Impairment of Pseudomonas aeruginosa Biofilm Resistance to Antibiotics by Combining the Drugs with a New Quorum-Sensing Inhibitor

ABSTRACT Pseudomonas aeruginosa plays an important role in chronic lung infections among patients with cystic fibrosis (CF) through its ability to form antibiotic-resistant biofilms. In P. aeruginosa, biofilm development and the production of several virulence factors are mainly regulated by the rhl and las quorum-sensing (QS) systems, which are controlled by two N-acyl-homoserine lactone signal molecules. In a previous study, we discovered an original QS inhibitor, N-(2-pyrimidyl)butanamide, called C11, based on the structure of C4-homoserine lactone, and found that it is able to significantly inhibit P. aeruginosa biofilm formation. However, recent data indicate that P. aeruginosa grows under anaerobic conditions and forms biofilms in the lungs of CF patients that are denser and more robust than those formed under aerobic conditions. Our confocal microscopy observations of P. aeruginosa biofilms developed under aerobic and anaerobic conditions confirmed that the biofilms formed under these two conditions have radically different architectures. C11 showed significant dose-dependent antibiofilm activity on biofilms grown under both aerobic and anaerobic conditions, with a greater inhibitory effect being seen under conditions of anaerobiosis. Gene expression analyses performed by quantitative reverse transcriptase PCR showed that C11 led to the significant downregulation of rhl QS regulatory genes but also to the downregulation of both las QS regulatory genes and QS system-regulated virulence genes, rhlA and lasB. Furthermore, the activity of C11 in combination with antibiotics against P. aeruginosa biofilms was tested, and synergistic antibiofilm activity between C11 and ciprofloxacin, tobramycin, and colistin was obtained under both aerobic and anaerobic conditions. This study demonstrates that C11 may increase the efficacy of treatments for P. aeruginosa infections by increasing the susceptibility of biofilms to antibiotics and by attenuating the pathogenicity of the bacterium.

[1]  K. M. Lee,et al.  Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[2]  E. Pesci,et al.  Dueling quorum sensing systems in Pseudomonas aeruginosa control the production of the Pseudomonas quinolone signal (PQS). , 2004, FEMS microbiology letters.

[3]  E. Greenberg,et al.  Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[4]  D. Pritchard,et al.  Quorum sensing and the population-dependent control of virulence. , 2000, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[5]  M. Bally,et al.  Production of elastase, exotoxin A, and alkaline protease in sputa during pulmonary exacerbation of cystic fibrosis in patients chronically infected by Pseudomonas aeruginosa , 1995, Journal of clinical microbiology.

[6]  P. Seed,et al.  Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa , 1997, Journal of bacteriology.

[7]  R. Hancock,et al.  Pseudomonas aeruginosa: all roads lead to resistance. , 2011, Trends in microbiology.

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

[9]  L. Leibovici,et al.  Colistin: new lessons on an old antibiotic. , 2012, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

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

[11]  J. Costerton,et al.  The involvement of cell-to-cell signals in the development of a bacterial biofilm. , 1998, Science.

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

[13]  Jian Li,et al.  Structure--activity relationships of polymyxin antibiotics. , 2010, Journal of medicinal chemistry.

[14]  A. Oliver,et al.  Antimicrobial therapy for pulmonary pathogenic colonisation and infection by Pseudomonas aeruginosa in cystic fibrosis patients. , 2005, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[15]  F. Baquero,et al.  In vitro prevention of Pseudomonas aeruginosa early biofilm formation with antibiotics used in cystic fibrosis patients. , 2012, International journal of antimicrobial agents.

[16]  T. B. Rasmussen,et al.  Quorum-sensing inhibitors as anti-pathogenic drugs. , 2006, International journal of medical microbiology : IJMM.

[17]  N. Høiby,et al.  Synergistic antibacterial efficacy of early combination treatment with tobramycin and quorum-sensing inhibitors against Pseudomonas aeruginosa in an intraperitoneal foreign-body infection mouse model. , 2012, The Journal of antimicrobial chemotherapy.

