Growing old together: processes that lead to biofilm maturation
Raising the shields: the composition and function of the extracellular matrix
One of the key changes that initiate/accompany irreversible attachment to the surface is the production of an encapsulating ECM by the adherent bacteria. The ECM is considered a hallmark feature of biofilms and serves to protect and hold resident bacteria together (Fig. 3.2(d)).
Composition of the extracellular matrix
The composition of the ECM varies by bacterial species and can also vary among strains of the same species. ECM composition is subject to the environmental conditions in which a bacterial biofilm is grown including, but not limited to temperature, nutrient resources, and oxygen concentration. Typically the ECM comprises a combination of lipids, polysaccharides, eDNA, and proteins (Flemming and Wingender, 2010; Sutherland, 2001; Branda et al., 2005; Izano et al., 2008; Vilain et al., 2009; Tetz et al., 2009; Koo et al., 2010; Bokranz et al., 2005; Thomas et al., 2009; Friedman and Kolter, 2004; Steinberg and Kolodkin-Gal, 2015; Zogaj et al., 2001; Hung et al., 2013; McCrate et al., 2013). Almost all adhesive components discussed in Section 3.3 of this chapter can also be found as part of the bacterial ECM under the appropriate growth conditions.
In many important biofilm-forming pathogens, the exopolysaccharide component is the most prevalent ECM constituent. Studies in P. aeruginosa have uncovered the presence of up to three types of matrix exopolysaccharides secreted by different strains: Psl, Pel, and alginate (Hatch and Schiller, 1998; Hentzer et al., 2001; Ma et al., 2006, 2007; Yang et al., 2007, 2011; Leid et al., 2005; Whitchurch et al., 2002; Friedman and Kolter, 2004; Toyofuku et al., 2012; Jackson et al., 2004; Vasseur et al., 2005; Zhao et al., 2013). Psl is a galactose- and mannose-rich exopolysaccharide (Ma et al., 2007). The structure and synthesis of the Pel polysaccharide was only elucidated in 2015. Experimental evidence supports that Pel is composed of partially acetylated linked glucosamine and galactosamine sugars (Jennings et al., 2015) and has been demonstrated to facilitate cell-to-cell adhesion within the P. aeruginosa biofilm ECM to maintain biomass structure and integrity (Colvin et al., 2013). To achieve this function, Pel has been shown to crosslink extracellular DNA within the P. aeruginosa
ECM. The presence of Pel can compensate for the absence of Psl to maintain biofilm integrity in some strains of P. aeruginosa (Jennings et al., 2015; Colvin et al., 2012). This redundancy highlights the necessity and variability of polysaccharides within the ECM. Alginate is a polymeric polysaccharide made up of guluronate and mannuro- nate moieties (Ramsey and Wozniak, 2005). Alginate has been shown to be a major constituent of primarily mucoid strains of P. aeruginosa that have a detrimental impact on opportunistic infection of patients with cystic fibrosis (Ramsey and Wozniak, 2005; Wozniak et al., 2003).
The exopolysaccharide component of the ECM produced by E. coli (Rowe et al., 2010; Hung et al., 2013; McCrate et al., 2013) and K. pneumoniae (Huertas et al., 2014; Zogaj et al., 2003) is primarily comprised of cellulose. Gram-positive pathogens produce an array of polysaccharides to incorporate within their ECM. S. aureus and S. epidermidis incorporate poly-N-acetylglucosamine (PNAG) into their matrix (Maira-Litran et al., 2002; Gokgen et al., 2013; Lin et al., 2015; Cerca et al., 2011).
eDNA has also been shown to be a component of the biofilm ECM formed by many bacterial species (Okshevsky and Meyer, 2015). This DNA can be self-secreted or come from lysed bacteria within the community, and serves to bolster biofilm infrastructure. For example, Gram-positive pathogens such as E. faecalis (Barnes et al., 2012), Listeria monocytogenes (Harmsen et al., 2010), Bacillus cereus (Vilain et al.,
2009), S. pneumoniae (Moscoso et al., 2006), and S. aureus (Schwartz et al., 2015; Grande et al., 2014; Izano et al., 2008; Dengler et al., 2015), as well as Gram-negative pathogens such as P. aeruginosa (Steinberger and Holden, 2005; Wilton et al., 2015), E. coli (Sanchez-Torres et al., 2010; Devaraj et al., 2015), Campylobacter jejuni (Svensson et al., 2014; Brown et al., 2015a,b), and N. menengitidis (Lappann et al., 2010), have all been shown to incorporate eDNA in their ECM under certain environmental conditions.
While largely playing a role in maintaining structure and adhesion within the ECM, eDNA has also been shown to enhance biofilm tolerance to antimicrobials. Specifically, in P. aeruginosa biofilms, high levels of eDNA increase the acidity of the matrix, which signals the bacteria to increase expression of resistance mechanisms to antimicrobial aminoglycosides through the PhoPQ and PmrAB two-component systems (Wilton et al., 2015).
Proteins constitute another critical constituent of the ECM for many biofilm-forming bacterial species. Under certain growth conditions, the ECM produced by a UPEC cystitis strain was determined to consist of ~85% adhesive curli amyloid fibers (McCrate et al., 2013). Some strains of S. epidermidis may utilize the polysaccharide intracellular adhesin (PIA) consisting of PNAG, or the accumulation-associated protein (Aap) to maintain biofilm integrity (Rohde et al., 2005, 2007). Aap has also been shown to play a key role in S. epidermidis biofilms that have been isolated from prosthetic hip and knee joints in patients (Rohde et al., 2007). The ECM from biofilms of other Gram-positive bacteria, such as those formed by Bacillus subtilis, have been shown to consist primarily of a protein component known as TasA (Branda et al., 2006). These examples demonstrate how in some cases a primarily proteinaceous matrix is responsible for maintaining community integrity; however, the organization and foundation of the ECM often includes a complex interplay between the different types of matrix constituents (Payne and Boles, 2015). In both S. aureus (Dengler et al., 2015) and E. coli (Justice et al., 2012; Devaraj et al., 2015), DNA-binding proteins have been shown to maintain the interactions of eDNA with the ECM for proper adhesion. In some P. aeruginosa strains, eDNA and the polysaccharide Psl have been shown to interact with one another to form the web-like infrastructure of the ECM (Wang et al., 2015). These examples demonstrate that while the ECM is made up of different constituents, the interplay between these constituents is vital for optimal function of the matrix.