Aquaculture is the fastest growing food industry in the world and it accounts for roughly half of the world's fish supply. The majority of global aquaculture production occurs in freshwater systems that are increasingly subject to multiple uses by different stakeholders. Given the overall scarcity of freshwater on a global scale, freshwater aquaculture will face increasing environmental constraints that will demand an ever better understanding of its potential impacts on the aquatic environment and human health. This thesis consists of a series of studies that, collectively, contribute to further our understanding on the effects of freshwater aquaculture effluents on aquatic ecosystems, on the effects and environmental safety of antibiotics used in freshwater aquaculture on aquatic bacterial communities and on the link between antibiotic pollution and antibiotic resistance. Chapter 2 reviews the effects of freshwater aquaculture effluents on stream ecosystems using land-based salmonid farms as a case study. In this chapter I discuss relevant considerations related to the temporal and spatial scales of effluent discharge and ecological effects that highlight the need to characterize the patterns of stressor discharge when assessing environmental impacts and designing ecological effects studies. I also discuss the potential role of multiple stressors - with an emphasis on veterinary medicines - in disrupting ecosystem structure and function. Overall, the critical analysis presented in this chapter indicates that further research on the effects of veterinary medicines using relevant exposure scenarios would significantly contribute to our understanding of their impact in relation to other effluent stressors. Chapter 3 is a general methods chapter that describes the stream microcosm system used to assess the effects of erythromycin thiocyanate (ERT) and florfenicol (FFC) on bacterial communities of stream biofilms. This chapter presents the results of preliminary experiments whose results provided relevant information on the overall operation of the microcosms and on the variability of major physical and biological variables. This information guided the experimental designs used to assess the effects of FFC and ERT on the bacterial community structure of stream biofilms. Chapter 4 presents the results of the experiment conducted to assess the effects of FFC on the bacterial community structure of developing biofilms. The objective was to assess changes in bacterial community structure along a gradient of FFC concentrations that could provide insight into the type and magnitude of effects that could be expected from episodic exposure of stream biofilms to FFC in headwater streams. At 10 and 20 days of biofilm development, bacterial community structure differentiated in a pattern consistent with the FFC concentration gradient and there was a positive relationship between bacterial richness and bacterial diversity with FFC concentration. At 15 days of biofilm development there was also a positive relationship between FFC concentration and the surface coverage of bacteria and extracellular polymeric substances. These trends declined as the biofilm developed a more complex architecture, in terms of thickness and in the surface coverage of algae. The results are consistent with an initial stimulatory effect of FFC on biofilm formation that triggered changes in bacterial community structure that were gradually compressed as the development of a complex biofilm architecture increased the relative importance of autogenic ecological processes. The results suggest that the co-occurrence of FFC with bacterial pathogens in effluents and wastewaters may favour their persistence in the environment by enhancing biofilm formation. Chapter 5 presents the results of the experiment conducted to assess the effects of ERT on the bacterial community structure of developing biofilms. Currently, Aquamycin® 100 - a Type A medicated article (i.e., Premix) containing 100 g ERT lb-1 and used to produce a Type C medicated feed - is a candidate drug for approval by the US FDA to control mortality associated with bacterial kidney disease in freshwater salmonids. The objective of this experiment was to assess the effects of ERT on the bacterial community structure of stream biofilms using an exposure period consistent with the 28-day treatment regime suggested for Aquamycin® 100. The results provide no evidence to suggest that a 30-day exposure to ERT concentrations in the range of 10 μg L-1 (i.e., 7.3 ± 3.9 μg L-1) would lead to changes in the bacterial community structure or overall bacterial abundance of stream biofilms, while they suggest that these effects may occur at concentrations in the range of 100 