Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Microorganisms show a tendency to accumulate on surfaces in aqueous environments to form biofilms. Microbial biofilms represent a significant problem in public health microbiology as the development of these microbial communities, especially in water distribution systems, may lead to (i) the enhanced growth of opportunistic pathogens, (ii) the development of organoleptic problems, (iii) the reduction in the flow rate and (iv) the regrowth of microorganisms. In this project, biofilm monitors were installed in a large water distribution system to study biofilm phenomena in drinking water systems, and to deduce the biological stability and quality of the potable water. Measurements of biofilm formation potential showed that biofilms did not reach a steady state after 100 to 150 days. The microbial cells in these biofilms were mostly non-culturable. The contribution of the heterotrophic colony count to active biomass, as determined with cell numbers based on ATP measurements were often < 1%, while the ratio of heterotrophic plate counts and direct acridine orange counts were also <1%. The ratio between cell numbers based on ATP measurements and direct acridine orange counts were often < 100%. Results also showed that under certain conditions, such as those investigated in the present study, 1 pg of ATP may not be equal to approximately 104 active bacteria/cells, as stipulated by previous investigations, and that the average ATP content per active bacterial cell is indeed less than 10-16 - 10-15 g. It was calculated that threshold values for assimilable, and dissolved organic carbon below -5 IJg Gil and -0.5 mg Gil, respectively, should be target values for the control of biofilm formation in this system. It was shown that polyethylene, polyvinylchloride, teflon, plexiglass, copper, zinc-coated steel and aluminium provide favourable attachment surfaces that allowed primary colonisation and subsequent biofilm formation. Significant (p < 0.05) differences in surface colonisation on the materials were observed, indicating that the composition of the material has a direct influence on microbial colonisation. The two grades of stainless steel evaluated in this study were the least favourable materials for biofilm formation. It was further demonstrated that the nature of the surface of these materials, flow conditions and water type all had a direct influence on biofilm formation. While modification of the attachment surface did not result in significant differences (p > 0.05) in disinfection efficiency of two commonly used biocides, the concentration of the biocide, as well as the material to which the biofilm is attached, greatly influenced biocidal efficiency. The results show that biofilm monitoring needs to be implemented at the water treatment plants in addition to common biostability measurements. / AFRIKAANSE OPSOMMING: Mikro-organismes neig om te akkumuleer aan oppervlaktes in akwatiese omgewings om biofilms te vorm. Mikrobiese biofilms verteenwoordig In betekenisvolle probleem in publieke gesondheidsmikrobiologie omdat die ontwikkeling van hierdie mikrobiese gemeenskappe in waterverspreidingsisteme mag lei tot (i) die verhoogde groei van opportunistiese patogene, (ii) ontwikkeling van organoleptiese probleme, (iii) die vermindering in die vloeitempo en (iv) die hergroei van mikro-organismes. In hierdie projek was biofilm monitors geïnstalleer in In groot waterverspreidingsisteem om biofilm fenomene in drinkwatersisteme to bestudeer, en om die biologiese stabiliteit en kwaliteit van drinkwater af te lei. Bepalings van biofilmvormingspotensiaal het aangetoon dat biofilms nie In stabiele stadium na 100 tot 150 dae bereik nie. Die mikrobiese selle in hierdie biofilms was meestal niekweekbaar. Die bydrae van die heterotrofiese kolonie tellings tot aktiewe biomassa, soos bepaal deur seltellings gebaseer op ATP metings was dikwels < 1%, terwyl die verhouding van die heterotrofiese plaatteIIings en direkte akridien oranje tellings ook < 1% was. Die verhouding tussen seltellings, gebaseer op ATP metings en direkte akridien oranje tellings was dikwels < 100%. Resultate het ook aangetoon dat onder sekere omstandighede, soos dié wat ondersoek was in die huidige studie, 1 pg ATP nie gelyk is aan min of meer 104 aktiewe bakterieë/selle soos gestipuleer deur vorige ondersoeke nie, en dat die gemiddelde ATP inhoud per aktiewe bakteriële sel inderdaad minder as 10-16 tot 10-15 g is. Dit was bereken dat die drempelwaardes vir assimileerbare en opgeloste organiese koolstof onder -51-1g C/l en -0.5 mg C/l, onderskeidelik, teikens moet wees vir die beheer van biofilmvorming in hierdie sisteem. Dit was aangetoon dat polyetileen, polyvinielchlroried, teflon, plexiglas, koper, sink-bedekte staal en aluminium gunstige aanhegtings oppervlaktes voorsien wat primêre kolonisering en daaropvolgende biofilmvorming toelaat. Betekinisvolle (p <0.05) verskille in oppervlak kolinisering op die materiale was waargeneem, wat aandui dat die samestelling van die materiaal In direkte invloed op mikrobiese kolonisering het. Die twee tipes vlekvryestaal wat geëvalueer was in hierdie studie, was die minder gunstige materiale vir biofilmvorming. Dit was verder gedemonstreer dat die aard van die oppervlak van hierdie materiale, vloeitoestande, en water tipe almal In direkte invloed het op biofilmvorming. Terwyl die aanpassing van aanhegtingsoppervlak nie die ontsrnettinqsdoeltreffendheid resultaat van die twee algemeen-gebruikte biosiede betekinisvol (p > 0.05) beïnvloed het nie, het die konsentrasie van die biosiede doeltreffendheid grootliks beïnvloed. asook die aanhegtings-materiaal, biosied Die resultate het aangetoon dat biofilm monitering geïmplementeer moet word by waterbehandelingsaanlegte as In alternatief vir algemene biostabiliteit metings.

