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

Jacobs, Anelet (Stellenbosch : Stellenbosch University, 2007-03)

Thesis (MSc)--University of Stellenbosch, 2007.

Thesis

ENGLISH ABSTRACT: 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, fish- and 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.

AFRIKAANSE OPSOMMING: Opportunistiese patogene kom gereeld in akwakultuur sisteme voor as deel van die akwatiese mikroflora wat dikwels biofilms vorm op oppervlaktes in hierdie sisteme. Visinfeksies veroorsaak deur hierdie patogene lei tot ernstige ekonomiese verliese vir akwakultuur industrieë. Chryseobacterium, Elizabethkingia, Myroides en Empedobacter spp. is reeds voorheen van verskeie geïnfekteerde visspesies geïsoleer hierdie bakterieë is verantwoordelik vir sekondere visinfeksies, die bederf van vis- en kosprodukte, asook menslike siektes. Vier-en-dertig Chryseobacterium en Elizabethkingia spp. en 5 Myroides en Empedobacter spp. isolate, geïsoleer vanaf verskeie geïnfekteerde visspesies en biofilm-groei in Suid Afrikaanse akwakultuur-sisteme, is geneties met behulp van 16S rRNS geen PKR restriksie fragment lengte polimorfisme (RFLP), toevallig geamplifiseerde polimorfiese DNS (TGPD) PKR, heel-sel protein (HSP) en buitemembraan protein (BMP) analise gekarakteriseer. BMP analise het getoon dat die Myroides en Empedobacter spp. isolate geneties heterogeen is, alhoewel 16S rRNS TGPD-PKR, TGPD-PKR en HSP analise nie tussen die isolate kon onderskei nie. BMP analise, 16S rRNS TGPD-PKR met MspI en TGPD-PKR met inleier P2 was meer suksesvol as HSP analise, 16S rRNS TGPD-PKR met CfoI en MspI, en TGPD-PKR met inleier P1, om onderskeid te tref tussen die Chryseobacterium en Elizabethkingia spp. isolate en het gedui op ‘n hoë vlak van genetiese heterogeniteit tussen hierdie isolate. Beide die Chryseobacterium en Elizabethkingia spp. en Myroides en Empedobacter spp. isolate het ‘n hoë vlak van antibiotika weerstand getoon wat dui op ‘n menigvuldigde antibiotika weerstands-fenotiepe. Primêre vashegting vermoëns en die invloed van omgewingsfaktore op vashegting is met behulp van ‘n gemodifiseerde mikrotiterplaat vashegtings toets ondersoek. Vashegting van die isolate is beïnvloed deur variasies in die samestelling van die medium, temperatuurveranderings en verskillende hidrodinamiese inkubasie kondisies. Inteenstelling met die sterk vashegtingsvermoë van die Myroides en Empedobacter spp. isolate, het die vermoë om vas te heg grootliks tussen die Chryseobacterium en Elizabethkingia spp. isolate gevarieer. Verder is ondersoek ingestel op die invloed van seloppervlak eienskappe soos die teenwoordigheid van kapsules en hidrofobisiteit op die isolate se vermoë om aan oppervlaktes te heg. Die Myroides en Empedobacter spp. isolate en verskeie Chryseobacterium en Elizabethkingia spp. isolate is omring deur dik kapsules, maar geen verband tussen vashegting en die teenwoordigheid van kapsules kon bepaal word nie. Die sout aggregasie toets (SAT) en bakteriële vashegting aan koolwaterstowwe (BVAK) toets was gebruik om die hidrofobisiteit van die isolate se seloppervlaktes te bepaal. Die Myroides en Empedobacter spp. isolate en 74% van die Chryseobacterium en Elizabethkingia spp. isolate het ‘n baie hidrofiliese seloppervlak getoon. Slegs die hidrofobisiteit bepaal deur die SAT toets het ‘n positiewe verwantskap met die aanhegtingsvermoë van die Chryseobacterium en Elizabethkingia spp. isolate getoon. Mede-aggregasie tussen die isolate en verskeie belangrike mediese en akwakultuur mikroörganismes is ook ondersoek. Die Myroides en Empedobacter spp. isolate het ‘n sterk assosiasie met E. faecalis en S. aureus getoon Die Chryseobacterium en Elizabethkingia spp. isolate het sterk met E. faecalis, S. aureus, S. enterica serovar Arizonae en Listeria spp. geassosieer. Vloei-sel studies is uitgevoer om die biofilm-vormingsvermoë van die isolate te ondersoek. Vir beide die Myroides en Empedobacter spp. en Chryseobacterium en Elizabethkingia spp. isolate is tipiese kegelagtige biofilm stukture waargeneem. Die invloed van verhoogde hidrodinamiese kondisies in die vloei-sel het vernouing van die biofilm strukture en die vorming van enkel-sel kettings tot gevolg gehad. Vanuit hierdie studie is afgelei dat die Myroides en Empedobacter spp. en Chryseobacterium en Elizabethkingia spp. isolate onder verskeie kondisies aan oppervlaktes kan vasheg en dus potensiële primêre biofilm-vormings organismses is. Hierdie organismes besit ook die vermoë om met ‘n verskeidenheid ander organismes te assosieer, wat waarskynlik hulle suksesvolle oorlewing in akwakultuursisteme verseker.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/19634
This item appears in the following collections: