Microorganisms are all around us. Some of them even live in our body and are essential for our healthy being. Study of microbial communities based on their genetic content has become very popular with the development of new technologies, which enable easy reading of DNA or RNA. The key role of these studies is usually to characterize significant microbial patterns of an environment. However, currently used visualization tools have many drawbacks for such analyses. The subject of this thesis is to design a R/Bioconductor package for simple creation of bipartite graphs from microbial data. This type of visualization brings many advantages for microbiome analysis. Benefits of bipartite graphs are further demonstrated by analysis of main parameters affecting computer processing of microbial data.

Ciliates are the most diversified protists in suboxic and anoxic habitats where they often form symbioses with prokaryotes. Although the diversity of anaerobic ciliates has been overlooked for a long time, anaerobic representatives can be found in most ciliate classes. This study focuses on anaerobic ciliates from the subclass Scuticociliatia, a neglected lineage, which belongs to the species-rich class Oligohymenophorea. One of the main outcomes resulting from this study is the discovery of a novel anaerobic clade of ciliates, from which only one species has been described molecularly to date. We have shown that the clade represents a diversified lineage, likely a new order. Thanks to the sampling of many freshwater and marine anoxic sediments, we have established the largest culture collection of anaerobic scuticociliates in the world. This has enabled us to determine the 18S rRNA gene sequences of 55 cultured anaerobic scuticociliates and to study their morphology both in-vivo and using various silver- impregnation methods. Besides, we applied transmission and scanning electron microscopy techniques to study the ultrastructure of both ciliates and symbionts. To identify the symbionts, we also employed other methods including microbiome sequencing and fluorescence in-situ hybridization. Since all...

Die Methan (CH4) Produktion der Milchkühe wird durch eine Vielzahl von umwelt- und wirtsspezifischen Faktoren beeinflusst, wobei Trockensubstanzaufnahme und Rationszusammensetzung die größte Auswirkung haben. Der größte Teil des CH4 wird von Archaeen im Pansen produziert. Auch die kurzkettige Fettsäure (SCFA) Acetat wird im Pansen durch mikrobielle Fermentation gebildet und kann vom Wirtstier zur Milchfettsynthese im Euter verwendet werden. Die Acetatbildung im Pansen korreliert mit der CH4 Produktion. Allerdings kann Milchfett auch aus nicht veresterten Fettsäuren (NEFA) und Triacylgylcerolen endogenen Ursprungs synthetisiert werden, insbesondere aus mobilisiertem Körperfett. In dieser Studie wurde die Hypothese überprüft, dass eine Verdrängung des zur Milchfettbildung genutzten Acetats durch eine höhere Körperfettmobilisation in der Frühlaktation die ruminale Acetatproduktion senkt und damit die Bildung von CH4 verringert. Ein weiteres Ziel war zu untersuchen, ob der Anstieg der CH4 Produktion im Laktationsverlauf mit einer Veränderung des Mikrobioms assoziiert ist, und ob sich Kühe mit hoher oder niedriger CH4 Emission in ihrer Bakterien- und Archaeen-Zusammensetzung unterscheiden. 20 Holstein Kühe wurden in ihrer ersten Laktation untersucht; ihre Futteraufnahme und Rationszusammensetzung wurde analysiert. Im Verlauf des Versuchs wurden mehrfach Blut- und Pansensaftproben gewonnen. Die Plasma-NEFA-Konzentrationen wurden photometrisch, die Pansen-SCFA-Konzentrationen mittels Gaschromatographie analysiert. Während des Beobachtungszeitraums wurde an 4 Zeitpunkten die individuelle CH4 Produktion in Respirationskammern erfasst. In einer Untergruppe von 9 Kühen wurden Pansensaftproben von 3 Zeitpunkten während der Laktation einer DNA-Extraktion unterzogen und bakterielle und archaeale 16S rRNA Amplicons wurden sequenziert. Die Bakterien- und Archaeenpopulation im Pansensaft wurden beschrieben und Pansenmikrobiom der CH4 Ausbeute gegenübergestellt. Statistische Auswertungen wurden mit repeated measurements ANOVA und Tukey Tests, sowie mit der Pearsons‘ Korrelation für ausgewählte Parameter durchgeführt. Mikrobielle Daten wurden mit multivariaten Analysen (PERMANOVA) weiterverarbeitet und Bray-Curtis-Unähnlichkeiten ermittelt. Die gesamte CH4 Produktion stieg signifikant von durchschnittlich 208 l/Tag in der Trockenperiode auf 516 l/Tag in der Spätlaktation an. Der Grad der Körperfettmobilisation, ausgedrückt als Plasma NEFA Konzentration, und die CH4 Ausbeute waren in der Frühlaktation negativ korreliert (p = 0,002). Kühe mit hoher Fettmobilisation (NEFA > 580 μmol/l) neigten nur vor der Geburt, aber nicht während