[18]  A. Furiga,et al.  Growth inhibition of adherent Pseudomonas aeruginosa by an N-butanoyl-L-homoserine lactone analog. , 2010, Canadian journal of microbiology.

[19]  Kathrine B. Christensen,et al.  Identity and effects of quorum-sensing inhibitors produced by Penicillium species. , 2005, Microbiology.

[20]  E. Greenberg,et al.  Acyl-homoserine lactone quorum sensing in gram-negative bacteria: a signaling mechanism involved in associations with higher organisms. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[21]  S. Kjelleberg,et al.  Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors , 2003, The EMBO journal.

[22]  A. Brooks,et al.  Microarray Analysis of Pseudomonas aeruginosa Quorum-Sensing Regulons: Effects of Growth Phase and Environment , 2003, Journal of bacteriology.

[23]  L. Eberl,et al.  Screening for Quorum-Sensing Inhibitors (QSI) by Use of a Novel Genetic System, the QSI Selector , 2005, Journal of bacteriology.

[24]  G. Baziard-Mouysset,et al.  Interactions between Biocide Cationic Agents and Bacterial Biofilms , 2002, Antimicrobial Agents and Chemotherapy.

[25]  B. Iglewski,et al.  Analysis of the Pseudomonas aeruginosa elastase (lasB) regulatory region , 1996, Journal of bacteriology.

[26]  M. Hentzer,et al.  Azithromycin Blocks Quorum Sensing and Alginate Polymer Formation and Increases the Sensitivity to Serum and Stationary-Growth-Phase Killing of Pseudomonas aeruginosa and Attenuates Chronic P. aeruginosa Lung Infection in Cftr−/− Mice , 2007, Antimicrobial Agents and Chemotherapy.

[27]  C. van Delden,et al.  Biofilm formation by Pseudomonas aeruginosa: role of the C4-HSL cell-to-cell signal and inhibition by azithromycin. , 2003, The Journal of antimicrobial chemotherapy.

[28]  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.

[29]  S. Ichiyama,et al.  Relationship between morphological changes and endotoxin release induced by carbapenems in Pseudomonas aeruginosa. , 1999, Journal of medical microbiology.

[30]  S. Kjelleberg,et al.  Impact of Pseudomonas aeruginosa quorum sensing on biofilm persistence in an in vivo intraperitoneal foreign-body infection model. , 2007, Microbiology.

[31]  J. Collins,et al.  How antibiotics kill bacteria: from targets to networks , 2010, Nature Reviews Microbiology.

[32]  T. B. Rasmussen,et al.  Rational design and synthesis of new quorum-sensing inhibitors derived from acylated homoserine lactones and natural products from garlic. , 2005, Organic & biomolecular chemistry.

[33]  D. Pritchard,et al.  Synthetic analogues of the bacterial signal (quorum sensing) molecule N-(3-oxododecanoyl)-L-homoserine lactone as immune modulators. , 2003, Journal of medicinal chemistry.

[34]  L. Lutz,et al.  Macrolides decrease the minimal inhibitory concentration of anti-pseudomonal agents against Pseudomonas aeruginosa from cystic fibrosis patients in biofilm , 2012, BMC Microbiology.

[35]  H. Vogel,et al.  In vivo regulation of virulence in Pseudomonas aeruginosa associated with genetic rearrangement. , 1991, The Journal of infectious diseases.

[36]  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.

[37]  B. Iglewski,et al.  Azithromycin Retards Pseudomonas aeruginosa Biofilm Formation , 2004, Journal of Clinical Microbiology.

[38]  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.

[39]  E. Greenberg,et al.  A network of networks: quorum-sensing gene regulation in Pseudomonas aeruginosa. , 2006, International journal of medical microbiology : IJMM.

[40]  Daniel Yordanov,et al.  Pseudomonas aeruginosa - a phenomenon of bacterial resistance. , 2009, Journal of medical microbiology.

