Early detection of pathogenic bacteria in food, drinking water and medicine products is one of the essential tasks of routine microbiological analysis. Through analytics, outbreaks can be discovered and consequently, countermeasures can be initiated to minimize health and economic damage. Cultivation of pathogens from contaminated specimens is routinely performed in microbiological laboratories worldwide. The procedure is easy to perform, requires little equipment and provides simple quantitative data in colony-forming units (CFUs) per sample volume. Only the time between preparation and confirmation of a positive (contaminated) sample usually extends over several days. The desired goal should be a technique that can retain the simplicity of cultivation while providing real-time information about the sample under investigation for early detection of potential contamination. Therefore, in the framework of this thesis, systematic heat flow measurements were performed on two model strains, Lactobacillus plantarum DSM 20205 and Pseudomonas putida mt-2 KT2440. The influence of cultivation techniques (in liquid, on solid and membrane filter placed onto solid medium) in static ampoule systems on calorimetric detection was investigated. In particular, the effect of contamination level (initial bacterial cell number), substrate amount (nutrients and oxygen), and detection limits were systematically evaluated. In addition, microcalorimetric measurements of Legionella pneumophila ATCC 33152, a waterborne pathogen, were conducted for the first time. Heat flow profiles demonstrated that high contamination levels (> 1000 CFU) were detected within 24 h. Compared to detection times of up to 10 days by ISO 11731:2017, calorimetric detection can serve as an early warning system. With this knowledge, a uniquely manufactured micro(bio-)calorimetric test system was designed to meet the requirements for detecting bacterial contaminations. In particular, the sample vessel geometry and the operating temperature perfectly matched the microbiological analysis. Within this development work, numerical models were established to investigate the temperature distribution of selected compounds as well as the complete calorimetric system. Based on these models, modifications to the test system were numerically simulated in advance to improve the instrument's performance stepwise. This thesis presents the methodological principles and a calorimetric test system designed as an early warning and detection tool for microbiological samples.

No description available.

Legionella pneumophila est un pathogène intracellulaire, agent de la Légionellose, et dont le réservoir dans l'environnement est constitué d'amibes aquatiques comme Acanthamoeba castellani. Mes objectifs de thèse étaient l'identification de mécanismes moléculaires contrôlant la virulence et la multi-résistance chez L. pneumophila, et en particulier l'exploration du rôle des protéines " GGDEF/EAL ". Les domaines GGDEF et EAL sont retrouvés dans des enzymes permettant respectivement de synthétiser (diguanylate cyclase, DGC) ou dégrader (phosphodiestérase, PDE) le di-GMP cyclique, un second messager spécifique des bactéries, qui participe au contrôle de fonctions clés comme la virulence ou la mobilité. L. pneumophila Lens possède 22 gènes codant des protéines GGDEF/EAL, et dont la plupart sont exprimés lorsqu'elle est dans sa phase virulente. L'activité enzymatique des 22 protéines " GGDEF/EAL " a été analysée in vitro : sur 10 protéines purifiées, 6 sont des DGC, dont 2 présentes une double activité DGC/PDE. L'inactivation de 5 gènes des 22 gènes et la surexpression de 2 autres entrainent une baisse de la virulence vis-à-vis d'A. castellanii. De plus, nous avons montré que l'activité DGC d'au moins 2 de ces protéines est requise lors du cycle infectieux. Enfin, nous avons décrit un système à deux composants original comprenant l'histidine kinase (HK) Lpl0330, capable de s'autophosphoryler sur un nouveau domaine HisKA, retrouvé dans 64 autres HK potentielles, et Lpl0329, le premier régulateur de réponse à double activité enzymatique caractérisé, dont la phosphorylation conduit à moduler le taux de di‐GMPc en favorisant une de ses deux activités (Levet-Paulo et al., 2011).