der Laktation zu höheren Pansenacetat Konzentrationen als Tiere mit niedriger Mobilisation (NEFA < 580 μmol/l). Trotz einer möglichst gleichbleibenden Rationszusammensetzung während der Laktation änderte sich das Mikrobiom mit der Zeit signifikant, was sich in einer Abnahme des Artenreichtums und der Biodiversität zeigte. In der Spätlaktation, als die CH4 Ausbeute am höchsten war, gab es keinen Unterschied in der bakteriellen oder archealen Populationsstruktur zwischen den drei Kühen mit der schwächsten und den dreien mit der stärksten CH4 Ausbeute. Parallel zum Anstieg der CH4 Produktion von 434,3 l/Tag auf 540,5 l/Tag veränderte sich das Verhältnis von (Acetat + Butyrat) / Propionat im Pansensaft mit dem Fortschreiten der Laktation von 3,5 auf 4,4. Dennoch war kein Zusammenhang zwischen der Konzentration der ruminalen SCFA und der CH4 Ausbeute festzustellen. Der Stoffwechselzustand des Tieres, insbesondere der Grad der Körperfettmobilisierung bei negativer Energiebilanz, nahm Einfluss auf die CH4-Ausbeute. Die Zusammensetzung des Mikrobioms im Pansen und dessen Stoffwechselnetzwerk veränderte sich mit der Zeit. Es war jedoch in dieser Studie nicht möglich, einzelne Mikroorganismen als Prädiktor für die CH4-Emission von Milchkühen zu identifizieren. Vielmehr scheinen Verschiebungen der mikrobiellen Gemeinschaften insgesamt für die Veränderung der CH4 Ausbeute verantwortlich zu sein.:1 Introduction 1 2 Background 2 2.1 Greenhouse Gases 2 2.2 Dairy cows and their importance to food production 3 2.3 Rumen functions 5 2.3.1 Anatomy and Physiology 5 2.3.2 Rumen microbes 7 2.3.2.1 Bacteria 8 2.3.2.2 Archaea 11 2.3.3 Short-chain fatty acids 12 2.3.4 Methane formation 15 2.4 Interrelationship between methane and host animal physiology 15 2.4.1 Physiologic aspects affecting methane formation 15 2.5 Effects of feed composition and feed contents on methane production 16 2.5.1 Relationship of ruminal short-chain fatty acids and methane production 17 2.5.2 Milk fatty acids to estimate methane emission 19 2.6 Description of methods 20 2.6.1 Methane Measurement 20 2.6.2 Sampling of rumen contents 21 2.6.3 Methods to identify microbes 22 2.7 Objective and realization of the studies 23 3 Publications 26 3.1 First Publication 26 3.1.1 Supplement first Publication 40 3.2 Second Publication 42 3.2.1 Supplement second Publication 56 4 Discussion 60 4.1 Assessment of experimental design 60 4.1.1 Animals 60 4.1.2 Feed 61 4.1.3 Rumen fluid 61 4.1.4 Blood and milk metabolites 62 4.2 Assessment of results 62 4.2.1 Variance of methane emissions 62 4.2.2 Rumen short-chain fatty acids and methane 65 4.2.3 Acetate in the cows’ metabolism and methane production 66 4.2.4 Fat mobilization in early lactation 67 4.2.5 NEFA in the context of metabolism 68 4.2.6 Rumen microbes 69 4.2.6.1 Microbial community change over time 70 4.2.6.2 Community differences between individuals 71 4.2.6.3 Relationship between microbes and methane production levels 72 4.2.7 Further considerations 74 5 Conclusions 75 6 Summary 77 7 Zusammenfassung 79 8 References 81 / Methane (CH4) production in dairy cows is influenced by a variety of environmental and host-specific factors, among which dry matter intake and ration composition have the greatest impact. The major part of CH4 is produced in the rumen by Archaea. The short-chain fatty acid (SCFA) acetate is also produced in the rumen by microbial fermentation and can be used by the host to synthesize milk fat in the mammary gland. The production of acetate is correlated with ruminal CH4 production. Milk fat can also be synthesized from non-esterified fatty acids (NEFA) and triacylglycerol that originate from endogenous fat stores of dairy cows, especially during times of fat mobilization. This study checked the hypothesis that a higher fat mobilization during early lactation decreases ruminal acetate production by replacing acetate for milk fat synthesis and, thus, decreases synthesis of CH4. Another aim of this study was to test the hypothesis that increases in CH4 yield over the course of lactation are associated with changes in rumen microbial community profile, and that high and low CH4 emitting cows differ in their bacterial and archaeal community structure. A herd of 20 Holstein cows was studied during the course of their first lactation; feed intake and diet composition was monitored. Blood and rumen fluid were repeatedly sampled throughout the trial. Plasma NEFA concentrations were analyzed by photometrical analysis, and rumen SCFA concentrations by gas chromatography. Individual CH4 production was measured in respiration chambers at four times during the observation period. In a subgroup of 9 cows, rumen fluid samples from 3 timepoints during lactation were subjected to DNA extraction and