Ecological Dynamics in Compost-Amended Soils and the Resulting Effects on Escherichia coli Survival

Escherichia coli (E. coli) are common and typically innocuous copiotrophic bacteria found in the mammalian gut microbiome. However, over the past 30 years, pathogenic E. coli have been responsible for several outbreaks of foodborne illness linked to contaminated produce. The introduction of Escherichia coli to an agricultural soil, via contaminated water, compost, or raw manure, exposes the bacterium to a medley of ecological forces not found in a mammalian gut environment. This study assesses a variety of abiotic and biotic soil factors that influence the ability of an invasive copiotrophic coliform bacterium to survive in compost-amended agricultural soil. The study included both field and laboratory components. In the lab experiment, a cocktail of rifampicin-resistant generic E.coli strains was added to sterile and non-sterile extracts of eight different composts and one soil sample from the field sites. E. coli abundance was monitored over a one-week period and composts were analyzed for their nutrient profile. In the field experiment, the same E. coli cocktail was sprayed on plots with the following treatments: 1) dairy windrow compost, 2) dairy vermicompost, 3) poultry windrow compost, or 4) no compost. E. coli abundance, soil water potential, soil temperature, extracellular enzyme activity, microbial respiration, phospholipid fatty acid biomarker abundance, and genetic sequencing of the microbial community were measured over a six-month field season. The lab experiment showed that E. coli were able to grow well in sterile compost extracts, without microbial competition for nutrients. Conversely, E. coli populations were only able to survive in non-sterile soil extracts. These results suggest that copiotrophic organisms adapted for high-nutrient environments may depend on the extracellular enzyme activity of native oligotrophic organisms to acquire sufficient nutrients to survive in soils. Results of the field experiment showed clear and interdependent effects of soil moisture and nutrient availability on microbial community dynamics and E. coli survival. Data suggest that saturated soils cause a decrease in microbial extracellular enzyme activity, and drying-rewetting cycles can cause respiration bursts, nutrient mineralization, and shifts in community composition. The saturation of soils, which mobilizes nutrients and may result in a decrease in competition from aerobic organisms, correlated directly with increased survival of E. coli. Additionally, amendment with ammonium-rich poultry compost resulted in the maintenance of high levels of E. coli throughout the field season. Despite an increase in microbial biomass from dairy vermicompost amendment, poultry compost was the only compost that had a significant effect on E. coli survival. The results suggest that nitrogen availability and water potential are strong drivers of E. coli\u27s survival in soils. Correlations among abiotic factors, community composition, and E. coli survival reveal insights into the complex relationships that occur in disturbed agricultural soil environments. Further research on E. coli\u27s response to targeted organisms, abiotic soil properties, and nutrient inputs could have implications for agricultural considerations in food safety and microbial ecology

Quantitative physiology of bacterial survival under carbon starvation and temperature stress

