Les moisissures d’altération sont à l’origine de pertes alimentaires et économiques importantes et certaines espèces peuvent présenter un danger pour la santé humaine et animale avec la production de mycotoxines. Dans ce contexte, la maîtrise de la qualité et de la sécurité des aliments passe par une bonne connaissance des espèces impliquées. Cette connaissance repose sur une identification fiable et rapide et l’obtention d’informations sur les facteurs abiotiques impactant leur développement, tels que les conservateurs, largement utilisés dans l’industrie. Dans ce cadre, les objectifs de thèse étaient de développer l’utilisation de la spectrométrie de masse MALDI-TOF pour l’identification des moisissures et d’évaluer son application à la résolution de complexe d’espèces et au typage, et enfin d’évaluer la néphélométrie laser pour mesurer en haut-débit leur croissance en présence de conservateurs. Dans un premier temps, une base de données robuste a été construite avec près de 6500 spectres correspondant à 136 espèces fongiques. Dans un deuxième temps, la technique MALDI-TOF a été appliquée avec succès à la différenciation de 23 espèces du complexe Aspergillus section Flavi et a permis de différencier des isolats de Penicillium roqueforti appartenant à 3 populations génétiquement différenciées. Enfin, la néphélométrie laser a permis un suivi haut-débit de la croissance de 14 espèces fongiques d’altération en présence de 3 conservateurs et ainsi d’obtenir des informations sur les concentrations minimales inhibitrices de ces derniers. Ces travaux ont démontré l’applicabilité de techniques alternatives permettant d’identifier et de caractériser les moisissures d’altération. / Spoilage fungi represent a major cause of food and economic losses and certain species, which may produce mycotoxins, may also pose a threat to human and animal health. Thus, food safety and quality management relies notably on a good knowledge of the involved species. This knowledge is notably based on their fast and reliable identification and on the study of abiotic factors affecting their growth such as food preservatives, which are commonly used in the food industry. In this context, the objectives of this PhD. thesis were to develop MALDI-TOF mass spectrometry for mold identification and to evaluate its potential for species complex differentiation and strain typing, and finally, to evaluate the use of laser nephelometry to monitor fungal growth in the presence of food preservatives.First, a robust database was developed with 6500 spectra corresponding to 136 spoilage fungi. Then, MALDI-TOF MS was successfully applied to differentiate 23 species of Aspergillus section Flavi and Penicillium roqueforti isolates belonging to 3 genetically distinct populations.Finally, in 14 fungal species, laser nephelometry allowed a high-throughput monitoring of their growth after exposition to 3 different food preservatives and the determination of their associated minimal inhibitory concentrations.Overall, the obtained results demonstrate the usefulness of alternative techniques for identification and characterization of food spoilage fungi.

The soybean crop is one of the main agricultural crops, with high global economic relevance. The large area under soybean cultivation in Brazil, including the incorporation of new areas in the northern and midwestern regions, mostly under monoculture and non-tillage system, has been affected the prevalence and the intensity of diseases. Among these, one of most prominent is anthracnose, mainly associated with the fungal species Colletotrichum truncatum. Knowledge of the genetic structure of plant pathogen populations can be used to infer their life histories and the evolutionary processes that shape populations in the agroecosystems, which can help to implement effective disease management strategies. However, the genetic structure of C. truncatum populations associated with soybean remains unknown. We collected C. truncatum isolates from 10 sites representing two of main areas of soybean producing in Brazil and used microsatellite markers and whole-genome sequencing to investigate the population biology and evolutionary history of this important pathogen. The multilocus microsatellite typing of 237 isolates revealed high gene and haplotypic diversity within populations, as well low genetic differentiation and sharing of multilocus haplotypes among populations and regions. In addition, three distinct genetic clusters were detected, coexisting in syntopy in the soybean fields, without evidence of admixture between them. Such finding suggesting that Brazilian C. truncatum populations resulted from at least three founder events, which led to three genetic lineages that spread throughout the country. However, the genetic makeup of these lineages remains unknow, and their extreme geographic proximity raises the question of the maintenance of their genetic integrity in the face of admixture. In order to gain insights into the evolutionary history of C. truncatum lineages and to investigate in more details the possibility of a lack of genetic exchanges between them, we employed a population genomic approach. For that, we produced a draft genome sequence of a typical strain of the species associated with soybean anthracnose, which was used as the reference genome. Eighteen representative C. truncatum isolates from the three lineages were submitted to whole genome sequencing, aligned against the reference genome, and variants were identified. Our population genomic analyzes revealed that the genetic structure of C. truncatum pathogen causing soybean anthracnose is formed by three deeply divergent lineages with levels of genetic diversity consistent with repeated introduction events for each lineage. We also found evidence for sexual recombination within and between lineages, with multiples isolates displaying signatures of admixture. Our findings support a scenario in which the three lineages initially diverged in allopatry before experiencing hybridization following secondary contact. Monitoring of the pathogen\'s diversity over time is needed to reveal whether these lineages maintain or fuse, which can impact the disease control methods currently employed. / A soja é uma das principais culturas agrícolas, com alta relevância econômica global. A grande área sob cultivo de soja no Brasil, incluindo a incorporação de novas áreas nas regiões norte e centro-oeste, principalmente sob monocultura e plantio direto, tem afetado a prevalência e a intensidade das doenças. Entre elas, uma das mais proeminentes é a antracnose, principalmente associada à espécie fúngica Colletotrichum truncatum. O conhecimento