Pyocyanin secreted by<em> Pseudomonas aeruginosa</em> inhibits growth of cystic fibrosis isolates of <em>Staphylococcus aureus and Escherichia coli, </em>but not<em> Burkholderia cenocepacia</em> — ASN Events
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  2. Human microbial ecology
  3. Human Microbial Ecology
  4. Pyocyanin secreted by Pseudomonas aeruginosa inhibits growth of cystic fibrosis isolates of Staphylococcus aureus and Escherichia coli, but not Burkholderia cenocepacia

Pyocyanin secreted by Pseudomonas aeruginosa inhibits growth of cystic fibrosis isolates of Staphylococcus aureus and Escherichia coli, but not Burkholderia cenocepacia (#20)

Edwina Buckle 1 , Mariana Campos-Atherino 2 , William Klare 1 , Stuart Cordwell 3 , Theerthankar Das 1 , Jim Manos 1
  1. Department of Infectious Diseases and Immunology, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia
  2. Department of Basic Pathology, Universidade Federal do Paraná, Paraná, Brazil and the CAPES Foundation, Ministry of Education of Brazil, Brazil, Brasilia
  3. Mass Spectrometry Core Facility, The Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia

Prevalence of key bacterial pathogens in the cystic fibrosis (CF) lung changes over time. Pseudomonas aeruginosa and Staphylococcus aureus are isolated in relatively equal proportions early in life, but P. aeruginosa becomes dominant later. To identify P. aeruginosa-secreted inhibitors, we grew strains PAO1, PA14, AES-1R(acute) and AES-1M(chronic) isogens to mid-log-phase at 37oC in Luria Broth (LB) and filtered out the cells. S. aureus, E. coli and Burkholderia cenocepacia were then inoculated into serial dilutions of the filtrate. Neat broth from PAO1, PA14 and AES-1R inhibited growth of all species. In contrast, AES1-M-filtered broth did not inhibit S. aureus or B. cenocepacia. P. aeruginosa siderophores have been shown to be inhibitory; therefore we supplemented LB with ferritin in CF-relevant concentrations to reduce siderophore load. AES-1M with added ferritin inhibited S. aureus and B. cenocepacia. To investigate if pyocyanin was the inhibitor, a pyocyanin-negative mutant (PA14ΔphzA-G) was used and S. aureus, E. coli and B. cenocepacia grew in its neat-filtered broth. To assess strain sensitivity to pyocyanin, S. aureus, B. cenocepacia and E.coli were grown for 24hrs in the presence pyocyanin, pyocyanin+ferritin, and hydrogen peroxide, and absorbance measured over time in separate experiments. S. aureus and E. coli were inhibited by low concentrations of pyocyanin, whilst being resistant to H2O2. Presence of ferritin potentiated the effect of pyocyanin on E. coli but not S. aureus. B. cenocepacia was unaffected by 150μM pyocyanin with or without ferritin, yet inhibited by 30μM H2O2. To assess the effect of Type VI secretions (T6SS) in P. aeruginosa inhibition, we assayed E. coli and B. cenocepacia growth in a PA14ΔT6SS growth filtrate. Both species grew, suggesting that B. cenocepacia inhibition is due to T6SS rather than pyocyanin. Results indicate pyocyanin dictates overall microbial abundance in presence of P. aeruginosa in CF lung with the exception of B. cenocepacia, and CF sputum iron enhances pyocyanin expression as measured by qPCR and quantification of phenazines in P. aeruginosa supernatant.

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