Thesis (MSc)--University of Stellenbosch, 2007. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/19634 |
| Date | 03 1900 |
| Creators | Jacobs, Anelet |
| Contributors | Chenia, H.Y., Rawlings, D.E., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 217 leaves : ill. |
| Rights | Stellenbosch University |
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