bacterial and archaeal 16S rRNA amplicons were sequenced. The bacterial and archaeal community structures in the rumen fluid were described, and the rumen microbiome composition linked to CH4 yield. Statistical analysis was conducted using repeated measurement ANOVA and Tukey tests, as well as Pearsons’ correlation for selected parameters. Microbial data was further treated with multivariate analyses (PERMANOVA) and Bray-Curtis dissimilarities were determined. Total CH4 production increased significantly over time from an average 208 L/day during the dry period to 516 L/day in late lactation. The level of fat mobilization, expressed as blood plasma NEFA concentrations, and CH4 yield showed an inverse relationship in early lactation (p = 0.002). High mobilizing cows (NEFA > 580 μmol/L) tended to show higher ruminal acetate concentrations than low mobilizing cows (NEFA < 580 μmol/L) only before parturition and not during lactation. Despite a diet composition that was kept as constant as possible throughout the lactation, the microbial community changed significantly over time as indicated by a decrease in species richness and species evenness. However, in late lactation when CH4 yield was highest, no difference in bacterial or archaeal community structure could be detected between the three highest CH4 yielding cows and the three lowest CH4 yielding cows. The ratio of (acetate + butyrate) / propionate in rumen fluid changed significantly with progressing lactation from 3.5 to 4.4, accompanied by an increase in CH4 production from 434.3 L/d to 540.5 L/d. However, no correlation between the concentration of ruminal SCFA and CH4 yield was found. The metabolic state of the animal, especially the degree of fat mobilization during times of negative energy balance, had an impact on CH4 yield. Also, the microbial community composition in the rumen and its metabolic network is adaptable and changes over time. However, in this study individual microorganisms could not be identified to serve as predictor for CH4 emission from dairy cows at the moment. Rather, shifts in the microbial communities as a whole appear to be responsible for the changes in CH4 yield.:1 Introduction 1 2 Background 2 2.1 Greenhouse Gases 2 2.2 Dairy cows and their importance to food production 3 2.3 Rumen functions 5 2.3.1 Anatomy and Physiology 5 2.3.2 Rumen microbes 7 2.3.2.1 Bacteria 8 2.3.2.2 Archaea 11 2.3.3 Short-chain fatty acids 12 2.3.4 Methane formation 15 2.4 Interrelationship between methane and host animal physiology 15 2.4.1 Physiologic aspects affecting methane formation 15 2.5 Effects of feed composition and feed contents on methane production 16 2.5.1 Relationship of ruminal short-chain fatty acids and methane production 17 2.5.2 Milk fatty acids to estimate methane emission 19 2.6 Description of methods 20 2.6.1 Methane Measurement 20 2.6.2 Sampling of rumen contents 21 2.6.3 Methods to identify microbes 22 2.7 Objective and realization of the studies 23 3 Publications 26 3.1 First Publication 26 3.1.1 Supplement first Publication 40 3.2 Second Publication 42 3.2.1 Supplement second Publication 56 4 Discussion 60 4.1 Assessment of experimental design 60 4.1.1 Animals 60 4.1.2 Feed 61 4.1.3 Rumen fluid 61 4.1.4 Blood and milk metabolites 62 4.2 Assessment of results 62 4.2.1 Variance of methane emissions 62 4.2.2 Rumen short-chain fatty acids and methane 65 4.2.3 Acetate in the cows’ metabolism and methane production 66 4.2.4 Fat mobilization in early lactation 67 4.2.5 NEFA in the context of metabolism 68 4.2.6 Rumen microbes 69 4.2.6.1 Microbial community change over time 70 4.2.6.2 Community differences between individuals 71 4.2.6.3 Relationship between microbes and methane production levels 72 4.2.7 Further considerations 74 5 Conclusions 75 6 Summary 77 7 Zusammenfassung 79 8 References 81

Water crises are predicted to be amongst the risks of highest concern for the next ten years, due to availability, accessibility, quality and management issues. Knowledge of the microbial communities indigenous to drinking water is essential for treatment and distribution process control, risk assessment and infrastructure design. Drinking water distribution systems (DWDSs) ideally should deliver to the consumer water of the same microbial quality as that leaving a treatment plant (“biologically stable” according to WHO). At the start of this Ph.D. program water microbiology comprised conventional culturedependent methods, and no studies were available on microbial communities from source to tap. A method combining 16S rRNA gene pyrosequencing with flow cytometry was developed to accurately detect, characterize, and enumerate the microorganisms found in a water sample. Studies were conducted in seven fullscale Dutch DWDSs which