Braunschweig, Techn. Universiẗat, Diss., 1998.

Zugl.: Braunschweig, Techn. Univ., Diss., 2008

Zugl.: Braunschweig, Techn. Univ., Diss., 2008

joruiz@clinic.ub.es / Introduction: Bartonella bacilliformis is the etiological agent of Carrion’s disease, which is a neglected disease linked to people in low-socioeconomic populations in Andean valleys. An outbreak of B. bacilliformis was reported in a rural area of the Peruvian Amazon region. The aim of this study was to characterize this outbreak using molecular techniques. Methodology: Fifty-three blood samples from patients diagnosed with Carrion’s disease were analyzed by molecular tools, using both a Bartonella-specific polymerase chain reaction (PCR) and an universal PCR, both based on 16S rRNA gene amplification. Additional water samples from the area were also analyzed. Results: Unexpectedly, the samples were positive only when the universal PCR was used. Although environmental contamination cannot be ruled out, the results showed that Sphingomonas faeni was the possible causative agent of this outbreak, and that water was the most feasible infection source. Conclusions: Diagnosis by clinical criteria or microscopy may lead to misdiagnosis. There is a need to include molecular tools in the routine diagnosis of febrile syndromes, including Carrion’s disease.

The biodegradability and physicochemical properties of unsubstituted and methylated polycyclic aromatic hydrocarbons (PAHs) were investigated. The focus was on the development of models expressing the influence of molecular structure and properties on observed behavior. Linear free energy relationships (LFERs) were developed for the estimation of aqueous solubilities, octanol/water partition coefficients, and vapor pressures as functions of chromatographic retention time. LFERs were tested in the estimation of physicochemical properties for twenty methylated naphthalenes containing up to four methyl substituents. It was determined that LFERs can accurately estimate physicochemical properties for methylated naphthalenes. Twenty unsubstituted and methylated PAHs containing up to four aromatic rings were biodegraded individually by Sphingomonas paucimobilis strain EPA505, and Monod-type kinetic coefficients were estimated for each PAH using the integral method. Estimated extant kinetic parameters included the maximal specific biodegradation rate, the affinity coefficient, and the inhibition coefficient. The generic Andrews model adequately simulated kinetic data. The ability of PAHs to serve as sole energy and carbon sources was also evaluated. Quantitative structure-biodegradability relationships (QSBRs) were developed based on the estimates of the kinetic and growth parameters. A genetic algorithm was used for QSBR development. Statistical analysis and validation demonstrated the predictive value of the QSBRs. Spatial and topological molecular descriptors were essential in explaining biodegradability. Mechanistic interpretation of the kinetic data and the QSBRs provided evidence that simple or facilitated diffusion through the cell membranes is the rate-determining step in PAH biodegradation by strain EPA505. A kinetic experiment was conducted to investigate biodegradation of PAH mixtures by strain EPA505. The investigation focused on 2-methylphenanthrene, fluoranthene, and pyrene, and their mixtures. Integrated material balance equations describing different interaction types were fitted to the depletion data and evaluated on a statistical and probabilistic basis. Mixture degradation was most adequately described by a pure competitive interaction model with mutual substrate exclusivity, a fully predictive model utilizing parameters estimated in the sole-PAH experiments only. The models developed in this research provide insight into how molecular structure and properties influence physicochemical properties and biodegradability of PAHs. The models have considerable predictive value and could reduce the need for laboratory testing.

No description available.

Estrogens are a group of endocrine disrupting compounds capable of causing abnormalities in the reproductive systems of the wildlife. Wastewater is a major source of environmental estrogens, in part due to incomplete removal of estrogens in biological wastewater treatment processes. This dissertation investigated factors affecting estrogen biodegradation in bioreactors. Specifically, research efforts were placed on characterization of several bacterial estrogen degraders (model strains: Aminobacter strains KC6 and KC7, and a Sphingomonas strain KC8) and examination of the effects of operating parameters on estrogen removal and estrogen-degrading microbial community structure. Sphingomonas strain KC8 can use 17beta-estradiol as a sole carbon source, suggesting that estrogen degradation by KC8 is a growth-linked, metabolic reaction; however, estrogen degradation by strains KC6 and KC7 might be a non-growth linked, cometabolic reaction. One important finding was that strain KC8 can also degrade and further utilize testosterone as a growth substrate. Strain KC8 was characterized in terms of its utilization kinetics toward estrogens and testosterone with the results that showed relatively smaller kinetic parameters than the typical values for heterotrophs in activated sludge. Strain KC8 can also grow on other organic constituents (glucose, succinate, and acetate). Strain KC8 retained its ability to degrade both 17beta-estradiol and estrone (after 15 d of growth on a complex nutrient medium without 17beta-estradiol). Effective removals (>98.7 %) of 17beta-estradiol with no significant differences were observed in sequencing batch reactors (SBRs) under three solid retention times (SRTs of 5, 10, 20 d). The population ratios of known estrogen degraders (strains KC8 and ammonia-oxidizing bacteria (AOB)) and amoA gene (associated with ammonia oxidation) to total bacteria decreased as SRT increased in SBRs. These observations correspond to the decreasing percentages of 17 beta-estradiol biodegraded in SBR when SRT increased from 5 to 20 d, when the sorption of 17 beta-estradiol onto biomass was considered. Real-time terminal restriction fragment length polymorphism showed that more ribotypes were observed in SBR-20d than SBR-5d. The species evenness (E) in microbial community structures in SBRs was not affected by SRT. However, diversity indices (Shannon-Weaver diversity index (H) and the reciprocal of Simpson?s index (1/D)) suggest that longer SRTs might lead to a more diverse microbial community structure.

