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The Biofilm Matrix Destabilizers, EDTA and Dnasei, Enhance The ORIGINAL RESEARCH The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin Rosalia Cavaliere1, Jessica L. Ball2, Lynne Turnbull1 & Cynthia B. Whitchurch1 1The ithree Institute, University of Technology Sydney, Sydney, New South Wales, Australia 2Department of Microbiology, Monash University, Clayton, Victoria, Australia Keywords Abstract Antibiotic, biofilm, cations, eDNA, matrix. Nontypeable Hemophilus influenzae (NTHi) is a Gram-negative bacterial patho- Correspondence gen that causes chronic biofilm infections of the ears and airways. The biofilm Cynthia B. Whitchurch, P.O. Box 123, matrix provides structural integrity to the biofilm and protects biofilm cells Broadway, NSW 2007, Australia. from antibiotic exposure by reducing penetration of antimicrobial compounds Tel: +61 2 9514 4144; into the biofilm. Extracellular DNA (eDNA) has been found to be a major Fax: +61 2 9514 4143; matrix component of biofilms formed by many species of Gram-positive and E-mail: [email protected] Gram-negative bacteria, including NTHi. Interestingly, the cation chelator ethy- Funding Information lenediaminetetra-acetic acid (EDTA) has been shown to reduce the matrix C. B. W. was supported by an Australian strength of biofilms of several bacterial species as well as to have bactericidal National Health and Medical Research activity against various pathogens. EDTA exerts its antimicrobial activity by Council Senior Research Fellowship (571905). chelating divalent cations necessary for growth and membrane stability and by L. T. was supported by a UTS Chancellor’s destabilizing the matrix thus enhancing the detachment of bacterial cells from Postdoctoral Fellowship. Confocal laser the biofilm. In this study, we have explored the role of divalent cations in scanning microscopy was performed at the NTHi biofilm development and stability. We have utilized in vitro static and UTS Microbial Imaging Facility. This study was supported through funding from the continuous flow models of biofilm development by NTHi to demonstrate that Australian National Health and Medical magnesium cations enhance biofilm formation by NTHi. We found that the Research Council (491126) and the Financial divalent cation chelator EDTA is effective at both preventing NTHi biofilm for- Markets Foundation for Children. mation and at treating established NTHi biofilms. Furthermore, we found that the matrix destablilizers EDTA and DNaseI increase the susceptibility of NTHi Received: 12 January 2014; Revised: 9 May biofilms to ampicillin and ciprofloxacin. Our observations indicate that DNaseI 2014; Accepted: 28 May 2014 and EDTA enhance the efficacy of antibiotic treatment of NTHi biofilms. These MicrobiologyOpen 2014; 3(4): 557–567 observations may lead to new strategies that will improve the treatment options available to patients with chronic NTHi infections. doi: 10.1002/mbo3.187 Introduction patients with cystic fibrosis, otitis media, and COPD have been shown to form biofilms in vitro and in vivo (Greiner Nontypeable Hemophilus influenzae (NTHi) is an oppor- et al. 2004; Starner et al. 2006; Moriyama et al. 2009; tunistic pathogen associated with respiratory and ear Swords 2012). infections in both children and adults. It has been espe- Biofilms are structured aggregates of bacteria that are cially implicated in acute exacerbations and chronic infec- encased in a self-produced polymeric matrix (Costerton tions in patients with conditions such as otitis media with et al. 1995, 1999). The matrix which holds the bacterial effusion, chronic obstructive pulmonary disease (COPD), biofilm together is a complex mixture of macromolecules and cystic fibrosis (Ehrlich et al. 2002; Hall-Stoodley et al. including exopolysaccharides, proteins, and DNA (Suther- 2006; Starner et al. 2006). Strains of NTHi isolated from land 2001), which are collectively called extracellular ª 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 557 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Magnesium Cations Enhance NTHi Biofilm Development R. Cavaliere et al. polymeric substances (EPS). The EPS matrix is highly Extracellular DNA (eDNA) has been shown to be hydrated and has various roles, including adhesion of the required for biofilm development by P. aeruginosa biofilm to surfaces, cell–cell cohesion, sequestering of (Steinberger et al. 2002; Whitchurch et al. 2002; Matsuk- toxic substances from the environment, and protection awa and Greenberg 2004; Allesen-Holm et al. 2006). from predators and the host immune system (Costerton Addition of the eDNA degrading enzyme DNase I inhib- et al. 1981; Donlan and Costerton 2002). its biofilm