This master thesis deals with typing of Klebsiella pneumoniae isolates. The first part of the thesis introduces molecular typing methods. Then bacterial genomes and Klebsiella pneumoniae are characterized. Following part describes data validation, assembly of genomes and proposed algorithm for finding genes with high variability. In last part obtained results are presented and validated on other genomes of Klebsiella pneumoniae.

Descoberto e caracterizado em 1909 por Carlos Chagas, o Trypanosoma cruzi é o agente etiológico da Doença de Chagas. Durante a fase crônica da doença de Chagas, onde a parasitemia é baixa, o diagnóstico se baseia na busca de anticorpos contra os antígenos de T. cruzi no sangue. Para aumentar a certeza do resultado recomenda-se o uso de pelo menos dois métodos sorológicos (geralmente ensaio ELISA, Imunofluorescência Indireta ou Hemaglutinação Indireta) para a confirmação de diagnóstico. Embora os ensaios mencionados sejam de uso amplamente difundido, nenhum deles possui suficiente especificidade para definir o diagnóstico isoladamente, especialmente em pacientes provenientes de regiões onde há sobreposição geográfica com outros parasitos, especialmente do gênero Leishmania ou T. rangeli. Uma alternativa de interesse que foi levantada por alguns autores na década de oitenta é a busca de moléculas de interesse diagnóstico (anticorpos, antígenos ou imunocomplexos) na urina de pacientes. Durante os últimos anos técnicas de proteômica têm sido aplicadas a muitos campos da medicina. Uma das aplicações é na nefrologia para o melhor entendimento da fisiologia renal, explorar a complexidade de mecanismos de doenças e para identificar novos biomarcadores. Além do mais, a maioria das proteínas na urina são glicosiladas e suas propriedades são únicas fazendo delas uma importante fonte de biomarcadores. De maneira geral, percebe-se que apesar dos avanços significativos nos métodos de diagnósticos para a detecção da infecção por T. cruzi, há algumas lacunas que ainda precisam ser preenchidas. O fato de que a sorologia convencional para busca de anticorpos IgGs manter-se-á positiva ao longo da vida do paciente, mesmo após tratamento devido a persistência da resposta imune humoral, é uma limitação, já que não permite ter um critério confiável de cura. Por outro lado, a falta de um sistema rápido e confiável para acompanhar a evolução do tratamento, gera sérias limitações para avaliar o desempenho de novos protocolos de tratamento com fármacos já existentes ou de novos fármacos. Em função dessas limitações, propõe-se desenvolver e validar novos métodos de diagnóstico baseado em espectrometria de massas para a detecção da infecção pelo T. cruzi utilizando a urina como fonte de biomarcadores. / Discovered and characterized in 1909 by Carlos Chagas, Trypanosoma cruzi is the etiologic agent of Chagas\' disease. During the chronic phase of Chagas\' disease, where parasitemia is low, the diagnosis is based on the search for antibodies against T. cruzi antigens in the blood. To increase the certainty of the result, is recommended two serological methods (usually ELISA, Indirect Immunofluorescence or Indirect Hemagglutination) for diagnostic confirmation. Although the aforementioned assays are widely used, none of them has sufficient specificity to define the diagnosis alone, especially in patients from regions where there is geographical overlap with other parasites, especially of the genus Leishmania or T. rangeli. An interesting alternative that was raised by some authors in the 1980s is the use of molecules of diagnostic interest (antibodies, antigens or immunomplexes) in the patients\' urine. During the last years techniques of proteomics have been applied to many fields of medicine. One of the applications is nephrology for the better understanding of renal physiology, to explore the completeness of disease mechanisms and identify new biomarkers. Moreover, most of the proteins in the urine are glycosylated. Their properties are unique, making them an important source of biomarkers. In general, perceived that despite significant advances in diagnostic methods for the detection of T. cruzi infection, there are some gaps that need to be filled. The fact that conventional IgG antibody serology will remain positive over the life of the patient, despite the persistence of the humoral immune response, is a limitation, since it does not allow a reliable criterion of cure. On the other hand, the lack of a reliable system to follow the evolution of the treatment generates serious limitations to evaluate the performance of new treatment protocols with existing drugs or new drugs. Due to these limitations, we propose to develop new methods of diagnosis based on mass spectrometry for the detection of T. cruzi infection using urine as a source of biomarkers.