A large number of the bacteria on Earth live for long periods in states of very low metabolic activity and little or no growth due to starvation and other environmental stresses. Within millions of years, bacteria have developed several strategies to adapt to many different environments, where they survive and evolve to optimize their fitness and to undergo rapid division cycles when conditions become favourable. However, many of these survival strategies are still a puzzle and relatively little is known about the mechanisms that underpin the dominant modes of bacterial existence. This is particularly alarming, as the growth-arrest phase has become crucial to understand the contribution of microorganisms to human physiology and predisposition to disease as well as microbial tolerance and resistance to antibiotics. The dearth of information is mainly due to the difficulties in defining, reproducing and measuring bacterial behaviours in growth-arrest states, which may often seem erratic and unpredictable, while cell physiology is similarly diverse and often specific to the particular environmental conditions. Thus, determining how molecular contributions affect survival is challenging. This explains why, in the last century, bacteria have been mainly studied during the exponential growth phase, which is, on the contrary, a well-defined and reproducible steady state of constant growth, gene expression and molecular compositions. As a result, an increasing combined use of experiments and predictive models focused on this phase has provided a deep understanding of bacterial physiology and gene regulation during growth. A similar quantitative approach that focuses on the growth-arrest phase is largely missing. In this thesis, we contribute to fill this gap by developing new quantitative approaches to investigate bacterial physiology in hostile environments where stresses, such as lack of nutrients and additional environmental perturbations, like temperature increase, force the cells to activate strategies of survival. To do so, we choose to work with the bacterium Escherichia coli (E. coli) that, among the estimated 10^12 microbial species living in our planet, is one of the most studied thanks to its hardiness, versatility and ease of handling. In Chapter 1, we provide an overview of the physiology of E. coli life cycle and of the main quantitative methods so far used to study it, especially focusing on its behaviour during the growth-arrest phase. In Chapter 2, we establish the missing quantitative approach to study E. coli physiology in the death phase. We show that in carbon starvation, an exponential decay of viability emerges as a collective phenomenon, with viable cells recycling nutrients from dead cells to maintain viability. The observed collective death rate is determined by the maintenance rate of viable cells and the amount of nutrients recovered from dead cells, the yield. Using this relation, we study the cost of a wasteful enzyme during starvation and the benefit of the stress response sigma factor RpoS. While the enzyme activity increases maintenance and thereby the death rate, RpoS improves biomass recycling, decreasing the death rate. Our approach thus enables quantitative analyses of how cellular components affect the survival of non-growing cells. In Chapter 3, we use the quantitative approach developed in the previous chapter to study how survival of E. coli in carbon starvation depends on the previous culture conditions. We show that environments that support only slow growth lead to longer survival in starvation because of a decrease of maintenance rate, meaning that slower growing cells need less energy to survive. Our results suggest a physiological trade-off between the ability to proliferate fast and the ability to survive long that could shed light on the long-standing question of why bacteria outside of laboratory environments are not optimized for fast growth. In Chapter 4, we study E. coli physiology under the combined stresses of carbon starvation and high temperatures, characterizing a thermal fuse that leads to a dormant and antibiotic persistent sub-population. This fuse is implemented by a thermally unstable enzyme, MetA, in the methionine synthesis pathway. The combination of a positive feed-back in the methionine system and a dual-use of methionine for protein synthesis and as a methyl-donor results in the bacterial population splitting into two distinct states at elevated temperatures, growing and dormant. We then reveal that these dormant bacteria not only survive antibiotic treatment, but also heat shocks, suggesting that the thermal fuse has originally evolved as a ''bet-hedging'' strategy to ensure survival in heat shocks. Our findings, summarized in Chapter 5, pave the way for the development of a new theoretical framework and experimental approach to understand bacterial physiology in the growth-arrest phase, by linking phenomenological modeling to molecular mechanisms.Eine große Anzahl der Bakterien auf der Erde lebt über große Zeiträume in einem Zustand mit sehr geringer Stoffwechselaktivität und nur geringem oder keinem Wachstum. Ein Grund dafür sind widrige Umwelteinflüsse und die damit einhergehenden Belastungen wie beispielsweise Ressourcenmangel. Innerhalb von Millionen von Jahren haben Bakterien diverse Strategien zur Anpassung an verschiedene Umgebungen, in denen sie überleben und sich weiterentwickeln, entwickelt, um ihre Fitness zu optimieren und bei günstigen Bedingungen schnelle