Background: Critically ill patients are at particular risk of developing bloodstream infection. Such infections are associated with the development of sepsis, leading to a marked increase in mortality rate. Early detection of the causative organism and appropriate antibiotic treatment are therefore critical for optimum outcome of patients with nosocomial infection. Current infection diagnosis is based on standard blood culture techniques. However, microbiological culture has a number of limitations, not least that it takes several days to confirm infection and is therefore not useful in directing the early treatment with antibiotics. New techniques based on the detection of pathogen DNA using real-time polymerase chain reaction (PCR) technology have the potential to address these limitations but their clinical utility is still to be proved. Objectives: Develop and evaluate novel PCR-based approaches to bloodstream infection diagnosis in critical illness based on detection and identification of bacterial and fungal DNA in blood. Methods: A range of commercial and 'in-house' PCR-based assays for detection of bacterial and fungal DNA were developed and/or optimised for use in clinical blood samples. These included LightCycler SeptiFast, a CE-marked multi-pathogen assay for common bloodstream pathogens, BactScreen and GramScreen, broad spectrum bacterial assays based on 16S rRNA gene and real-time PCR assays developed to detect a range of clinically important fungal pathogens. Novel approaches to speciation of pathogen DNA using melting temperature (Tm) profiling and high resolution melting analysis (HRMA) were developed. Clinical evaluation of assays was either on blinded clinical isolates or blood samples from critically ill patients with clinical suspicion of bloodstream infection against conventional microbiological culture. Several techniques aimed at improving extraction of pathogen DNA from blood were also investigated. Results: The CE-marked commercial assay SeptiFast showed analytical sensitivity and specificity of 79% and 83% respectively. Concordance with positive culture results was good but high levels of 'false positives' were detected possibly attributed to detection of free pathogen DNA not associated with viable pathogens. The predictive value of a negative SeptiFast test was 98% suggesting that absence of pathogen DNA is a strong indicator of absence of infection. Further studies were aimed at detailed optimisation and validation of 16S rRNA gene real-time PCR assays for bacterial DNA. BactScreen and GramScreen were able to detect a broad range of clinically important bacteria down to <50 CFU/ml blood. A preliminary comparative evaluation against SeptiFast showed BactScreen gave excellent concordance with blood culture results with minimal false positive results compared to SeptiFast. Efficient extraction of pathogen DNA was shown to be a key factor in determining analytical sensitivity and several protocols were evaluated. Low cost approaches to speciation of bacterial DNA were developed by combining broad range real-time PCR with HRMA. A novel HRMA method based on Tm profiling was shown to identify 89% and 96% of blinded clinical isolates at species or genus level respectively. Real-time PCR/HRMA approaches were also successfully developed for detection and identification of fungal pathogens including a range of Candida and Aspergillus species associated with bloodstream fungal infection. Conclusions: These studies have highlighted some of the key factors that need to be considered when developing and validating PCR based assays for pathogen DNA detection in blood. A set of novel tools have been developed for rapid detection and identification of bacterial and fungal pathogens that could address the challenges of infection diagnosis based on pathogen DNA detection. Further work is required, not least in development of more efficient pathogen DNA extraction and detailed clinical validation but the tools described here have the potential to provide cost effective solutions to aid infection diagnosis that would be complementary to current culture-based methods. The provision of time critical information could have a positive impact on clinical decision-making leading to more effective management and treatment of patients with suspected bloodstream infection.