μg L-1 (i.e., 87.2 ± 31.1 μg L-1). Chapter 6 attempts to determine whether environmental concentrations of antibiotics and concentrations representing action limits used in environmental risk assessment may exert a selective pressure on clinically relevant bacteria in the environment. In this chapter I use bacterial inhibition as an assessment endpoint to link antibiotic selective pressures to the prevalence of resistance in bacterial populations. Species sensitivity distributions were derived for three antibiotics by fitting log-logistic models to endpoints calculated from minimum inhibitory concentration (MIC) distributions based on worldwide data collated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Bacteria represented in these distributions were placed in a broader context by performing a brief phylogenetic analysis. The potentially affected fraction of bacterial genera at measured environmental concentrations of antibiotics and environmental risk assessment action limits was used as a proxy for antibiotic selective pressure. Measured environmental concentrations and environmental risk assessment action limits were also directly compared to wild-type cut-off values. Results suggest that measured environmental concentrations of antibiotics and concentrations representing environmental risk assessment action limits are high enough to exert a selective pressure on clinically relevant bacteria that may lead to an increase in the prevalence of resistance. Chapter 7 presents the results of an exploratory analysis conducted to assess the abundance of class 1 integrons in stream biofilms exposed to FFC and ERT. There was no pattern in the abundance of intI1 genes consistent with the treatment of FFC and ERT, suggesting either the absence of gene cassettes involved in dealing with selective pressures caused by these antibiotics or that the concentrations tested were below those required to give them a selective advantage. Chapter 8 is a brief general discussion that brings together the findings of the thesis and makes suggestions for future research. Key areas identified for future research include assessing in further detail the stimulatory effect of FFC on biofilm formation in complex bacterial communities, the interactive effects of multiple aquaculture effluent stressors on aquatic bacterial communities and their potential effects on the development of antibiotic resistance, the fate of FFC and ERT in stream ecosystems, and further developing the analysis based on MIC distributions presented in chapter 6 to assess the potential effects of antibiotic pollution on the selection of multi-drug resistance in the environment.

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.

This thesis is an investigation of novel mono-ADP-ribosylating toxins. In the current data-rich era, making the leap from sequence data to knowledge is a task that requires an elegant bioinformatics toolset to pinpoint questions. A strategy to expand important protein-family knowledge is required, particularly in cases in which primary sequence identity is low but structural conservation is high. For example, the mono-ADP-ribosylating toxins fit these criteria and several approaches have been used to accelerate the discovery of new family members. A newly developed tactic for detecting remote members of this family -- in which fold recognition dominates -- reduces reliance on sequence similarity and advances us toward a true structure-based protein-family expansion methodology. Chelt, a cholera-like toxin from Vibrio cholerae, and Certhrax, an anthrax-like toxin from Bacillus cereus, are among six new bacterial protein toxins identified and characterized using in silico and cell-based techniques. Medically relevant toxins from Mycobacterium avium and Enterococcus faecalis were also uncovered. Agriculturally relevant toxins were found in Photorhabdus luminescens and Vibrio splendidus. Computer software was used to build models and analyze each new toxin to understand features including: structure, secretion, cell entry, activation, NAD+ substrate binding, intracellular target binding and the reaction mechanism. Yeast-based activity tests have since confirmed activity. Vibrio cholerae produces cholix – a potent protein toxin of particular interest that has diphthamide-specific ADP-ribosyltransferase activity against eukaryotic elongation factor 2. Here we present a 2.1Å apo X-ray structure as well as a 1.8Å X-ray structure of cholix in complex with its natural substrate, nicotinamide adenine dinucleotide (NAD+). Hallmark catalytic residues were substituted and analyzed both for NAD+ binding and ADP-ribosyltransferase activity using a fluorescence-based assay. These new toxins serve as a reference for ongoing inhibitor development for this important class of virulence factors. In addition to using toxins as targets for antivirulence compounds, they can be used to make vaccines and new cancer therapies. / Natural Sciences and Engineering Research Council (CGS-D), Canadian Institutes of Health Research, Cystic Fibrosis Canada, Human Frontier Science Program, Ontario government (OGSST), University of Guelph (Graduate Research Scholarship)