Teilungszyklen zu durchlaufen. Viele dieser Überlebensstrategien sind jedoch immer noch ein Rätsel und es ist nur relativ wenig über die Mechanismen bekannt, die den dominanten Formen der bakteriellen Existenz zu Grunde liegen. Dies ist von besonderer Bedeutung, da die Phase unterdrückten Wachstums entscheidend ist, um den Beitrag von Mikroorganismen zur menschlichen Physiologie und Anfälligkeit für Krankheiten, sowie zur mikrobiellen Verträglichkeit und Antibiotikaresistenz zu verstehen. Der Mangel an Informationen ist hauptsächlich auf die Schwierigkeiten bei der Definition, Reproduktion und Messung des Verhaltens von Bakterien in Zuständen des Wachstumsstillstands zurückzuführen, die oft unberechenbar und unvorhersehbar erscheinen, während die Zellphysiologie ähnlich vielfältig und oft spezifisch für die jeweiligen Umgebungsbedingungen ist. Daher ist es schwierig zu bestimmen, wie sich molekulare Mechanismen auf das Überleben auswirken. Dies erklärt, warum im letzten Jahrhundert Bakterien hauptsächlich während der exponentiellen Wachstumsphase untersucht wurden, die im Gegenteil ein genau definierter und reproduzierbarer Gleichgewichtszustand des konstanten Wachstums, der Genexpression und der molekularen Zusammensetzung ist. Infolgedessen hat eine zunehmende Kombination von Experimenten und Vorhersagemodellen, die sich auf diese Phase konzentrieren, ein tiefes Verständnis der bakteriellen Physiologie und Genregulation während des Wachstums geliefert. Ein ähnlicher quantitativer Ansatz, der sich auf die Phase der Stagnation konzentriert, fehlt weitgehend. In dieser Doktorarbeit tragen wir dazu bei, diese Lücke durch die Entwicklung neuer quantitativer Ansätze zur Untersuchung der bakteriellen Physiologie in ungünstigen Umgebungen zu füllen, in denen Stressfaktoren, wie beispielsweise Nährstoffmangel, auftreten und zusätzliche umweltbedingte Störungen, wie eine Temperaturerhöhung, die Zellen zwingen, Strategien zum Überleben zu aktivieren. Dazu arbeiten wir mit dem Bakterium Escherichia coli (E. coli), das unter den circa 10^12 mikrobiellen Spezies, die auf unserem Planeten leben, wegen seiner Widerstandsfähigkeit, Vielseitigkeit und einfachen Handhabung eines der am besten untersuchten Bakterien darstellt. In Kapitel 1, geben wir einen Überblick über die Physiologie des Lebenszyklus von E. coli und über die wichtigsten bisher verwendeten quantitativen Methoden, wobei wir uns auf das Verhalten während der Wachstumsphase konzentrieren. In Kapitel 2, stellen wir den fehlenden quantitativen Ansatz zur Untersuchung der Physiologie von E. coli während der Sterbephase fest. Wir zeigen, dass bei Kohlenstoffmangel ein exponentieller Zerfall der Lebensfähigkeit als kollektives Phänomen auftritt, wobei lebensfähige Zellen Nährstoffe aus toten Zellen recyceln, um die Lebensfähigkeit aufrechtzuerhalten. Die beobachtete kollektive Sterberate wird durch die Erhaltungsrate lebensfähiger Zellen und die Menge an Nährstoffen, die aus toten Zellen als Ertrag gewonnen werden, bestimmt. Unter Verwendung dieser Beziehung untersuchen wir die Kosten einer verschwenderischen Enzymaktivität während des Hungerns und den Nutzen des Sigma Faktors RpoS für die Stressreaktion. Während diese Aktivität die Instandhaltung und damit die Sterblichkeitsrate erhöht, verbessert RpoS das Recycling der Biomasse und senkt die Sterblichkeitsrate. Unser Ansatz ermöglicht daher quantitative Analysen darüber, wie sich zelluläre Komponenten auf das Überleben nicht wachsender Zellen auswirken. In Kapitel 3, verwenden wir den im vorherigen Kapitel entwickelten quantitativen Ansatz, um zu untersuchen, wie das Überleben von E. coli bei Kohlenstoffmangel von den vorherigen Kulturbedingungen abhängt. Wir zeigen, dass Umgebungen, die nur langsames Wachstum unterstützen, aufgrund einer verringerten Erhaltungsrate zu einem längeren Überleben führen, was bedeutet, dass langsamer wachsende Zellen weniger Energie zum Überleben benötigen. Unsere Ergebnisse legen einen physiologischen Kompromiss zwischen der Fähigkeit, sich schnell zu vermehren, und der Fähigkeit, lange zu überleben, nahe, der Auschluss darüber geben könnte, warum Bakterien außerhalb von Laborumgebungen nicht für schnelles Wachstum optimiert sind. In Kapitel 4, untersuchen wir die Physiologie von E. coli unter dem kombinierten Stress von Kohlenstoffmangel und hohen Temperaturen und charakterisieren eine thermische Sicherung, die zu einer ruhenden und antibiotisch persistierenden Subpopulation führt. Diese Sicherung wird durch ein thermisch instabiles Enzym, MetA, im Methioninsyntheseweg implementiert. Die Kombination aus einer positiven Rückkopplung im Methioninsystem und einer doppelten Verwendung von Methionin für die Proteinsynthese und als Methyldonor führt dazu, dass sich die Bakterienpopulation bei erhöhten Temperaturen in zwei verschiedene Zustände aufspaltet, wobei jeweils eine Subpopulation wächst und die Andere schläft. Wir zeigen dann, dass diese ruhenden Bakterien nicht nur eine Antibiotikabehandlung, sondern auch Hitzeschocks überstehen, was darauf hindeutet, dass sich die thermische Sicherung ursprünglich als eine ''bet-hedging'' Strategie entwickelt hat, um das Überleben bei Hitzeschocks sicherzustellen. Unsere Ergebnisse, die in Kapitel 5 zusammengefasst sind, ebnen den Weg für die Entwicklung eines neuen theoretischen Rahmens und experimentellen Ansatzes zum Verständnis der Bakterienphysiologie in der Phase des Wachstumsstopps, indem phänomenologische Modelle mit molekularen Mechanismen verknüpft werden

