exclusive., 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. 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. Biofilms architecture of P. aeruginosa is specifically sensitive to iron, (which can facilitate its conversion from nonmucoid to mucoid phenotype).,
| 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,, pellicle (PeI),, and polysaccharide synthesis locus.,
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.,, It is a shiny, high-molecular mass, unbranched heteropolymer consisting of 1,4-linked uronic residues of b-D-mannuronate and a-L-guluronate. These components are arranged in homopolymeric blocks of polymannuronate and heteropolymeric sequences with a random distribution of guluronate and partially O-acetylated mannuronate residues. 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., 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. 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. 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.,, 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.
In the nonmucoid not expressing alginate biosynthesis genes, Pel and Psl are involved in the establishment of biofilms., Pel is a glucose-rich polysaccharide, whereas Psl consists of a repeating pentasaccharide containing d-mannose, d-glucose, and l-rhamnose. Pel is essential for the formation of biofilms (called PeIs) at air-liquid interfaces and biofilms that are attached to a surface,, 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.,, 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.
In P. aeruginosa, the association of extracellular lactonizing lipase (lipA; also, known as lip) with alginate is based on weak-binding forces., 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.
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., 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. Galactose-specific and fucose-specific lectin LecA and LecB, respectively, 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.
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., Due to this characteristic, DNase therapy can be used to inhibit biofilm formation.,, 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. The localization of eDNA can vary widely between biofilms. In P. aeruginosa, biofilms eDNA forms a grid-like structure. 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. 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. Recent studies have shown that PMN-derived genomic DNA can be incorporated into P. aeruginosa biofilms and confer increased bacterial resistance to aminoglycoside treatment. eDNA can limit anti-microbial peptides, membrane damage, and killing., eDNA can also confer antibiotic resistance by acidifying biofilm cultures.
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. 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.
| 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., 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. 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. 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. 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, 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. 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., The subpopulation of cells constituting the stalk is less metabolically active in comparison to the cells in the cap., 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]., 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: Biofilm formation by Pseudomonas aeruginosa proceeds through four different stages, wherein dispersal constitutes the fifth stage. Planktonic bacteria weakly attach to surface (within a host or abiotic) to form microcolonies. Microcolonies aggregate further to form macrocolonies with robust adhesion. Macrocolonies evolve into mature mushroom-shaped biofilms. Cells associated with biofilms are then dispersed to once again resume the planktonic lifestyle. Cyclic dimeric guanosine monophosphate is a cellular second messenger whose levels are intricately regulated during the different stages of biofilm formation. The upper panel illustrates the various diguanylate cyclase (red), phosphodiesterase (blue), different enzymatic recruiters are activated at different stages of biofilm formation, (as indicated in the panel). Phosphodiesterases are maximally induced in mature biofilms, where cyclic dimeric guanosine monophosphate levels are reduced for dispersion to occur|
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| Transcriptional Regulators of Biofilm Formation|| |
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.,,, LasI synthase is responsible for synthesizing N-(3-oxododecanoyl)-L-homoserine lactone, which is recognized by the LasR receptor.,, In the Rhl system, N-butanoyl homoserine lactone (C4-HSL) is produced by the RhlI synthase which is sensed by the RhlR receptor.,, 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. Recently, a fourth QS system, termed the IQS system, has shown to connect environmental stress cues with the QS networks. 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. 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.,,,,
Control by sRNA and Two-component systems in Pseudomonas aeruginosa biofilms
P. aeruginosa biofilm is regulated by sRNAs, rsmY, and rsmZ. Three sensor kinases are involved, are involved in this pathway, namely, RetS, LadS, but mainly GacS. GacS phosphorylates GacA, 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, and also downregulate another effector, RsmN, controlling the same functions as RsmA. In addition, alternative sigma factor, RpoS expression was also significantly increased in P. aeruginosa biofilms acts as a positive regulator of the expression of the psl gene.,
| 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., 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. 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., 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 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). 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., In P. aeruginosa, biofilms, NO is produced by anaerobic respiration, which in turn induced dispersal. 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,, other studies have shown that dispersal was induced with low, sublethal concentrations (25–500 nM) of the NO donor sodium nitroprusside, 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)., 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. 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. 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., 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. 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, 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.,,,
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., 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. 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. 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., In turn, the WspR phosphorylation triggers subcellular WspR oligomerization and cluster formation, which further increases the DGC activity. 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. 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.,
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., GcbA and NicD DGCs or the DipA (Pch), RbdA, BifA, and NbdA PDEs have been linked to biofilm dispersal.,,,, 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.,
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., These are slow growing and antibiotic resistant.
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. 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.
| 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. 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., Mixed-species biofilms also demonstrate the occurrence of cooperative behavior in biofilms formed by P. aeruginosa, P. protegens and Klebsiella pneuomoniae. This scenario can partly explain the high tolerance of these communities to toxin accumulation, particularly of their planktonic counterparts. P. aeruginosa can also associate with bacteria such as Staphylococcus epidermis and Staphylococcus aureus 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., The fatty acid cis-2-decenoic acid, produced by P. aeruginosa, can induce dispersal in other bacterial species, including B. subtilis and S. aureus. P. aeruginosa rhamnolipids when added exogenously can disperse biofilms produced by Bordetella bronchiseptica and Salmonella More Details Typhimurium.,
| 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. 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., Biofilm formation in co-culture models of P. aeruginosa with host cells such as epithelial cells and neutrophils can also be studied. 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.,, 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. 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.
For detecting biofilms of multispecies such as P. aeruginosa and S. aureus laser ablation electrospray ionization mass spectrometry can be employed. 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. Raman microscopy and microspectroscopy are upcoming approaches that are used in combination with CLSM-based lectin-binding analysis. 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.
In vivo systems
Nonmammalian hosts such as Zebrafish, Drosophila, melanogaster, Caenorhabditis elegans, and mammalian hosts such as guinea pigs and minks 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, 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.
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. 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.,, 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). 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. 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. 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. 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.,, We have recently in depth reviewed the host response to P. aeruginosa biofilms and therapeutic approaches that can modulate biofilm formation.
| 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.
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