The objectives of this work are: 1) to agarose-stabilize fragile biofilms for quantitative structure analysis; 2) to understand the influences of LuxS on biofilm formation; 3) to improve teacher quality by preparing Georgia’s middle school science teachers to integrate inquiry-based, hands-on research modules in the classroom. Quantitative digital image analysis demonstrated the effectiveness of the agarose stabilization technique for generating reproducible measurements of three dimensional biofilm structure. The described method will also benefit researchers who transport their flow cell-cultivated biofilms to a core facility for imaging. AI-2-dependent and independent effects of LuxS on biofilm-related phenotypes were revealed, suggesting that LuxS is a versatile enzyme, possessing multiple functions in E. coli ecology that could assist E. coli in adapting to diverse conditions. Overall, the work presented in this dissertation supported the concept that quorum sensing, biofilm formation, and cell adhesion are largely related. Additionally, through this project, teachers enhanced content knowledge and confidence levels, mastered innovative teaching strategies and integrated inquiry-based, inter-disciplinary, hands-on activities in the classroom. As a result, student learning was enhanced, and teachers are better equipped to give Georgia’s students a solid foundation in the sciences.

Thesis (M.S.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains ix, 74 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 64-74).

Thesis (Ph. D.)--University of California, Riverside, 2009. / Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed March 16, 2010). Includes bibliographical references. Also issued in print.

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The growth of micro-organisms in foods is different from that in axenic liquid culture in laboratory media. In natural environments, including food, micro-organisms generally grow in mixed culture and in close proximity to each other, because of which antagonistic or synergistic interactions can occur. To elucidate the behaviour of bacteria within food matrices an understanding of the food structure is required, as foods are complex ecosystems on the micrometer scale. Most processed foods are emulsions and as such are highly structured heterogeneous environments. Antibody-linked probes can be used for the immuno-location of micro-organisms or their products within food matrices to demonstrate the sites at which growth occurs and elucidate the possible bacterial interactions with food components. The aim of the project was to raise antibodies to spoilage Pseudomonas species and to use the developed antibody-linked probes to follow psychrotrophic spoilage Pseudomonas within heterogeneous foods. By using antibody-linked probes the natural spoilage of milk and milk products can be followed along traditional lines examining extrinsic parameters but with the additional benefit that the major spoilage organisms can be located within the mixed natural flora. The use of antibodies in this way facilitated the study of a defined natural population and surmounted any adaptive problems associated with introduced organisms. An oil-in-water near-foodgrade model was developed to investigate the growth of Pseudomonas as it overcame some of the technical problems of using natural cream. Pseudomonas species, which grew as colonies within the near-food-grade model, were visualised using fluorescently-labeled antibody-linked probes. Pseudomonas used to raise the antisera were isolated from psychrotrophically spoiled food and characterised together with isolates retrieved from the environment. The phenotypic characterisation of Pseudomonas using classical biochemical tests and API 20NE test strips (BioMerieux) did not produce definitive identifications of the unknown isolates. Nutritional screening of the Pseudomonas isolates using commercially produced standardised test microtitration plates (Biolog MicroPlate TM), that contained 95 carbon sources, was carried out. The data produced from the test microtitration plates were analysed using numerical taxonomic methods. The relatedness of the Pseudomonas isolates was strongly influenced by the source from which the test isolates originated and did not definitively identify all of the unknown isolates tested. Molecular techniques, ribotyping and amplified ribosomal DNA restriction analysis (ARDRA), based on the genomic fingerprinting of the 16S rRNA gene were evaluated to aid the definitive identification of the Pseudomonas isolates but needed a more extensive data base to be useful. The difficulties encountered in phenotypically identifying food and environmentally isolated Pseudomonas species stems from the fact that the Pseudomonas genus is now classified according to its ribosomal DNA homology. The classification of the species within the Pseudomonas genus is still under review. Robust phenotypic criteria for the identification of all the species within the genus have not to date been defined. In this study, the association of phenotype with environmental source of isolation (whether characterised by nutritional studies or by antibody cross-reaction) demonstrates clearly that more appropriate phenotypic characterisation is required to allow identification schemes to reflect the underlying phylogeny of this group.

Chitin belongs to the most abundant biopolymers on earth where it has an important role as a structural element in crustaceans, insects, fungi and some phytoplankton. Missing evidence for long-term accumulation of chitin in nature implies fast turnover and as chitin is composed of aminosugar subunits it holds central roles in both carbon and nitrogen cycles. The aim of this thesis was to contribute to a better understanding of organic matter cycling by learning more about the diversity, function and ecology of bacteria that degrade chitin. A metagenome-enabled study of the spatial distribution of chitinolytic bacteria in aquatic ecosystems identified salinity as the major environmental factor for shaping their community composition. To address the role of alternative environmental variables controlling chitinolytic communities, a temporally resolved study was completed in a dimictic freshwater lake. Pronounced seasonal change in the indigenous chitinolytic community was observed and parallel measured environmental parameters pointed to the availability and crystalline form of chitin as significant controlling factors.  The different ecological niches occupied by microbes that utilize chitin for growth were studied in an experimental study. Single-cell quantification of chitinolytic cells and cells incorporating chitin hydrolysis products suggested that commensal use of chitin hydrolysis products without simultaneous chitinase activity could be an important ecological strategy in freshwater bacterioplankton communities. Members of the ubiquitous and often quantitatively dominant group of freshwater Actinobacteria Ac1 were identified as particularly active in this “cheater” lifestyle. Further experiments based on artificially created gradients in bacterial diversity demonstrated the importance of specific bacterial populations and community composition rather than overall community richness in controlling more specific functions such as chitin and cellulose degradation. To conclude, results of this thesis provide insight into the biogeography, niche-separation and species interactions of the functional community of chitin degraders and the influence of general bacterial diversity to the respective system functioning. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 700