Um método baseado na extração de rRNA, seguido de RT-PCR rRNA 16S, clonagem e ARDRA foi otimizado e validado para a identificação das bactérias ativas em biofilmes. O método foi analisado primeiro com consórcios artificiais de três organismos. As etapas de clonagem e RT não causaram variações importantes na composição destes consórcios, do contrário da etapa de PCR, onde foi necessária a redução de 30 para 10 ciclos para limitar a distorção da proporção de templates. A análise de biofilmes reais indicou que clones dominantes podem ser identificados com o critério de ocorrência de >2% na biblioteca, mas que a reprodutibilidade de análises ainda é insatisfatória, possivelmente devido a fatores como a micro heterogeneidade espacial do biofilme, viés na reação de PCR e formação de mais de um clone de ARDRA por organismo. O armazenamento do biofilme a -20 °C por 2 meses não levou à alterações expressivas em sua composição. O perfil de clones detectado com o kit (Mo Bio) de extração de RNA foi muito diferente do perfil detectado com o método otimizado neste trabalho. / A method based on extraction of rRNA, followed by RT-PCR of 16S rRNA, cloning and ARDRA was optimized and validated for identification of bacteria active in biofilms. The method was first tested with artificial three-membered consortia. Cloning and RT did not lead to significant changes in the composition of the artificial consortia, but a reduction in cycle number in the PCR reaction from 30 to 10 was necessary for limiting the distortion in the proportion of amplicons relative to that of the templates. Analysis of real biofilms revealed that clones from active organisms occurred in frequencies >2% in the clone library, but reproducibility of analysis was unsatisfactory, probably due to factors such as the spatial heterogeneity of colonization of biofilms by microbes, PCR bias and more than one ARDRA clone per organism. Storage of biofilm samples at -20 °C for 2 months did not lead to important changes in composition. Very different clone profiles were obtained in the analysis of the same biofilm sample with the optimized method and with a kit (Mo Bio) for extraction of RNA.

Tato práce se zabývá problematikou automatizované klasifikace a rozpoznávání bakterií po získání jejich DNA procesem sekvenování. V rámci této práce je navržena a popsána nová metoda klasifikace založená na základě segmentu 16S rRNA. Představený princip je vytvořen podle stromové struktury taxonomických kategorií a používá známé algoritmy strojového učení pro klasifikaci bakterií do jedné ze tříd na nižší taxonomické úrovni. Součástí práce je dále implementace popsaného algoritmu a vyhodnocení jeho přesnosti predikce. Přesnost klasifikace různých typů klasifikátorů a jejich nastavení je prozkoumána a je určeno nastavení, které dosahuje nejlepších výsledků. Přesnost implementovaného algoritmu je také porovnána s několika existujícími metodami. Během validace dosáhla implementovaná aplikace KTC více než 45% přesnosti při predikci rodu na datových sadách BLAST 16S i BLAST V4. Na závěr je zmíněno i několik možností vylepšení a rozšíření stávající implementace algoritmu.

With its unique consortium of microorganisms from all domains of life, termite gut is considered one of the most efficient lignocellulose degrading systems in nature. Recently, host diet and taxonomy as well as gut microenvironmental conditions have emerged as main factors shaping microbial communities in termite guts. The aim of this thesis was to investigate this highly efficient lignocellulolytic system at holobiont level, with a particular focus on gut microbiome function and composition in relation to the host diet. As a starting point, we optimised a complete framework for an accurate termite gut prokaryote-oriented metatranscriptomics, which was at the basis of all subsequent sequencing assay designs and analyses performed in the course of the work. Afterwards, we characterised the compositions and functions of biomass-degrading bacterial communities in guts of plant fibre- and soil-feeding higher termites, proving the existence of functional equivalence across microbial populations from different termite hosts. We also showed that each termite is a reservoir of unique microorganisms and their accompanying genes. We further extended above approach to metagenomics and bacterial genomes reconstruction and we applied it to explore the process of biomass digestion in the different sections of the highly compartmented gut of soil feeding Labiotermes labralis. We showed that primarily cellulolytic activity of the termite host was restricted to foregut and midgut, while bacterial contribution was most pronounced in P1 and P3 hindgut compartments and included activities targeting broad range of lignocellulose components. Finally, we investigated the adaptation of a laboratory-maintained grass-feeding higher termite colony of Cortaritermes spp. to Miscanthus diet at host and symbiont levels. A natural system of a termite gut was shown to progressively change in composition to yield a consortium of microbes specialised in degradation of a specific biomass. Overall, the integrative omics approach proposed here provide a framework for a better understanding of a complex lignocellulose degradation by a higher termite gut system and pave a road towards its future bioprospecting. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

