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.

Aquarium biofilters are designed to promote the growth and activity of nitrifying microorganisms, which are primarily responsible for the removal of toxic nitrogen-cycle intermediates. Ammonia is a natural waste product excreted by fish that is lethal to aquatic life at relatively low concentrations. Ammonia-oxidizing archaea (AOA) outnumber ammonia-oxidizing bacteria (AOB) in biofilters of mature freshwater aquaria with low-ammonia conditions. However, no study has investigated the early establishment of AOA and AOB within biofilter communities, especially when aquarium ammonia concentrations are elevated. My thesis research investigated the relative abundance of AOA and AOB in freshwater aquarium biofilters through early aquarium establishment. AOA and AOB genes were detected in DNA extracts from the biofilters of 14 start-up freshwater aquaria with increasing fish biomass loads (Experiment 1), as well as from 12 biofilters of start-up aquaria treated with AOA and AOB supplements (Experiment 2). In start-up aquaria, early ammonia concentrations increased with fish biomass, and AOB amoA genes were strongly detected over AOA marker genes in all filters without initial AOA inoculation. Inoculation of AOA-dominated supplements into newly established biofilters improved early ammonia oxidation rates in comparison to filters supplemented with AOB or those lacking supplements. Inoculated AOA thrived in filter biofilm during and beyond stabilization of low-ammonia conditions in aquaria. Microbial activity experiments demonstrated that AOA were present and active in the biofilters eight months after inoculation, when aquaria were fully established. In addition, AOB and AOA populations were monitored in new aquaria in three unregulated home environments. Thaumarchaeal 16S rRNA genes were detected in all aquarium filters within one month of aquarium development. In one filter, AOA were the only ammonia-oxidizers detected in the biofilm during aquarium development, suggesting that AOA were the sole contributors to nitrification in this aquarium. The results from these experiments suggest that AOA may be key players in early aquarium nitrification once introduced into the aquarium environment. Further, this research provides insight into the ecology of AOB and AOA in engineered freshwater environments

Secondary plant metabolites have recently been gaining interest in livestock production systems following the ban of in-feed antibiotics within the European Union. The rise in antimicrobial resistance found in pathogenic and non-pathogenic bacteria has lead to increased interest in the research community regarding the use of phythochemicals as an alternative to antibiotics. The purpose of this research was to evaluate the impact of including phytochemicals in a livestock production system. Specifically, a high tannin-containing forage, sainfoin (Onobrychis viciifolia), was evaluated in vitro for its antimicrobial effect on Escherichia coli. We determined that phytochemicals alone are not as inhibitory as synthetic antibiotics. Thus, the use of combination therapy to deter the development of antimicrobial resistance was evaluated. A myriad of plant compounds were screened for their synergistic interactions with ciprofloxacin. Geraniol, an essential oil, was identified to possess good antimicrobial activity and synergistic interactions with ciprofloxacin. Therefore the effect of long term exposure to both ciprofloxacin and geraniol were examined. Results demonstrated that once an antimicrobial concentration threshold was reached, resistance to ciprofloxacin increased markedly in the presence of both geraniol and ciprofloxacin. Finally, an in vivo trial was conducted in which forty steers were fed sainfoin or alfalfa over a 9-week period to evaluate its ability to reduce E. coli shedding and its impact on gut microbiota in the context of popular theoretical ecology concepts. Results from the in vivo study indicate that sainfoin was able to promote a slight decrease in generic E. coli shedding which could be maintained throughout the trial. Using high-throughput sequencing, the effect of sainfoin on the microbial ecosystem of the ruminant gut was evaluated. Sainfoin induced a significant shift in the microbial community structure of the rumen and to a lesser extent in the hindgut. Using ecology theories, a hypothesis was formulated regarding the mechanisms that mediate the development of tolerance and the fundamental ecological processes controlling microbial population shifts. Understanding how the gut ecosystem functions and predicting its behaviour in the presence of various fluctuating environmental conditions will enable more efficient manipulation of the rumen and promote best management practices in livestock production.