da estrutura genética das populações de patógenos de plantas pode ser usado para inferir suas histórias de vida e os processos evolutivos que moldam as populações nos agroecossistemas, o que pode ajudar a implementar estratégias eficazes de manejo da doença. No entanto, a estrutura genética das populações de C. truncatum associadas à soja permanece desconhecida. Coletamos isolados de C. truncatum em 10 áreas, representando duas principais regiões produtoras de soja no Brasil. Utilizamos marcadores microssatélites e sequenciamento do genoma completo para investigar a biologia populacional e a história evolutiva desse importante patógeno. A tipagem de microssatélites multilocus de 237 isolados revelou alta diversidade genética e haplotípica nas populações, bem como baixa diferenciação genética e compartilhamento de haplótipos entre populações e regiões. Além disso, foram detectados três grupos genéticos distintos, coexistindo nas mesmas áreas, sem evidência de mistura entre eles. Isto sugere que as populações C. truncatum no Brasil resultaram de pelo menos três eventos fundadores, o que levou á formação das três linhagens genéticas que se espalharam pelo país. No entanto, a composição genética dessas linhagens permanece desconhecida, e sua extrema proximidade geográfica levanta a questão sobre a manutenção de sua integridade genética em face a mistura entre elas. A fim de analisar a história evolutiva das linhagens de C. truncatum e investigar a possibilidade de ausência de trocas genéticas entre elas, empregamos uma abordagem genômica populacional. Para isso, produzimos uma versão preliminar do genoma completo de um isolado típico da espécie, o qual foi utilizado como genoma de referência. Dezoito isolados representativos das três linhagens foram submetidos ao sequenciamento completo, alinhados ao genoma de referência, e variantes foram identificados. Nossas análises genômicas populacionais revelaram que a estrutura genética do patógeno é formada por três linhagens profundamente divergentes, com níveis de diversidade consistentes com repetidos eventos de introdução para cada linhagem. Também encontramos evidências de recombinação sexual dentro e entre linhagens, com múltiplos isolados apresentando assinaturas de mistura. Nossas descobertas sugerem um cenário no qual as três linhagens divergiram inicialmente em alopatria antes de experimentar hibridação, após contato secundário. O monitoramento da diversidade do patógeno ao longo do tempo é necessário para revelar se essas linhagens se mantêm geneticamente separadas ou se fundem, o que pode afetar os métodos de controle da doença atualmente empregados.

<p>In today´s labour market, men and women are segregated, both vertically and horizontally. Exceptions to the rule of gender segregation are few. There is, however, one managerial group where women and men are equal in numbers: school principals. In a short period of time, the distribution in terms of sex among principals in Sweden has changed dramatically. How does gender equality in numbers affect gender equality in a qualitative sense? That is the focus of this thesis. In order to find out, I interviewed eight senior level school principals, on the topics of sex/gender, leadership and gender equality.</p><p>Three important conclusions were drawn from the interviews. The first one is that there is a strong connection between masculinity and leadership among the principals. When they talk about establishing boundaries and discussing right and wrong, they focus on male principals. In certain situations, no woman is good enough, no matter how good a principal she is. Male principals are described as different, in a positive way. The second conclusion has to do with the way men and women are described. The male principals describe themselves as intuitive and focused on relationship issues – features traditionally labelled as female. The women, however, describe themselves as masculine in some ways. It appears that who and what is labelled as male or female is negotiable among the principals in this study.</p><p>The final conclusion concerns gender equality. It appears that the strive for gender equality in a quantitative sense has led to a focus on men and masculinity. The strive for gender equality in numbers, seems to put egual opportunitys for men and women at risk.</p>

The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing. Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension. The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results. Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses. Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results. <b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis

Yogurt is a characteristic fermented dairy product of Turkey and Bulgaria and its popularity has been increasing all over the world. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus) are used together as starter culture in production of yogurt. The objective of this study was to isolate and characterize yogurt cultures from traditionally produced yogurts (i.e. produced without using commercial starter cultures) and to search the genotypic diversity within traditional S. thermophilus isolates. Yogurt cultures were isolated from traditionally produced yogurts collected from different regions of Turkey and identified biochemically. Acidification ability of the isolates was examined and the cultures giving best acidifying rates were further subjected to a selection in terms of their acetaldehyde production ability. Then, phage resistance and proteolytic activity of chosen isolates were tested. Finally, twenty-five L. bulgaricus and twenty-two S. thermophilus isolates were selected as cultures having best technological properties. Furthermore, subtyping studies were carried out to indicate strain diversity among isolates. S. thermophilus was selected as target organism for subtyping in this study. Clustered regularly interspaced short palindromic repeats (CRISPR) loci are highly polymorphic genetic regions, which are composed of partially palindromic direct repeats interspaced by sequences called spacers. In order to characterize S. thermophilus isolates genotypically, CRISPR1 locus of the isolates were analyzed. Additionally, nineteen isolates selected after CRISPR1 analysis were characterized using multilocus sequence typing (MLST). This provided to compare CRISPR1 analysis with MLST as a typing method. According to CRISPR1 analysis S. thermophilus isolates were grouped into 6 main clusters with a total of 15 sub-clusters. MLST results demonstrated an evolutionary relationship among these strains compatible with that derived from the CRISPR1 analysis.