The persistence and propagation of bacterial antibiotic resistance presents significant challenges to the treatment of drug resistant bacteria with current antimicrobial chemotherapies, while a dearth in replacements for these drugs persists. The thiopeptide family of antibiotics may represent a potential source for new drugs and thiostrepton, the prototypical member of this antibiotic class, is the primary subject under study in this thesis. Using a facile semi-synthetic approach novel, regioselectively-modified thiostrepton derivatives with improved aqueous solubility were prepared. In vivo assessments found these derivatives to retain significant antibacterial ability which was determined by cell free assays to be due to the inhibition of protein synthesis. Moreover, structure-function studies for these derivatives highlighted structural elements of the thiostrepton molecule that are important for antibacterial activity. Organisms that produce thiostrepton become insensitive to the antibiotic by producing a resistance enzyme that transfers a methyl group from the co-factor S-adenosyl-L-methionine (AdoMet) to an adenosine residue at the thiostrepton binding site on 23S rRNA, thus preventing binding of the antibiotic. Extensive site-directed mutagenesis was performed on this enzyme to generate point mutations at key active site residues. Ensuing biochemical assays and co-factor binding studies on these variants identified amino acid residues in the active site that are essential to the formation of the AdoMet binding pocket and provided direct evidence for the involvement of an active site arginine in the catalytic mechanism of the enzyme. Certain bacteria that produce neither thiostrepton nor the resistance methyltransferase express the thiostrepton binding proteins TIP-AL and TIP-AS, that irreversibly bind to the antibiotic, thereby conferring resistance by sequestration. Here, it was found that the point mutation of the previously identified reactive amino acid in TIP-AS did not affect covalent binding to the antibiotic, which was immediately suggestive of a specific, high affinity non-covalent interaction. This was confirmed in binding studies using chemically synthesized thiostrepton derivatives. These studies further revealed structural features from thiostrepton important in this non-covalent interaction. Together, these results indicate that thiostrepton binding by TIP-AS begins with a specific non-covalent interaction, which is necessary to properly orient the thiostrepton molecule for covalent binding to the protein. Finally, the synthesis of a novel AdoMet analogue is reported. The methyl group of AdoMet was successfully replaced with a trifluoromethyl ketone moiety, however, the hydrated form (germinal diol) of this compound was found to predominate in solution. Nevertheless, the transfer of this trifluoroketone/ trifluoropropane diol group was demonstrated with the thiopurine methyltransferase.

No description available.

Glycopeptides are the so-called last-resort antibiotics in clinical practice used to treat heavier, predominantly nosocomial infections caused by multi-resistant coagulase-negative staphylococci. The origin and genetic basis of resistance to glycopeptide antibiotics has not yet been elucidated within coagulase-negative staphylococci. Research on Staphylococcus aureus has shown, that intermediate resistance to glycopeptide antibiotics is associated with the presence of one or more mutations, rather than being conditioned by the support of a particular genetic element, such as in enterococci. By using various types of in vitro resistant mutant selection, we were able to obtain isogenic pairs of glycopeptide sensitive and resistant strains of Staphylococcus epidermidis and Staphylococcus haemolyticus. By sequencing the genomes of these pairs, one nucleotide polymorphisms were identified and predominantly found in metabolic and cell wall control systems. Phenotypic analysis did not reveal a direct association of glycopeptide resistance with increased biofilm formation. In clinical practice, the cross-resistance of glycopeptides and other antibiotics is problematic. For the non-glycopeptide antibiotics imipenem and rifampicin, the incidence of cross-resistance with glycopeptide antibiotics in S. aureus...

