The 1,1-dichloroethene (1,1-DCE) cometabolic transformation abilities of indigenous and bioaugmented microorganisms were compared in microcosms constructed with groundwater and aquifer solids from the Moffett Field site, CA. Microbial community structure in the microcosms and possible community shifts due to 1,1-DCE transformation stress was evaluated by terminal restriction fragment length polymorphism method (T-RFLP). An existing biotransformation model was used to simulate the experimental data using parameter values determined by Kim et al. (2002) and Rungkamol (2001) with small adjustments to the parameter values. The laboratory microcosm studies showed that both indigenous and bioaugmented butane utilizers were capable of transforming 1,1-DCE when fed butane as a primary substrate. A butane-grown enriched culture was bioaugmented into the microcosms and exposed to several repeated additions of butane and/or 1,1-DCE, ranging from 7.1 to 76 ��mol and from 0.17 to 1.99 ��mol, respectively. The bioaugmented butane-utilizers showed a reduced lag period compared to the indigenous butane-utilizers. The greatest ability to transform 1,1-DCE was observed in bioaugmented microcosms, simultaneously exposed to butane and 1,1-DCE. Very little 1,1-DCE was transformed in the bioaugmented microcosms that were not fed butane, presumably due to lack of reductant supply and/or product toxicity of 1,1-DCE transformation. Microbial community analyses revealed similar results for replicate microcosms and differences in the community structure in microcosms subjected to different patterns of substrate addition and 1,1-DCE cometabolism. 1,1-DCE transformation resulted in temporal fluctuations in specific bacterial groups in the bioaugmented microcosms. It could be inferred that microorganisms, correlated with the T-RFL of 183 base pair (bp) were generally predominant in butane-fed bioaugmented microcosms simultaneously exposed to 1,1-DCE. Bioaugmented microcosms that were pre-exposed to 1,1-DCE for 29 days in the absence of growth substrate, followed by the addition of butane showed a significantly different microbial community from bioaugmented microcosms fed butane and 1,1-DCE simultaneously. Microorganisms with T-RFL of 179 or 277.8 bp dominated in these microcosms. These differences were possibly the result of extensive 1,1-DCE transformation product toxicity during the pre-exposure phase of the tests. A model developed by Kim et al. (2002) was used to mathematically describe the rate and extent of butane utilization and the cometabolic transformation of 1,1-DCE in the microcosm tests. Using the kinetic parameter values previously determined by Kim et al. (2002) and Rungkamol (2001), heuristic fits were obtained between the experimental data and model simulations. The model successfully predicted the trend of the butane utilization and 1,1-DCE transformation. The model outputs were statistically quantified for their fit to the experimental data by estimating Standard Error of Estimate (SEE). A reasonable fit between model predictions and experimental observations was achieved. A significant contribution of this study was developing the laboratory methods to evaluate the microbial abilities to cometabolize 1,1-DCE and determining the communities of microorganisms correlated with those biotransformation activities. Furthermore, the model comparison to experimental data indicated that there was a potential in using the existing model to predict and improve bioremediation strategies. The results showed the successful bioaugmentation of a butane-utilizing culture to improve transformation performance. / Graduation date: 2003

Graduation date: 1997

This study was conducted to assess the incidence of infectious drug resistance among animal strains of Escherlchia coli. Fecal samples were examined from a cattle feedlot southeast of Muncie, Indiana. The use of tetracycline and sulfonamide compounds as feed additives was a common practice on this farm, and theoretically could provide the antibiotic pressure necessary to select for resistant strains. A total of 32 feedlot E. coli were isolated from the feedlot floor. In addition, 10 strains of E. coli isolated from patients with urinary tract infections were introduced into this investigation for comparative purposes. All isolates were tested for antibiotic susceptibility using disc-diffusion methods. Multiple resistant strains were designated as donor organisms and were used in conjugation experiments with an antibiotic sensitive recipient E. coli; wild type K-12 F-.Multiple drug resistance occurred among 21.8 percent of the feedlot E. coli isolated, with a predominance of resistance to chlorotetracycline, tetracycline, and sulfathiazole. Three out of seven multiple resistant donors isolated were able to transfer their antibiotic resistance markers to the recipient K-12 strain. Transferable drug resistance among the clinical isolates occurred in 6 of the 10 donor resistant strains, with tetracycline and ampicillin being the most frequently encountered resistance.Ball State UniversityMuncie, IN 47306

In some areas the effect of pathogenicity of microorganisms upon the European corn borer have been explored. The microorganisms studied were: Nosema, pyraustae, Nosema necatrix, and Bacillus thuringlensis var, thuringiensie. Most studies have been limited to either laboratory or field work. In this thesis, a comparative study of mortality, percent infection, and tune intervals in which infection occurs have been explored, in the field and the laboratory studies.It is concluded: (1) field recovered borers at different day intervals showed interaction between the experimental microorganisms and time intervals (2) that the microorganisms used in the field, especially N. necatrix, may be used as effective microbial control agents against Ostrinia nubilalis.Ball State UniversityMuncie, IN 47306

