Flavobacterium johnsoniae and othermembers of the phylum Bacteroidetes play a specialized role in the degradation and uptake of dissolved organic material, especially complex biopolymers such as cellulose and chitin, which form part of the high molecular mass fraction of dissolved organic matter in aquatic environments (Kirchman, 2002). F. johnsoniae typically inhabits moist soil and aquatic environments rich in organic matter and the ability of this organism to break down chitin, especially in soil, is believed to play a role in the degradation process of invertebrate carcasses (Bernardet and Bowman, 2006). F. johnsoniae is also frequently isolated from water samples, indicating its ability to survive in the free-living form in aquatic environments. The survival and persistence of Vibrio cholerae in the natural environment is linked to its ability to adhere to and form biofilms on chitinaceous surfaces (Pruzzo et al., 2008; Reguera and Kolter, 2005). The attachment of V. cholerae cells to chitinaceous surfaces and subsequent biofilm formation is mediated through pili, such as type IV pili (Pruzzo et al., 2008; Reguera and Kolter, 2005). The V. cholerae-chitin relationship encompasses several important characteristics of this aquatic pathogen, including physiological responses such as biofilm formation, induction of competence, symbiotic relationship with higher organisms, cycling of nutrients in the aquatic environment and most importantly, pathogenicity to humans and aquatic animals (Pruzzo et al., 2008). It may well be that the colonization of chitinaceous surfaces and detritus-associated biofilm communities gives rise to the abundance of F. johnsoniae in fresh water (Kirchman, 2002). This may serve as a possible explanation for F. johnsoniae- associated fish disease, since water would be mostly deprived of organic matter, compared to soil or detritus aggregates, forcing this organism to enter an attached, pathogenic lifestyle. F. johnsoniae cells lack pili, however, surface filaments have been associated with the ability of this organism to glide on solid surfaces (Liu et al., 2007). Additionally, chitin degradation of F. johnsoniae has been linked to the ability of this organism to glide on solid surfaces. Chang et al. (1984) observed that non-gliding F. johnsoniae mutants lacked the ability to degrade chitin. Disruption of the gldA, gldB, gldD, gldF, gldG and gldI genes involved in F. johnsoniae gliding motility also disrupted its ability to degrade chitin (McBride et al., 2003; McBride and Braun, 2004). The abilities of F. psychrophilum to glide and form biofilms have been found to be antagonistic properties (Álvarez et al., 2006). Similarly, F. johnsoniae-like isolates displaying strong gliding motility exhibit a weak biofilm phenotype when grown on abiotic surfaces (Basson et al., 2008). In addition to a correlation between motility and the chitin degrading ability (McBride and Braun, 2004), a significant negative correlation between biofilm formation and gliding motility of these organisms has been observed (Álvarez et al., 2006; Basson et al., 2008). However, no correlation has been established as yet between the biofilm-forming ability and chitin degradation of F. johnsoniae-like isolates and this requires further investigation. Biofilm formation is generally associated with the production of EPS, especially during the early stages of biofilm development (Danese et al., 2000; Donlan, 2002; Hall-Stoodley and Stoodley, 2002; Jefferson, 2004; Stanley and Lazazzera, 2004; Wang et al., 2004b). EPS often constitute large amounts of extracellular carbohydrate, aiding in cellular attachment and protection of biofilm cells. Kives et al. (2006) found compositional differences in carbohydrate content of biofilm and planktonic P. fluorescence EPS, respectively. A combination of glucuronic and guluronic acid were the main components in biofilm EPS, besides rhamnose, glucose and glusosamine, whereas only glucuronic acid was present as the main carbohydrate in planktonic EPS (Kives et al., 2006). Quantifying the amount of EPS produced by bacteria can be troublesome, not only due to the complex nature of EPS but also because of the difficulty involved in separation of true EPS and other polysaccharides such as LPS (Ryu and Beuchat, 2003). Ryu and Beuchat (2003) developed an assay to estimate the total amount of carbohydrate present in the extracellular layer of carbohydrate produced by cells grown on an agar medium. Fraction I obtained during the isolation of extracellular carbohydrate complexes (ECC) consists of slimy EPS, traces of capsular EPS and other cell surface polysaccharides, including mono- and oligosaccharides secreted by the cell. Fraction II would mainly consist of capsular EPS and cell surface-associated polysaccharides (Ryu and Beuchat, 2003). In V. cholerae, Vibrio polysaccharide (VPS) is associated with the rugose colony phenotype and its biofilm-forming ability (Beyhan et al., 2007). Strains that lack VPS appear as smooth colonies and lack the ability to form complex, mature biofilms (Beyhan et al., 2007). A similar difference in colony morphology, i.e., hazy and smooth, has been observed amongst the F. johnsoniae-like isolates (Flemming et al., 2007). Flavobacterium spp. are known to produce excessive EPS during colonization of solid surfaces and have previously been associated with paper spoilage due to the production of this