Acidiphilium cryptum is a heterotrophic acidophile commonly found in acidic environments and industrial bioleaching operations. Attachment to mineral surfaces may serve to maintain this organism in aqueous environments where it is subject to removal by hydrodynamic forces. Using indirect and direct methods we have looked at the binding of A.cryptum to chalcopyrite (CuFeS₂) and other mineral ores to determine whether specific adhesins mediate binding. A modified ELISA binding assay (the Ore ELISA) was developed to measure direct adherence. Finely ground chalcopyrite was bound irreversibly to the walls of an ELISA plate, the organisms were added and after incubation and washing, the number of attached bacteria were assessed by reacting with anti-A.cryptum antibody followed by goat anti-rabbit IgG conjugated to alkaline phosphatase. This assay was found to be sensitive, rapid and reproducible. The Ore ELISA allowed direct binding measurement in the presence of various inhibitors and provided a rapid screening method for adherence-defective mutants. Adherence was shown to be saturable and increased slightly as pH decreased. A moderate increase in binding affinity was recorded in the presence of monovalent and divalent cations and EDTA. Various bactericidal agents and pentose and hexose sugars had no effect on chalcopyrite attachment. Reducing agents had little effect on cell adherence. A strong increase in adherence was observed in the presence of surface active agents. Bovine serum albumin and gelatin were both found to markedly reduce mineral surface binding. Competition for attachment sites between A.cryptum and the autotrophic acidophile, Thiobacillus ferrooxidans, showed that each organism binds to unique sites on the chalcopyrite surface. A.cryptum mutant strains displaying reduced adherence to chalcopyrite were shown to lack a 31.6 kDa outer membrane protein. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

A semi-synthetic medium for use in determining adherent growth with Staphylococcus epidermidis and Staphylococcus saprophyticus was developed. Production of an adherent biofilm was dependent upon the presence of hematin in the growth medium. Clinical strains of Staphylococcus epidermidis were tested for production of an adherent biofilm in trypticase soy broth, the semi-synthetic medium and the hyperalimentary nutrient solution used in the neonatal hospital unit. An adherent biofilm was obtained when Staphylococcus epidermidis was cultured m hematin supplemented hyperalimentary solution. Growth in the hyperalimentary nutrient solution diluted with fetal calf serum showed the same growth rate as when the nutrient solution was diluted with water. The final growth yield was always higher in serum diluted nutrients. There was no effect of hematin on the growth rate of the organisms.

The adhesion of seven meat spoilage bacteria to model meat surfaces (thin fat and tendon slices) was studied in a specially designed flow chamber. In general, adhesion was not influenced by the physiological age of the cells and was not correlated with the cell surface characteristics (electrical charge, hydrophobicity) of the organisms. Also, adhesion data did not corroborate the predictions based on changes in the free energy of adhesion, calculated from contact angles and surface tension measurements. A more detailed study of the adhesion of Pseudomonas fluorescens to tendon was subsequently undertaken. Neither physiological activity nor the presence of flagella was found to be essential for adhesion. Selective chemical alterations of the cell surface pointed to no direct implication of carboxyl or amino groups in an adhesive bond with tendon. These groups may participate in adhesion, however, through electrostatic interactions as suggested from the variations of adhesion with ionic strength.

