Investigation and comparison of adherence- and biofilm-forming capacities of yellow-pigmented Chryseobacterium, Elizabethkingia and Myroides spp. isolated from South African aquaculture systems

In the aquaculture setting, opportunistic pathogens are present as part of the normal aquatic microflora, colonizing surfaces in fish tanks as part of biofilm communities, and often causing severe economic losses to the aquacultural industry. Isolates belonging to the genera Chryseobacterium, Elizabethkingia, Myroides and Empedobacter have been isolated from diseased fish, and are responsible for causing secondary fish infections, fishand food-product spoilage, and have been described as etiological agents of various human diseases. Thirty-four Chryseobacterium and Elizabethkingia spp. and five Myroides and Empedobacter spp. isolates, obtained from various diseased fish species and biofilm growth in South African aquaculture systems, were characterised genetically using 16S rRNA gene PCR restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD) PCR, whole cell protein (WCP) and outer membrane protein (OMP) analyses. Genetic heterogeneity was displayed by the Myroides and Empedobacter spp. study isolates following OMP analysis, although 16S rRNA gene RFLP, RAPD-PCR and WCP analysis did not allow for differentiation of these isolates. A high degree of genetic heterogeneity was displayed by the Chryseobacterium and Elizabethkingia spp. study isolates following OMP analysis, 16S rRNA gene RFLP with MspI, and RAPD-PCR with primer P2. However, based on the results obtained by WCP analysis, 16S rRNA gene RFLP with CfoI and TaqI, and RAPD-PCR with primer P1 the isolates appeared genetically very homogeneous. High MAR indices and potential multi-drug resistance phenotypes were obtained for the Myroides and Empedobacter spp. and some of the Chryseobacterium and Elizabethkingia spp. isolates by antimicrobial susceptibility testing. Primary adherence and the influence of environmental changes on adherence was investigated by a modified microtitre-plate adherence assay. Nutrient composition, temperature and hydrodynamic incubation conditions were observed to influence adherence abilities of all study isolates. In addition, adherence varied greatly among isolates of the genera Chryseobacterium and Elizabethkingia, as opposed to a consistent strong adherence profile observed for the Myroides and Empedobacter spp. isolates. The influence of cell surface properties such as capsule presence and cell surface hydrophobicity, on primary adherence of the isolates was also investigated. Quantitative analysis of capsular material revealed the presence of thick capsular material surrounding the Myroides and Empedobacter spp. and some of the Chryseobacterium and Elizabethkingia spp. isolates, but could not be directly associated with adherence. Hydrophobicity were investigated using the salt aggregation assay (SAT) and bacterial adherence to hydrocarbon test (BATH). A very hydrophilic cell surface was observed for all of the Myroides and Empedobacter spp. isolates, and majority (74%) of the Chryseobacterium and Elizabethkingia spp. isolates. Cell surface hydrophobicity could not be correlated to the adherence of the Myroides and Empedobacter spp. isolates, and only SAT-determined hydrophobicity could be positively correlated to adherence of Chryseobacterium and Elizabethkingia spp. isolates under certain conditions. Coaggregation studies were performed between the study isolates and various important clinical and aquacultural microorganisms. High coaggregation indices were observed between the Myroides and Empedobacter spp. isolates and E. faecalis and S. aureus, and between E. faecalis, S. enterica serovar Arizonae, S. aureus and Listeria spp. and the Chryseobacterium and Elizabethkingia spp. isolates. Biofilm-forming capacity of the study isolates in an environment simulating their natural environment was investigated microscopically using a flow cell system. Typical ‘cone-like’ biofilm structures were observed for selected strains of both Myroides and Empedobacter spp. and Chryseobacterium and Elizabethkingia spp. isolates. The effect of increased hydrodynamics on biofilm architecture was seen through the narrowing of the biofilm structures and the formation of single cell chains towards the increased hydrodynamic area of the flow chambers. Chryseobacterium and Elizabethkingia spp. and Myroides and Empedobacter spp. appear to be potential primary biofilm-formers associating with a variety of microbes thus perpetuating their survival in a variety of aquatic habitats.