slimy exudate (Oppong et al., 2003). The differences in extracellular carbohydrate production of smooth and hazy colonial phenotypes are unknown and the role of EPS in Flavobacterium spp. biofilm formation is unclear. Moreover, extracellular polysaccharides and proteins present in bacterial capsules have been suggested to act as adhesins (Decostere et al., 1999a and b; Kroncke et al., 1990). However, the function of this carbohydrate capsular material in F. johnsoniae adherence, virulence and biofilm formation is poorly understood. In F. columnare, the carbohydrate content of capsule is associated with adherence to gill tissue. High virulence strains, with increased ability to adhere to gill tissue, have a thicker capsule layer compared to low virulence strains (Decostere et al., 1999a and b). As with F. columnare, capsule presence has also been observed for F. johnsoniae-like isolates (Flemming, 2006). Comparative quantification of the exopolysaccharide production of F. johnsoniae- like isolates grown in the planktonic and sessile state may reveal the role and relevance of EPS in Flavobacterium spp. biofilm formation. Apart from their application for the rapid identification of many pathogenic bacteria from infectious disease outbreaks, molecular typing techniques are also effective for the classification of bacterial species and phylogeny, as well as for distinguishing between specific phenotypic traits among bacterial strains (Borucki et al., 2003; Somers et al., 2001). The food-borne pathogen, Listeria monocytogenes, has been classified into two major phylogenetic divisions, Division I and II, with the use of molecular analysis and typing techniques, including pulsed-field gel electrophoresis (PFGE) (Borucki et al., 2003; Brosch et al., 1994). Borucki et al. (2003) observed that L. monocytogenes strains from Division II displayed increased biofilm formation compared to strains from Division I. Similarly, genomic profiling with PFGE analysis verified the presence of biofilm-forming strains of nonstarter lactic acid bacteria in cheese spoilage (Somers et al., 2001). Described as the gold standard of molecular typing techniques for bacterial pathogens (Barett et al., 2005), PFGE facilitates the typing of large groups of a broad range of bacterial species (Tenover et al., 1995). The restriction patterns generated by PFGE are highly specific to different strains of bacteria and its high discrimination power adds significant value to investigations of pathogenic organisms responsible for disease outbreaks (Gautom, 1997; Tenover et al., 1995). PFGE has been used to differentiate amongst F. columnare and F. psychrophilum isolates obtained from a diversity of diseased fish species (Arai et al., 2007; Chen et al., 2008; Soto et al., 2008). Following intra-species typing of F. columnare strains obtained from channel catfish, large- mouth bass, red pacu, carp and brown trout, PFGE was found to be a highly reproducible and powerful epidemiological tool for discriminating between F. columnare isolates, regardless of the fish host species (Soto et al., 2008). Arai et al. (2007) demonstrated that PFGE typing of F. psychrophilum isolates, obtained from diseased ayu, using restriction endonucleases BlnI and XhoI, enabled more accurate classification of the isolates compared to conventional RFLP analysis. Although the genetic diversity among South African F. johnsoniae-like isolates has been previously investigated by 16S rRNA gene sequence analysis, 16S rRNA gene PCR restriction fragment length polymorphism (RFLP) analysis, randomly amplified polymorphic DNA (RAPD) PCR and repetitive extragenic palindromic (REP) PCR (Flemming et al., 2007), the genetic relatedness of these isolates still remains unclear. F. johnsoniae-like isolates are known to form biofilms in aquaculture tanks (Basson et al., 2008). The presence of these bacteria in aquaculture and aquatic systems in the biofilm state may contribute to recurrent disease outbreaks in fish, especially trout (Basson et al., 2008; Flemming et al., 2007). Although previously described as an opportunistic fish pathogen, this organism is the leading cause of flavobacterial disease, being the primary infectious agent in trout in South Africa (Flemming et al., 2007). Besides the continuous release of bacterial cells from abiotic surface-associated colonies present in aquaculture settings, detritus- associated communities of microorganisms abundant in freshwater aquatic systems also continuously release bacteria into the surrounding environment (Kirchman, 2002). In the present study, the ability of F. johnsoniae-like isolates to degrade chitin and the amount of ECC produced by these isolates in planktonic phase and from agar surface-associated growth was investigated. The association between these characteristics and the biofilm-forming phenotypes was assessed. Additionally, F. johnsoniae-like isolates were differentiated on the basis of genetic diversity by using PFGE. Since 16S rRNA gene PCR-RFLP analysis provided poor discrimination of F. johnsoniae-like isolates (Flemming et al., 2007), RFLP analysis of the entire genome using PFGE was used for detailed genotyping. The relationship between PFGE patterns and specific biofilm phenotypes was also investigated. In document Comparative proteomic and genomic analysis of Flavobacterium johnsoniae-like biofilm, planktonic and agar surface-associated cells (Page 52-57)