Attachment strength and on-farm die-off rate of Escherichia coli on watermelon surfaces

Pre-harvest contamination of produce has been a major food safety focus. Insight into the behavior of enteric pathogens on produce in pre-harvest conditions will aid in developing pre-harvest and post-harvest risk management strategies. In this study, the attachment strength (SR) and die-off rate of E. coli on the surface of watermelon fruits and the efficacy of aqueous chlorine treatment against strongly attached E. coli population were investigated. Watermelon seedlings were transplanted into eighteen plots. Prior to harvesting, a cocktail of generic E. coli (ATCC 23716, 25922 and 11775) was inoculated on the surface of the watermelon fruits (n = 162) and the attachment strength (SR) values and the daily die-off rates were examined up to 6 days by attachment assay. After 120 h, watermelon samples were treated with aqueous chlorine (150 ppm free chlorine for 3 min). The SR value of the E. coli cells on watermelon surfaces significantly increased (P\u3c0.05) from 0.04 to 0.99 in the first 24 h, which was primarily due to the decrease in loosely attached population, given that the population of strongly attached cells was constant. Thereafter, there was no significant change in SR values, up to 120 h. The daily die-off rate of E. coli ranged from -0.12 to 1.3 log CFU/cm2. The chlorine treatment reduced the E. coli level by 4.2 log CFU/cm2 (initial level 5.6 log CFU/cm2) and 0.62 log CFU/cm2 (initial level 1.8 log CFU/cm2), on the watermelons that had an attachment time of 30 min and 120 h respectively. Overall, our findings revealed that the population of E. coli on watermelon surfaces declined over time in an agricultural environment. Microbial contamination during pre-harvest stages may promote the formation of strongly attached cells on the produce surfaces, which could influence the efficacy of post-harvest washing and sanitation techniques

Sanitation Assessment of Food Contact Surfaces and Lethality of Moist Heat and a Disinfectant Against Listeria Strains Inoculated on Deli Slicer Components

The overall objectives of this study were to: evaluate the efficacy of different cleaning cloth types and cloth-disinfectant combinations in reducing food contact surface contamination to acceptable levels; determine the optimum moist heat and moist heat + sanitizer treatments that can significantly reduce the number of Listeria strains on deli slicer components; and investigate if the moist heat treatment used in this study induced the viable-but-non-culturable (VBNC) state in Listeria cells. The efficacy of wiping cloths was measured using ATP-bioluminescence and total plate count methods using four different wiping cloths and silver dihydrogen citrate sanitizer on food contact surfaces. The lethality study of moist heat and silver dihydrogen citrate disinfectant against Listeria strains was done using deli slicer components and the viable-but-non-culturable state of Listeria strains subjected to sub-lethal moist heat and silver dihydrogen citrate disinfectant stresses was measured using BacLight bacterial viability test kit. In the first study we demonstrated that the cleaning effects of wiping cloths on food contact surfaces can be enhanced when used with the SDC sanitizer and stated that the ATP-B measurements can be used for real-time hygiene monitoring in the public sector with inclusion of microbial contamination testing (total plate count) for more reliable measure of cleanliness. In the moist heat lethality study, the internal moist heat only treatment and both the internal and external moist heat + disinfectant treatments yielded non-detectable levels of Listeria strains on stainless steel and cast aluminum coupons. Moist heat only and moist heat + disinfectant treatments at 150 °F (66°C) and at least 20% relative humidity (RH) for 5 h was adequate to attain non-detectable levels of a Listeria strains cocktail on both stainless steel and cast aluminum deli meat slicer components. The BacLight bacterial viability test demonstrated that the moist heat treatment applied in this study was effective in inactivating Listeria strains. However, the absence of growth on nutrient agar plates and detection of live cells by the viability test demonstrated that the sub-lethal temperature used in this study could induce the VBNC state in Listeria strains

