Evaluation of Adherence, Hydrophobicity, Aggregation, and Biofilm Development of Flavobacterium johnsoniae-Like Isolates

Abstract

Flavobacterium spp. isolates have been identified in diverse biofilm structures, but the mechanism of adherence has not been elucidated. The absence of conventional biofilm-associated structures such as fimbriae, pili, and flagella suggest that surface hydrophobicity, and/or autoaggregation and coaggregation may play an important role in adherence and biofilm formation. The biofilm-forming capacity of 29 Flavobacterium johnsoniae-like isolates obtained from South African aquaculture systems was assessed using microtiter plate assays. The role of hydrophobicity [salting aggregation test (SAT) and bacterial adherence to hydrocarbons (BATH) assays], autoaggregation, and coaggregation on biofilm formation by Flavobacterium spp. was also investigated, while biofilm structure was examined using flow cells and microscopy. All isolates displayed a hydrophilic nature, but showed varying levels of adherence in microtiter assays. Significant negative correlations were observed between adherence and biofilm-forming capacity in nutrient-poor medium at 26 degrees C and BATH hydrophobicity and motility, respectively. Isolates displayed strain-to-strain variation in their autoaggregation indices and their abilities to coaggregate with various Gram-negative and Gram-positive organisms. Microcolony and/or biofilm development were observed microscopically, and flavobacterial isolates displayed stronger biofilm structures and interaction with a Vibrio spp. isolate than with an Aeromonas hydrophila isolate. The role of extracellular polysaccharides and specific outer membrane proteins will have to be examined to reveal mechanisms of adherence and coaggregation employed by biofilm-forming F. johnsoniae-like strains.

Effect of physiochemical factors on autoaggregation and adhesion of Flavobacterium johnsoniae-like isolates

“…Twelve (12) previously characterized F. johnsoniae-like isolates (YO12, YO15, YO19, YO34, YO45, YO51, YO53, YO59, YO60, YO63, YO64, and YO67) isolated from eel, koi-carp and trout were selected for study based on their biofilm-forming abilities (Flemming et al, 2007;Basson et al, 2008). Isolates were divided into two groups (Table 1) based on their colony morphotypes (Flemming et al, 2007), that is, smooth (YO19, YO34, YO51, YO59, YO63, YO64 and YO67) and hazy (YO12, YO15, YO45, YO53 and YO60).…”

“…Isolates were divided into two groups (Table 1) based on their colony morphotypes (Flemming et al, 2007), that is, smooth (YO19, YO34, YO51, YO59, YO63, YO64 and YO67) and hazy (YO12, YO15, YO45, YO53 and YO60). The associated motility characteristics, biofilm phenotypes and hydrophobicity of isolates are indicated in Table 1 (Flemming et al, 2007;Basson et al, 2008 …”

“…Percentage of autoaggregation was measured by transferring a 1 ml sample of bacterial suspension to a sterile 2 ml cuvette and measuring the OD after 60 min at a wavelength of 660 nm (Basson (Flemming et al, 2007;Basson et al, 2008Basson et al, ). et al, 2008.…”

“…Sixty min after the OD 0 was obtained, the cells suspensions were centrifuged at 2 000 rpm for 2 min and the optical density (OD 60 ) determined. Experiments were carried out in triplicate on two separate occasions (Basson et al, 2008). The inhibitory effects of four treatments on autoaggregation were investigated for bacteria that were grown in 50 ml EAOB at 26°C with agitation on a flatbed shaker (120 rpm) for 36 h, harvested, washed three times with sterile 0.1 M phosphate buffer (pH 7), and resuspended in sterile dH 2 O to an OD 660 nm of 0.3.…”

“…Members of the genus Flavobacterium are known to form biofilms (Álvarez et al, 2006;Basson et al, 2008), not only as a stress response but as an adaptation to allow persistence of cells on the outer surface of the fish host during the early stages of infection (Staroscik and Nelson, 2008) or to survive as saprophytes in the fish farm environment (Kunttu et al, 2009). Adhesion to the host's surface is an essential step in the primary colonization of the host by a pathogenic bacterium, and pathogenic Flavobacterium spp.…”