[41]  G. Pier,et al.  Inactivation of the rhlA gene in Pseudomonas aeruginosa prevents rhamnolipid production, disabling the protection against polymorphonuclear leukocytes , 2009, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica.

[42]  T. Coenye,et al.  Synergistic antibacterial efficacy of early combination treatment with tobramycin and quorum-sensing inhibitors against Pseudomonas aeruginosa in an intraperitoneal foreign-body infection mouse model , 2012 .

[43]  R. Hancock,et al.  Interactions of Bacterial Cationic Peptide Antibiotics with Outer and Cytoplasmic Membranes ofPseudomonas aeruginosa , 2000, Antimicrobial Agents and Chemotherapy.

[44]  E. Pesci,et al.  Regulation of Pseudomonas Quinolone Signal Synthesis in Pseudomonas aeruginosa , 2005, Journal of bacteriology.

[45]  B. Iglewski,et al.  Cell-to-cell signaling and Pseudomonas aeruginosa infections. , 1998, Emerging infectious diseases.

[46]  A. Prince,et al.  Pseudomonas aeruginosa Cell-to-Cell Signaling Is Required for Virulence in a Model of Acute Pulmonary Infection , 2000, Infection and Immunity.

[47]  R. Hancock,et al.  Alternative mechanisms of action of cationic antimicrobial peptides on bacteria , 2007, Expert review of anti-infective therapy.

[48]  C. van Delden,et al.  Detection of Pseudomonas aeruginosa cell-to-cell signals in lung tissue of cystic fibrosis patients. , 2002, Microbial pathogenesis.

[49]  M. Elkins,et al.  Antibiotic Susceptibilities of Pseudomonas aeruginosa Isolates Derived from Patients with Cystic Fibrosis under Aerobic, Anaerobic, and Biofilm Conditions , 2005, Journal of Clinical Microbiology.

[50]  K. Miller,et al.  Elastase of Pseudomonas aeruginosa: Inactivation of Complement Components and Complement-Derived Chemotactic and Phagocytic Factors , 1974, Infection and immunity.

[51]  Alfred Pingoud,et al.  Real‐Time Polymerase Chain Reaction , 2003, Chembiochem : a European journal of chemical biology.

[52]  R. Duval,et al.  Nanoscale effects of antibiotics on P. aeruginosa. , 2012, Nanomedicine : nanotechnology, biology, and medicine.

[53]  J. Rolain,et al.  Colistin: an update on the antibiotic of the 21st century , 2012, Expert review of anti-infective therapy.

[54]  Russell M. Taylor,et al.  A physical linkage between cystic fibrosis airway surface dehydration and Pseudomonas aeruginosa biofilms , 2006, Proceedings of the National Academy of Sciences.

[55]  M. Rosenthal,et al.  Long term azithromycin in children with cystic fibrosis: a randomised, placebo-controlled crossover trial , 2002, The Lancet.

[56]  Miguel Cámara,et al.  Direct detection of N-acylhomoserine lactones in cystic fibrosis sputum. , 2002, FEMS microbiology letters.

[57]  Mary Jane Ferraro,et al.  Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically : approved standard , 2000 .

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

[59]  M. Son,et al.  In Vivo Evidence of Pseudomonas aeruginosa Nutrient Acquisition and Pathogenesis in the Lungs of Cystic Fibrosis Patients , 2007, Infection and Immunity.

[60]  N. Høiby,et al.  Pharmacokinetics/Pharmacodynamics of Colistin and Imipenem on Mucoid and Nonmucoid Pseudomonas aeruginosa Biofilms , 2011, Antimicrobial Agents and Chemotherapy.

[61]  S. Chhibber,et al.  Azithromycin and ciprofloxacin: a possible synergistic combination against Pseudomonas aeruginosa biofilm-associated urinary tract infections. , 2015, International journal of antimicrobial agents.