Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Martina Klaučová Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Consultant: PharmDr. Ivona Pávková, Ph.D. Diploma thesis title: Ames test in the drug development Background: Thesis objective is the determination of potential genotoxicity of newly developed drugs within primary testing and the introduction of the Ames microfluctuation test which can be used in common laboratory conditions. Methods: I used commercially supplied kit based on the principles of Ames test which detects reverse mutation through colour changes of the samples using bacterial strains S. typhimurium. At first I had to study literary sources and then I could design the procedures of the Ames microfluctuation test, preparation of the chemicals and storage of the strains which are optimal for all laboratories. Results: The drug samples T6445 and T6447 with 30 µM concentration tested by metabolic activation S9 on bacterial strain ST TA 98 show genotoxicity. The sample UOCHB1 with 30 µM concentration tested without activation shows possible genotoxicity on both strains ST TA 98 and ST TA 100. Other samples do not show any toxicity. I used 3 different procedures during the designation of assay. The most suitable version of the...

The propensity of Salmonella to persist in water environments under unfavourable conditions is of concern as these water environments serve as contamination reservoirs. The role of contaminated water in the transmission of Salmonella in developing countries is largely unknown. The fate and persistence of non-typhoidal Salmonella in water environments and the specific influence of the indigenous microbiota on the survival and growth of Salmonella is poorly understood. A tagged Salmonella strain distinguishable in vivo from a mixed bacterial community would greatly facilitate the study of Salmonella in water environments. The clinically relevant S. enterica subsp. enterica ser. Typhimurium isolate was chromosomally tagged using the pUT mini–Tn5 Km transposon with the green fluorescent protein gene gfpmut3b*. Southern Blot hybridisation confirmed that the gfp gene had integrated into the chromosome. The gfp gene was stably maintained and the gfp-labelled recombinants were not growth rate impaired under low nutrient conditions. No significant changes were observed between the wild-type and the tagged strain. The survival fitness studies indicated the incorporation of the gfp gene did not have any noted detrimental effects on the survival and behaviour of the tagged strains. These tagged strains could therefore be used to study the fate and survival of Salmonella in biofilms of drinking water distribution systems. Genetic tagging of the target organism with the gfp gene, encoding the green fluorescent protein, allows in situ detection of undisturbed cells and is ideally suited for monitoring Salmonella as a monospecies or in a complex mixed community. The fate and persistence of non-typhoidal Salmonella in drinking water biofilms was investigated. The ability of Salmonella to form biofilms independently and the fate and persistence of Salmonella in an aquatic biofilm was examined. </p.> In monoculture S. Typhimurium formed loosely structured biofilms. Salmonella colonized established multi-species drinking water biofilms within 24 hours, growing to form micro-colonies within the biofilm. S. Typhimurium was also released at high levels from the drinking water-associated biofilm into the flow, and was seen to re-colonize elsewhere. Results showed that Salmonella can enter into, survive and grow within, and be released from a drinking water biofilm. Once Salmonella has entered into a distribution system, it will be able to colonize an existing biofilm, grow in it and be released into the flow for re-colonization elsewhere, and possible subsequent infection of consumers. / Dissertation (MSc (Microbiology ))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

Publicación a texto completo no autorizada por el autor / Identifica molecularmente los serotipos de cepas sospechosas de Salmonella spp., aisladas mediante protocolos microbiológicos estandarizados, a partir de 272 muestras pareadas de hisopados rectales y vaginales de reproductoras primerizas clínicamente sanas de un criadero del distrito de Pachacamac. El ADN de los aislados sospechosos, evaluados hasta pruebas bioquímicas, es extraído y analizado mediante la técnica de PCR múltiple, para detectar la presencia de los genes inv A, prot6E y fliC específicos para Salmonella spp., Salmonella Enteritidis y Salmonella Typhimurium, respectivamente; encontrándose en 12 cepas aisladas que amplifican al gen inv A; de éstas, 10 (83.3%) amplifican para el gen fliC y ningún aislado amplifica el gen prot6E. Estos resultados confirman que Salmonella Typhimurium es el patógeno predominante en cobayos reproductoras al primer parto en esta crianza comercial. / Tesis

