Use of the rotating wall vessel technology to study the effect of shear stress on growth behaviour of Pseudomonas aeruginosa PA01.

The biofilm phenotype of Pseudomonas aeruginosa enables this opportunistic pathogen to develop resistance to the immune system and antimicrobial agents. Pseudomonas aeruginosa biofilms are generated under varying levels of shear stress, depending on the infection site. In the lung mucus of cystic fibrosis (CF) patients, P. aeruginosa forms matrix-enclosed microcolonies which cause chronic infections representing the major cause of mortality in CF patients. The lung mucus of CF patients is probably characterized by low fluid shear as the main shear-causing factor, i.e. mucociliary clearance, is absent. In this study, the influence of fluid shear on the growth behaviour of P. aeruginosa PA01 was investigated using a low-shear suspension culture device, the rotating wall vessel (RWV). Cultivation in low shear induced a self-aggregating phenotype of P. aeruginosa PA01, resulting in the formation of biofilms in suspension similar to what has been described in CF mucus. The addition of a ceramic bead to the culture medium in the RWV created a higher-shear condition which led to the formation of surface-attached rather than suspension biofilms. In low-shear culture conditions, a significant increase of the rhl N-butanoyl-l-homoserine lactone (C(4)-HSL) directed quorum sensing (QS) system, and the psl polysaccharide synthetic locus was demonstrated using gene expression analysis. Accordingly, the low-shear condition induced a higher production of rhamnolipids, which is controlled by the C(4)-HSL QS-system and is known to play a role in CF lung pathology. These results indicate that fluid shear has an impact on the growth phenotype of P. aeruginosa which might play a role in CF lung infections caused by this bacterium.

[1]  G. Pessi,et al.  Dual control of hydrogen cyanide biosynthesis by the global activator GacA in Pseudomonas aeruginosa PAO1. , 2001, FEMS microbiology letters.

[2]  P. Cornelis,et al.  Characterization of a new efflux pump, MexGHI-OpmD, from Pseudomonas aeruginosa that confers resistance to vanadium. , 2002, Microbiology.

[3]  David A. D'Argenio,et al.  Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Diggle,et al.  Biosensor-based assays for PQS, HHQ and related 2-alkyl-4-quinolone quorum sensing signal molecules , 2007, Nature Protocols.

[5]  N. Schiller,et al.  Inhibition of Macrophage Phagocytosis by Pseudomonas aeruginosa Rhamnolipids In Vitro and In Vivo , 1996, Current Microbiology.

[6]  G. Pessi,et al.  The Global Posttranscriptional Regulator RsmA Modulates Production of Virulence Determinants andN-Acylhomoserine Lactones in Pseudomonas aeruginosa , 2001, Journal of bacteriology.

[7]  Richard C Boucher,et al.  Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. , 2002, The Journal of clinical investigation.

[8]  B. Iglewski,et al.  Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes , 1997, Journal of bacteriology.

[9]  P Ducheyne,et al.  Dynamics of a microcarrier particle in the simulated microgravity environment of a rotating-wall vessel. , 1997, Microgravity science and technology.

[10]  L. Eberl,et al.  Quorum sensing: the power of cooperation in the world of Pseudomonas. , 2005, Environmental microbiology.

[11]  A. Demain,et al.  Shear stress enhances microcin B17 production in a rotating wall bioreactor, but ethanol stress does not , 2001, Applied Microbiology and Biotechnology.

[12]  B. Rehm,et al.  Expression of the psl Operon in Pseudomonas aeruginosa PAO1 Biofilms: PslA Performs an Essential Function in Biofilm Formation , 2005, Applied and Environmental Microbiology.

[13]  T. Pitt,et al.  Widespread pyocyanin over-production among isolates of a cystic fibrosis epidemic strain , 2007, BMC Microbiology.

[14]  G. Taylor,et al.  Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and assessment of their contribution to sputum sol toxicity for respiratory epithelium , 1988, Infection and immunity.

[15]  B. Tümmler,et al.  RHAMNOLIPID OF PSEUDOMONAS AERUGINOSA IN SPUTUM OF CYSTIC FIBROSIS PATIENTS , 1987, The Lancet.

[16]  R. Kolter,et al.  Quorum-Sensing Regulation of the Biofilm Matrix Genes (pel) of Pseudomonas aeruginosa , 2007, Journal of bacteriology.