Depuis son isolement en 1977, les épidémies de pneumopathies à Legionella pneumophila (Lp) sont en constante progression. La contamination se fait essentiellement par inhalation d'aérosols contaminés, générés à partir de l'eau des réseaux de distribution. L'écologie de Lp est complexe, faisant intervenir des protozoaires dans lesquels elle se multiplie et des biofilms dans lesquels elle persiste à l'état viable mais non cultivable.<br />L'objectif de ce travail était de mieux comprendre cette écologie. Des incubateurs permettant le prélèvement de biofilm ont été installés in situ dans un réseau, la colonisation en eau froide et en eau chaude a été suivie pendant 1 an. Par ailleurs, un pilote reproduisant un réseau intérieur permis de comparer l'efficacité de différents traitements de désinfection. Enfin, la recherche de molécules auto-inductrices qui pourraient expliquer l'exacerbation de la virulence lors de la multiplication intra-cellulaire a été menée dans différentes conditions.<br />Les incubateurs ont permis l'amplification de la quantité de légionelles, rendant possible la détection par culture en eau froide et entraînant une stabilisation de la population en eau chaude. L'utilisation de sondes spécifiques n'a pas permis d'améliorer la détection dans les biofilms.<br />Lors des essais sur pilote, les populations des biofilms ont été analysées grâce à des méthodes moléculaires (FISH, marquage de vitalité) et par culture. Le classement des traitements par ordre d'efficacité décroissante est le suivant : dioxyde de chlore > chlore > ozone > monochloramine > cuivre-argent. Seul le dioxyde de chlore montre une activité résiduelle dans les bras morts rincés quotidiennement avec de l'eau traitée. Les amibes résistent aux traitements, et Lp retrouve son niveau de population initial quelques jours seulement après leur interruption.<br /> Enfin, aucune sécrétion d'auto-inducteur n'a été mise en évidence, et l'incubation de Lp avec ces molécules ne semble pas modifier l'invasion des amibes.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references.

This diploma thesis deals with microbiological quality of hot water in a hospital in Cesky Krumlov as well as hot water piping condition with respect to Legionella. It also deals with arrangements for improving the quality of hot water in this hospital. The subject and target of this thesis are the findings of the mentioned parameters based on own observations, technical documentation and analysis of warm water performed by an accredited laboratory. The hospital in Cesky Krumlov, the construction of which began in 1909, was built in 1911 as the 'Nemocnice Císaře Františka Josefa I.' As the time went by, new buildings were built up. The hospital was renamed to 'Všeobecná veřejná nemocnice' in 1942. In the following years the hospital expanded until the current condition. Water quality and presence of Legionella is a world-wide issue, which we must fight against. The thesis is divided into two parts. First, theoretical part, which includes both general and specific knowledge about Legionella, its health risks, the environment in which it occurs, possibilities of eliminating, suitability of the material for hot water pipes, gained in professional literature and a part of the Czech legislation dealing with public health protection, the law 'No. 258/2004 Coll.' in actual version and quality of drinking water and hot water. In the second, empirical part, I focus on the situation of the hospital in Cesky Krumlov from 2009 till 2015, concerning newly built boiler room and the whole distribution system of hot water regarding Legionella. Particularly I focused on materials used for ecologisation of the gas boiler room and materials used for hot water pipes and actual state of water distribution in individual buildings of this health care facility. I mentioned the analysis of the water samples performed by an accredited laboratory with corresponding technical documentation, which concerns the water distribution system, provided by the medical facility. I used secondary data analysis to evaluate these documents. Regarding the results from 2009 to 2015 the microbiological quality of warm water doesn't meet the limits defined in the Attachment No. 3 of the above mentioned law Order 252/2004 Coll. Limit value for colony forming units of Legionella is set to maximum of 100 CFU. In most of the samples this limit was exceeded by series of units up to hundred thousands of units. The last results in 2015 prove improvement of the unfavorable situation. In that year the analysis was tested three times. First in February, where the CFU units were 'only' in hundreds, with the top value of 550 CFU in the Postacute Care ward. Second analysis was performed in June that same year. Results were acceptable. The highest value was 64 CFU. Last analysis in the hospital was performed in December. Increased concentration of Legionella was again in the Surgery building. There were established of 1400 colony forming units. There were 300 CFU in the Postacute Care ward and the limit value of 100 CFU was in the ward of Internal medicine. The answers to research questions 'How to prevent reproduction of Legionella peumophylis in the water distribution system of the hospital in Cesky Krumlov?' and 'How to ensure a sufficient amount of quality hot water in the medical facility?' are included at the end of the stated recommendations. A risk factor causing the fast reproduction of Legionella is the old water distribution system in most of the buildings and low water temperature of water leaving the boiler, which is 55°C. All mentioned results and evaluations are included in this thesis as well as attached pictures, which show the environment and situation within the Health Care Institutions.