<p>The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing.</p><p>Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension.</p><p>The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results.</p><p>Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses.</p><p>Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results.</p><p><b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis</p>

Η διατήρηση της μικροβιολογικής ποιότητας του υδάτινου περιβάλλοντος είναι υψίστης σημασίας δεδομένων των κινδύνων που ενέχονται για τη δημόσια υγεία. Η αξιολόγηση της μικροβιολογικής ποιότητας των υδάτων πραγματοποιείται με την ανίχνευση της κοπρανώδους μόλυνσης και με τον έλεγχο της παρουσίας και συγκέντρωσης συγκεκριμένων μικροοργανισμών – δεικτών, όπως είναι η Escherichia coli. Ωστόσο, η απλή ανίχνευση κοπρανώδους μόλυνσης δεν επαρκεί για την υπόδειξη τρόπων εξυγίανσης και αντιμετώπισης του εκάστοτε προβλήματος. Οι δύο κύριες ομάδες στις οποίες διακρίνεται η κοπρανώδης μόλυνση είναι η ανθρώπινη και η ζωική, οι οποίες υποδηλώνουν πιθανή παρουσία διαφορετικών κάθε φορά παθογόνων μικροοργανισμών για τον άνθρωπο. Έτσι, προκειμένου να οριοθετηθεί ο κίνδυνος για τη δημόσια υγεία και να καθοριστούν μέτρα αντιμετώπισης της μόλυνσης ενδείκνυται ο προσδιορισμός της ανθρώπινης ή ζωικής προέλευσης της κοπρανώδους μόλυνσης. Στην παρούσα μελέτη αναπτύχθηκαν, εφαρμόστηκαν και αξιολογήθηκαν οι μέθοδοι: α)Έλεγχος πολλαπλής ανθεκτικότητας σε αντιβιοτικά (Multiple Antibiotic Resistance – MAR – φαινοτυπική μέθοδος) και β) PCR με τυχαία ενισχυμένα τμήματα πολυμορφικού DNA - Random Amplified Polymorphic DNA-PCR (RAPD-PCR – γονοτυπική μέθοδος), ως τεχνικές προσδιορισμού και διάκρισης προέλευσης μικροοργανισμών. Κατά το πρώτο στάδιο καθορίστηκαν οι παράμετροι των μεθόδων για το διαχωρισμό στελεχών E. coli γνωστής προέλευσης (60 στελέχη απομονωμένα από ζωικά κόπρανα και 68 στελέχη από ανθρώπινα). Για το διαχωρισμό και κατηγοριοποίηση των στελεχών εφαρμόστηκαν η Ιεραρχική Ανάλυση Κατά Συστάδες και η Διαχωριστική Ανάλυση. Με τη MAR ανάλυση τα στελέχη E. coli εμφάνισαν διαφορετικούς συνδυασμούς ανθεκτικότητας και διαχωρίστηκαν βάσει της προέλευσής τους με μέσο ποσοστό σωστής ταξινόμησης (ARCC) 99,2%. Με την RAPD-PCR χρησιμοποιήθηκαν δύο εκκινητές ξεχωριστά (1254 & 1290) και τα 128 στελέχη E. coli γνωστής προέλευσης διαχωρίστηκαν σε ανθρώπινης και ζωικής πηγής με ARCC 98,4% και με τους δύο εκκινητές. Η διακριτική ικανότητα της RAPD-PCR με τους δύο εκκινητές ήταν D1254=0,97 & D1290=0,90. Επιπλέον, η αξιολόγηση της επαναληψιμότητας της RAPD-PCR και με τους δύο εκκινητές έδωσε ικανοποιητικά αποτελέσματα με την εμφάνιση ίδιων ηλεκτροφορητικών εικόνων για τα ίδια βακτηριακά στελέχη. Στη συνέχεια οι επιλεγμένες τεχνικές εφαρμόστηκαν για την ταξινόμηση και κατηγοριοποίηση στελεχών E. coli άγνωστης προέλευσης εκτιμώντας την ανθρώπινη ή ζωική πηγή τους βάσει του μοντέλου διαχωρισμού των E. coli γνωστής προέλευσης. Οι E. coli άγνωστης προέλευσης (234 στελέχη) απομονώθηκαν από δείγματα πόσιμου νερού δικτύου από 11 περιοχές και δείγματα μη επεξεργασμένων λυμάτων από τις εισόδους τεσσάρων σταθμών βιολογικού καθαρισμού (ΚΕΡΕΦΥΤ – Νομός Αττικής, ΨΥΤΤΑΛΕΙΑ – Νομός Αττικής, ΡΙΟ – Νομός Αχαΐας και ΠΑΤΡΑ - Νομός Αχαΐας). Τα 234 στελέχη με τη MAR ανάλυση ταξινομήθηκαν ως ανθρώπινα και ζωικά σε ποσοστά 46,6% και 53,4% αντίστοιχα. Τα αποτελέσματα ταξινόμησης ήταν διαφορετικά με τη μέθοδο RAPD-PCR. Με τον εκκινητή 1254 τα άγνωστα στελέχη προσδιορίστηκαν ως ανθρώπινα κατά το 64,9% και ως ζωικά κατά το 35,1%. Αντίστοιχα, με τον εκκινητή 1290 τα ποσοστά ήταν 60,3% ανθρώπινα και 39,7% ζωικά. Τα στελέχη του πόσιμου νερού που προέρχονταν από τους σταθμούς δειγματοληψίας που ήταν αστικά κέντρα χαρακτηρίστηκαν εξ ολοκλήρου ως ανθρώπινης προέλευσης. Αντίθετα, στις περιοχές δειγματοληψίας με ανεπτυγμένη κτηνοτροφία βρέθηκαν και στελέχη ζωικής προέλευσης, γεγονός που υποδηλώνει την είσοδο στο δίκτυο κοπρανώδους υλικού προερχόμενου από ζώα των συγκεκριμένων περιοχών, τα οποία ενδεχομένως