[17]  A. Jeanes,et al.  A new modification of the carbazole analysis: application to heteropolysaccharides. , 1968, Analytical biochemistry.

[18]  M. Mazzola,et al.  Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats , 1992, Applied and environmental microbiology.

[19]  D. Pierson,et al.  Effect of simulated microgravity and shear stress on microcin B17 production by Escherichia coli and on its excretion into the medium , 1997, Applied and environmental microbiology.

[20]  U. Römling,et al.  Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung. , 2005, Journal of medical microbiology.

[21]  K. Edwards,et al.  Real‐time PCR used to measure stress‐induced changes in the expression of the genes of the alginate pathway of Pseudomonas aeruginosa , 2001, Journal of applied microbiology.

[22]  Fritz Wagner,et al.  New method for detecting rhamnolipids excreted by Pseudomonas species during growth on mineral agar , 1991 .

[23]  M. Parsek,et al.  Analysis of Pseudomonas aeruginosa Conditional Psl Variants Reveals Roles for the Psl Polysaccharide in Adhesion and Maintaining Biofilm Structure Postattachment , 2006, Journal of bacteriology.

[24]  S. Weinbaum,et al.  A hydrodynamic mechanosensory hypothesis for brush border microvilli. , 2000, American journal of physiology. Renal physiology.

[25]  Matthew R. Parsek,et al.  Characterization of Colony Morphology Variants Isolated from Pseudomonas aeruginosa Biofilms , 2005, Applied and Environmental Microbiology.

[26]  S. Diggle,et al.  The MexGHI-OpmD multidrug efflux pump controls growth, antibiotic susceptibility and virulence in Pseudomonas aeruginosa via 4-quinolone-dependent cell-to-cell communication. , 2005, Microbiology.

[27]  D. Hassett,et al.  Pseudomonas aeruginosa sodA and sodB mutants defective in manganese- and iron-cofactored superoxide dismutase activity demonstrate the importance of the iron-cofactored form in aerobic metabolism , 1995, Journal of bacteriology.

[28]  V. Venturi.  Regulation of quorum sensing in Pseudomonas. , 2006, FEMS microbiology reviews.

[29]  T G Hammond,et al.  Optimized suspension culture: the rotating-wall vessel. , 2001, American journal of physiology. Renal physiology.

[30]  I. Sutherland,et al.  The biofilm matrix--an immobilized but dynamic microbial environment. , 2001, Trends in microbiology.

[31]  Matthew R. Parsek,et al.  Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms , 2000, Nature.

[32]  Marina S. Kuznetsova,et al.  Functions Required for Extracellular Quinolone Signaling by Pseudomonas aeruginosa , 2002, Journal of bacteriology.

[33]  S. M. Kirov,et al.  Anaerobic culture conditions favor biofilm-like phenotypes in Pseudomonas aeruginosa isolates from patients with cystic fibrosis. , 2006, FEMS immunology and medical microbiology.

[34]  A. Filloux,et al.  The pel genes of the Pseudomonas aeruginosa PAK strain are involved at early and late stages of biofilm formation. , 2005, Microbiology.

[35]  V. Deretic,et al.  Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. , 1996, Microbiological reviews.

[36]  Matthew R. Parsek,et al.  Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[37]  S. Wuertz,et al.  Effect of flow regime on the architecture of a Pseudomonas fluorescens biofilm. , 2002, Biotechnology and bioengineering.

[38]  N. Høiby.  Pseudomonas aeruginosa infection in cystic fibrosis. Relationship between mucoid strains of Pseudomonas aeruginosa and the humoral immune response. , 2009 .

[39]  A. Kirkham,et al.  Pseudomonas aeruginosa Quorum-Sensing Systems May Control Virulence Factor Expression in the Lungs of Patients with Cystic Fibrosis , 2002, Infection and Immunity.

[40]  J. Blake.  A note on mucus shear rates. , 1973, Respiration physiology.

[41]  G. Pier,et al.  Nonmucoid Pseudomonas aeruginosa expresses alginate in the lungs of patients with cystic fibrosis and in a mouse model. , 2005, The Journal of infectious diseases.

[42]  S. Kjelleberg,et al.  A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms , 2006, Molecular microbiology.