Flyer zur am 13. Dezember 2012 in Kraft getretenen Zweiten Verordnung zur Änderung der Trinkwasserverordnung. Hauptbestandteil ist die verpflichtende Untersuchung der Warmwasserinstallation in Mietshäusern auf eine mögliche Belastung mit Legionellen. In öffentlichen Gebäuden wie Schulen, Pflegeeinrichtungen oder Kliniken sind diese Kontrollen längst Pflicht und sichern so die Gesundheit von Patienten und Bewohnern.

No description available.

All cells maintain essential metal nutrients at optimal levels by metal homeostasis. P-type ATPases, a crucial superfamily of integral membrane proteins, are involved in the active transport of metal ions across biological membranes driven by the motive force of ATP- hydrolysis. The PIB-type ATPase subfamily, also called CPx-ATPases, fulfills a key role in heavy metal homoeostasis among the most widespread species from bacteria to human. In humans, the defect in copper transporters is the direct cause of severe neurological and hepatic disorders such as Wilson and Menkes diseases, therefore, understanding the molecular function of these pumps is of paramount importance in human health. Cu+-ATPases have two transmembrane metal binding sites (TM-MBS) and three cytosolic domains, namely the actuator (A-domain) and phosphorylation and nucleotide-binding domain (PN), and regulatory N-terminal heavy metal binding domain (HMBD). Here, we have studied the Legionella pneumophila CopA (LpCopA) and its isolated cytosolic domains to improve our understanding of the functional interaction of the protein domains during metal transport relate this to the known structure of this ATPase. To elucidate how cytosolic ligands (Cu+ and nucleotide) stimulate the interactions among the cytosolic domains and may transmit conformational changes to the TM-MBS, the interactions among recombinant isolated cytosolic domains were first examined biochemically by co-purification and spectroscopically by circular dichroism, time-resolved fluorescence and site-directed fluorescent labeling assays. The Cu+-dependent interaction between the A-domain and HMBD has been postulated as a mechanism for activating the ATPase cycle. This question was addressed here by studying copper-dependent interactions between the isolated expressed domains. Spectroscopic evidence is provided that an HMBD-A complex is formed in the presence of Cu+ which binds with 100-200 nM affinity to the recombinant HMBD. In contrast, the A-domain interacts with the PN domain in a nucleotide-dependent fashion. This molecular recognition is required for the dephosphorylation step in the catalytic cycle. The interaction was investigated in more detail by the use of a decameric peptide derived from the PN-binding interface of the A-domain and carrying the conserved TGE-motif involved in dephosphorylation. Its binding to the isolated PN domain in a weakly nucleotide-dependent manner, is demonstrated here by stopped-flow fluorescence spectroscopy. Several ATPase assays were modified to assess the functionality of the PN-domain and full length LpCopA. The peptide was found to reduce the catalytic turnover of full length LpCopA. This agrees with the expected slowing down of the reformation of the PN-A-domain interaction since the peptide occupies their binding interface. Thus, the synthetic peptide provides a means to study specifically the influence of PN-A-domain interactions on the structure and function of LpCopA. This was done by time-correlated single photon counting (TCSPC) method. The time-dependent Stokes shift of the environmentally sensitive fluorophore BADAN which was covalently attached to the conserved CPC-motif in the TM-MBS was measured. The data indicate that the interior of the ATPase is hydrated and the mobility of the intra-protein water varies from high to low at C382 at the “luminal side” and C384 at the “cytosolic side” of the TM-MBS, respectively. This finding is consistent with the recent MD simulation of LpCopA, bringing the first experimental evidence on a luminal-open conformation of E2~P state. The A-domain-derived decapeptide, although binding to the cytosolic head piece, induces structural changes also at the TM-MBS. The peptide-stabilized state (with a disrupted PN-A interface) renders the C384 environment more hydrophobic as evidenced by TCSPC. Taken together, the data from cytosolic domain interactions, ATPase assays and of time-dependent Stoke shift analyses of BADAN-labeled LpCopA reveal the presence of hydrated intramembraneous sites whose degree of hydration is regulated by the rearrangement of cytosolic domains, particularly during the association and dissociation of the PN-A domains. Copper affects this arrangement by inducing the linkage of the A-domain to the HMBD. The latter appears to play not only an autoinhibitory but also a chaperone-like role in transferring Cu+ to the TM-MBS during catalytic turnover.