Ethanol is used as fuel in neat form in some countries (Brazil and India) or blended with gasoline (Europe, Canada and the United States). The benefits of ethanol use include octane enhancement, a cleaner environment and a secure renewable energy supply. BTEX compounds (benzene, toluene, ethylbenzene, m-xylene, p-xylene and o-xylene) are aromatic hydrocarbons present in gasoline. The fate of these compounds in the environment is of great health concern due to their carcinogenic (benzene) and toxic properties, and due to their high solubility in water compared to the other gasoline hydrocarbons. Ethanol present in gasoline may affect BTEX degradation, in an event of a spill into the subsurface environment. To address the effects of ethanol on subsurface microorganisms, microbial activity and growth in the presence of ethanol (concentrations ranging 0 to 70% v/v) were assessed. Microcosms studies showed that ethanol at concentration ranging 0. 5 to3% (v/v) enhanced microbial activity and did not interfere inmicrobial growth at 10oC temperature, when another source of carbon was present (glucose). Ethanol at 0. 5% concentration enhanced microbial activity over water soluble gasoline components and R2A medium combined. Both microbialactivity and growth were not detected at ethanol concentrations equal and above 5%. Biodegradation study was conducted, in which subsurface material and ground water were exposed to BTEX and ethanol at 0. 5 and 1. 5% (v/v) concentration. The controls had BTEX alone and ethanol alone, sterile and nutrient-free. Total BTEX degradation was observed whenever ethanol was absent. Ethanol and BTEX were simultaneously degraded, however in microcosms containing 0. 5% ethanol, BTEX degradation was slowed, compared to microcosms without ethanol. Competition for inorganic nutrients was the major problem in slowed BTEX degradation in the presence of ethanol. In microcosms where 1. 5% ethanol was present, BTEX compounds and ethanol degradation were not observed.

Ethanol is used as fuel in neat form in some countries (Brazil and India) or blended with gasoline (Europe, Canada and the United States). The benefits of ethanol use include octane enhancement, a cleaner environment and a secure renewable energy supply. BTEX compounds (benzene, toluene, ethylbenzene, m-xylene, p-xylene and o-xylene) are aromatic hydrocarbons present in gasoline. The fate of these compounds in the environment is of great health concern due to their carcinogenic (benzene) and toxic properties, and due to their high solubility in water compared to the other gasoline hydrocarbons. Ethanol present in gasoline may affect BTEX degradation, in an event of a spill into the subsurface environment. To address the effects of ethanol on subsurface microorganisms, microbial activity and growth in the presence of ethanol (concentrations ranging 0 to 70% v/v) were assessed. Microcosms studies showed that ethanol at concentration ranging 0. 5 to3% (v/v) enhanced microbial activity and did not interfere inmicrobial growth at 10oC temperature, when another source of carbon was present (glucose). Ethanol at 0. 5% concentration enhanced microbial activity over water soluble gasoline components and R2A medium combined. Both microbialactivity and growth were not detected at ethanol concentrations equal and above 5%. Biodegradation study was conducted, in which subsurface material and ground water were exposed to BTEX and ethanol at 0. 5 and 1. 5% (v/v) concentration. The controls had BTEX alone and ethanol alone, sterile and nutrient-free. Total BTEX degradation was observed whenever ethanol was absent. Ethanol and BTEX were simultaneously degraded, however in microcosms containing 0. 5% ethanol, BTEX degradation was slowed, compared to microcosms without ethanol. Competition for inorganic nutrients was the major problem in slowed BTEX degradation in the presence of ethanol. In microcosms where 1. 5% ethanol was present, BTEX compounds and ethanol degradation were not observed.

Thesis--M. Phil., University of Hong Kong. / Typewritten.