[43]  S. Diggle,et al.  The Pseudomonas aeruginosa quinolone signal molecule overcomes the cell density‐dependency of the quorum sensing hierarchy, regulates rhl‐dependent genes at the onset of stationary phase and can be produced in the absence of LasR , 2003, Molecular microbiology.

[44]  G. Mortier,et al.  qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data , 2007, Genome Biology.

[45]  N. Høiby,et al.  Role of alginate in infection with mucoid Pseudomonas aeruginosa in cystic fibrosis. , 1992, Thorax.

[46]  B. Rehm,et al.  The role of polyhydroxyalkanoate biosynthesis by Pseudomonas aeruginosa in rhamnolipid and alginate production as well as stress tolerance and biofilm formation. , 2004, Microbiology.

[47]  J. Sha,et al.  Alterations in the Virulence Potential of Enteric Pathogens and Bacterial–Host Cell Interactions Under Simulated Microgravity Conditions , 2006, Journal of toxicology and environmental health. Part A.

[48]  Lina M. Nilsson,et al.  Elevated Shear Stress Protects Escherichia coli Cells Adhering to Surfaces via Catch Bonds from Detachment by Soluble Inhibitors , 2006, Applied and Environmental Microbiology.

[49]  R. Tompkins,et al.  Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell communication. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[50]  M. Denis,et al.  Release of interleukin-8, interleukin-6, and colony-stimulating factors by upper airway epithelial cells: implications for cystic fibrosis. , 1993, American journal of respiratory cell and molecular biology.

[51]  Roberto Kolter,et al.  Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms , 2003, Molecular microbiology.

[52]  Zhenglun Li,et al.  Shear Stress Affects the Kinetics of Staphylococcus aureus Adhesion to Collagen , 2000, Biotechnology progress.

[53]  C. Mark Ott,et al.  Microgravity as a Novel Environmental Signal Affecting Salmonella enterica Serovar Typhimurium Virulence , 2000, Infection and Immunity.

[54]  Peter Gilligan,et al.  A bacterial cell to cell signal in the lungs of cystic fibrosis patients. , 2002, FEMS microbiology letters.

[55]  D. Pierson,et al.  Novel Quantitative Biosystem for Modeling Physiological Fluid Shear Stress on Cells , 2006, Applied and Environmental Microbiology.

[56]  A. Chakrabarty,et al.  Isolation and assay of Pseudomonas aeruginosa alginate. , 1994, Methods in enzymology.

[57]  R. Kolter,et al.  Two Genetic Loci Produce Distinct Carbohydrate-Rich Structural Components of the Pseudomonas aeruginosa Biofilm Matrix , 2004, Journal of bacteriology.

[58]  B. Rehm,et al.  PslD Is a Secreted Protein Required for Biofilm Formation by Pseudomonas aeruginosa , 2006, Applied and Environmental Microbiology.

[59]  Samuel I. Miller,et al.  Quantitative proteomic analysis indicates increased synthesis of a quinolone by Pseudomonas aeruginosa isolates from cystic fibrosis airways , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[60]  J. Aarnoudse,et al.  POST-PARTUM PULMONARY ŒDEMA ASSOCIATED WITH PREVENTIVE THERAPY FOR PREMATURE LABOUR , 1979, The Lancet.

[61]  Paul Stoodley,et al.  Viscoelasticity of Staphylococcus aureus Biofilms in Response to Fluid Shear Allows Resistance to Detachment and Facilitates Rolling Migration , 2005, Applied and Environmental Microbiology.

[62]  J. Costerton,et al.  Influence of Hydrodynamics and Cell Signaling on the Structure and Behavior of Pseudomonas aeruginosa Biofilms , 2002, Applied and Environmental Microbiology.

[63]  Gloria Soberón-Chávez,et al.  The Pseudomonas aeruginosa RhlA enzyme is involved in rhamnolipid and polyhydroxyalkanoate production , 2005, Journal of Industrial Microbiology and Biotechnology.

[64]  T. Tolker-Nielsen,et al.  Multiple Roles of Biosurfactants in Structural Biofilm Development by Pseudomonas aeruginosa , 2007, Journal of bacteriology.

[65]  Viola Vogel,et al.  Shear‐dependent ‘stick‐and‐roll’ adhesion of type 1 fimbriated Escherichia coli , 2004, Molecular microbiology.

[66]  G. Pugliese,et al.  Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms , 2002, Infection Control & Hospital Epidemiology.