Evalúa la capacidad de detección de Salmonella Typhimurium y Enteritidis mediante el método de la Reacción en Cadena de la Polimerasa (PCR) Múltiple a partir de muestras en pre-enriquecimiento no selectivo (utilizando secuencias blanco de los genes InvA, fliC y prot6E) y hallar la concordancia entre ésta y el método de detección microbiológico convencional que consta de preenriquecimiento no selectivo, enriquecimiento selectivo, aislamiento en agar diferencial y pruebas bioquímicas. Se analizan simultáneamente mediante ambos métodos un total de 111 muestras de hígado de cobayos con diagnóstico presuntivo de Salmonelosis provenientes de Chancay (Lima) y El Mantaro (Junín), llegando a detectar Salmonella Typhimurium por PCR Múltiple en 54% (60/111) de muestras y por análisis microbiológico en 41% (45/111) de ellas. Las pruebas muestran una concordancia substancial con un valor de Kappa de 0.64 y la prueba de McNemar demuestra que los resultados de ambas pruebas son estadísticamente diferentes (p>0.05), por lo tanto se concluye que la PCR Múltiple a partir de muestras en pre-enriquecimiento no selectivo sirve como una prueba tamiz que detecta Salmonella Typhimurium en volúmenes grandes de muestras de hígado de cobayos, siendo esta menos laboriosa y más rápida que la detección microbiológica convencional, sin embargo se recomienda utilizar ambas técnicas en combinación para mejorar los resultados. / Tesis

Bacteria inhabit diverse environmental niches and consequently, must modulate their metabolism to adapt to stress. The nucleotide second messengers guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (collectively referred to as (p)ppGpp) are essential for survival during nutrient starvation. (p)ppGpp is synthesized by the RelA-SpoT homologue (RSH) protein family and coordinates the control of cellular metabolism through its combined effect on over 50 proteins. While the role of (p)ppGpp has largely been associated with nutrient limitation, recent studies have shown that (p)ppGpp and related nucleotides have a previously underappreciated effect on different aspects of bacterial physiology, such as regulating bacterial interactions with its host. This thesis focuses on the coordination of virulence gene expression and evasion of host immunity by (p)ppGpp in Salmonella enterica serovar Typhimurium. In the first data chapter, I describe the role of (p)ppGpp in mediating bacterial resistance to killing by the human complement system. I identified that (p)ppGpp activates ppnN, a nucleotide metabolism associated enzyme, and the biosynthesis of lipopolysaccharide O-antigen to protect Salmonella from cell lysis by complement. The second data chapter compares and contrasts the stringent response of an invasive clinical isolate of Salmonella Typhimurium to a strain of Salmonella Typhimurium that causes acute gastroenteritis using RNA-sequencing. Critical analysis of our transcriptomics dataset showed that flagellar-based motility is differentially regulated by (p)ppGpp in the two strains of Salmonella. Together, these findings demonstrate that (p)ppGpp has significant functional roles beyond mediating adaptation to nutrient limitation. / Thesis / Doctor of Philosophy (PhD)

Innate immunity operates independently of prior exposure to pathogens. There are several signal transduction pathways that play a key role in inflammatory and immune responses. Critical signaling cascade in the interest of my research is type-1 interferon (IFN) signaling pathway in response to infection with Salmonella Typhimurium (ST). The role of type-I interferons is well established in the context of a viral infection; however, their role in bacterial infections is not clear. In my thesis I aimed to understand the role of type-1 IFNs in bacterial pathogenesis, and scrutinize the mechanism adopted by various components of type-1 IFN signaling, especially ISGF3 complex in response to Salmonella Typhimurium. My results indicate that type-I IFN signaling is detrimental to host survival. I further investigated the mechanism through which type-1 IFN signaling results in host susceptibility against Salmonella. My results indicated that the three transcription factors downstream of IFNAR1 have different impacts in mounting an innate immune response against ST. IRF9 and STAT2 promote susceptibility against ST whereas STAT1 through IFNAR1-signaling, promotes enhanced expression of pro inflammatory cytokines and protection against ST. I also observed that the monocytes/macrophages lineage in Ifnar1⁻ᐟ⁻ mice is responsible for conferring the enhanced resistance against ST. Furthermore, my work determined that expression of type-I IFN signaling compromises the fitness of macrophages by reducing mitochondrial respiration, glycolysis and myelopoiesis.