transport low-AOC water without disinfectant residuals, produced from fresh water applying conventional treatment. Full-scale studies were also conducted at the desalination plant and DWDS of KAUST, Saudi Arabia where drinking water is produced from seawater applying RO membrane treatment and then transported with chlorine residual. Furthermore, biological stability was evaluated in a wastewater reuse application in the Netherlands. When low-AOC water was distributed without disinfectant residuals, greater bacterial richness was detected in the networks, however, temporal and spatial variations in the bacterial community were insignificant and a substantial fraction of the microbiome was still shared between the treated and transported water. This shared fraction was lower in the system transporting water with chlorine residual, where the eukaryotic community changed with residence time. The core microbiome was characterized and dominant members varied between the two systems. Biofilm and deposit-associated communities were found to drive tap water microbiology regardless of water source and treatment scheme. Network flushing was found to be a simple method to assess water microbiology. Biological stability was not associated with safety. The biological stability concept needs to be revised and quantified. Further research is needed to understand microbial functions and processes, how water communities affect the human microbiome, and what the “drinking” water microbiome is like in undeveloped countries. / The research presented in this doctoral dissertation was financially supported by and conducted in collaboration with Delft University of Technology (TU Delft) and Evides Waterbedrijf in the Netherlands.

<p>The most common food and supplement prebiotic fiber is inulin – most commonly extracted from chicory root. Fructo-oligosaccharide (FOS) is a smaller version of inulin, both containing mainly fructose units with β-1,2 linkages. FOS/inulin has been used, and studied, as a prebiotic for decades. The potential of alternative prebiotics intrinsic in whole foods, such as in tubers, grains, vegetables, and pulses – the world’s most common staple crops – are not as commonly recognized as prebiotics, though have this potential if fermentable in the gut. If such alternative sources of prebiotic ingredients could be established it would allow for cheaper, possibly more effective, and more diverse food product development options beyond FOS/inulin. </p> <p>This study demonstrates the potential of tubers, grains, and pulses as prebiotics in relation to their <i>in vitro</i> human fecal fermentation rate, short chain fatty acid (SCFA) production, and microbiota indicators of alpha diversity and impact on key bacterial genera. Fecal samples were obtained from three diverse healthy human donors and used as the initial bacterial inoculum to simulate conditions in the human gut (colon). Substrates (n=35), after undergoing an upper gastrointestinal tract simulated digestion, were fermented by each individual donors’ inoculum separately, and measurements after 6, 12, and 24 h of fermentation were made on gas production, SCFA metabolite production, and microbiome composition. </p> <p>The results of this study establish high fermentability and potential prebiotic effects of dietary fibers from tubers, grains, and pulses. Whole foods, ground and cooked the same way, produced dietary fibers that were largely insoluble, but surprisingly fermentable with high SCFA levels, mostly slow fermentation profiles indicating high tolerability, and mostly promoting diverse microbiota responses compared to FOS. Generally, whole food fibers had higher fermentability than similar isolated fibers. Overall, the processing steps, such as atmospheric or pressure cooking, tested in some pulses did not detract, or add to, the prebiotic abilities of the substrates. Each food fiber substrate had unique effects on the gut microbiota parameters tested. Gut microbiome compositional responses to the same substrate varied significantly among the three donors, but notably SCFA metabolite responses were similar among donors. </p>