να έχουν άμεση πρόσβαση στις πηγές και γεωτρήσεις. Όσον αφορά στο χαρακτηρισμό των E. coli που καταλήγουν στους αναφερόμενους βιολογικούς καθαρισμούς, η πλειοψηφία ανίχνευσης ανθρωπίνων στελεχών δηλώνει την πιθανή παρουσία στα ακατέργαστα λύματα πολλών ανθρωπίνων εντερικών παθογόνων σημαντικών για τη δημόσια υγεία. Δεδομένου ότι τα τελευταία χρόνια οι ερευνητές έχουν αποδυθεί σε μια προσπάθεια επαναχρησιμοποίησης επεξεργασμένων λυμάτων επισημαίνεται η ανάγκη επεξεργασίας τους σε διάφορα στάδια για τη διασφάλιση της δημόσιας υγείας. Παρατηρήθηκε συμφωνία αποτελεσμάτων με τη χρήση των δύο εκκινητών καθώς η διαφορά στα ποσοστά δεν ήταν στατιστικά σημαντική (P>0,05). Συγκρίνοντας τα αποτελέσματα που ελήφθησαν με τις δύο μεθόδους, τη φαινοτυπική (MAR ανάλυση) και τη γονοτυπική (RAPD-PCR), υπήρξε στατιστικά σημαντική διαφορά (P<0,05), με συνέπεια να τίθεται θέμα επιλογής της πιο ενδεδειγμένης μεθόδου τυποποίησης και διάκρισης περιβαλλοντικών μικροοργανισμών. H παρούσα μελέτη αναδεικνύει την RAPD-PCR ως μια γονοτυπική μέθοδο με ικανοποιητική διακριτική ικανότητα, ευαισθησία, επαναληψιμότητα υπό αυστηρά καθορισμένες συνθήκες και χαμηλού κόστους. Η ευκολία εφαρμογής για την τυποποίηση μεγάλου αριθμού βακτηριακών στελεχών, χωρίς την απαίτηση γνώσης της νουκλεοτιδικής αλληλουχίας του γενετικού υλικού την καθιστούν ιδιαίτερα προσιτή σε εργαστήρια μοριακής μικροβιολογίας, ως τεχνική διάκρισης προέλευσης της κοπρανώδους μόλυνσης στο υδάτινο περιβάλλον. / Maintenance of the microbiological quality and safety of water systems is imperative, as their faecal contamination may exact high risks to human health as well as result in significant economic losses. The microbiological quality of water systems is evaluated by detecting their faecal pollution and especially specific faecal indicators such as Escherichia coli. Simple detection of faecal pollution is not sufficient in order to apply appropriate management plans to remedy the problem and to prevent any further contamination. Human faecal material is generally perceived as constituting a grater human health risk than animal faecal material, considering that it is more likely to contain human-specific enteric pathogens. Thus, it would be desirable to determine the source of the faecal material, especially for the assessment of risk for public health and for the development of monitoring plans. In the present study the development and assessment of Multiple Antibiotic Resistance Analysis (MAR – phenotypic method) and Randomly Amplified Polymorphic DNA-PCR Analysis (RAPD-PCR – genotypic method) were established as microbial source tracking methods. Firstly, parameters of the two selected methods were determined for the discrimination of E. coli isolates of known source (60 isolates from animal faecal material & 68 isolates from human faecal material). Hierarchical Cluster Analysis and Discriminant Analysis were applied for the classification of the isolates. With MAR analysis E. coli isolates developed different resistance profiles and were discriminated according to their source with an average rate of correct classification (ARCC) of 85.2%. With RAPD-PCR analysis two different 10-nt primers of arbitrary sequence were used (1254 & 1290) and the 128 E. coli isolates of known origin were classified as human and animal with the following ARCC: ARCC1254= 87.5% & ARCC1290= 81.3%. The discriminatory power of RAPD-PCR with the two selected primers was D1254=0.97 & D1290=0.90. Furthermore, the assessment of reproducibility of RAPD-PCR analysis provided satisfactory results with both primers, as RAPD profiles were identical for the same bacterial isolates. The assessment of specificity of the method resulted in the discrimination among RAPD profiles of E. coli isolates and other reference bacteria. The selected methods were applied for the classification and the source tracking of E. coli isolates, derived from tap water and raw sewage samples. In total 234 E. coli strains were isolated from tap