Iron storage and regulation at a molecular level

Iron is an essential element for the proper functioning of the metabolic network in a living system. However, it is also toxic in physiological conditions. Apart from precipitation it can damage and compromise cellular macromolecules by Fenton reactions. Thus, ferritins, hollow spherical proteins, comes to solve this problem by storing iron in its inner cavity. Dps (DNAbinding protein in starved cells), focused in this study, has a detoxifying function, protecting DNA from ROS. The reaction catalyzed by ferritins can be divided in the following stages: iron intake, oxidation, storage and release. The latter is the least explored and known function of this protein. The M. hydrocarbonoclasticus WrbA flavoprotein, present in the same genome, was used as a P. nauticas Dps redox partner, to reduced and release iron from the iron core. Mössbauer spectroscopy was used to investigate the kinetics properties of WrbA(FMN):NADH:Dps in anaerobic conditions. To determine kinetic parameters it was needed to acquire spectra for different reaction times. The iron release for wild-type, Q14E and Δ15 Dps variants follow a first-order kinetic, with rate constants very similar. Was also explored a more inexpensive and faster kinetic assay based on the ophenanthroline method, monitored by Visible spectroscopy. The result showed that the three Dps variants have no significant difference regarding the kinetic profile obtained, but rate constants were significantly lower than those obtained by Mössbauer spectroscopy probed kinetic measurements. Phenanthroline might cause an inhibitor effect and in order to understand that effect, the kinetic assays were repeated in the absence of phenanthroline. Using bioinformatic tools (docking, modeling and others), was possible to conclude that exist conserved amino acid (G43, L74, P78 e W149) in Dps that appear to participate and are in the electron transfer pathway

Proteome-wide measurement of non-canonical bacterial mistranslation by quantitative mass spectrometry of protein modifications.

The genetic code is virtually universal in biology and was likely established before the advent of cellular life. The extent to which mistranslation occurs is poorly understood and presents a fundamental question in basic research and production of recombinant proteins. Here we used shotgun proteomics combined with unbiased protein modification analysis to quantitatively analyze in vivo mistranslation in an E. coli strain with a defect in the editing mechanism of leucyl-tRNA synthetase. We detected the misincorporation of a non-proteinogenic amino acid norvaline on 10% of all measured leucine residues under microaerobic conditions and revealed preferential deployment of a tRNA(Leu)(CAG) isoacceptor during norvaline misincorporation. The strain with the norvalylated proteome demonstrated a substantial reduction in cell fitness under both prolonged aerobic and microaerobic cultivation. Unlike norvaline, isoleucine did not substitute for leucine even under harsh error-prone conditions. Our study introduces shotgun proteomics as a powerful tool in quantitative analysis of mistranslation

Quantitative physiology of bacterial survival under carbon starvation and temperature stress