In vitro gut models provide several advantages over in vivo models for the study of the human gut microbiota. However, because communities developed in these models are simplified simulations of the in vivo environment it is necessary to characterize the reproducibility, repeatability and stability of cultured communities. We also need to broadly define the differences between in vitro consortia and the communities from which they are derived. In this study we characterized and validated a twin-vessel (independent, identical) single-stage chemostat model of the human distal gut. Samples were analyzed using a molecular fingerprinting technique (Denaturing Gradient Gel Electrophoresis) to compare and monitor changes in the overall structure of the communities while a phylogenetic microarray (Human Intestinal Tract Chip) was used to obtain phylogenetic information. We found that twin-vessels inoculated with feces developed and maintained diverse communities that reached stable compositions by at most 36 days post-inoculation. Communities were enriched in Bacteroidetes but not Clostridium cluster XIVa, Bacilli or other Firmicutes relative to the fecal inocula. Vessels were very reproducible when inoculated with identical fecal inocula, less similar when inoculated with consecutive fecal donations from the same donor, and maintained donor-specific identities when inoculated with feces from different donors. Norepinephrine exposure (undefined perturbation) did not appear to have a substantial effect on the structure of chemostat communities, while clindamycin treatment (defined perturbation) caused large changes in the structure of chemostat communities. Packed-column biofilm reactors incorporated a simulated mucosal environment into our chemostat system, allowing us to simultaneously culture biologically relevant planktonic and biofilm communities that were complex, reproducible, and distinct. Defined communities were comparable to fecal communities at the phylum/class-level but established stable compositions more rapidly. While it was difficult to assess the persistence of synthetic stool in a healthy fecal chemostat community (+/- antibiotic perturbation), mixing communities from two donors resulted in a mixed community that more closely resembled one donor over the other. Although future experimentation is required, the results presented here show our twin-vessel single-stage chemostat model represents a valid simulation of the human distal gut environment and can support complex, representative microbial communities ideal for experimental manipulation. / Canadian Institutes of Health Research; Ontario Ministry of Agriculture, Food and Rural Affairs; Ontario Ministry of Research and Innovation; Canada Foundation for Innovation; Ontario Ministry of Training, Colleges and Universities

A polyphasic approach was used to examine the impact of contamination on soil microbial community structure. Two systems were examined using a combined biochemical and molecular biological approach. Petroleum hydrocarbon contaminated soils from two Northern Canadian sites, representing long-term contamination, were examined using Biolog GN plates and PCR-denaturing gradient gel electrophoresis (DGGE) analysis of total community 16S rDNA. Results obtained using both methods demonstrated a positive correlation between samples that was based on the geographical origin of the samples, not on contamination level. In the second system, non-contaminated soil was contaminated with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to monitor the effect of short- to medium-term contamination. Changes in the soil microbial community were examined using PCR-DGGE of total community 16S rDNA combined with RDX mineralization and chemical analysis of intermediates. The non-contaminated loam soil had an inherent RDX degradative capability and contamination of soil columns with 1000 mg RDX/kg soil did not significantly change the 16S rDNA bacterial community profile. The bacterial diversity remained high as estimated by the number of bands present in the DGGE and by NQ-78704 statistical rarefaction analysis of 16S rDNA clone RFLPs. The same soil, used in 10% soil slurries (w/v), demonstrated two apparently different RDX degradation mechanisms based on mineralization and chemical analysis. The differences were based on aerobic versus anaerobic conditions and the presence/absence of Na3 citrate. PCR-DGGE performed on 16S rDNA from aerobic slurries amended with Na3-citrate detected the stimulation of 3 operational taxonomic units, identified as Stenotrophomonas sp., Sphingomonas sp. and a member of the Alcaligenaceae. The results from the two systems examined (short- to medium-term and long-term contamination) demonstrated the utility of a polyphasic approach in the examina

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.

Feeding sub-therapeutic levels of antibiotics to livestock has been associated withdevelopment and spread of antibiotic resistant bacteria. The present experiment was conductedto investigate the effect of antibiotic alternatives (caffeic acid, chlorogenic acid, and carbadox)on the microbial ecology of swine feces in vitro.Minimum inhibitory concentrations of caffeic and chlorogenic acids were determined forseveral pathogens using macrobroth and agar dilution techniques. Gram-negative bacteria werenot inhibited. Caffeic acid inhibited four Staphylococcus aureus strains at 200 ppm or less, andtwo Clostridium perfringens strains at 300 ppm. Chlorogenic acid inhibited all S. aureus strainsat 500 ppm, and one C. perfringens strain at 400 ppm.Effects of antibiotic alternatives on fecal microbial ecology were determined using an invitro incubation. Caffeic acid lowered total anaerobes, Bifidobacteria, Escherichia coli, andpercent E. coli (pandlt;0.01). Chlorogenic acid lowered total anaerobes, Bifidobacteria, andlactobacilli (pandlt;0.01), and increased acetate concentration (pandlt;0.0001). Carbadox lowered totalanaerobes, Bifidobacteria, E. coli, and coliforms (pandlt;0.01), and lowered acetate, propionate,butyrate, valerate, and total volatile fatty acid concentrations (pandlt;0.01). It can be concluded thataddition of caffeic acid, chlorogenic acid, or carbadox effected bacterial and chemicalcomponents of the microbial ecology of swine feces.