water from 11 areas and raw sewage samples from four treatment plants (KEREFYT – prefecture of Attiki, PSITALIA - prefecture of Attiki, RIO - prefecture of Achaia and PATRA - prefecture of Achaia). With MAR analysis the 234 isolates were classified as human and animal in percentages of 46.6% & 53.4%, respectively. Classification results were different with RAPD-PCR analysis. With primer 1254 the classification was: 64.9% of human origin and 35.1% of animal origin and with primer 1290 the classification was: 60.3% of human origin and 39.7% of animal origin. Isolates derived from tap water of urban areas were classified in total as of human origin. On the contrary, in areas with many farm breeders many isolates were classified as of animal origin, indicating presence of faecal material in the water systems derived animal activities. As far as E. coli isolates from raw sewage samples are concerned, the majority of them were classified as of human source, indicating the possible presence of other human enteric pathogens as well. Taking into account the fact that there has been an effort in order to reuse treated sewage, it seems necessary a multi-stage process to renovate wastewater before it re-enters a body of water. There was an agreement of results of classification obtained form the use of the two different primers as the percentages did vary statistically (P>0.05). Comparing results obtained from the two selected methods, the difference was statistically significant (P<0.05), raising a question of the appropriate method for the typing and discrimination of environmental microorganisms. The present study demonstrates RAPD analysis as a simple, cost effective genotypic method with satisfactory discriminatory power, sensitivity and reproducibility. It can be applied for the analysis of a large number of bacterial isolates without the prior knowledge of nucleotide sequence of DNA to be necessary. Finally, it may fulfil environmental for the determination of origin of faecal pollution protecting water resources and public health.

Σκοπός της παρούσας ερευνητικής εργασίας ήταν η επιδημιολογική μελέτη των σταφυλοκοκκικών λοιμώξεων και κυρίως των λοιμώξεων από MRSA, τόσο στο ενδονοσοκομειακό περιβάλλον, όσο και στην κοινότητα, το διάστημα 2001-2006. Κατά τη διάρκεια της μελέτης, συλλέχθηκαν συνολικά 1922 στελέχη Staphylococcus aureus από όλες τις κλινικές και από διαφορετικούς ασθενείς στο Πανεπιστημιακό Γενικό Νοσοκομείο Πατρών (ΠΓΝΠ). Στη συνέχεια τα στελέχη ελέγχθηκαν για την παραγωγή της πρωτεΐνης PBP2α και την ύπαρξη του γονιδίου mecA για τον προσδιορισμό των ανθεκτικών στη methicillin στελεχών S. aureus (Methicillin-Resistant S. aureus, MRSA). Από το σύνολο των 1922 S. aureus, τα 757 (39.4%) χαρακτηρίσθηκαν ως MRSA. Γενικά, παρατηρήθηκε αύξηση του αριθμού των λοιμώξεων από S. aureus, με παράλληλη αύξηση του ποσοστού των MRSA λοιμώξεων, από 23% το 2001 στο 49% το 2006. Οι MRSA αποτελούν σοβαρό πρόβλημα για τα νοσοκομεία, αλλά και γενικότερα σε όλο το πληθυσμό, καθώς απομονώνονται με αυξανόμενη συχνότητα από την κοινότητα. Στο διάστημα που καλύπτει η παρούσα μελέτη, 2001-2006, το ποσοστό των στελεχών που απομονώθηκαν από την κοινότητα (Community-acquired MRSA, CA-MRSA) αυξήθηκε από 3% το 2001, σε 37% το 2006, ενώ εκείνο των ενδονοσοκομειακών στελεχών (Hospital-acquired MRSA, HA-MRSA), είναι ενθαρρυντικό ότι μειώθηκε από 20% το 2001, στο 12% το 2006. Για την επιδημιολογική μελέτη των λοιμώξεων που οφείλονται σε MRSA στελέχη, μέθοδος αναφοράς είναι η PFGE, κυρίως σε συνδυασμό και με υβριδισμό του χρωμοσωμικού DNA με ειδικούς ανιχνευτές. Oι μέθοδοι αυτές παρουσιάζουν δυσκολία στη σύγκριση των κλώνων μεταξύ των χωρών και έτσι αναπτύχθηκε η MLST, που βασίζεται στη εύρεση της νουκλεοτιδικής αλληλουχίας επτά συντηρημένων γονιδίων και επιτρέπει τον προσδιορισμό των κλωνικών συμπλεγμάτων (Clonal Complex, CC) με δυνατότητα άμεσης ταυτοποίησής τους. Τα MRSA που απομονώθηκαν κατατάχθηκαν σε επτά ST/SCCmec κλώνους, τους