A large number of the bacteria on Earth live for long periods in states of very low metabolic activity and little or no growth due to starvation and other environmental stresses. Within millions of years, bacteria have developed several strategies to adapt to many different environments, where they survive and evolve to optimize their fitness and to undergo rapid division cycles when conditions become favourable. However, many of these survival strategies are still a puzzle and relatively little is known about the mechanisms that underpin the dominant modes of bacterial existence. This is particularly alarming, as the growth-arrest phase has become crucial to understand the contribution of microorganisms to human physiology and predisposition to disease as well as microbial tolerance and resistance to antibiotics. The dearth of information is mainly due to the difficulties in defining, reproducing and measuring bacterial behaviours in growth-arrest states, which may often seem erratic and unpredictable, while cell physiology is similarly diverse and often specific to the particular environmental conditions. Thus, determining how molecular contributions affect survival is challenging. This explains why, in the last century, bacteria have been mainly studied during the exponential growth phase, which is, on the contrary, a well-defined and reproducible steady state of constant growth, gene expression and molecular compositions. As a result, an increasing combined use of experiments and predictive models focused on this phase has provided a deep understanding of bacterial physiology and gene regulation during growth. A similar quantitative approach that focuses on the growth-arrest phase is largely missing. In this thesis, we contribute to fill this gap by developing new quantitative approaches to investigate bacterial physiology in hostile environments where stresses, such as lack of nutrients and additional environmental perturbations, like temperature increase, force the cells to activate strategies of survival. To do so, we choose to work with the bacterium Escherichia coli (E. coli) that, among the estimated 10^12 microbial species living in our planet, is one of the most studied thanks to its hardiness, versatility and ease of handling. In Chapter 1, we provide an overview of the physiology of E. coli life cycle and of the main quantitative methods so far used to study it, especially focusing on its behaviour during the growth-arrest phase. In Chapter 2, we establish the missing quantitative approach to study E. coli physiology in the death phase. We show that in carbon starvation, an exponential decay of viability emerges as a collective phenomenon, with viable cells recycling nutrients from dead cells to maintain viability. The observed collective death rate is determined by the maintenance rate of viable cells and the amount of nutrients recovered from dead cells, the yield. Using this relation, we study the cost of a wasteful enzyme during starvation and the benefit of the stress response sigma factor RpoS. While the enzyme activity increases maintenance and thereby the death rate, RpoS improves biomass recycling, decreasing the death rate. Our approach thus enables quantitative analyses of how cellular components affect the survival of non-growing cells. In Chapter 3, we use the quantitative approach developed in the previous chapter to study how survival of E. coli in carbon starvation depends on the previous culture conditions. We show that environments that support only slow growth lead to longer survival in starvation because of a decrease of maintenance rate, meaning that slower growing cells need less energy to survive. Our results suggest a physiological trade-off between the ability to proliferate fast and the ability to survive long that could shed light on the long-standing question of why bacteria outside of laboratory environments are not optimized for fast growth. In Chapter 4, we study E. coli physiology under the combined stresses of carbon starvation and high temperatures, characterizing a thermal fuse that leads to a dormant and antibiotic persistent sub-population. This fuse is implemented by a thermally unstable enzyme, MetA, in the methionine synthesis pathway. The combination of a positive feed-back in the methionine system and a dual-use of methionine for protein synthesis and as a methyl-donor results in the bacterial population splitting into two distinct states at elevated temperatures, growing and dormant. We then reveal that these dormant bacteria not only survive antibiotic treatment, but also heat shocks, suggesting that the thermal fuse has originally evolved as a ''bet-hedging'' strategy to ensure survival in heat shocks. Our findings, summarized in Chapter 5, pave the way for the development of a new theoretical framework and experimental approach to understand bacterial physiology in the growth-arrest phase, by linking phenomenological modeling to molecular mechanisms.Eine große Anzahl der Bakterien auf der Erde lebt über große Zeiträume in einem Zustand mit sehr geringer Stoffwechselaktivität und nur geringem oder keinem Wachstum. Ein Grund dafür sind widrige Umwelteinflüsse und die damit einhergehenden Belastungen wie beispielsweise Ressourcenmangel. Innerhalb von Millionen von Jahren haben Bakterien diverse Strategien zur Anpassung an verschiedene Umgebungen, in denen sie überleben und sich weiterentwickeln, entwickelt, um ihre Fitness zu optimieren und bei günstigen Bedingungen schnelle