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The nutritional value of an amino acid (AA) is determined by its bioavailability, however concept of digestibility is mostly used in practical situations. Four studies were conducted to test 2 exogenous factors that were hypothesized to affect the AA digestibility in non-ruminant animals. In study 1, broiler chicks were randomly allotted to 4 dietary treatments of control and distillers dried grains with solubles (DDGS, 20%) diets supplemented or not with a novel mixture of carbohydrases. Results indicated the ability of carbohydrase mixture to increase energy utilization of the DDGS diet, with significant improvements in AA digestibility, consequently improving growth performance of broilers. Study 2 examined effect of the carbohydrase mixture in pigs fed a high DDGS diet. Ileal cannulated growing pigs (n = 8, 64.3 ± 0.5 kg) were allotted to 4 dietary treatments in a replicated 4 Ã 4 Latin Square design. Control and DDGS (40%) diets were supplemented or not with a mixture of carbohydrases. Numeric increases for AA digestibility, along with a decreased tendency of urinary energy output suggested a possibility for improved nutrient utilization in pigs when carbohydrases were supplemented to 40% corn DDGS diet. Collectively, carbohydrase seems less effective for swine applications due to greater water content and consequently a lower viscosity in pig digesta. Next, study 3 showed changes in AA digestibility and endogenous AA losses (EAAL) when pigs were challenged orally with Salmonella enterica serovar Typhimurium. Nursery pigs (n = 48, 17.9 ± 0.5 kg) were randomly assigned to a 2 Ã 2 factorial arrangement consisting of two diets (control or N-free) and inoculation (sterile broth or 9.8 Ã 10^9 CFU of Salmonella). Measurements at 24 and 72 h post-inoculation indicated that AA digestibility of pigs is impaired through the initial phase of Salmonella infection and gradually restored, but not fully by 72 h. Finally, study 4 determined the dynamic fluctuations of EAAL and subsequent AA digestibility in response to Salmonella Typhimurium measured at multiple time points. Ileal cannulated pigs (n = 8, 76.0 ± 1.4 kg) were randomly assigned to either a control or a N-free diet and challenged orally with 1.3 Ã 10^10 CFU of Salmonella. Inflammatory diarrhea was associated with reduced AA digestibility and increased EAAL showing respective peak values at 8-16 h post-inoculation. Alterations in AA digestibility and EAAL were gradually recovered to near pre-inoculation values by 56-64 h post-inoculation, but showed impaired digestibility at 72-80 h post-inoculation. / Ph. D.

According to the WHO, foodborne derived enteric infections are a global disease burden and often manifest in diseases that can potentially reach life threatening levels, especially in developing countries. These diseases are caused by a variety of enteric pathogens and affect the gastrointestinal tract, from the gastric to the intestinal to the rectal tissue. Although the complex mucosal structure of these organs is usually well prepared to defend the body against harmful agents, specialised pathogens such as Salmonella enterica can overcome the intestinal defence mechanism. After ingestion, Salmonella are capable of colonising the gut and establishing their proliferative niche, thereby leading to inflammatory processes and tissue damage of the host epithelium. In order to understand these processes, the scientific community in the last decades mostly used cell line based in vitro approaches or in vivo animal studies. Although these approaches provide fundamental insights into the interactions between bacteria and host cells, they have limited applicability to human pathology. Therefore, tissue engineered primary based approaches are important for modern infection research. They exhibit the human complexity better than traditional cell lines and can mimic human-obligate processes in contrast to animal studies. Therefore, in this study a tissue engineered human primary model of the small intestinal epithelium was established for the application of enteric infection research with the exemplary pathogen Salmonella Typhimurium. To this purpose, adult stem cell derived intestinal organoids were used as a primary human cell source to generate monolayers on biological or synthetic scaffolds in a Transwell®-like setting. These tissue models of the intestinal epithelium were examined for their comparability to the native tissue in terms of morphology, morphometry and barrier function. Further, the gene expression profiles of organotypical mucins, tight junction-associated proteins and claudins were investigated. Overall, the biological scaffold-based tissue models showed higher similarity to the native tissue - among others in morphometry and polarisation. Therefore, these models were further characterised on cellular and structural level. Ultrastructural analysis demonstrated the establishment of characteristic microvilli and tight-junction