The influence of iron concentration on the production of pyoverdine by Pseudomonas aeruginosa in mono and mixed biofilm cultures

Abstract

Most species of bacteria have the ability to form biofilms, communities of bacterial cells that aggregate in a self-made matrix of extracellular polymeric substances (EPS), that allow them to adhere to inert and organic substances (Banin, Vasil and Greenberg, 2005; Lin et al., 2012). The biofilm is perceived to be the dominant form of bacterial life in the environment (Donlan and Costerton, 2002) and in comparison to planktonic or free swimming cells, sessile or biofilm cells often have a higher tolerance to antibiotics and host defense mechanisms, alluding to their importance in human health and disease (Hentzer, Eberl and Givskov, 2005). One of the many factors regulating biofilm growth and formation is the presence or absence of iron (Lin et al., 2012). Iron is an essential nutrient for growth, in order to acquire iron many bacteria produce siderophores to sequester iron from host proteins and the environment (Re'dly and Poole, 2003). Pyoverdine (PVD) is one of two hydroxamate classed siderophores produced by P. aeruginosa (Meyer et al., 1997; Schalk et al., 2001) and regulated in part by Fur (Imperi, Banin). While iron concentration, pyoverdine production and biofilm formation have been studied in relation to P. aeruginosa, studies of this complex relationship has not yet been conducted in relation to mixed cultures. The aim of this research was to study the influence of iron concentration on the production of pyoverdine by Psuedomonas aeruginosa in mono and mixed biofilm cultures with Staphylococcus aureus Newman strain. An iron assay kit determined the concentration of iron in 1% TSB to be 1.62µM. Pyoverdine levels in P. aeruginosa biofilms were higher in 0.1% TSB biofilms than 1% TSB biofilms. In planktonic growth, pyoverdine concentration was higher in 1% TSB. Crystal violet assays were used to determine the biofilm forming capabilities of P. aeruginosa and S. aureus Newman in differing iron conditions. P. aeruginosa formed more biofilm in 1% TSB, and formed more biofilm than S. aureus Newman in both media. S. aureus, surprisingly, formed more biofilm in 1% TSB as opposed to 0.1%TSB regardless of the fact that S. aureus Newman was been proven to promote biofilm formation only in low iron environments (Johnson, Cockayne and Morrissey, 2008; Lin et al., 2012). The amount of CFU of bacteria in mixed cultures was determined through sonicating biofilm cells into buffer and spread plating. P. aeruginosa dominated the biofilm growth in all 0.1% TSB mixed biofilms, however in 1% TSB mixed culture, S. aureus Newman produced more biofilm than P. aeruginosa when inoculated first. Due to interference within the co-cultures, the pyoverdine levels within the mixed culture biofilms were not established. Further investigation of pyoverdine concentrations in mixed culture biofilms of different iron concentrations would allow for a better understanding of the complex interrelationship between pyoverdine production, biofilm formation and iron concentration. Three factors that contribute significantly to the bacteria's ability to cause infection and mortality.

Date of Award	2018
Original language	English