ST80/IV, ST5/IV, ST377/V, ST30/IVvar, ST239/III, ST225/NT και ST217/NT και πέντε κλωνικά συμπλέγματα (Πίνακας 46). Στα CA-MRSA επικρατεί ο ST80/IV, με μικρό ποσοστό να ανήκει και στον πρόσφατο τύπο ST377/V, ενώ στα HA-MRSA απαντώνται κυρίως οι ST239/III και ST30/IVvar. Η PVL είναι μια τοξίνη που καταστρέφει τα λευκοκύτταρα ανοίγοντας πόρους στην κυτταρική τους μεμβράνη, προκαλεί νέκρωση ιστών και νεκρωτική πνευμονία, κυρίως σε μικρά παιδιά, και κωδικοποιείται από τα γονίδια lukS-PV και lukF-PV. Το 74% των MRSA που μελετήθηκαν το διάστημα 2001-2006, έφεραν τα γονίδια αυτά, καταγράφοντας αύξηση από 33% το 2001, σε 88% το 2006. Τα PVL-θετικά MRSA ανήκουν στους κλώνους ST80/IV και ST377/V. Τα περισσότερα PVL-θετικά CA-MRSA της μελέτης μας απομονώθηκαν από λοιμώξεις δέρματος και μαλακών μορίων, ενώ τα PVL-θετικά HA-MRSA προέρχονταν από χειρουργικά τραύματα, κυρίως σε περιπτώσεις χρήσης προσθετικών υλικών. Ένα PVL-θετικό στέλεχος, που απομονώθηκε από αιματοκαλλιέργεια, προερχόταν από οστεομυελίτιδα. Πρόκειται για πρώτη αναφορά περιστατικών οξείας οστεομυελίτιδας, με αίτιο στελέχη CA-MRSA και MSSA που παράγουν PVL. Φαίνεται ότι η παρουσία των γονιδίων lukS-PV και lukF-PV συνδέεται κυρίως με λοιμώξεις που προκύπτουν δευτερογενώς σε τραύματα και επιπολής λοιμώξεις δέρματος και μαλακών μορίων, ενώ η απομόνωση PVL-θετικού στελέχους από οστεομυελίτιδα αποτελεί ένδειξη για την εμπλοκή της τοξίνης αυτής και στη παθογένεια εν τω βάθει λοιμώξεων. Εκτός από την PVL, σημαντικοί λοιμογόνοι παράγοντες και αίτια σοβαρών κλινικών συνδρόμων στον άνθρωπο θεωρούνται και οι τοξίνες της οικογένειας των υπεραντιγόνων, όπως η τοξίνη του συνδρόμου τοξικής καταπληξίας (TSST-1) και οι εντεροτοξίνες (SEs). Το γονίδιο tst κωδικοποιεί την παραγωγή της TSST-1, ενώ το οπερόνιο egc (enterotoxin gene cluster) την έκφραση πρωτεϊνών που μοιάζουν στη δομή και αλληλουχία με τις εντεροτοξίνες. Στα MRSA της συλλογής μας, τα γονίδια tst και egc2 ανιχνεύθηκαν μόνο στον κλώνο ST30/IV και δε βρέθηκαν ποτέ να συνυπάρχουν με τα γονίδια της PVL. Ανιχνεύθηκαν και συνδυασμοί των γονιδίων tst και του οπερονίου egc στους κλώνους ST80/IV και ST239/III, που σημαίνει ότι τα γονίδια αυτά διασπείρονται με οριζόντια μεταφορά, ενώ τα γονίδια lukS-PV και lukF-PV, που εντοπίστηκαν αποκλειστικά στους κλώνους ST80/IV ST377/V και χωρίς να συνυπάρχουν με γονίδια υπεραντιγόνων, φαίνεται ότι μεταφέρονται πιο ειδικά. Προϊόν της παρούσας ερευνητικής εργασίας ήταν η ανάπτυξη μεθοδολογίας για την ποσοτικοποίηση με αλυσιδωτή αντίδραση πολυμεράσης πραγματικού χρόνου, του γονιδίου tst σε στελέχη S. aureus που ήταν ανθεκτικά στη methicillin, κατατάσσονταν σε διαφορετικούς κλώνους και έφεραν ποικίλα γονίδια τοξινών. Για τη σήμανση και παρακολούθηση των προϊόντων χρησιμοποιήθηκε το SYBR Green I (SG), που είναι μη ειδικός τρόπος σήμανσης. Η χρήση του SYBR Green I κάνει τη μέθοδο εύχρηστη, αφού μπορεί η χρωστική να προστεθεί απλά στο υπόλοιπο μίγμα της αντίδρασης και φθηνή, επειδή δε χρειάζεται να σχεδιαστούν καινούριοι, ειδικοί εκκινητές. Οι αντιδράσεις απόλυτης ποσοτικοποίησης, που πραγματοποιήθηκαν με τη συγκεκριμένη μέθοδο, είχαν υψηλή απόδοση (2.04) και τα αποτελέσματα ήταν συνεχή και επαναλαμβανόμενα, γεγονός που σημαίνει ότι μπορεί να εφαρμοσθεί στη ρουτίνα του κλινικού εργαστηρίου για τη γρήγορη ανίχνευση και ποσοτικοποίηση του γονιδίου tst στα κλινικά στελέχη. Επί πλέον αποδείχθηκε με τη στατιστική ανάλυση, ότι στελέχη που απομονώθηκαν από λοιμώξεις μαλακών μορίων συνέθεταν υψηλότερα ποσά tst. Για τον υπολογισμό των λόγων έκφρασης του γονιδίου tst, στη σχετική ποσοτικοποίηση εφαρμόσθηκαν δυο μαθηματικά μοντέλα (2-ΔΔCt και Pfaffl). Συγκρίνοντας τα αποτελέσματα από τους δυο διαφορετικούς τρόπους υπολογισμού, βρέθηκαν διαφορές στα επίπεδα έκφρασης ίδιων στελεχών και καταλήξαμε στο συμπέρασμα ότι το μαθηματικό μοντέλο του Pfaffl είναι πιο ακριβές και αξιόπιστο, καθώς συνυπολογίζει την απόδοση της αντίδρασης. / The purpose of this study was to establish the clonality and evolution of CA-MRSA (Community-acquired