Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sun, 25 Nov 2018 01:19:10 Iron-binding compounds impair Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions Che Y. O’May, 1 Kevin Sanderson, 1 Louise F. Roddam, 1 Sylvia M. Kirov 2 3 and David W. Reid 1 3 Correspondence David W. Reid D.E.C.Reid@utas.edu.au 1 Menzies Research Institute, University of Tasmania Clinical School, 43 Collins Street, Hobart, Tasmania 7001, Australia 2 School of Medicine, University of Tasmania Clinical School, 43 Collins Street, Hobart, Tasmania 7001, Australia Received 12 August 2008 Accepted 6 January 2009 The success of Pseudomonas aeruginosa in cystic fibrosis (CF) and other chronic infections is largely attributed to its ability to grow in antibiotic-resistant biofilm communities. This study investigated the effects of limiting iron levels as a strategy for preventing/disrupting P. aeruginosa biofilms. A range of synthetic and naturally occurring iron-chelating agents were examined. Biofilm development by P. aeruginosa strain PAO1 and CF sputum isolates from chronically infected individuals was significantly decreased by iron removal under aerobic atmospheres. CF strains formed poor biofilms under anaerobic conditions. Strain PAO1 was also tested under anaerobic conditions. Biofilm formation by this model strain was almost totally prevented by several of the chelators tested. The ability of synthetic chelators to impair biofilm formation could be reversed by iron addition to cultures, providing evidence that these effective chelating compounds functioned by directly reducing availability of iron to P. aeruginosa. In contrast, the biological chelator lactoferrin demonstrated enhanced anti-biofilm effects as iron supplementation increased. Hence biofilm inhibition by lactoferrin appeared to occur through more complex mechanisms to those of the synthetic chelators. Overall, our results demonstrate the importance of iron availability to biofilms and that iron chelators have potential as adjunct therapies for preventing biofilm development, especially under low oxygen conditions such as encountered in the chronically infected CF lung. INTRODUCTION Cystic fibrosis (CF) individuals are highly susceptible to chronic lung infection by the opportunistic environmental bacterium Pseudomonas aeruginosa (Lyczak et al., 2002). This organism is thought to persist by forming biofilms within the hypoxic mucus of the CF lung. Bacteria growing in biofilms exhibit increased resistance to antimicrobials and the host immune response compared to their free- living, planktonic counterparts. Once established, biofilm- dwelling bacteria are virtually impossible to eradicate with existing therapies (Ceri et al., 1999; Singh et al., 2000; Worlitzsch et al., 2002). To improve the quality of life for CF individuals, novel strategies for preventing or delaying P. aeruginosa differentiation into the biofilm growth mode are urgently needed. Reducing iron availability has been proposed as a potential means to impair P. aeruginosa biofilm development (Banin et al., 2006; Musk et al., 2005). We recently demonstrated a strong positive correlation between sputum iron content and quantitative load of P. aeruginosa infection. Moreover, we found that the iron content of airway secretions in CF is elevated in patients from whom P. aeruginosa has not been isolated, suggesting that increased iron in the CF lung may be a critical factor in facilitating initial acquisition and then chronicity (Reid et al., 2007). These in vivo observations support the hypothesis that limiting bacterial iron availability in the CF lung may be a strategy that could be used to disrupt the success of P. aeruginosa. The recent publication by Moreau-Marquis et al. (2008) demonstrating that cultured CF airway epithelial cells promote P. aeruginosa biofilm formation because of an inherent problem with epithelial cell iron sequestration and luminal loss support our avenue of investigation into iron chelation. There are also emerging reports on the importance of iron to P. aeruginosa biofilm formation, Abbreviations: 2DP, 2,29-dipyridyl; CF, cystic fibrosis; DM, deferoxamine mesylate; DTPA, diethylenetriaminepentacetic acid; EDDA, ethylenedia- mine-N,N9-diacetic acid. 3These authors contributed equally to this work. Journal of Medical Microbiology (2009), 58, 765–773 DOI 10.1099/jmm.0.004416-0 004416 G 2009 SGM Printed in Great Britain 765