[67]  Viola Vogel,et al.  Bacterial Adhesion to Target Cells Enhanced by Shear Force , 2002, Cell.

[68]  B. Iglewski,et al.  Quorum-Sensing Genes in Pseudomonas aeruginosa Biofilms: Their Role and Expression Patterns , 2001, Applied and Environmental Microbiology.

[69]  George M. Hilliard,et al.  Pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis. , 2002, Developmental cell.

[70]  Freya Harrison,et al.  Microbial ecology of the cystic fibrosis lung. , 2007, Microbiology.

[71]  S. Fleiszig,et al.  Mutation of lasA and lasB reduces Pseudomonas aeruginosa invasion of epithelial cells. , 2003, Microbiology.

[72]  S. Molin,et al.  Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation. , 2004, Journal of medical microbiology.

[73]  T. Schwartz,et al.  Use of quantitative real-time RT-PCR to analyse the expression of some quorum-sensing regulated genes in Pseudomonas aeruginosa , 2007, Analytical and bioanalytical chemistry.

[74]  S. Diggle,et al.  4-quinolone signalling in Pseudomonas aeruginosa: old molecules, new perspectives. , 2006, International journal of medical microbiology : IJMM.

[75]  P. Gilligan,et al.  Microbiology of airway disease in patients with cystic fibrosis , 1991, Clinical Microbiology Reviews.

[76]  G. O’Toole,et al.  Rhamnolipid Surfactant Production Affects Biofilm Architecture in Pseudomonas aeruginosa PAO1 , 2003, Journal of bacteriology.

[77]  C. Harwood,et al.  Responses of Pseudomonas aeruginosa to low oxygen indicate that growth in the cystic fibrosis lung is by aerobic respiration , 2007, Molecular microbiology.

[78]  S. Kurioka,et al.  Effect of the Hemolysin of Pseudomonas aeruginosa on Phosphatides and on Phospholipase c Activity , 1967, Journal of bacteriology.

[79]  S. Diggle,et al.  The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa. , 2006, Environmental microbiology.

[80]  B. Tümmler,et al.  Highly adherent small-colony variants of Pseudomonas aeruginosa in cystic fibrosis lung infection. , 2003, Journal of medical microbiology.

[81]  D. Newman,et al.  The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa , 2006, Molecular microbiology.

[82]  Don P. Giddens,et al.  Blood Flow in Major Blood Vessels—Modeling and Experiments , 2005, Annals of Biomedical Engineering.

[83]  F. Lépine,et al.  rhlA is required for the production of a novel biosurfactant promoting swarming motility in Pseudomonas aeruginosa: 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), the precursors of rhamnolipids. , 2003, Microbiology.

[84]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[85]  M. Molyneux,et al.  Adhesion of Plasmodium falciparum-infected erythrocytes to hyaluronic acid in placental malaria , 2000, Nature Medicine.

[86]  R. Miller,et al.  Rhamnolipid (biosurfactant) effects on cell aggregation and biodegradation of residual hexadecane under saturated flow conditions , 1997, Applied and environmental microbiology.

[87]  L. Mizrahi,et al.  Purification of the galactose‐binding hemagglutinin of Pseudomonas aeruginosa by affinity column chromatography using sepharose , 1972, FEBS letters.

[88]  J. Costerton,et al.  Production of mucoid microcolonies by Pseudomonas aeruginosa within infected lungs in cystic fibrosis , 1980, Infection and immunity.

[89]  D M Klaus,et al.  Clinostats and bioreactors. , 2007, Gravitational and space biology bulletin : publication of the American Society for Gravitational and Space Biology.

[90]  Leo Eberl,et al.  Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. , 2002, Microbiology.

[91]  P. Williams,et al.  Rapid necrotic killing of polymorphonuclear leukocytes is caused by quorum-sensing-controlled production of rhamnolipid by Pseudomonas aeruginosa. , 2007, Microbiology.

[92]  M. Knowles,et al.  Mucus clearance as a primary innate defense mechanism for mammalian airways. , 2002, The Journal of clinical investigation.

[93]  Frederick M. Ausubel,et al.  Molecular Mechanisms of Bacterial Virulence Elucidated Using a Pseudomonas Aeruginosa– Caenorhabditis Elegans Pathogenesis Model , 2022 .