<p>Bovine respiratory disease (BRD) is an ongoing health and economic issue in the dairy and beef cattle industry. Also, there are multiple risk factors that make an animal susceptible to BRD and it's diagnosis and treatment is a challenge for producers. Four bacterial species, <em>Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, </em>and<em> Mycoplasma bovis</em> have been associated with BRD mortalities. Hence, this study aims to characterize the cattle nasal microbiome as a potential additional diagnostic method to identify animals suspected to have a lung infection. Quantitative PCR and 16S rRNA gene sequencing were used to determine the bacterial load of these four bacterial pathogens in the nasal microbiome of apparently healthy (N=75) and (N=58) affected by BRD Holstein steers. We then sought to identify a value or equation that could be used to discriminate between BRD and healthy animals using a Linear Discriminant Model (LDA). Additionally, co-occurrence between commensal bacterial and BRD-pathogens were also identified. Cattle diagnosed with BRD presented lower richness, evenness and phylogenetic diversity than healthy pen-mates. Bacterial species and genera <em>Truperella pyrogenes </em>and <em>Bibersteina</em> were increased in the BRD group, and the species <em>Mycoplasma bovirhinis</em> and <em>Clostridium sensu stricto</em> increased in the healthy group. Prevalence of <em>H. somni </em>(98%)<em> </em>and <em>P. multocida </em>(97%) were the highest regardless of disease diagnosis in all the samples. Prevalence of <em>M. haemolytica </em>(81 vs. 61%) and<em> M. bovis </em>(74 vs. 50.7%) were higher in the BRD group. The bacterial density of <em>M. haemolytica</em> and<em> M. bovis </em>was also higher in the BRD group, whereas <em>Histophilus somni</em> was lower in the BRD group. Five different models were tested using LDA, and one model produced a sensitivity and specificity of 60% and 81% agreement with diagnosis based on animal symptoms. Co-occurrence analysis demonstrated that the nasal microbiome members are more likely to interact with each other than associations between BRD-pathogens and nasal microbiome members. This study offers insight into the BRD-pathogens prevalence and difference in nasal microbiome between healthy and BRD animals and provides a potential platform for future studies and potential pen-side diagnostic testing.</p>