MRSA, CA-MRSA) and HA-MRSA (Hospital-acquired MRSA, HA-MRSA), as well as the epidemiology of MRSA (Methicillin-Resistant S. aureus, MRSA) infections, during 2001-2006. In total 1922 Staphylococcus aureus strains were collected from patients with different pathologies admitted at the University Hospital of Patras. Among them 757 (39.4%) strains were MRSA. The prevalence of MRSA infections rose from 23% in 2001 to 49% in 2006. MRSA is a major problem worldwide in the nosocomial setting and the community. During 2001-2006 CA-MRSA isolated with an increasing rate from 3% in 2001 to 37% in 2006, while HA-MRSA decreased from 20% in 2001 to 12% in 2006. The epidemiological study of MRSA infections was based on PFGE, the “gold standard” of typing methods and hybridization with specific DNA probes. However, for the full characterization of a strain it is recommended the application of the multilocus sequence typing (MLST), since it is a highly discriminatory method and permits to compare the results from different laboratories. MLST represents a major advance since it relates organisms on the basis of the nucleotide sequences of ~450 bp internal fragments of seven conserved housekeeping genes resulting to the determination of Sequence Types (ST) and Clonal Complexes (CC). MRSA of our collection belonged to seven ST/SCCmec clones (ST80/IV, ST5/IV, ST377/V, ST30/IVvar, ST239/III, ST225/NT and ST217/NT) and five Clonal Complexes. Most CA-MRSA isolates belonged to ST80/IV clone and a small percentage to the newly described clone, ST377/V, while HA-MRSA strains were mainly characterized as ST239/III and ST30/IVvar clones. PVL is a bicomponent toxin associated with skin and soft tissue infections, but also with necrotizing pneumonia, especially in children. In total 74% of MRSA were positive for the PVL genes rising from 33% in 2001 to 88% in 2006. PVL-positive MRSA strains belonged to ST80/IV and ST377/V clones. Most PVL-positive CA-MRSA isolated from skin and soft tissue infections, while PVL-positive HA-MRSA isolated from surgical wounds, especially when prosthetic devices were used. In one patient acute staphylococcal osteomyelitis (AO) was diagnosed, due to MRSA carrying the PVL genes. This is the first description of CA-MRSA producing PVL as causative agents of AO suggesting that PVL-positive S. aureus can be isolated from patients with invasive musculo-skeletal infections, including acute childhood osteomyelitis, as well as among patient with skin and soft-tissue infections. Staphylococcal enterotoxins, enterotoxin-like superantigens (enterotoxin gene operon, egc) and the toxic shock syndrome toxin-1 that belong to the pyrogenic toxin superantigens (PTSAgs) are considered major virulence factors. The genotype tst/egc belonged only to ST30/IV clone and never coexisted with the PVL genes. Other combinations of genes were also detected belonging to clones ST80/IV and ST239/III, suggesting the horizontal transfer of those genes. On the contrary, the PVL genes were detected only in ST80/IV and ST377/V clones, meaning a more specific way of spread. A real-time PCR assay was developed for the quantification of tst gene in methicillin-resistant S. aureus using SYBR Green I (SG) chemistry, which is an easy and cost-effective approach to real-time, since it does not require the design of sequence specific probes and new primers. By the developed method of absolute quantification the results were reproducible and constant, meaning that the assay can be applied in the routine laboratory. The statistically significant difference of tst gene expression among strains associated with SSTIs, suggests that such strains may be the cause of TSS among patients. For the calculation of expression ratios in the relative quantification we applied two mathematical models (2-ΔΔCt and Pfaffl). Comparing ratios derived from the two mathematical methods we found variations, allowing us to suggest the use of the Pfaffl model as the more precise and reliable method.