Dans le milieu clinique, Stenotrophomonas maltophilia est décrite comme bactérie pathogène opportuniste, responsable d'infections nosocomiales principalement chez des patients immunodéprimés ou présentant des pathologies sévères ou chroniques. L'impressionnant bouclier de résistance aux antibiotiques observé chez les souches cliniques rend les traitements particulièrement complexes pour les patients atteints. Les souches de S. maltophilia représentent une réelle menace pour la santé humaine. De plus, les fortes potentialités d'adaptation des S. maltophilia leur permettent une dispersion dans un éventail très large de biotopes cliniques mais aussi environnementaux. En effet, les S. maltophilia colonisent aussi abondamment les niches écologiques environnementales telles que les sols rhizosphériques. Le niveau des connaissances sur ces souches environnementales est particulièrement limité face à celui disponible du milieu médical. Les propriétés en tant que pathogène opportuniste de ces souches environnementales restent encore peu connues et controversées tant au niveau génétique que phénotypique. Afin de mieux évaluer le potentiel danger sanitaire que représentent les souches environnementales face aux souches cliniques, il a été envisagé lors de ce projet de thèse d'évaluer des caractéristiques phénotypiques d'un groupe de souches de S. maltophilia provenant de contextes différemment en contact avec l'homme et l'environnement. Des souches de S. maltophilia fortement impactées par le contact de l'homme ont été isolées de patients atteints de pathologies variables (mucoviscidose, infections nosocomiales, pathologies sévères). Ce groupe de souches considérées comme les plus à risque pour l'homme, a été comparé à un groupe de souches de S. maltophilia environnementales provenant de contextes ayant pu favoriser des acquisitions/maintiens de résistances aux molécules antimicrobiennes tels que les sols rhizosphériques, les sols pollués aux métaux lourds ou encore les sols soumis aux activités répétées de l'homme. Tout d'abord, les signatures métaboliques (croissance, dégradations de substrats) et les capacités de résistance à diverses molécules antibiotiques cliniques ont été évaluées pour la collection de souches de S. maltophilia. Dans un deuxième volet, ont été étudiées les potentialités de virulence de ces souches telles que la mobilité, les sécrétions enzymatiques, la formation de biofilm et la virulence envers des amibes. Enfin, une analyse croisée statistique a mis en lien les différentes signatures obtenues à partir des données métaboliques, de résistance aux antibiotiques et de virulence en confrontant les origines des souches et les influences qu'elles ont subies vis-à-vis de l'homme. D'après le jeu de données du projet, quatre signatures distinctes émergent entre les souches de S. maltophilia structurées par les effets dus la proximité de l'homme et à leur origine. Des souches environnementales potentiellement les plus impactées par les contacts avec l'homme possèdent des caractéristiques similaires aux souches cliniques ; elles sont donc potentiellement aussi dangereuses que les souches cliniques / In the clinical settings, Stenotrophomonas maltophilia is described as an opportunistic bacterial pathogen responsible for nosocomial infections mainly in immunocompromised patients or with severe or chronic diseases. The heavy shield of antibiotic resistances observed in clinical strains lead to particularly complex treatments for patients. S. maltophilia strains represent a real threat to human health. Moreover, the high potential for adaptation of S. maltophilia allow their dispersion in a wide range of clinical habitats but also environmental. Indeed, S. maltophilia strains also colonize widely environmental niches such as the rhizospheric soils. The knowledge about these environmental strains is particularly limited compared to the available medical data. The properties as opportunistic pathogenic of environmental strains remain poorly known and controversial. To better assess the potential health hazard of these environmental S. maltophilia compared to the clinical ones, were assessed in this Ph-D project phenotypic characteristics of a group of S. maltophilia strains from contexts differentially affected by human and environment imprints. S. maltophilia heavily impacted by human contacts have been isolated from patients with varying disease (cystic fibrosis, nosocomial infections, severe pathologies). This group of strains considered as the most at risk to humans, was compared to a group of S. maltophilia from environmental contexts that could promote acquisition/maintaining of resistances to antimicrobial molecules such as rhizospheric soils, heavy metal-contaminated soils or agricultural soils. Firstly, metabolic signatures (growth, substrate degradations) and antibiotic resistance capacities were evaluated among the collection of S. maltophilia strains. In a second part, were studied pathogenic potentialities of these strains such as mobility, enzyme secretions, biofilm formation and virulence to amoebae. Finally, a statistical analysis made connections on the different signatures obtained from the metabolic data, antibiotic resistance and virulence with the origins of the strains and human impacts. According to the datasets of the project, four distinct signatures emerged between S. maltophilia strains structured by the effects of human proximity and origin of the strains. Environmental strains potentially the most impacted by contact with humans showed similar characteristics with the clinical strains; they could potentially be as dangerous as clinical strains

