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published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

Little is known about the distribution of bacterial indicators and pathogens in biofilms on different surface types in natural aquatic environments. This study was conducted to examine the distribution of pathogens and indicator bacteria in biofilms in an agricultural watershed. The study particularly focused on whether biofilms can act as sinks for pathogenic organisms and could be monitored to protect public health. To do so, we monitored the presence of faecal contamination indicators (heterotrophic plate counts, faecal coliforms, enterococci, and E. coli) and particular pathogens (E. coli 0157, Campylobacter sp. and Salmonella sp.) in water, sediment, and in biofilms on river and slate rock, wood, sandpaper, and Lexan™ in Elk Creek (British Columbia, Canada) from December 2005 to April 2007. Faecal indicator concentrations and pathogen presence were evaluated using standard culturing and isolation methods. The results showed that both faecal indicators and pathogens were present at the headwaters and that the use of water column grab samples underestimated faecal indicator numbers. Also, water column grab samples during the dry season were not representative of pathogens present in the creek. This indicates that biofilms might be the main reservoir of Salmonella sp. and pathogenic E. coli O157 in the summer when rainfall (which results in flow changes and sloughing) is limited. Campylobacter sp. was not retrieved in the dry season. Campylobacter on sediment, slate rock and wood showed high correlations with nitrates and enterococci, which could be used as faecal contamination surrogates. Numbers of indicator organisms and pathogens in one-month biofilms were compared to those in long-term biofilms (colonized 12 and 24 weeks) and short-term biofilms (colonized one to three weeks). The comparison showed that surface type, colonization period and water quality all affected the concentration of indicator organisms and pathogens present in biofilms. Finally, results showed high levels of phenotypic antibiotic resistance of E. coli and pathogenic E. coli O157 isolated from the watershed (even at the headwaters), particularly to tetracycline, ampicillin and streptomycin. This study highlights the potential biofilms could play in prediction of water quality changes, the improvement of sampling methods, and the study of aquatic environments.

The number of superficial candidal infections has grown due to an increase in the elderly cohort and ever-increasing immunocompromised population, and the increased prevalence of antifungal drug resistance. The aim of this research was to investigate the antifungal efficacy of a citrus fruit extract, Biosecur c320c (B320), against two strains of Candida albicans (nystatin sensitive and resistant). The viability of C. albicans strain treated with 10% B320 was reduced by 90-99% depending on biofilm age. A B320/nystatin combination demonstrated even greater efficacy at killing biofilm cells in either strain. The nystatin sensitive strain did not develop resistance to B320 while resistance was developed following long-term exposure to nystatin. Scanning electron micrographs of C. albicans biofilms revealed cellular debris after treatment with combined B320/nystatin. Citrus fruit extracts containing polyphenols might contribute to the development of novel therapeutics for the treatment of oral candidiasis, particularly in those refractory to nystatin therapy.

The number of superficial candidal infections has grown due to an increase in the elderly cohort and ever-increasing immunocompromised population, and the increased prevalence of antifungal drug resistance. The aim of this research was to investigate the antifungal efficacy of a citrus fruit extract, Biosecur c320c (B320), against two strains of Candida albicans (nystatin sensitive and resistant). The viability of C. albicans strain treated with 10% B320 was reduced by 90-99% depending on biofilm age. A B320/nystatin combination demonstrated even greater efficacy at killing biofilm cells in either strain. The nystatin sensitive strain did not develop resistance to B320 while resistance was developed following long-term exposure to nystatin. Scanning electron micrographs of C. albicans biofilms revealed cellular debris after treatment with combined B320/nystatin. Citrus fruit extracts containing polyphenols might contribute to the development of novel therapeutics for the treatment of oral candidiasis, particularly in those refractory to nystatin therapy.

Large infected bone defects (IBD) are very complicated to treat due to their high variability; they often require multiple procedures. Bone autografts are the gold standard for treatment but have several drawbacks, such as a need for a second surgery site, limited grafting material, and donor site morbidity. The objective of this research was to develop a moldable synthetic bone grafting material capable of releasing both antimicrobial and osteogenic drugs over a clinically relevant time course for the treatment of IBDs. Current treatment methods for large IBDs require two separate procedures to treat the bone defect and the infection. This research sought to combine these two procedures into one implantable composite bone graft substitute for the treatment IBDs. To begin, the degradation and mechanical properties of the calcium sulfate (CS) based composite material were evaluated for different compositions. Next, the controlled drug release profiles from the composite was achieved by using a shell and core system incorporating poly(lactic-co-glycolic acid) microspheres (PLGAms). The release of vancomycin from the shell began immediately and continued over the course of 6 weeks, while the release of simvastatin from the core was delayed before being released over 4 weeks. Next, an infected, critically-sized rat femoral defect model was used to test different treatment methods with and without the composite bone graft substitute. Animals treated with locally released antibiotics had survivorship rates 24% higher than those treated with systemic antibiotics, and animals that received both antibiotics and an osteogenic drug had an increased amount of bone formation at 12 weeks compared to controls. Finally, several different anti-biofilm agents were evaluated for their ability to inhibit and/or disrupt the growth of Staphylococcus aureus (S. aureus) biofilms in vitro. Lysostaphin was the only drug investigated that was able to both inhibit and disrupt S. aureus biofilms. Furthermore, lysostaphin encapsulated into PLGAms maintained its bioactivity and may be useful for future incorporation into biofilm-combating materials. The bone grafting material developed here can be used to locally deliver drugs in a temporally controlled manner to reduce the number of procedures necessary for the treatment of complex IBDs.