Teilungszyklen zu durchlaufen. Viele dieser Überlebensstrategien sind jedoch immer noch ein Rätsel und es ist nur relativ wenig über die Mechanismen bekannt, die den dominanten Formen der bakteriellen Existenz zu Grunde liegen. Dies ist von besonderer Bedeutung, da die Phase unterdrückten Wachstums entscheidend ist, um den Beitrag von Mikroorganismen zur menschlichen Physiologie und Anfälligkeit für Krankheiten, sowie zur mikrobiellen Verträglichkeit und Antibiotikaresistenz zu verstehen. Der Mangel an Informationen ist hauptsächlich auf die Schwierigkeiten bei der Definition, Reproduktion und Messung des Verhaltens von Bakterien in Zuständen des Wachstumsstillstands zurückzuführen, die oft unberechenbar und unvorhersehbar erscheinen, während die Zellphysiologie ähnlich vielfältig und oft spezifisch für die jeweiligen Umgebungsbedingungen ist. Daher ist es schwierig zu bestimmen, wie sich molekulare Mechanismen auf das Überleben auswirken. Dies erklärt, warum im letzten Jahrhundert Bakterien hauptsächlich während der exponentiellen Wachstumsphase untersucht wurden, die im Gegenteil ein genau definierter und reproduzierbarer Gleichgewichtszustand des konstanten Wachstums, der Genexpression und der molekularen Zusammensetzung ist. Infolgedessen hat eine zunehmende Kombination von Experimenten und Vorhersagemodellen, die sich auf diese Phase konzentrieren, ein tiefes Verständnis der bakteriellen Physiologie und Genregulation während des Wachstums geliefert. Ein ähnlicher quantitativer Ansatz, der sich auf die Phase der Stagnation konzentriert, fehlt weitgehend. In dieser Doktorarbeit tragen wir dazu bei, diese Lücke durch die Entwicklung neuer quantitativer Ansätze zur Untersuchung der bakteriellen Physiologie in ungünstigen Umgebungen zu füllen, in denen Stressfaktoren, wie beispielsweise Nährstoffmangel, auftreten und zusätzliche umweltbedingte Störungen, wie eine Temperaturerhöhung, die Zellen zwingen, Strategien zum Überleben zu aktivieren. Dazu arbeiten wir mit dem Bakterium Escherichia coli (E. coli), das unter den circa 10^12 mikrobiellen Spezies, die auf unserem Planeten leben, wegen seiner Widerstandsfähigkeit, Vielseitigkeit und einfachen Handhabung eines der am besten untersuchten Bakterien darstellt. In Kapitel 1, geben wir einen Überblick über die Physiologie des Lebenszyklus von E. coli und über die wichtigsten bisher verwendeten quantitativen Methoden, wobei wir uns auf das Verhalten während der Wachstumsphase konzentrieren. In Kapitel 2, stellen wir den fehlenden quantitativen Ansatz zur Untersuchung der Physiologie von E. coli während der Sterbephase fest. Wir zeigen, dass bei Kohlenstoffmangel ein exponentieller Zerfall der Lebensfähigkeit als kollektives Phänomen auftritt, wobei lebensfähige Zellen Nährstoffe aus toten Zellen recyceln, um die Lebensfähigkeit aufrechtzuerhalten. Die beobachtete kollektive Sterberate wird durch die Erhaltungsrate lebensfähiger Zellen und die Menge an Nährstoffen, die aus toten Zellen als Ertrag gewonnen werden, bestimmt. Unter Verwendung dieser Beziehung untersuchen wir die Kosten einer verschwenderischen Enzymaktivität während des Hungerns und den Nutzen des Sigma Faktors RpoS für die Stressreaktion. Während diese Aktivität die Instandhaltung und damit die Sterblichkeitsrate erhöht, verbessert RpoS das Recycling der Biomasse und senkt die Sterblichkeitsrate. Unser Ansatz ermöglicht daher quantitative Analysen darüber, wie sich zelluläre Komponenten auf das Überleben nicht wachsender Zellen auswirken. In Kapitel 3, verwenden wir den im vorherigen Kapitel entwickelten quantitativen Ansatz, um zu untersuchen, wie das Überleben von E. coli bei Kohlenstoffmangel von den vorherigen Kulturbedingungen abhängt. Wir zeigen, dass Umgebungen, die nur langsames Wachstum unterstützen, aufgrund einer verringerten Erhaltungsrate zu einem längeren Überleben führen, was bedeutet, dass langsamer wachsende Zellen weniger Energie zum Überleben benötigen. Unsere Ergebnisse legen einen physiologischen Kompromiss zwischen der Fähigkeit, sich schnell zu vermehren, und der Fähigkeit, lange zu überleben, nahe, der Auschluss darüber geben könnte, warum Bakterien außerhalb von Laborumgebungen nicht für schnelles Wachstum optimiert sind. In Kapitel 4, untersuchen wir die Physiologie von E. coli unter dem kombinierten Stress von Kohlenstoffmangel und hohen Temperaturen und charakterisieren eine thermische Sicherung, die zu einer ruhenden und antibiotisch persistierenden Subpopulation führt. Diese Sicherung wird durch ein thermisch instabiles Enzym, MetA, im Methioninsyntheseweg implementiert. Die Kombination aus einer positiven Rückkopplung im Methioninsystem und einer doppelten Verwendung von Methionin für die Proteinsynthese und als Methyldonor führt dazu, dass sich die Bakterienpopulation bei erhöhten Temperaturen in zwei verschiedene Zustände aufspaltet, wobei jeweils eine Subpopulation wächst und die Andere schläft. Wir zeigen dann, dass diese ruhenden Bakterien nicht nur eine Antibiotikabehandlung, sondern auch Hitzeschocks überstehen, was darauf hindeutet, dass sich die thermische Sicherung ursprünglich als eine ''bet-hedging'' Strategie entwickelt hat, um das Überleben bei Hitzeschocks sicherzustellen. Unsere Ergebnisse, die in Kapitel 5 zusammengefasst sind, ebnen den Weg für die Entwicklung eines neuen theoretischen Rahmens und experimentellen Ansatzes zum Verständnis der Bakterienphysiologie in der Phase des Wachstumsstopps, indem phänomenologische Modelle mit molekularen Mechanismen verknüpft werden