Recentemente, o bis-(3\',5\')-di-guanosina monofosfato cíclico (c-di-GMP) surgiu como uma importante molécula sinalizadora nas bactérias. Essa molécula foi identificada como uma das responsáveis pelo controle do comportamento bacteriano e está relacionada com a patogenicidade e a adaptação de diversas bactérias, coordenando a expressão de genes envolvidos com virulência, motilidade e formação de biofilme. O mecanismo pelo qual c-diGMP atua vem sendo motivo de estudo de vários grupos de pesquisa nos últimos anos. Já foi demonstrado o papel dessa molécula em diferentes etapas do controle da expressão gênica. Acredita-se que a manipulação dos níveis de c-di-GMP pode ser uma nova abordagem terapêutica contra bactérias patogênicas. Pseudomonas aeruginosa é uma proteobactéria do grupo gama, que atua como um patógeno oportunista, causando infecções em pacientes imunocomprometidos, sendo o maior causador de infecções crônicas em pacientes portadores de fibrose cística. O genoma de P. aeruginosa PA14 apresenta vários genes que codificam proteínas envolvidas no metabolismo e/ou ligação de c-di-GMP, o que pode indicar um amplo papel regulatório deste nucleotídeo nessa bactéria. Uma associação infundada entre níveis elevados de c-di-GMP e a resistência aos antibióticos é geralmente assumida, já que altos níveis de c-di-GMP levam à formação de biofilme, que é comprovadamente um modo de crescimento mais resistente. Nesse trabalho, utilizando uma abordagem proteômica, mostramos que Pseudomonas aeruginosa PA14 regula a expressão de cinco porinas em resposta a variações nos níveis de c-di-GMP, independentemente dos níveis de mRNA. Uma dessas porinas, OprD, é responsável pela entrada do antibiótico &#946;-lactâmico imipenem na célula e é menos abundante em condições de alto c-di-GMP. Também demonstramos que linhagens com altos níveis de c-di-GMP apresentam uma vantagem competitiva de crescimento em relação a linhagens com níveis mais baixo de c-di-GMP quando crescidas em meio contendo imipenem. Em contraste, observamos que células planctônicas com elevados níveis c-di-GMP são mais sensíveis a tobramicina. Em conjunto, estes resultados mostram que c-di-GMP pode regular a resistência a antibióticos em sentidos opostos, e independentemente do crescimento em biofilme / Following the genomic era, a large number of genes coding for enzymes predicted to synthesize and degrade 3\'-5\'-cyclic diguanylic acid (c-di-GMP) was found in most bacterial genomes and this dinucleotide emerged as an important intracellular signal molecule controlling bacterial behavior. Diverse molecular mechanisms have been described as targets for c-di-GMP, but several questions remain to be addressed. An association between high c-di-GMP levels and antibiotic resistance is largely assumed, since high c-di-GMP upregulates biofilm formation and the biofilm mode of growth leads to enhanced antibiotic resistance; however, a clear understanding of this correlation is missing. Pseudomonas aeruginosa is a versatile gamma-proteobacterium that behaves as an opportunistic pathogen to a broad range of hosts. The ability of P. aeruginosa to form biofilms contributes to its virulence and adaptation to different environments. The P. aeruginosa PA14 genome presents several genes encoding proteins involved in metabolism or binding to c-di-GMP, which may indicate a wide regulatory role of this nucleotide in this bacterium. Here, using a proteomic approach, we show that Pseudomonas aeruginosa PA14 regulates the amount of five porins in response to c-di-GMP levels, irrespective of their mRNA levels. One of these porins is OprD, decreased in high c-di-GMP conditions, which is responsible for the uptake of the &#946;-lactam antibiotic imipenem. We also demonstrate that this difference leads strains with high c-di-GMP to be more resistant to imipenem even when growing as planktonic cells, giving them a competitive advantage over cells with low c-di-GMP. Contrastingly, we found that planktonic cells with high c-di-GMP levels are more sensitive to aminoglycosides antibiotics. Together, these findings show that c-di-GMP levels can regulate the antibiotic resistance to different drugs in opposite ways and irrespective of a biofilm mode of growth.