Aeromonas spp. are important biofilm-forming fish pathogens causing great economic loss in aquaculture. Bacterial cells within biofilms communicate with each other via the production of quorum sensing (QS) signalling molecules called acyl-homoserine lactones (AHLs), which influence biofilm development and production of virulence factors. QS together with efflux pumps, extracellular polymeric substances (EPS) and eDNA are associated with resistance of bacteria to antimicrobial agents. These mechanisms provide a target for different control strategies. The objectives of this study were to: (i) determine effective antimicrobial agents and exposure concentrations against aeromonad biofilms; (ii) ascertain whether Aeromonas spp. produce QS molecules or display efflux pump phenotypes, and (iii) investigate the effect of antimicrobial agents, lytic enzymes, efflux pump inhibitors and QS inhibitors on biofilm formation by Aeromonas spp. isolates.signalling MICs of azithromycin, ciprofloxacin, ceftazidime, and tetracycline ranged between 0.064-64 μg/ml. Gentamicin had the lowest MICs which ranged between 0.0048-32 μg/ml.The highest MBIC at which antimicrobial agents exhibited inhibition was 4096 μg/ml. Majority of the isolates displayed MIC levels ranging from 2-32 μg/ml, and thus a ≥ 128-fold increase was observed for MBICs. Of the sub-MIC, MIC and supra-MIC exposures tested, MIC exposure of biofilms was the most effective. Gentamicin MIC exposures inhibited initial attachment of 100% (28/28) of isolates tested, while azithromycin MIC exposure detached 82.1% (23/28) of isolates. Carbonyl cyanide 3-chlorophenylhydrazone completely inhibited efflux of cefpodoximeby 14.8% of isolates. However, 1-(1-naphthylmethyl)-piperazinewas more effective, decreasing adherence of 98.1% (53/54) of isolates and increasing detachment of 100% (54/54) of isolates. DNase I was more effective against the mature biofilm,where it increased biofilm detachment of 64.8% of isolates. Of the 48 Aeromonas spp. and six Plesiomonas spp. isolates used, only a single isolate induced the production of violacein by the C. violaceum CV026 biosensor, while all isolates induced the utilization of X-gal to produce a visible blue colour with the A.tumefaciens A136 biosensor. Based on the reaction to the two biosensors, aeromonads appeared to produce long-chain acylhomoserine lactones. By blocking QS, S-adenosyl homoserinewas more effective in inhibiting both initial attachment (72.2% of isolates) and pre-formed biofilms (detached 74.1% of isolates). The investigated strategies are promising for Aeromonas spp. biofilm inhibition. Thesecould be explored aspotential therapeutic measures in aquaculture systems to limit aeromonad pathogenicity and overcome antimicrobial resistance. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