Beef tendons were sectioned into 60 mum-thick slices (1cm 2) and deposited onto glass cover slips. These meat slices were flooded with a cell suspension of either Pseudomonas fluorescens, Enterobacter agglomerans, or Moraxella osloensis in distilled water and adhesion allowed to occur. The adhesion experiments were also conducted on agar-covered slides to evaluate the effect of the nature of the substratum on adhesion. The non-adherent organisms found on either surface tested (meat or agar) were removed by flushing liquid over the slide. The slides were then incubated in a moist atmosphere at 30°C. Once the presence of microcolonies had been established microscopically, the slides were mounted into flow chambers and the surface flushed with distilled water to ascertain the effects of bacterial proliferation on adhesion. In other experiments, the influence of acetic, citric, and lactic acid rinses on cell adhesion and subsequent cell proliferation was evaluated. Microcolony formation was shown to reduce the adhesion strength of Enterobacter agglomerans and, to a lesser extent, that of Moraxella osloensis while increasing that of Pseudomonas fluorescens. The probable determinant of adhesion strengths of microcolonies is exopolymer synthesis. A minimal decrease in bacterial adhesion and microcolony formation was observed with the use of organic acid rinses. The bactericidal activity and effect on bacterial proliferation increased with increasing concentration and rinse times of the organic acids. The extent of the adhesion reductions suggests that the preservation action of organic acids is due to cell death and not adhesion inhibition.

No description available.

No description available.

β-lactoglobulin (β-Lg), bovine serum albumin (BSA), α-lactalbumin (α-Lac), and β-casein were adsorbed onto silanized silica surfaces of low and high hydrophobicity for 8 h, and β-Lg and BSA for 1 h. The surfaces were incubated in buffer for 0, 5, 10, or 15 h and then contacted with Listeria monocytogenes for 3 h. Cell adhesion was quantified using image analysis. Following 8 h of protein contact, adhesion to both surfaces was greatest when β-Lg was present and lowest when BSA was present. Preadsorption of α-Lac and β-casein showed an intermediate effect on cell adhesion. Adsorption of β-Lg for 1 h resulted in lower numbers of cells adhered as compared to the 8 h adsorption time, while the opposite was observed with BSA, but adhesion to BSA was observed to decrease slowly with film age to values comparable to the 8 h tests. The adsorption of BSA and β-Lg to both surfaces was also carried out where each protein was allowed to contact the surface in sequence and simultaneously. In sequential tests performed with an 8 h contact/protein, cell numbers on each surface were near that expected for the bare hydrophobic surface when β-Lg contact preceded introduction of BSA, whereas adhesion was reduced to values below that expected for the bare hydrophilic surface when BSA preceded β-Lg contact. In short-term sequential tests (1 h contact/protein), adhesion was lower than that recorded on bare hydrophilic surfaces in each case. Adhesion to each surface following contact with an equimolar mixture of β-Lg and BSA was lower than that measured on the bare hydrophilic surface in each case, with adhesion following 1 h contact being greater than that following 8 h contact. Adhesion following competitive adsorption was greater to hydrophobic than to hydrophilic surfaces. These results were explained with reference to the surface passivating character of BSA, and its ability to rapidly attain a nonexchangeable state upon adsorption, relative to β-Lg. / Graduation date: 1995

Graduation date: 1992

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.

Protection of potable water supplies demands a better understanding of the factors controlling migration of disease causing bacteria in subsurface environments. In this study, the migration behaviour of the waterborne pathogenic microorganisms Escherichia coli O157:H7 and Yersinia enterocolitica was investigated in water saturated granular systems. Both facultative bacteria were grown under aerobic and anaerobic conditions and further acclimatized to a microaerophilic or fully aerated environment for 21 h. Experiments were conducted using laboratory-scale packed columns over controlled extreme dissolved oxygen (DO) concentrations. The observed differences in the transport potential of these pathogens were found to depend strongly on the antecedent growth conditions under the tested environmental settings as well with the environmental DO in certain conditions. Further microbial characterization using cell titrations and FTIR spectroscopy gave a greater insight on the source of the surface charge that was found to dominate the attachment phenomena in sand packed columns. Techniques also revealed a probable role of other cell surface macromolecules (LPS) that could account for non-DLVO behaviour. The results illustrate the importance of considering physicochemical conditions relevant to the natural subsurface environment when designing laboratory transport experiments as evidenced by variations in microbe migration as a function of the DO under growth and acclimation. / Keywords: bacterial adhesion, bacterial transport, DLVO, physicochemical characterization, dissolved oxygen, porous media.