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Streptomycetes, primary soil saprophytic microorganisms are at the center of interest in many research groups, mainly because of their ability to produce a wide range of biologically active substances useful in medicine, biotechnology and agriculture. The marginal, and little explored areas are the interactions that streptomycetes create with humans. Recent metagenomic studies have shown that streptomycetes colonize the skin, the respiratory and possibly the urogenital tract of humans. In addition to apparent pathogens such as S. somaliensis and S. sudanensis, the clinical impact of these streptomycetes on human health is unknown. For this reason, a unique collection of non-pathogenic streptomycetes isolated from human clinical specimens was developed. The isolates were collected by the National Reference Laboratory for Pathogenic Actinomycetes in Trutnov, Czech Republic. On the basis of pilot studies, an isolate labeled TR42 was selected from the Trutnov collection, showing a very broad spectrum of biological activities. This strain was isolated from sputum from a patient with unknown respiratory diagnosis. The TR42 strain exhibits considerable biotechnological potential and after following a thorough study, it could be a source of new biologically active substances with pharmaceutically...

<div>The first 1000 days of life, from conception until the child’s second birthday, constitute a critical window for child growth and development. During infancy and early childhood, significant and rapid physical changes occur, including increases in weight, height, and brain size and organ development accompanied by cognitive and psychomotor development. Adequate infant feeding, including breastfeeding and complementary feeding, that meets the infants’ energy and nutrient requirements can help protect against growth faltering, infant and child morbidity and mortality, and delayed mental and motor development. Adequate nutrition during this critical period can also protect against adverse health outcomes and chronic diseases later in life according to the hypothesis of developmental origins of health and disease.</div><div><br></div><div>A web of factors that are country- and culture- specific influence infant feeding practices and child growth. Further, the microbiome has been suggested as a strong potential player in the association between infant nutrition and child growth. Therefore, the overarching theme of the current dissertation is to investigate hypotheses that can provide evidence to inform the paradigm linking socio-demographic, maternal, and child determinants including prenatal intake to infant feeding, the breast milk and infant gut microbiome, and child growth within the first 1000 days of life. Specifically, aims one and two examine the socio-demographic, maternal, and child determinants of child growth and breastfeeding in a cross-sectional survey of mother-child dyads in Lebanon, a middle-income country undergoing nutrition transition in the Middle East. The third aim focuses on the CHILD cohort study, a multi-center longitudinal prospective birth cohort study, to examine the associations between prenatal diet and supplement intake and the breast milk microbiome. Finally, the fourth aim is to review the evidence for the potential of the infant gut microbiome as a promising target linking complementary feeding to child undernutrition in low- and middle- income countries (LMIC) with the highest burden of undernutrition.</div><div><br></div><div>The results for aim one revealed sex-specific determinants of child growth in Lebanon. The determinants examined through a hierarchical conceptual framework included: maternal and paternal education among boys and crowding index among girls at the distal sociodemographic level, and maternal obesity among girls at the intermediate maternal level. The proximal child determinants included birth length, number of children in the household and breastfeeding duration among girls, birthweight among boys and child’s age among boys and girls.</div><div><br></div><div>In the analysis for aim two, breastfeeding practices were suboptimal in Lebanon as less than half (41.5%) of the infants were exclusively breastfed during the 40-day rest period and 12.3% were exclusively breastfed during the 6-month duration recommended by the World Health Organization. Higher socioeconomic status, as reflected by a larger number of cars owned, and C-section delivery were consistently inversely associated with lower odds of exclusive breastfeeding for 40 days and 6 months. Belonging to a family with more children was associated with higher odds of exclusive breastfeeding for 40 days; while maternal overweight and obesity were associated with lower odds of exclusive breastfeeding for 6 months.</div><div><br></div><div>Findings from aim three suggested that prenatal supplement use, but not prenatal dietary quality and patterns, modulate the breast milk microbiota composition in the CHILD cohort in Canada. This project was exploratory and utilized one of the largest birth cohort studies with available breast milk microbiome data. Specifically, use of vitamin C and D supplements plus multivitamins during any trimester in pregnancy was consistently associated with milk microbial diversity and genus composition before and after adjustment for socio-demographic, maternal, and child covariates. Use of other supplements such as fish oil, folate, and calcium was less consistently associated with the breast milk microbiome.