In dieser Arbeit wurden PCR-Primer für den Nachweis von Genen des bakteriellen Chloraromaten-Abbaus entwickelt. Als Zielgene wurden hierfür die Gene der Chlorcatechol-1,2-Dioxygenasen und Chlormuconat-Cycloisomerasen des modifizierten ortho-Weges ausgesucht. Die entwickelten Primer wurden an verschiedenen Chloraromaten abbauenden Bakterien getestet. Es gelang dabei erstmals, Fragmente von Chlormuconat-Cycloisomerase-Genen aus alpha-Proteobakterien zu erhalten. Mit den neu entwickelten Primern zum Nachweis der Chlorcatechol-1,2-Dioxygenase-Gene wurden aus den Stämmen Burkholderia sp. 3CB-1 und Rhodococcus opacus 1CP auch Fragmente amplifiziert, die nur relativ geringe Ähnlichkeiten zu bereits bekannten Genen aufwiesen. Um die Sequenzdatenbasis für das Primerdesign zu erweitern, wurden außerdem Chlorcatechol-Gencluster aus zwei Vertretern der alpha-Proteobakterien kloniert und sequenziert. Aus dem Stamm Sphingomonas sp. TFD44 konnten dabei zwei verschiedene Gencluster charakterisiert werden, von denen nur eines einen kompletten Satz der Chlorcatechol-Gene enthielt. Die beiden Gencluster aus dem anderen Stamm, Sphingomonas sp. EML146, wiesen Homologien zu diesem Gencluster auf. Die Konstruktion einer Knockout-Mutante und Proteinanreicherungen ergaben Hinweise auf weitere Chlorcatechol-Abbaugene in Sphingomonas sp. TFD44.

abstract: This thesis discusses the use of mass spectrometry and polymerase chain reaction (PCR), among other methods, to detect biomarkers of microorganisms in the environment. These methods can be used to detect bacteria involved in the degradation of environmental pollutants (bioremediation) or various single-celled pathogens, including those posing potential threats as bioterrorism agents. The first chapter introduces the hurdles in detecting in diverse environmental compartments in which they could be found, a select list of single-celled pathogens representing known or potential bioterrorism agents. These hurdles take the form of substances that interfere either directly or indirectly with the detection method. In the case of mass spectrometry-based detection, many of these substances (interferences) can be removed via effective sample pretreatment. Chapters 2 through 4 highlight specific methods developed to detect bioremediation or bioterrorism agents in environmental matrices. These methods are qualitative mass spectrometry, quantitative PCR, and quantitative mass spectrometry, respectively. The targeted organisms in these methods include several bioremediation agents, e.g. Pseudomonas putida F1 and Sphingomonas wittichii RW1, and bioterrorism agents, e.g. norovirus and Cryptosporidium parvum. In Chapter 2, I identify using qualitative mass spectrometry, biomarkers for three bacterial species involved in bioremediation. In Chapter 3, I report on a new quantitative PCR method suitable for monitoring of a key gene in yet another bioremediation agent, Sphingomonas wittichii RW1; furthermore, I apply this method to track the efficacy of bioremediation in bioaugmented environmental microcosms. In Chapter 4, I report on the development of new quantitative mass spectrometry methods for two organisms, S. wittichii RW1 and Cryptosporidium parvum, and evaluate two previously published methods for their applicability to the analysis of complex environmental samples. In Chapter 5, I review state-of-the-art methods for the detection of emerging biological contaminants, specifically viruses, in environmental samples. While this summary deals exclusively with viral pathogens, the advantages and remaining challenges identified are also applicable to all single-celled organisms in environmental settings. The suggestions I make at the end of this chapter are expected to be valid not only for future needs for emerging viruses but also for bacteria, eukaryotic pathogens, and prions. In general, it is advisable to continue the trend towards quantification and to standardize methods to facilitate comparison of results between studies. / Dissertation/Thesis / Ph.D. Biological Design 2012

In dieser Arbeit wurden PCR-Primer für den Nachweis von Genen des bakteriellen Chloraromaten-Abbaus entwickelt. Als Zielgene wurden hierfür die Gene der Chlorcatechol-1,2-Dioxygenasen und Chlormuconat-Cycloisomerasen des modifizierten ortho-Weges ausgesucht. Die entwickelten Primer wurden an verschiedenen Chloraromaten abbauenden Bakterien getestet. Es gelang dabei erstmals, Fragmente von Chlormuconat-Cycloisomerase-Genen aus alpha-Proteobakterien zu erhalten. Mit den neu entwickelten Primern zum Nachweis der Chlorcatechol-1,2-Dioxygenase-Gene wurden aus den Stämmen Burkholderia sp. 3CB-1 und Rhodococcus opacus 1CP auch Fragmente amplifiziert, die nur relativ geringe Ähnlichkeiten zu bereits bekannten Genen aufwiesen. Um die Sequenzdatenbasis für das Primerdesign zu erweitern, wurden außerdem Chlorcatechol-Gencluster aus zwei Vertretern der alpha-Proteobakterien kloniert und sequenziert. Aus dem Stamm Sphingomonas sp. TFD44 konnten dabei zwei verschiedene Gencluster charakterisiert werden, von denen nur eines einen kompletten Satz der Chlorcatechol-Gene enthielt. Die beiden Gencluster aus dem anderen Stamm, Sphingomonas sp. EML146, wiesen Homologien zu diesem Gencluster auf. Die Konstruktion einer Knockout-Mutante und Proteinanreicherungen ergaben Hinweise auf weitere Chlorcatechol-Abbaugene in Sphingomonas sp. TFD44.