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.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Fish infections caused by pathogenic Flavobacterium spp. are a major problem in the aquaculture industry worldwide, often leading to large economic losses. Thirty-two Flavobacterium spp. isolates, obtained from various diseased fish species and biofilm growth, were characterised genetically using 16S rRNA gene sequencing, 16S rRNA gene PCR restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) element PCR, plasmid profiling, whole cell protein (WCP) and outer membrane protein (OMP) analyses. The biofilm-forming capability of five genetically heterogeneous Flavobacterium spp. study isolates was investigated using a modified microtiter-plate adherence assay, as well as flow cell studies. Experimental infection studies with Mozambique tilapia (Oreochromis mossambicus) were carried out in order to determine the virulence of the Flavobacterium spp. study isolates. 16S rRNA gene sequence analysis showed the Flavobacterium spp. study isolates were closely related, and 97% sequence similarity was shared with published F. johnsoniae sequences. A high degree of genetic heterogeneity was displayed by the Flavobacterium spp. study isolates following RAPD-PCR, REP-PCR and OMP analysis, however, based on the results obtained by plasmid profiling and WCP analysis, the isolates appeared genetically very homogeneous. The biofilm phenotype was displayed by all five Flavobacterium spp. isolates tested and varied from weakly to strongly adherent. No specific correlation was observed between the RAPD, REP and/or OMP profiles and degree of adherence displayed by Flavobacterium spp. isolates. However, a specific WCP profile (profile B), exhibited by 48% of the Flavobacterium spp. isolates, was linked to strong adherence. Experimental infection studies showed that Flavobacterium spp. isolates displayed variable levels of virulence, which could not be linked to biofilm formation, nor specific genotypes. This is the first reported isolation and characterisation of Flavobacterium spp. isolated from diseased fish in Southern Africa, and there appears to be significant diversity amongst the isolates which is not geographically linked nor host related. / AFRIKAANSE OPSOMMING: Visinfeksies veroorsaak deur Flavobacterium spp. is problematies in die akwakultuur industrie wêreldwyd en lei tot groot ekonomiese verliese. Twee en dertig Flavobacterium spp. isolate, geïsoleer vanaf verskye geïnfekteerde visspesies en biofilm groei, was geneties gekarakteriseer met behulp van 16S rRNS geenvolgorde, 16S rRNS geen PKR restriksie fragment lengte polimorfisme (RFLP), toevallig geamplifiseerde polimorfiese DNS (TGPD) PKR, herhaalde ekstrageniese palindromiese (HEP) element PKR, plasmied profilering, heelsel protein (HSP) en buite membraan protein (BMP) analise. Die vermoë van vyf geneties heterogene Flavobacterium spp. isolate om biofilms te vorm was ondersoek met behulp van ‘n gemodifiseerde mikrotiterplaat vashegtings toets asook vloei-sel studies. Eksperimentele infeksie studies was uitgevoer op bloukurpers (Oreochromis mossambicus) om die virulensie van die Flavobacterium spp. studie isolate te toets. 16S rRNS geenvolgorde analise het getoon dat die Flavobacterium spp. studie isolate naby verwant was, en het 97% ooreenstemming getoon met gepubliseerde F. johnsoniae volgordes. TGPD-PKR, HEP-PKR en BMP analise het ‘n hoë graad van heterogeniteit tussen die Flavobacterium spp. studie isolate aangetoon, egter, op grond van plasmied profilering en HSP analise, was die studie isolate geneties baie homogeen. Die biofilm fenotipe was getoon deur al die getoetsde Flavobacterium spp. isolate en het gevarieer van swak tot sterk vashegting. Geen spesifieke korrelasie was waargeneem tussen die TGPD, HEP en/of BMP profiele en graad van vashegting vertoon deur Flavobacterium spp. isolate nie, maar ‘n spesifieke HSP profiel (profiel B), getoon deur 48% van die Flavobacterium spp. isolate, was verbind met sterk vashegting. Eksperimentele infeksie studies het getoon dat Flavobacterium spp. isolate varierende grade van virulensie vertoon het en wat met biofilm formasie of spesifieke genotipes geassosieer kon word nie. Hierdie is die eerste gedokumenteerde isolasie en karakterisering van Flavobacterium spp. geïsoleer van geïnfekteerde vis in Suider Afrika, en daar is beduidende diversiteit tussen die isolate wat nie geografies of gasheer geassosieerd is nie.