connections between individual epithelial cells. Furthermore, the expression pattern of typical intestinal epithelial protein was addressed and showed in vivo-like localisation. Interested in the cell type composition, single cell transcriptomic profiling revealed distinct cell types including proliferative cells and stem cells, progenitors, cellular entities of the absorptive lineage, Enterocytes and Microfold-like cells. Cells of the secretory lineage were also annotated, but without distinct canonical gene expression patterns. With the organotypical polarisation, protein expression, structural features and the heterogeneous cell composition including the rare Microfold-like cells, the biological scaffold-based tissue model of the intestinal epithelium demonstrates key requisites needed for infection studies with Salmonella. In a second part of this study, a suitable infection protocol of the epithelial tissue model with Salmonella Typhimurium was established, followed by the examination of key features of the infection process. Salmonella adhered to the epithelial microvilli and induced typical membrane ruffling during invasion; interestingly the individual steps of invasion could be observed. After invasion, time course analysis showed that Salmonella resided and proliferated intracellularly, while simultaneously migrating from the apical to the basolateral side of the infected cell. Furthermore, the bacterial morphology changed to a filamentous phenotype; especially when the models have been analysed at late time points after infection. The epithelial cells on the other side released the cytokines Interleukin 8 and Tumour Necrosis Factor α upon bacterial infection in a time-dependent manner. Taken together, Salmonella infection of the intestinal epithelial tissue model recapitulates important steps of the infection process as described in the literature, and hence demonstrates a valid in vitro platform for the investigation of the Salmonella infection process in the human context. During the infection process, intracellular Salmonella populations varied in their bacterial number, which could be attributed to increased intracellular proliferation and demonstrated thereby a heterogeneous behaviour of Salmonella in individual cells. Furthermore, by the application of single cell transcriptomic profiling, the upregulation of Olfactomedin-4 (OLFM4) gene expression was detected; OLFM4 is a protein involved in various functions including cell immunity as well as proliferating signalling pathways and is often used as intestinal stem cell marker. This OLFM4 upregulation was time-dependent, restricted to Salmonella infected cells and seemed to increase with bacterial mass. Investigating the OLFM4 regulatory mechanism, nuclear factor κB induced upregulation could be excluded, whereas inhibition of the Notch signalling led to a decrease of OLFM4 gene and protein expression. Furthermore, Notch inhibition resulted in decreased filamentous Salmonella formation. Taken together, by the use of the introduced primary epithelial tissue model, a heterogeneous intracellular bacterial behaviour was observed and a so far overlooked host cell response – the expression of OLFM4 by individual infected cells – could be identified; although Salmonella Typhimurium is one of the best-studied enteric pathogenic bacteria. This proves the applicability of the introduced tissue model in enteric infection research as well as the importance of new approaches in order to decipher host-pathogen interactions with higher relevance to the host. / Nach Angaben der WHO stellen lebensmittelbedingte Darminfektionen eine globale Krankheitslast dar und äußern sich häufig in Krankheiten, die potenziell lebensbedrohliche Ausmaße annehmen können, insbesondere in Entwicklungsländern. Diese Krankheiten werden durch eine Vielzahl von enterischen Erregern verursacht und betreffen den Magen-Darm-Trakt, vom Magen über den Darm bis zum Enddarm. Obwohl die komplexe Schleimhautstruktur dieser Organe in der Regel gut darauf vorbereitet ist, den Körper vor schädlichen Reagenzien zu schützen, können spezialisierte Erreger wie Salmonella enterica den Abwehrmechanismus des Darms überwinden. Nach der Nahrungsaufnahme sind Salmonellen in der Lage, den Darm zu kolonisieren und ihre proliferative Nische zu etablieren, was letztlich zu entzündlichen Prozessen und Gewebeschäden des Wirtsepithels führt. Um diese Prozesse zu verstehen, hat die Wissenschaft in den letzten Jahrzehnten hauptsächlich auf Krebslinien basierende in vitro-Ansätze oder in vivo-Tierstudien verwendet. Obwohl diese Ansätze grundlegende Erkenntnisse über die Wechselwirkungen zwischen Bakterien und Wirtszellen lieferten, sind sie nur begrenzt auf die Pathologie des Menschen übertragbar. Daher sind Tissue engineering und primärzellbasierte Ansätze für die moderne Infektionsforschung wichtig. Sie spiegeln die menschliche Komplexität besser wider als Ansätze mit Krebszellen und können im Gegensatz zu Tierversuchen human-obligate