Natürliche Biofilme von zwei urankontaminierten Standorten, dem ehemaligen Uranbergwerk in Königstein (Sachsen) und dem Gebiet der ehemaligen Aufstandsfläche der Gessenhalde (Thüringen), wurden in dieser Arbeit näher untersucht. An beiden Standorten konnte in den Minenwässern die hochmobile, gelöste Uranspezies Uranylsulfat (UO2SO4) als dominierend nachgewiesen werden. Aufgrund der Instabilität vieler kommerzieller Fluoreszenzfarbstoffe bei niedrigen pH-Werten war eine gezielte Anfärbung der Mikroorganismen in den sauren Biofilmen nicht möglich, ohne den pH-Wert der Biofilmproben anzuheben, was die Probenchemie maßgeblich verändert. In Kooperation mit der Firma DYOMICS (Jena, Deutschland) wurden neue, kommerziell nicht erhältliche, säurestabile Farbstoffe erstmals hinsichtlich ihrer Eignung zur Anfärbung von Mikroorganismen in sauren Biofilmen ohne Veränderung des pH-Wertes sowie der sonstigen Probenchemie getestet. Die neuen Farbstoffe DY-601XL, V07-04118, V07-04146 und DY-613 zeigten eine Eignung für solche Färbungen, da sie eine intensive Anfärbung der Mikroorganismen bei niedrigen pH-Werten unter pH 3 – 4 herbeiführen und außerhalb des Emissionsbereiches von Uran fluoreszieren. In dieser Arbeit wurde die Fähigkeit von Euglena Mutabilis-Zellen zur Bioakkumulation des Urans im pH-Wertbereich 3 – 6 in den Hintergrundmedien Natriumperchlorat (9 g/l) oder Natriumsulfat (3,48 g/l) an lebenden Zellen untersucht. Unabhängig vom Medium konnte bei sauren pH-Werten um pH 3 – 4 über 90 % des vorgelegten Urans aus den Probelösungen abgetrennt werden. Die Speziation des an den Euglena-Zellen akkumulierten Urans, wurde mittels laserinduzierter Fluoreszenzspektroskopie (LIFS) untersucht. Es zeigte sich, dass unabhängig vom Hintergrundmedium, Lebenszustand und pH-Wert eine vergleichbare neue Uranspezies an den Zellen gebildet wird. Durch den Vergleich der Daten aus den LIFS-Messungen mit Referenzwerten, konnte die gebildete Uranspezies auf eine Anbindung durch (organo)phosphatische und/oder carboxylische funktionelle Gruppen eingegrenzt werden. Mit Hilfe der zeitaufgelösten FT-IR-Spektroskopie konnte die carboxylische Anbindung des Urans an toten Zellen nachgewiesen werden. Ein Ausschluss der (organo)phosphatischen Komplexierung konnte jedoch mit dieser Methode nicht geführt werden. Untersuchungen zur Lokalisation des Urans an bzw. in den Zellen, mittels der gekoppelten CLSM/LIFS-Technik zeigten erstmals ein Indiz für die intrazelluläre Akkumulation von Uran in den lebenden Zellen. Ergänzende TEM/EDX-Messungen bestätigten die intrazelluläre Aufnahme und belegen eine Akkumulation in runden bis ovalen Zellorganellen, bei denen es sich vermutlich um Vakuolen oder Vakuolen-ähnliche Vesikel handelt. An den toten Zellen konnte mit diesen Methoden kein Uran detektiert werden. Dies lässt auf eine passive, homogen verteilte Biosorption des Urans an die verfügbaren Bindungsplätze an der Zelloberfläche der toten Biomasse schließen.

This project researched the fungal and bacterial communities (i.e. biofilms) found on concrete infrastructure in Georgia. Various microbial communities were sampled from four geographically separated sites. The species present in these biofilms were identified through DNA analysis and cultured for testing. A new, rapid test method was developed to accurately simulate field growth conditions in a laboratory environment. Using the newly developed test method, these communities were grown on small mortar tiles, which varied in w/cm, surface roughness, cement type (including photocatalytic cement), and supplementary cementing materials. This research determined that photocatalytic cement was the most effective in decreasing biofilm growth under artificial daylight, but did not increase or decrease growth when not exposed to light. The next most effective ways to decrease growth were lowering w/cm and decreasing surface roughness. The supplementary cementing materials examined did not increase or decrease biofilm growth.

Little is known about the distribution of bacterial indicators and pathogens in biofilms on different surface types in natural aquatic environments. This study was conducted to examine the distribution of pathogens and indicator bacteria in biofilms in an agricultural watershed. The study particularly focused on whether biofilms can act as sinks for pathogenic organisms and could be monitored to protect public health. To do so, we monitored the presence of faecal contamination indicators (heterotrophic plate counts, faecal coliforms, enterococci, and E. coli) and particular pathogens (E. coli 0157, Campylobacter sp. and Salmonella sp.) in water, sediment, and in biofilms on river and slate rock, wood, sandpaper, and Lexan™ in Elk Creek (British Columbia, Canada) from December 2005 to April 2007. Faecal indicator concentrations and pathogen presence were evaluated using standard culturing and isolation methods. The results showed that both faecal indicators and pathogens were present at the headwaters and that the use of water column grab samples underestimated faecal indicator numbers. Also, water column grab samples during the dry season were not representative of pathogens present in the creek. This indicates that biofilms might be the main reservoir of Salmonella sp. and pathogenic E. coli O157 in the summer when rainfall (which results in flow changes and sloughing) is limited. Campylobacter sp. was not retrieved in the dry season. Campylobacter on sediment, slate rock and wood showed high correlations with nitrates and enterococci, which could be used as faecal contamination surrogates. Numbers of indicator organisms and pathogens in one-month biofilms were compared to those in long-term biofilms (colonized 12 and 24 weeks) and short-term biofilms (colonized one to three weeks). The comparison showed that surface type, colonization period and water quality all affected the concentration of indicator organisms and pathogens present in biofilms. Finally, results showed high levels of phenotypic antibiotic resistance of E. coli and pathogenic E. coli O157 isolated from the watershed (even at the headwaters), particularly to tetracycline, ampicillin and streptomycin. This study highlights the potential biofilms could play in prediction of water quality changes, the improvement of sampling methods, and the study of aquatic environments.