Einleitung Adipositas und die damit verbundenen Erkrankungen wie Hyperlipidämie, Insulindysregulation und Hufrehe stellen ein großes gesundheitliches Problem domestizierter Pferde und Ponys dar. Besonders Ponys, die als „leichtfuttrig“ gelten, sind öfter von Adipositas und den damit verbundenen Erkrankungen betroffen. Aktuelle Untersuchungen der intestinalen Mikrobiota bei Mäusen, Menschen und Pferden deuten darauf hin, dass es einen Zusammenhang zwischen der Mikrobiota und der Gewichtsentwicklung gibt und bei adipösen Individuen eine andere Zusammensetzung der Mikrobiota als bei normalgewichtigen Individuen vorliegt. Untersuchungen zu den Veränderungen der fäkalen equinen Mikrobiota während einer mehrmonatigen Gewichtszunahme bei Pferden und Ponys fehlen. Ziele der Untersuchungen Zweck der vorliegenden Studie war die Überprüfung der Effekte einer zweijährigen energiereichen Fütterung auf die fäkale Mikrobiota bei Pferden und Ponys. Dabei sollten folgende Hypothesen geprüft werden: (1) Die equine fäkale Mikrobiota verändert sich im Laufe der Gewichtszunahme hin zu einer Zusammensetzung wie sie für adipöse Individuen beschrieben ist. (2) Ponys besitzen eine andere Zusammensetzung der fäkalen Mikrobiota als Pferde. Tiere, Material und Methoden Zehn Shetlandponys (Alter 6 ± 3 Jahre) und zehn Warmblutpferde (Alter 10 ± 3 Jahre) erhielten zwei Jahre 200 % ihres Erhaltungsbedarfs an umsetzbarer Energie. Monatlich wurde der Energiebedarf an die aktuelle Körpermasse (KM) angepasst. Die Ration bestand zu 60 % aus Heu und zu 40 % aus einem Ergänzungsfutter (Rohprotein 13,4 %, Rohfett 14,4 %, Rohfaser 9,8 %, stickstofffreie Extraktstoffe 54,3 %). Morphometrische Daten (Body Condition Score (BCS), Cresty neck score (CNS) und Körpermasse (KM) wurden wöchentlich erfasst. Kotproben wurden 5 (t1), 11 (t2) und 23 (t3) Monate nach Beginn der energiereichen Fütterung bei den Pferden und Ponys gewonnen. Aus den Kotproben wurde die DNA extrahiert und die V3-V4 Region auf der 16S rRNA wurde mittels PCR vervielfältigt. Die PCR Produkte wurden mit Ilumina MiSequ sequenziert. Die Sequenzierungsdaten wurden mit FROGS ausgewertet und zur Beurteilung der Zusammensetzung der fäkalen Mikrobiota wurden die Diversität mittels Shannon und Simpson Index sowie das beobachtete Artenreichtum kalkuliert. Die Konzentrationen der gesamten kurzkettigen Fettsäuren (SCFA) sowie von Azetat, Propionat, Butyrat, Isobutyrat, Valerat und Isovalerat wurden mittels flüssig Gaschromatographie bestimmt. Der Laktatgehalt im Kot wurde mittels enzymatisch kolorimetrischer Messung ermittelt. Die statistische Auswertung erfolgte mit der Software Statistica ©. Der Zeiteffekt wurde mit einer Friedmanns ANOVA und anschließendem Wilcoxen Rangsummentest mit Bonferroni Korrektur ermittelt. Für die Rasseneffekte wurde ein Mann- Whitney- U Test verwendet. Das Signifikanzniveau lag bei p < 0,05. Ergebnisse KM, BCS und CNS stiegen signifikant während der zweijährigen hochkalorischen Fütterung an. Beim Pony kam es zu einem Anstieg des Phylums Firmicutes (p = 0,025) zwischen t2 und t3 und bei beiden Rassen wurde ein signifikanter Anstieg des Phylums Actinobacteria in der fäkalen Mikrobiota zwischen t1 und t3 beobachtet. Bei Pferden konnte während der größten Gewichtszunahme zwischen t1 und t2 ein Abfall des Phylums Fibrobacteres (p = 0,028) beobachtet werden. In der fäkalen Mikrobiota der Ponys zeigte sich ein signifikanter Anstieg des Phylums Proteobacteria zwischen t1 und t2, gefolgt von einem Abfall zwischen t2 und t3. Der einzige signifikante Unterschied zwischen der fäkalen Mikrobiota der Pferde und der Ponys zum Zeitpunkt t1 war ein vermehrtes Vorkommen der Familie F082 in der fäkalen Mikrobiota der Ponys. Zum Zeitpunkt t2 wurde das Phylum Proteobacteria seltener in der Mikrobiota der Ponys beobachtet (p = 0,01). Die größten Unterschiede in der fäkalen Mikrobiota von Pferden und Ponys konnten zum Zeitpunkt t3 beobachtet werden. Dort kam das Phylum Fibrobacteres (p = 0,026) häufiger in der fäkalen Mikrobiota der Pferde vor und 5 Bakterienfamilien unterschieden sich signifikant in ihrem Vorkommen bei Pferden und Ponys.Während der größten Gewichtszunahme zwischen t1 und t2 zeigte sich ein signifikanter Abfall in der Artenvielfalt in der fäkalen Mikrobiota der Ponys. Im Kot der Pferde konnten, verglichen mit dem der Ponys, signifikant höhere SCFA Konzentrationen gemessen werden: t1: Isobutyrat, t2: Isobutyrat und Azetat und t3: gesamt SCFA, Propionat und Isovalerat. Zwischen t1 und t2 zeigte sich ein signifikanter Anstieg der Laktat Konzentration in den Fäzes der Pferde und Ponys, der bei den Pferden anschließend von einem Abfall zwischen t2 und t3 begleitet wurde. Schlussfolgerungen Während der energiereichen Fütterung konnte ein Abfall der Artenvielfalt sowie Änderungen in den Phyla Firmicutes, Actinobacteria und Fibrobacteres beobachtet werden. Unterschiede in der Mikrobiota von Pferden und Ponys konnten vor allem gegen Ende der 23-monatigen hochkalorischen Fütterung beobachtet werden. Während beim Pony die Bakterienfamilien F082, Bacteroidales UCG- 001, Rikenellaceae und Ruminococcaceae vermehrt in der fäkalen Mikrobiota auftraten, kam beim Pferd das fibrolytischen Phylum Fibrobacteres und die fibrolytische Familie Fibrobacteraceae sowie verschiedene SCFAs häufiger vor. Dies könnte ein Hinweis auf eine verbesserte Faserfermentation der Pferde sein. Die funktionellen Konsequenzen dieser Mikrobiota Verschiebungen sollten zukünftig über Metabolomanalysen weiter abgeklärt werden.