Bacteria in Public Swimming Pools – Inactivation Kinetics, Prevalence of Pathogens and Value of Indicators

Swimming pools and other recreational areas (e.g. hot tubs, saunas) enjoy worldwide popularity. Although procedures like disinfection are mandatory, swimming pools are commonly inhabited by diverse microorganisms potentially hazardous to human health, including viruses, bacteria, protozoans and fungi. Yet, there is a lack of comprehensive knowledge on the inactivation of microbes by disinfectants, the prevalence of pathogens in swimming pool water and the value of indicators in assessing the dangers associated. One common class of disinfecting agents used in swimming pools are chlorine compounds, both because of their effectiveness in inactivating microbes and the accompanied low costs. Using chlorine, inactivation of microbes relies on the powerful oxidizing features of these compounds. Furthermore, as chlorine remains stable in water, these compounds may be used as residual agents. However, this common practice is accompanied by considerable disadvantages. Chlorine compounds readily react with a broad spectrum of potential partners, of which microbes are only a fraction. As a result, a large variety of disinfection by-products are released, some of which are significantly unhealthy. Considering the lack of knowledge on microbial hazards, the question arises, which concentrations of chlorine are necessary and preferable in public swimming pools. The present study addresses several aspects of environmental hygiene associated with the use of chlorine in swimming pools. Two main tools in maintaining hygienic conditions are assessed: chlorine disinfection and evaluation of pool water quality using indicator bacteria. Routine data was analyzed for the occurrence of bacterial indicators. Applying MALDI-TOF techniques, it was determined which cultivable and potentially hazardous bacteria may be found in (German) public swimming pools. In order to include also less accessible and therefore less controllable parts of the microbial ecosystem of swimming pools, sand filter material was sampled and examined. Furthermore, inactivation kinetics of bacteria by hypochlorous acid were evaluated based on experiments using a setup mimicking swimming pool conditions and three exemplary test strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus). Raw data was fitted with several mathematical models taken from literature. In a parallel approach, chlorine consumption during the disinfection process was examined. To date, regulations on swimming pool maintenance mainly rely on practical experience rather than on empiric data from scientific studies. The present thesis is motivated by the intention to fill this gap by providing information on the topics of disinfection and monitoring. Inactivation kinetics followed comprehensible trends comparable to the results achieved in other fields of research. Chlorine consumption during this process proceeded quite fast, which presumably influenced the outcome. The results on indicators and pathogens imply that the practice of using the first to assess the presence of the latter is questionable. Especially E. coli proved very susceptible to chlorine inactivation, making it a weak indicator. For P. aeruginosa, the occurrence of survival states (small colony variants, viable but non-culturable states) is assumed. Since the assessment of hygiene relies on culture-dependent methods, this has implications for swimming pool maintenance. The Gram-positive species S. aureus was considerably more resistant to chlorine than its Gram-negative counterparts, raising the question whether it would be more reasonable to use this species as an indicator. Also, the question arises if it is acceptable that normally only bacteria are used as indicators

Studies on stationary phase Vibrio sp. 2

Vibrio sp. 2 stationary phase cells are novel and interesting in that they are able to support phage growth in standing cultures, but not in shaken (aerated) cultures. Many physiological and morphological characteristics change when Vibrio sp. 2 stationary phase cells are removed from aeration: the relatively high levels of protein synthesis (Robb et al., 1977; 1978) decrease, with a concomitant increase in the levels of RNA synthesis; protein degradation rises from 1 %h⁻¹ to 2,9 %h⁻¹, and whilst the average cell length decreases, the range of cell lengths markedly increases. The magic spot nucleotides, ppGpp and pppGpp, which are present in stressed exponential phase Vibrio sp. 2 cells, are not detectable in stationary phase Vibrio cells. The specific proteolytic activity of shaking stationary phase cell-free extracts against the foreign protein [¹⁴C-me]globin was slightly higher than that of extracts from standing or exponential phase cells, while the specific proteolytic activity against [¹²⁵I]-insulin was slightly lower. On the basis of inhibitor studies and subcellular distribution, the proteolytic activities of the three types of extract differed. The addition of exogenous ATP to cell-free extracts either stimulated (Car & Woods, 1984) or depressed proteolytic activity depending on the procedure used to prepare the extracts. The proteolytic activity of fractions containing substantial amounts of membrane material, from all three types of extract, were markedly depressed by ATP. On preincubation of cell-free extracts from exponentially growing cells prior to assay of proteolytic activity, the activity was markedly stimulated (two- to four-fold). The stimulation,. however, varied, greatly between independently produced extracts. ATP had a much smaller stimulatory effect on preparations free of cell wall material from both types of stationary phase cells (the stimulation was less than two-fold), and the stimulation was not affected by preincubation of the extracts. Extracts prepared from starving cells, previously in exponential growth, were affected by the addition of ATP in a similar manner to that observed with stationary phase extracts (Car & Woods, 1984). Exponential and both types of stationary phase Vibrio sp. 2 cells have ATP-stimulated and ATP-depressed activities separable by ion-exchange chromatography, in addition to several other proteolytic activities. All types of Vibrio sp. 2 cells have a similar complement of proteolytic activities