published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

Drug efflux represents an important protection mechanism against antibiotics and environmental toxic compounds in bacteria. Efflux genes constitute from 6% to 18% of all transporters in bacterial genomes, yet their regulation, natural substrates, and physiological functions are poorly understood. Among the 20 chromosomally encoded efflux genes in Escherichia coli K-12, only the AcrAB-TolC efflux system is constitutively expressed under the ordinary laboratory growth of E. coli. To explore conditions and circumstances that trigger the expression of additional efflux genes as well as their physiological functions, I examined the expression of all 20 efflux genes under a physiologically relevant circumstance for E. coli, which is anaerobic condition in this study. I found that expression of an RND type efflux pump MdtEF is up-regulated more than 20 fold when E. coli is cultured under anaerobic conditions. Mutagenesis studies revealed that the anaerobically induced expression of mdtEF is subject to the regulation of the anaerobic global transcription factor ArcA. Direct drug efflux and tolerance assay showed that anaerobically grown E. coli cells display an increased efflux activity and enhanced drug tolerance in an MdtEF dependent manner, confirming the functional up-regulation of the efflux pump MdtEF in the anaerobic physiology of E. coli. Since the up-regulation of mdtEF by anaerobic growth occurs in the absence of antibiotics and drugs, I speculate that MdtEF has physiological functions under the anaerobic growth of E. coli. To explore this, I first compared the viability of ΔmdtEF and WT MG1655 strains and found that ΔmdtEF caused a decreased cell survival during prolonged anaerobic growth of E. coli. Interestingly, this defect became more pronounced when cells grow in the presence of 10 mM nitrate, but no defect was observed in ΔmdtEF strain when cells grow in the presence of 40 mM fumarate under the same anaerobic conditions, suggesting that MdtEF has physiological roles relevant to the anaerobic respiration of nitrate. I further found that E. coli cells harboring the deletion of mdtEF are susceptible to indole nitrosative derivatives, a class of toxic by-products formed and accumulated within E. coli when the bacterium respires nitrate under anaerobic conditions, and deletion of the genes responsible for the biosynthesis of indole, tnaAB, restores the growth defect of the ΔmdtEF strain during anaerobic respiration of nitrate. Taken together, I conclude that the multidrug efflux pump MdtEF expels the nitrosated indole derivatives out of E. coli cells under anaerobic conditions. Since the production and accumulation of nitrosyl indole derivatives is ascribed to the reactive nitrogen species elicited when E. coli consumes nitrate, I propose that the up-regulated multidrug efflux pump MdtEF functions to protect E. coli from nitrosative damage in its anaerobic ecological niches. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Increasing carbapenem resistance among clinical isolates of E. coli and K. pneumoniae has become a serious public health problem over the last decade. Molecular epidemiology studies have shown that there is a global dissemination of epidemic clones of carbapenem-resistant E. coli and K. pneumoniae. Besides, successful epidemic plasmids were reported to disseminate carbapenemase genes in Enterobacteriaceae. The wide spread of carbapenem-resistant E. coli and K. pneumoniae limits treatment options of the infection, poses severe challenges to clinical professionals and threatens our health. Recently, carbapenem-resistant E. coli and K. pneumoniae are increasingly reported in Hong Kong. In 2012, our group has documented the emergence of carbapenem-resistant clinical isolates in Hong Kong. The findings of the previous study showed that 26.1% of the Enterobacteriaceae isolates were confirmed to produce carbapenemase. Notably, a novel IncX3 plasmid was found to be involved in the dissemination of blaNDM-1 gene. However, the previous findings fail to explicate the carbapenem resistance mechanisms of the remaining non-carbapenemase producing isolates. Further investigation is needed to elucidate the situation. Firstly, we investigated the carbapenem resistance mechanism of carbapenem-resistant E. coli and K. pneumoniae isolates recovered from the Hong Kong West Cluster hospitals from 2010 to 2012. PCRs were used to detect carbapenemase genes (blaNDM, blaKPC, blaIMP, blaVIM and blaOXA-48), blaCTX-M ESBL genes and blaAmpC genes. SDS-PAGE was used to detect porin loss. Among the 92 isolates in this study, only nine (9.8 %) isolates were detected with carbapenemase genes. The blaCTX-M and/or blaAmpC β-lactamase genes plus porin loss were detected in 47 non-carbapenemase-producing isolates (16 E. coli and 31 K. pneumoniae). The resistance determinant profiles of these 16 E. coli included: blaCTX-M + porin loss (n= 10), blaCIT + porin loss (n = 1), blaCTX-M + blaCIT/DHA + porin loss (n = 5). The resistance determinant profiles of the 31 K. pneumoniae included: blaCTX-M + porin loss (n= 4), blaDHA + porin loss (n = 7), blaCTX-M + blaCIT/DHA + porin loss (n = 20). The results showed that apart from acquired carbapenemases, the production of AmpC β-lactamase and/or ESBLs plus porin loss played a main role in the carbapenem resistance mechanism of the carbapenem-resistant E. coli and K. pneumoniae isolates. Secondly, we accessed the clonal relatedness of the isolates. Multi-locus sequence typing results showed that 55 (77.5%) K. pneumoniae isolates fall into the clonal complex 37. Our results suggest that the CC37 K. pneumoniae are associated with the acquisition of DHA-1 β-lactamase, CTXM-1-group β-lactamase and porin alterations which could confer a high-level of resistance to carbapenems resulting in their predominance in this study. Finally, we characterized the plasmids that carry carbapenemase gene by S1-PFGE, Southern blot, plasmid replicon typing and whole plasmid sequencing. A novel IncX3 plasmid was found to carry blaKPC gene. Together with the previously reported blaNDM-1 carrying IncX3 plasmids, it shows that IncX3 plasmids might be new epidemic plasmids involved in the dissemination of carbapenemase genes. These novel IncX3 plasmids are worrisome. Nationwide surveillance and more epidemiological study of IncX3 plasmids are needed. (Word / published_or_final_version / Microbiology / Master / Master of Philosophy