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

[摘要] ENGLISH ABSTRACT: In the aquaculture setting, opportunistic pathogens are present as part of the normalaquatic microflora, colonizing surfaces in fish tanks as part of biofilm communities, andoften causing severe economic losses to the aquacultural industry. Isolates belonging tothe genera Chryseobacterium, Elizabethkingia, Myroides and Empedobacter have beenisolated from diseased fish, and are responsible for causing secondary fish infections,fish- and food-product spoilage, and have been described as etiological agents of varioushuman diseases. Thirty-four Chryseobacterium and Elizabethkingia spp. and fiveMyroides and Empedobacter spp. isolates, obtained from various diseased fish speciesand biofilm growth in South African aquaculture systems, were characterised geneticallyusing 16S rRNA gene PCR restriction fragment length polymorphism (RFLP), randomlyamplified polymorphic DNA (RAPD) PCR, whole cell protein (WCP) and outermembrane protein (OMP) analyses. Genetic heterogeneity was displayed by theMyroides and Empedobacter spp. study isolates following OMP analysis, although 16SrRNA gene RFLP, RAPD-PCR and WCP analysis did not allow for differentiation ofthese isolates. A high degree of genetic heterogeneity was displayed by theChryseobacterium and Elizabethkingia spp. study isolates following OMP analysis, 16SrRNA gene RFLP with MspI, and RAPD-PCR with primer P2. However, based on theresults obtained by WCP analysis, 16S rRNA gene RFLP with CfoI and TaqI, andRAPD-PCR with primer P1 the isolates appeared genetically very homogeneous. HighMAR indices and potential multi-drug resistance phenotypes were obtained for theMyroides and Empedobacter spp. and some of the Chryseobacterium and Elizabethkingiaspp. isolates by antimicrobial susceptibility testing. Primary adherence and the influenceof environmental changes on adherence was investigated by a modified microtitre-plateadherence assay. Nutrient composition, temperature and hydrodynamic incubationconditions were observed to influence adherence abilities of all study isolates. Inaddition, adherence varied greatly among isolates of the genera Chryseobacterium andElizabethkingia, as opposed to a consistent strong adherence profile observed for theMyroides and Empedobacter spp. isolates. The influence of cell surface properties suchas capsule presence and cell surface hydrophobicity, on primary adherence of the isolateswas also investigated. Quantitative analysis of capsular material revealed the presence of thick capsular material surrounding the Myroides and Empedobacter spp. and some of theChryseobacterium and Elizabethkingia spp. isolates, but could not be directly associatedwith adherence. Hydrophobicity were investigated using the salt aggregation assay(SAT) and bacterial adherence to hydrocarbon test (BATH). A very hydrophilic cellsurface was observed for all of the Myroides and Empedobacter spp. isolates, andmajority (74%) of the Chryseobacterium and Elizabethkingia spp. isolates. Cell surfacehydrophobicity could not be correlated to the adherence of the Myroides andEmpedobacter spp. isolates, and only SAT-determined hydrophobicity could bepositively correlated to adherence of Chryseobacterium and Elizabethkingia spp. isolatesunder certain conditions. Coaggregation studies were performed between the studyisolates and various important clinical and aquacultural microorganisms. Highcoaggregation indices were observed between the Myroides and Empedobacter spp.isolates and E. faecalis and S. aureus, and between E. faecalis, S. enterica serovarArizonae, S. aureus and Listeria spp. and the Chryseobacterium and Elizabethkingia spp.isolates. Biofilm-forming capacity of the study isolates in an environment simulatingtheir natural environment was investigated microscopically using a flow cell system.Typical 'cone-like' biofilm structures were observed for selected strains of both Myroidesand Empedobacter spp. and Chryseobacterium and Elizabethkingia spp. isolates. Theeffect of increased hydrodynamics on biofilm architecture was seen through thenarrowing of the biofilm structures and the formation of single cell chains towards theincreased hydrodynamic area of the flow chambers. Chryseobacterium andElizabethkingia spp. and Myroides and Empedobacter spp. appear to be potential primarybiofilm-formers associating with a variety of microbes thus perpetuating their survival ina variety of aquatic habitats.

[发布机构] Stellenbosch University