</div><div>The fourth aim of the review chapter focused on the infant gut microbiome. The effects of complementary feeding on the infant gut microbiome are less commonly studied than those of breastfeeding, with most research conducted in high-income countries but not LMIC. In contrast, associations between inadequate complementary feeding and undernutrition have been examined in LMIC where undernutrition is most prevalent. Further, a disrupted gut microbiota has been associated with child undernutrition. Indeed, animal studies have suggested a causal association although the direction of the causality is not clear and is potentially bi-directional depending on genetic and environmental conditions. In light of the current state of knowledge described in our review supporting the potential of the gut microbiota as a key player in the relation between complementary feeding and undernutrition, the development of microbiota-directed interventions during the complementary period offers a promising route for undernutrition management.</div><div><br></div><div>Findings from the studies presented in this dissertation highlight several culture-specific determinants of child growth and breastfeeding in Lebanon. The findings also highlight the need for future research using longitudinal prospective cohorts, intervention trails and animal models to provide evidence for the proposed links to enhance the understanding of the paradigm. Such a holistic view of the determinants of and pathways between infant feeding and child growth are of great public health significance to improve the health of children throughout their lives.</div>

When patients develop new-onset infections after hospital admission, the origin of the infection is typically assumed to be nosocomial; however, ambulances are potentially unexplored reservoirs for emerging pathogens. This study seeks to identify the scope of bacterial contamination in rural East Tennessee ambulances. Though universal precautions and cleaning procedures aim to reduce the spread of infectious diseases to provider and patient, little is known about the bacterial microbiome of ambulances. To the best of our knowledge, this is the first study of its kind to be performed in the state of Tennessee and the first since the introduction of UVGI units as an ambulance-based COVID-19 infection control measure. Our dissemination of post-pandemic findings may impact ambulance sanitation measures and will add to the national and global knowledge pertaining to the microbiome of emergency medical patient transport systems. Ambulances in East Tennessee were sampled using environmental sampling contact plates. At least one active ambulance unit for each EMS service underwent sampling. Three samples were obtained from each of three areas: the floor of the ambulance transport area, the rear door panel inside the transport area and stretcher. The plates were then incubated at 30-35C for 48 hours. Colony counts were manually performed before the plates were shipped for species identification via MALDI-TOF DNA analysis by MIDI laboratories (Newark, DE). One plate from each ambulance door and stretcher was sent for bacterial identification. Only one sample returned free of growth. All floor samples, several stretcher samples, and three door samples presented vast growth with colonies too numerous to count. The results from bacterial identification showed all flora were human commensal flora or environmental flora. The flora found on ambulance doors with opportunistic capabilities are as follows: Staphylococcus hominis, Staphylococcus epidermidis, Enterobacter cloacae, Enterobacter xinagfangensis, Bacillus cereus, Klebsiella oxytoca, and Bacillus subtilis; and the flora found on the stretchers with opportunistic capabilities are as follows: Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus cohnii ssp urealyticus, Bacillus cereus, Corynebaccterium mucifaciens, Staphylococcus pettenkoferi, Klebsiella oxytoca, Staphylococcus capitis, Bacillus subtillis, and Staphylococcus caprae. In this era of increasing antibiotic resistance, it is concerning that several microbes with pathogenicity were found, including species that often confer the spread of resistance such as Klebsiella oxytoca and Enterobacter cloacae. Overall, the finding of numerous diverse colonies does not support adequate sanitation of the ambulances. Further study is required to identify the most effective sanitation methods, and further metagenomic study is needed to explore the presence of genes that facilitate the spread of microbial resistance.