Enterobacteriaceae belong to the normal enteric flora in humans and may cause infections. Escherichia coli is the leading urinary tract pathogen with septicaemic potential, whereas Klebsiella pneumoniae causes opportunistic infections and often outbreaks in hospital settings. Beta-lactams are the first choice for treatment of infections caused by Enterobacteriaceae, and might be destroyed by extended-spectrum beta-lactamases, ESBLs. ESBLs hydrolyse all beta-lactams except cephamycin and carbapenems, and constitute a large heterogeneous group of enzymes with different origins. The phenotypic and molecular characteristics of a K. pneumoniae strain causing a major outbreak at Uppsala University Hospital between 2005 and 2008 were described. The strain was multiresistant and produced CTM-M-15, a common ESBL type in Europe. Due to the lack of obvious epidemiological links between patients, a case-control study was performed, which identified risk factors for the acquisition of the outbreak strain in urine cultures. The complex chain of transmission facilitated by patient overcrowding and the interventions applied to curb the outbreak, was revealed in the subsequent study. In the final study, the genetic background of the observed increase in ESBL-producing E. coli isolates during the K. pneumoniae outbreak was explored. The utility of six typing methods in epidemiological investigations of a local outbreak with ESBL-producing E. coli was compared. The increase of ESBL-producing E. coli isolates was not secondary to the K. pneumoniae outbreak. Twentytwo per cent belonged to the epidemic O25b-ST131 clone and only a limited number of infections were caused by nosocomial transmission. ESBL-producing Enterobacteriaceae are a challenge to clinical microbiology laboratories and infection control teams. To investigate their dissemination, typing methods need to be continuously adapted to the current situation. Proper hand disinfection and structural key problems such as over-crowding, under-staffing, lack of single rooms and bathrooms must be adressed to limit transmission.

Τρεις ευρέως χρησιμοποιούμενες μέθοδοι τυποποίησης, μια βιοχημική (API20NE), μια φαινοτυπική (SDS-PAGE) και μια μοριακή (RAPD) χρησιμοποιήθηκαν για την ταυτοποίηση και ταξινόμηση 160 περιβαλλοντικών ψευδομονάδων που απομονώθηκαν από το υδάτινο περιβάλλον της Νοτιοδυτικής Ελλάδας και συγκεκριμένα από εμφιαλωμένα νερά (46%), νερά δικτύου ύδρευσης (16%), κολυμβητικών δεξαμενών (9%) και θαλασσών (29%). Οι ψευδομονάδες ταυτοποιήθηκαν με βάση το βιοχημικό τους αποτύπωμα δια μέσου του συστήματος ΑΡΙ20ΝΕ, και στη συνέχεια υποβλήθηκαν σε ηλεκτροφόρηση των ολικών πρωτεϊνών τους (SDS-PAGE) και σε ανάλυση του γενετικού τους υλικού με τη μέθοδο RAPD (Random Amplified Polymorphic DNAs) με χρήση δύο διαφορετικών δεκαμερών εκκινητών (primers). Το σύστημα API20NE ταυτοποίησε το 88% των στελεχών διακρίνοντας 14 ομάδες-είδη, ενώ η SDS-PAGE ταξινόμησε το 98.1% σε 20 ομάδες και η RAPD το 94% των στελεχών σε 22 και 34 ομάδες, με εκκινητή τον OPA-13 και τον OPD-13 αντίστοιχα. Η ταξινόμηση προέκυψε με εφαρμογή της ανάλυσης κατά συστάδες (cluster analysis) των αποτυπωμάτων (πρωτεϊνικών και γενετικών) που παρήγαγαν τα στελέχη. Τα 20 στελέχη που δεν ταυτοποιήθηκαν σε επίπεδο είδους με το API20NE, ταξινομήθηκαν με την SDS-PAGE σε ποσοστό 100%, ενώ με την RAPD σε ποσοστό 90%. Οι τρεις μέθοδοι συγκρίθηκαν ως προς την επαναληψιμότητα (reproducibility), την ικανότητα τυποποίησης (typeability) και τη διακριτική