All cells maintain essential metal nutrients at optimal levels by metal homeostasis. P-type ATPases, a crucial superfamily of integral membrane proteins, are involved in the active transport of metal ions across biological membranes driven by the motive force of ATP- hydrolysis. The PIB-type ATPase subfamily, also called CPx-ATPases, fulfills a key role in heavy metal homoeostasis among the most widespread species from bacteria to human. In humans, the defect in copper transporters is the direct cause of severe neurological and hepatic disorders such as Wilson and Menkes diseases, therefore, understanding the molecular function of these pumps is of paramount importance in human health. Cu+-ATPases have two transmembrane metal binding sites (TM-MBS) and three cytosolic domains, namely the actuator (A-domain) and phosphorylation and nucleotide-binding domain (PN), and regulatory N-terminal heavy metal binding domain (HMBD). Here, we have studied the Legionella pneumophila CopA (LpCopA) and its isolated cytosolic domains to improve our understanding of the functional interaction of the protein domains during metal transport relate this to the known structure of this ATPase. To elucidate how cytosolic ligands (Cu+ and nucleotide) stimulate the interactions among the cytosolic domains and may transmit conformational changes to the TM-MBS, the interactions among recombinant isolated cytosolic domains were first examined biochemically by co-purification and spectroscopically by circular dichroism, time-resolved fluorescence and site-directed fluorescent labeling assays. The Cu+-dependent interaction between the A-domain and HMBD has been postulated as a mechanism for activating the ATPase cycle. This question was addressed here by studying copper-dependent interactions between the isolated expressed domains. Spectroscopic evidence is provided that an HMBD-A complex is formed in the presence of Cu+ which binds with 100-200 nM affinity to the recombinant HMBD. In contrast, the A-domain interacts with the PN domain in a nucleotide-dependent fashion. This molecular recognition is required for the dephosphorylation step in the catalytic cycle. The interaction was investigated in more detail by the use of a decameric peptide derived from the PN-binding interface of the A-domain and carrying the conserved TGE-motif involved in dephosphorylation. Its binding to the isolated PN domain in a weakly nucleotide-dependent manner, is demonstrated here by stopped-flow fluorescence spectroscopy. Several ATPase assays were modified to assess the functionality of the PN-domain and full length LpCopA. The peptide was found to reduce the catalytic turnover of full length LpCopA. This agrees with the expected slowing down of the reformation of the PN-A-domain interaction since the peptide occupies their binding interface. Thus, the synthetic peptide provides a means to study specifically the influence of PN-A-domain interactions on the structure and function of LpCopA. This was done by time-correlated single photon counting (TCSPC) method. The time-dependent Stokes shift of the environmentally sensitive fluorophore BADAN which was covalently attached to the conserved CPC-motif in the TM-MBS was measured. The data indicate that the interior of the ATPase is hydrated and the mobility of the intra-protein water varies from high to low at C382 at the “luminal side” and C384 at the “cytosolic side” of the TM-MBS, respectively. This finding is consistent with the recent MD simulation of LpCopA, bringing the first experimental evidence on a luminal-open conformation of E2~P state. The A-domain-derived decapeptide, although binding to the cytosolic head piece, induces structural changes also at the TM-MBS. The peptide-stabilized state (with a disrupted PN-A interface) renders the C384 environment more hydrophobic as evidenced by TCSPC. Taken together, the data from cytosolic domain interactions, ATPase assays and of time-dependent Stoke shift analyses of BADAN-labeled LpCopA reveal the presence of hydrated intramembraneous sites whose degree of hydration is regulated by the rearrangement of cytosolic domains, particularly during the association and dissociation of the PN-A domains. Copper affects this arrangement by inducing the linkage of the A-domain to the HMBD. The latter appears to play not only an autoinhibitory but also a chaperone-like role in transferring Cu+ to the TM-MBS during catalytic turnover.