development by P. aeruginosa and established Biofilm infections are particularly problematic, as they P. aeruginosa biofilms can be effectively dispersed by are not dealt with effectively by the host’s immune system treatment with eDNA degrading enzymes (Whitchurch and are recalcitrant to treatment with antimicrobials. et al. 2002; Nemoto et al. 2003). Interestingly, in P.aeru- Indeed, studies have shown that NTHi biofilm cells exhibit ginosa, eDNA has been shown to act as a cation chelator increased resistance to killing by antibiotics compared to due to its highly anionic character and electrostatic the resistance exhibited by planktonic cells (Slinger et al. interactions between negatively charged DNA, and 2006; Starner et al. 2006; Izano et al. 2009). Although the cations such as Mg2+ and Ca2+, are thought to stabilize basis of biofilm-associated antibiotic resistance is not fully the eDNA matrix (Mulcahy et al. 2008; Lewenza 2013). understood, it is likely that multiple mechanisms operate Thus it is conceivable that the ability of EDTA to dis- simultaneously in biofilms to contribute to antibiotic resis- perse P. aeruginosa biofilms occurs, at least, in part tance. One such protective mechanism is thought to be the through cation sequestration leading to destabilization of sequestration and restricted penetration of antibiotics the eDNA matrix. through the biofilm matrix (Brown et al. 1988; de Beer It is now becoming increasingly apparent that eDNA is et al. 1997; Donlan and Costerton 2002; Fux et al. 2005). a major matrix component in biofilms formed by both Many studies have demonstrated the roles of calcium, Gram-positive and Gram-negative bacteria and that the magnesium, and iron divalent cations in promoting cell eDNA in the biofilm matrix plays an important role in growth and cell-to-cell adhesion in biofilms. Divalent disease pathogenesis and resistance to antimicrobials cations appear to stabilize the biofilm matrix of a variety (Costerton et al. 1981; Mulcahy et al. 2008; Chiang et al. of organisms by enhancing structural integrity through 2013; Jakubovics et al. 2013; Lewenza 2013). The matrix electrostatic interactions that serve to cross-link the of NTHi biofilms is comprised largely of eDNA, which matrix. Divalent cations have also been shown to enhance constitutes the major volumetric component of NTHi the interaction of a mucoid strain of Pseudomonas aeru- biofilms (Jurcisek and Bakaletz 2007; Izano et al. 2009). ginosa with tracheal epithelium (Marcus et al. 1989). In Despite their potentially important role in stabilizing the some bacteria, divalent cations also enhance EPS produc- eDNA matrix of NTHi biofilms through electrostatic tion. For example, mutations in the magnesium up-take interactions, divalent cations have not been well studied system of Aeromonas hydrophilia cause a reduction of bio- with regard to their roles in NTHi biofilms. The purpose film formation and swarming motility (Merino et al. of this study was to firstly investigate the role of divalent 2001) and divalent cations, in particular Mg2+, were cations in biofilm formation in a clinical isolate of NTHi, found to greatly enhance EPS production by Staphylococ- when supplied at physiologically relevant concentrations. cus epidermidis strains (Ozerdem Akpolat et al. 2003). In Secondly, we wanted to identify factors that may contrib- contrast, in the presence of the cation chelator ethylenedi- ute to the breakdown of NTHi biofilms by destablilizing aminetetra-acetic acid (EDTA), EPS production by S. epi- the eDNA matrix and to explore the effects of combining dermidis was significantly reduced (Ozerdem Akpolat these matrix destablilizers with antibiotics to treat NTHi et al. 2003). As divalent cations also contribute to the biofilms. integrity and stability of the outer membrane of Gram- negative bacteria (Leive and Kollin 1967; Geesey et al. Methods 2000), EDTA likely exerts antimicrobial activity by chelat- ing cations necessary for growth and membrane stability Bacterial strains and culture conditions and may also display anti-biofilm activity by reducing EPS production and/or enhancing the detachment of bac- The NTHi strain used in this study (strain 502) was terial cells from the biofilm (Banin et al. 2006). EDTA obtained from an ear infection and was provided by the has been shown to have bactericidal activity against both Royal Children’s Hospital, Melbourne. NTHi 502 was cul- Staphylococcus aureus and P. aeruginosa biofilm cells and tured overnight in brain heart infusion (BHI) broth or in causes dispersal of P. aeruginosa cells from the biofilm chemically defined medium pH 9 (CDM) (Coleman et al. (Banin et al. 2006). Furthermore, a
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