ικανότητα (discriminatory power). Την μεγαλύτερη επαναληψιμότητα έδωσαν το API20NE και η RAPD με τον εκκινητή OPA-13, την μεγαλύτερη ικανότητα τυποποίησης η SDS-PAGE, ενώ τη μεγαλύτερη διακριτική ικανότητα έδωσε η RAPD με τον εκκινητή OPD-13. Η πλέον σωστή ταξινόμηση, όπως προέκυψε από τη διακριτή ανάλυση, επιτεύχθη με τη μέθοδο SDS-PAGE. Η παρούσα εργασία αποδεικνύει ότι τα βιοχημικά συστήματα ταυτοποίησης (όπως το API20NE) μπορούν να χρησιμοποιηθούν με αξιοπιστία μόνο για αδρή αναγνώριση των περιβαλλοντικών ψευδομονάδων. Πληροφορίες σε βάθος για την ταυτότητα και τη φύση τους μπορούν να εξαχθούν με τη περαιτέρω εφαρμογή ηλεκτροφορητικών και μοριακών μεθόδων. Δεδομένης της ευρείας διασποράς, της ετερογένειας και της, έστω και δυνητικής, παθογόνου δράσης των ψευδομονάδων, είναι σημαντικό, από πλευράς δημόσιας υγείας, ο προσδιορισμός της ταυτότητάς τους να γίνεται με συνδυασμένη εφαρμογή βιοχημικών, ηλεκτροφορητικών και μοριακών μεθόδων ώστε να καθίσταται δυνατή η αναγνώριση στελεχών που μπορούν να αποτελέσουν αιτιολογικούς παράγοντες ασθενειών, ιδιαίτερα σε ομάδες υψηλού κινδύνου. / Three broadly used typing techniques, one biochemical (API20NE), one phenotypic (SDS-PAGE) and one molecular (RAPD), were employed for the identification and taxonomy of 160 environmental pseudomonas isolated from the aquatic environment in Southwestern Greece. In particular, the isolates were obtained from bottled waters (46%), potable waters (16%), waters from swimming pools (9%) and seawaters (29%). The isolates were identified by the system API20NE and then subjected to whole-cell protein electrophoresis (SDS-PAGE) and Random Amplified Polymorphic DNAs (RAPD) using two 10-mer primers. The API20NE system identified 88% of the whole bacterial population and classified them in 14 species, while SDS-PAGE classified 98.1% of the isolates in 20 groups and RAPD classified 94% of the strains in 22 groups using the primer OPA-13 and 34 groups using the primer OPD-13. The classification was achieved by applying cluster analysis in the protein or RAPD fingerprints of the isolates. Twenty isolates that could not be identified by the API20NE system, at least to the species level, were classified by the SDS-PAGE and the RAPD in a percentage of 100% and 90%, respectively. The reproducibility, typeability and discriminatory power of the three methods were compared to evaluate their application. The API20NE and the RAPD assay with primer OPA-13 showed better reproducibility in comparison with the other methods; the higher typeability was achieved by the SDS-PAGE assay while the higher discriminatory power was that obtained by the RAPD method with the primer OPD-13. The SDS-PAGE gave the higher percentage of “correctly classified” isolates, as it was assessed by discriminant analysis. This study shows that the rapid identification systems, such as the API20NE, may be reliable only for a rough characterization of environmental Pseudomonas. In order to acquire further information about their identities, other phenotypic and molecular techniques have to be applied. Given the ubiquity, heterogeneity and pathogenicity, either established or potential, of the environmental pseudomonas it is important, from a public health point of view, to monitor the identities of environmental Pseudomonas isolates using the combination of specific methods, so as to be possible for strains, which can serve as causative agents of diseases, especially in high risk population, to be recognizable.