The purpose of this research was to investigate the microorganisms, genetics and biochemistry of anaerobic dechlorination of chlorinated ethanes, which are common groundwater contaminants. Specifically, this project used mixed microbial cultures to study the dechlorination of 1,2-dichloroethane (1,2-DCA), 1,1,2-trichloroethane (1,1,2-TCA) and 1,1,1-trichloroethane (1,1,1-TCA). A mixed microbial culture enriched from a contaminated multilayered aquifer in West Louisiana dechlorinated 1,2-DCA, 1,1,2-TCA, tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride (VC) to non-toxic ethene when amended with ethanol as the electron donor. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organisms Dehalobacter and Dehalococcoides spp. Denaturing gradient gel electrophoresis analysis and quantitative PCR (qPCR) with species-specific primers demonstrated that both organisms grew during the dichloroelimination of 1,2-DCA to ethene. Conversely, during the dichloroelimination of 1,1,2-TCA to VC only Dehalobacter grew, while during the reductive dechlorination of VC to ethene only Dehalococcoides grew. Further enrichment with 1,2-DCA, H2 and acetate yielded a co-culture of Dehalobacter and Acetobacterium spp. that did not dechlorinate other chlorinated ethanes or ethenes. Dehalobacter grew in the presence but not in the absence of 1,2-DCA, while Acetobacterium growth was not affected by 1,2-DCA. A novel putative Dehalobacter-associated 1,2-DCA reductive dehalogenase gene was identified and was shown to be transcribed only in the presence of 1,2-DCA. An enrichment microbial culture derived from a 1,1,1-TCA-contaminated site in the northeastern United States was also studied. This culture, referred to as MS, reductively dechlorinated 1,1,1-TCA to 1,1-dichloroethane (1,1-DCA) and then to monochloroethane (CA) when amended with methanol, ethanol, acetate and lactate. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organism Dehalobacter sp., whose growth during 1,1,1-TCA and 1,1-DCA dechlorination was confirmed by qPCR. In the presence of chlorinated ethenes, dechlorination 1,1,1-TCA by the culture MS was slowed, while dechlorination of 1,1-DCA was completely inhibited. Experiments with cell-free extracts and whole cell suspensions of culture MS suggested that chlorinated ethenes have direct inhibitory effects on 1,1,1-TCA reductive dehalogenase(s), while the inhibition of 1,1-DCA dechlorination may be due to effects on non-dehalogenase components of Dehalobacter sp. cells. Additionally, two novel reductive dehalogenase genes associated with 1,1,1-TCA reductive dechlorination were identified.

Microbial activity occurs year-round in Arctic soils, including during the winter when soils are frozen. From 2004 to 2008 I monitored soil microbial and nutrient dynamics in low Arctic wet and dry sedge meadows near Churchill, Manitoba. I documented a consistent annual pattern in which soil microbial biomass (MB) and soil nutrients peak in late winter, and decrease during the early stages of spring thaw, remaining in low abundance during the summer. Based on a series of experiments, resource shortages do not appear to be the cause of the microbial decline, as has been hypothesized. Observations and theoretical considerations regarding soil physical properties indicate that this decrease is driven by the influx of liquid water at thaw that brings about a rapid change in the chemical potential of water, leading to cell lysis. I have used 15N isotope tracing to show that inorganic nitrogen is taken up very quickly at thaw by the roots of the dominant plant, Carex aquatilis. This represents a critical window of opportunity for these plants, as nitrogen remains abundant only for a short time. The described annual pattern was pronounced in wet sedge sites, but some inter-annual variation is evident, for example a post-thaw soil nitrogen pulse in 2006, and low winter MB in 2008. In the dry sedge meadow, fluctuations in MB and nutrients were dampened relative to wet sites, and the annual pattern was variable, particularly after 2006. Over four years, peak winter values of soil MB and nutrient variables declined in both wet and dry sites, and this could be related to a drying trend. This work improves our understanding of the controls on decomposition and primary productivity in a system that is experiencing climate warming and increased precipitation. Changes to hydrology, carbon and nitrogen cycling, and primary productivity will have further effects on vegetation communities and higher trophic levels, including several species of migratory birds.

The stromatolites of Shark Bay Western Australia, located in a hypersaline environment, is an ideal biological system for studying survival strategies of cyanobacteria and halophilic archaea to high salt and their metabolic cooperation with other bacteria. To-date, little is known of the mechanisms by which these stromatolite microorganisms adapt to hypersalinity. To understand the formation of these sedimentary structures, detailed analysis of the microbial communities and their physiology for adaptation in this environment are crucial. In this study, microbial communities were investigated using culturing and molecular methods. Phylogenetic analysis of the 16S rRNA gene was carried out to investigate the diversity of microorganisms present. Unique phylotypes from the bacteria, cyanobacteria and archaea clone libraries were identified. Representative cyanobacteria isolates and Halococcus hamelinensis, a halophilic archaea isolated from in this study, were the focus for identifying osmoadaptation mechanisms. The presence of osmolytes in these microorganisms was detected by Nuclear magnetic resonance spectroscopy (NMR). It was found that the cyanobacterial isolates studied utilised different osmolytes. Glucosylglycerol, unique to marine cyanobacteria was not identified; instead various saccharides, glycine betaine and TMAO were the predominant solutes present. Thus cyanobacteria are likely to possess more complex mechanisms of adaptation to osmotic stress than previously thought. Findings here also indicated that H. hamelinensis accumulates glycine betaine and glutamate instead of potassium ions. DNA molecular methods were employed to identify candidate genes for the uptake of osmoprotectants. Three putative glycine betaine transporters from Halococcus hamelinensis were identified. Functionality of one of these glycine betaine transporters was determined by complementation studies. For the first time, an archaeal glycine betaine transporter was shown to be successfully complemented in a glycine betaine transport deficient mutant (E. coli MKH13). This study has increased our understanding of how microorganisms co-exist in fluctuating environments in response to solubilisation/precipitation or dilution/evaporation processes, resulting in a hypersaline environment. It also provides an excellent platform for the identification of any novel osmolytes/compatible solutes that might have been produced by these microorganisms that have been isolated for the first time from stromatolites.

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