In der vorliegenden Studie wurde die Sanierung einer durch S. aureus verursachten Eutergesundheitsstörung in einem hessischen Milchviehbestand mittels klassischer Sanierungsmaßnahmen über einen Zeitraum von 18 Monaten begleitet. Durch konsequente Einhaltung der Sanierungsmaßnahmen nach dem Fünf-Punkte-Plan und die räumliche Trennung der Herde in eine S. aureus-positive und -negative Gruppe sank die S. aureus-Prävalenz im Betrieb innerhalb von 15 Monaten von 30% auf <1%. Nach 18 Monaten waren erstmalig keine Neuinfektionen mehr mit S. aureus zu verzeichnen. Zusätzlich zu den im Abstand von drei Monaten durchgeführten Viertelanfangsgemelk (VAG)- Gesamtbestandsuntersuchungen wurden Umweltproben bakteriologisch auf das Vorhandensein von S. aureus untersucht. Diese Untersuchungen verliefen ausschließlich mit negativem Ergebnis. Die im Untersuchungszeitraum asservierten 218 S. aureus-Isolate aus diesem Betrieb wurden genotypisch mittels PCR untersucht. Thermonuklease-Gen, Protein A-Gen (IgG-bindende Region) und Polymorphismen bei Protein A-Gen (Xr-Region) sowie Koagulase-Gen ermöglichten die Unterscheidung in sechs Typen. Zusätzlich wurden 80 dieser Isolate mittels Pulsfeldgelelektrophorese (Pfge, Gold Standard) typisiert. Es konnten zwei Pfge-Typen gefunden werden: Pfge-Typ I mit insgesamt 10 Subtypen (bei 78 Isolaten) und Pfge-Typ II (bei zwei Isolaten). Der Pfge-Typ II wurde ausschließlich bei einem Zukaufstier nachgewiesen, das vor der Einstallung in diesen Betrieb nicht zytobakteriologisch untersucht worden war. Die 12 unterschiedlichen Pfge-Typen bzw. -Subtypen wurden zusätzlich mittels Staphylokokken-Protein A- (spa)-Typisierung untersucht. Dabei zeigte sich, dass alle Subtypen des Pfge-Typs I dem spa-Typ t2067 zugeordnet werden konnten und der Pfge-Typ II dem spa-Typ t2112 entsprach. 92 weitere bovine S. aureus-Mastitisisolate wurden möglichst randomisiert über das Landesgebiet Hessens entnommen und mittels dieser Typisierungsmethode untersucht. Die Isolate stammten ebenfalls aus S. aureus-Problembetrieben und wurden aus VAG von (sub-) klinischen Mastitiden in Reinkultur isoliert. Insgesamt konnten 28 spa-Typen unterschieden werden. Durch den Algorithmus BURP wurden 57 dieser Isolate dem Clonal Complex CC543 zugeordnet und waren demnach genetisch eng miteinander verwandt. Der in dem Sanierungsbetrieb vorherrschende spa-Typ t2067 war dem CC543 nicht zuzuordnen. Die Anwendung der spa-Typisierung in der Routinediagnostik und damit die Möglichkeit der laborübergreifenden, möglichst zentralisierten Datendokumentation der Ergebnisse könnten zukünftig die Identifizierung besonders häufig vorkommender und damit hochkontagiöser S. aureus-Stämme ermöglichen.

A schooling system that claims to offer its students the opportunities to develop their talents and help towards self-determination in their adult lives might be expected to have a career structure itself that demonstrated these virtues, one in which there was equality of the genders in positions of influence and leadership, and no gender stereotyping of roles. Apart from the fairness and consistency of that expectation, it is also reasonable to expect the neutral template of teacher employment and textbook selection in schools. Many children may grow up with few books in their homes but lots of those in their schools. Many of the textbooks used in elementary schools, according to recent studies, contain gender stereotypes. In these, females are rarely found as central characters and when they appear at all, they are often passive figures dependent on male characters. Women are frequently shown in domestic roles / in most textbooks it is assumed that only males &#039 / go out to work&#039 / whereas daughters are the best helpers of their mothers whose sons are allowed to do what they wish. In the light of those allegations, this research is designed as a case study which addresses itself to the aim of looking into stereotyped gender role presentations existing in elementary school textbooks used by the students studying at 1st-5th grades in 2001/2002 academic year of an elementary school placed in Burdur and to see whether these students are affected by the exposure of those stereotyped gender role presentations. For this purpose, the textbooks being studied are analyzed according to pre-set categories to deduce how they include stereotyped gender role presentations and the evaluation of the effects of that exposure on students are made by asking 1st-3rd grade students to draw and 4th-5th grade students to write compositions on a given topic. This study also attempts to find out both whether Turkish elementary school teachers teaching at 1st-5th grades are aware of stereotyped gender role presentations in those textbooks that they use and their own points of view about stereotyped gender role presentations via interviews carried out with them. In conclusion, stereotyped gender role presentations are encountered in those analyzed school textbooks studied at 1st- 5th grades in 2001/2002 academic year of the elementary school placed in Burdur and the perceptions of those presentations are also obtained in the drawn and written productions of the students studied at the same school. Through the teachers&#039 / interviews, various kinds of perceptions towards gender role concept and its stereotyped presentations that take place in those textbooks are observed in their sayings