Prozesse nachbilden. Daher wurde in dieser Studie ein tissue engineered humanes Primärmodell des Dünndarmepithels für die Anwendung in der enterischen Infektionsforschung am Beispiel des Erregers Salmonella Typhimurium etabliert. Zu diesem Zweck wurden aus adulten Stammzellen gewonnene Darmorganoide als primäre humane Zellquelle verwendet, um 2D-Monolayer auf biologischen oder synthetischen Trägestrukturen in einer Transwell®-ähnlichen Umgebung zu erzeugen. Die so erzeugten Gewebemodelle des Darmepithels wurden auf ihre Vergleichbarkeit mit dem nativen Gewebe in Bezug auf Morphologie, Morphometrie und Barrierefunktion untersucht. Weiterhin wurde die Genexpression von organtypischen Muzinen, Tight Junction-assoziierten Proteinen und Claudinen sowie das Expressionsmuster der Tight Junction-Proteine untersucht. Insgesamt wiesen die auf biologischen Matrizes basierenden Gewebemodelle eine größere Ähnlichkeit mit dem nativen Gewebe auf - unter anderem in Bezug auf Morphometrie und Polarisation -, weshalb diese Modelle auf zellulärer und struktureller Ebene tiefgehender charakterisiert wurden. Die ultrastrukturelle Analyse zeigte die Ausbildung charakteristischer Mikrovilli und Tight-Junction-Verbindungen zwischen einzelnen Epithelzellen. Darüber hinaus wurden die Expressionsmuster typischer Darmepithelproteine untersucht, die eine in vivo ähnliche Lokalisation aufwiesen. Im Hinblick auf die Zelltypenzusammensetzung ergab die Analyse des Transkriptoms auf Einzel-Zell-Ebene definierte Zelltypen. Dies waren Zellen mit proliferativem Profil, Stammzellen und Vorläuferzellen, und Zellen der absorptiven Linie, Enterozyten und Microfold-Zellen. Zellen der sekretorischen Linie wurden ebenfalls annotiert, jedoch ohne eindeutige kanonische Genexpression. Mit der organotypischen Polarisierung, der Proteinexpression, den strukturellen Merkmalen und der heterogenen Zellzusammensetzung, einschließlich der seltenen Microfold-Zellen, weist das auf einer biologischen Matrix basierende Gewebemodell des Darmepithels die wichtigsten Voraussetzungen für Infektionsstudien mit Salmonellen auf. Im zweiten Teil dieser Studie wurde ein geeignetes Infektionsprotokoll für das Epithelgewebemodell mit Salmonella Typhimurium erstellt, gefolgt von der Untersuchung der wichtigsten Merkmale des Infektionsprozesses. Salmonella hafteten an den epithelialen Mikrovilli und verursachten während der Invasion das typische Membran-Kräuseln; interessanterweise konnten die Schritte der Invasion einzeln beobachtet werden. Nach der Invasion zeigte die Zeitverlaufsanalyse der Infektion, dass die Salmonellen intrazellulär lokalisierten und replizierten, während sie gleichzeitig von der apikalen zur basolateralen Seite der infizierten Zelle migrierten. Darüber hinaus veränderte sich die Morphologie der Bakterien in der Spätphase der Infektion zu einem filamentösen Phänotyp. Die Epithelzellen auf der anderen Seite setzten nach der bakteriellen Infektion zeitabhängig die Zytokine Interleukin 8 und Tumor-Nekrose-Faktor-α frei. Insgesamt rekapituliert die Salmonelleninfektion des intestinalen Epithelgewebemodells wichtige Schritte des Infektionsprozesses, wie sie in der Literatur beschrieben sind und stellt somit eine valide in vitro Plattform für die Untersuchung des Salmonelleninfektionsprozesses in einem menschlichen Kontext dar. Interessanterweise variierten die intrazellulären Salmonellenpopulationen während des Infektionsprozesses in ihrer Bakterienzahl, was auf eine erhöhte intrazelluläre Proliferation zurückgeführt werden konnte und somit ein heterogenes Verhalten der Salmonellen in einzelnen Zellen demonstriert. Darüber hinaus wurde durch die Anwendung von Einzel-Zell-Transkriptom-Analysen die Hochregulierung der Genexpression von Olfactomedin-4 (OLFM4) nachgewiesen; OLFM4 ist ein Protein mit verschiedenen Funktionen, darunter Prozesse der Zellimmunität sowie proliferierende Signalwege, und es wird häufig als Darmstammzellmarker verwendet. Diese OLFM4-Hochregulierung war zeitabhängig, auf mit Salmonella infizierten Zellen beschränkt und schien mit der intrazellulären Bakterienmasse zuzunehmen. Bei der Untersuchung der OLFM4-Regulationsmechanismen konnte eine nuclear factor κB-induzierte Hochregulierung ausgeschlossen werden, während die Hemmung der Notch-Signalübertragung zu einem Rückgang der OLFM4-Gen- und Proteinexpression führte. Darüber hinaus führte die Hemmung von Notch zu einer verminderten Bildung von filamentösen Salmonella. Insgesamt konnte durch die Verwendung des hier eingeführten primären Epithelgewebemodells ein heterogenes intrazelluläres bakterielles Verhalten beobachtet und eine bisher übersehene Wirtszellantwort - die Expression von OLFM4 durch einzelne infizierte Zellen - bei einem der am besten untersuchten enterischen Pathogene identifiziert werden. Dies beweist die Anwendbarkeit des vorgestellten Gewebemodells in der enterischen Infektionsforschung sowie die Bedeutung neuer Ansätze zur Entschlüsselung von Wirt-Pathogen-Interaktionen mit höherer Relevanz für den Wirt.