Soil microbial communities are affected extensively by addition of amendments to their environment. Of particular concern is the addition of poultry litter, which contains a substantial C, energy, and nutrient supply, but also antibiotic resistance genes (ARG), antimicrobials, and a multitude of microbial species. This project seeks to primarily assess if there is a change in bacterial community structure in response to poultry litter amendments to pasture land across geographically independent land across northern Georgia. It may be that changes in the relative abundance of bacterial communities also result in alteration in ARGs, and the community resistance to antibiotics (“resistome”) which in turn increases the potential threat of antibiotic resistance genes. While another part of this study will determine changes in integrons and specific ARGs, this project will focus on changes in bacterial communities and the potential functional changes in the community, which in turn have consequences for ARG levels and its horizontal transfer to various members of the soil community. Addition of waste from livestock is a historical method for increasing nutrients needed in the soil for the cultivation of crops, and in turn causes pronounced shifts in soil microbial communities due to the addition of large amounts of carbon, nutrients, foreign microbes, and other material. This study is unique because it utilizes a novel and relatively large landscape-scale to determine if there are discernable and repeatable patterns of bacterial community structure change in response to amendment regardless of exact soil type or source of chicken litter amendment. In the future, these data can also provide insight into the changes in the relative abundance antibiotic related genes associated with community change. / M.S. / Soil is complicated, both in terms of its physical makeup and the organisms that live inside of it. Predicting changes in soil based on the addition of foreign material such as chemicals or biological waste is not an easy process, and whether or not it is even possible to reliably predict those changes is a matter of some dispute. This study is designed to illustrate that such changes can in fact be reliably and consistently predicted even with regard to the addition of complicated materials to the soil. In this study, specifically, the material in question is chicken litter. A mix of the bedding and waste produced by chickens, litter is commonly handled by composting and is added to soil in farms as a fertilizer rich in organic matter. It is possible to point at specific elements of the soil such as the chemistry and bacteria and see how it is changed with the addition of chicken litter, which allows us to determine the nature and extent of the change that chicken litter has on soil. This study is conducted on a larger scale than similar experiments conducted in the past, making it apparent that these relationships exist on a repeated basis. It is the object of this study to pave the way and make it easier for scientists in the future to determine these relationships in other unique contexts.

Polymerase chain reaction (PCR) is a method used in many research protocols to amplify a small amount of a short segment of DNA to millions of copies. PCR is used for many taxonomic studies, as well as for some medical diagnostic procedures. Through PCR, short DNA primers bind to the template DNA to allow the thermostable DNA polymerase to copy the DNA. Often, researchers create universal primers to target a conserved region of DNA in multiple species, for example, the 16S rRNA gene in bacteria. The problem with these universal primers is that they do not always perfectly match the target DNA. The mismatch primers can still bind to the template, but could affect the efficiency of the PCR amplification. The effect of mismatch primers on the efficiency of the amplification in PCR is the focus of this thesis. Four forward primers with various mismatch overhangs were generated and incorporated into a DNA template through an initial PCR. These primers contained the binding region complementary to the V3/V4 region of the 16S rRNA bacterial gene. Further quantitative PCR (qPCR) reactions were run on these newly-made templates using two sets of primers complementary to the 16S rRNA gene region – one with ambiguous base pairs, one with unambiguous base pairs. The qPCR amplification curves, the Cq values, and the initial concentrations of DNA products (seed values) were analyzed and compared. The results showed differences in the Cq values and seed values between the reactions containing mismatches and those not containing mismatches. Other variables including annealing temperature, addition of Illumina sequencing tails to the primers, and initial primer concentration were also tested to determine if these variables had an effect on the amplification. The results from these reactions using different variables were inconclusive.

No description available.

No description available.