O presente estudo visou determinar a prevalência de bactérias Gram-negativas produtoras de produzem b-lactamases de amplo espectro (ESBL) e resistência adquirida a quinolonas mediada por plasmídeos (PMQR) em animais de estimação, investigando o potencial papel destes hospedeiros como portadores assintomáticos. Em 2012, foram coletadas 216 amostras (fezes e saliva) de 108 animais de companhia (29 gatos e 79 cães) abrigados em casas de família, um centro de acolhimento de animais abandonados, e no Centro de Controle de Zoonoses da Cidade de São Paulo. Do total de cepas estudadas, 85% apresentaram fenótipo sugestivo de PMQR; enquanto que 62% dos isolados exibiram um fenótipo característico e sugestivo para produção de ESBL, sendo na sua maioria identificadas como E. coli. Dentre os isolados, 14 carregaram variantes do gene blaCTX-M, 9 foram positivos para o gene blaTEM, e 6 foram positivos para blaSHV. Em relação às cepas resistentes às Q/FQ, 56% (n= 43) foram positivas para a presença do gene qnr, o qual foi identificado em 11 espécies diferentes. Os resultados apresentados demostram que animais de companhia podem ser portadores assintomáticos de cepas produtoras de ESBL e PMQR. / The present study aimed to determine the prevalence of Gram-negative bacteria producing b-lactamases producing broad-spectrum (ESBL) and acquired resistance to quinolones mediated by plasmids (PMQR) in pets, investigating the potential role of these hosts as asymptomatic carriers. In 2012, 216 samples (feces and saliva) of 108 companion animals (29 cats and 79 dogs) housed in shelters or a Zoonosis Control Center were collected from São Paulo city. Of the total strains studied, 85% had a phenotype suggestive for PMQR; while 62 % of the isolates exhibited a characteristic phenotype and suggestive for ESBL-producing genes, with the most identified as E. coli. Among the isolates, 14 carried variants blaCTX -M gene 9 were positive for blaTEM gene, and 6 were positive for blaSHV. Regarding resistant Q/FQ isolates, 56% (n = 43) were positive for the presence of qnr gene, which was identified on 11 different species. The results presented demonstrate that pets can be asymptomatic carriers of ESBL producing strains and PMQR.

A resistência aos antibióticos é um problema de saúde pública global com sérias consequências para o tratamento de várias infecções bacterianas. Os fatores de transcrição da família MarR têm sido descritos controlando resistência a antibióticos e vários outros processos em bactérias. Neste trabalho, estudamos mecanismos de resistência a antibióticos em Chromobacterium violaceum, uma bactéria Gram-negativa ambiental que pode atuar como um patógeno oportunista em humanos. A estratégia envolveu a varredura de um painel de treze linhagens mutantes de fatores de transcrição da família MarR de C. violaceum disponíveis, por testes de susceptibilidade a 24 antibióticos. Estes ensaios revelaram que apenas o mutante ?emrR apresentou resistência aumentada ao antibiótico ácido nalidíxico em relação à linhagem selvagem. Esta resistência aumentada do mutante ?emrR ao ácido nalidíxico foi revertida em uma linhagem complementada deste mutante, conforme verificado por ensaios de viabilidade, ensaios de difusão em disco e concentração inibitória mínima (MIC). O fenótipo de diminuída produção de violaceína deste mutante, observado em meio líquido, também foi complementado. Além disso, foi realizado o isolamento de mutantes espontâneos de C. violaceum resistentes a ácido nalidíxico com mutação pontual em emrR. Os ensaios de microarranjo de DNA mostraram que EmrR reprime algumas dezenas de genes, incluindo o operon emrCAB, o qual codifica a bomba de efluxo EmrCAB. Os ensaios de Northern blot confirmaram que o EmrR reprime o operon emrCAB, e que a expressão desta bomba é induzida por salicilato, mas não outros compostos, como ácido nalidíxico ou brometo de etídeo. Os ensaios de alteração de mobilidade eletroforética (EMSA) mostraram que a proteína EmrR purificada se liga diretamente às 8 regiões promotoras de emrR, emrCAB e vários outros genes do regulon EmrR, para exercer uma regulação negativa direta sobre esses genes. Um mutante nulo ?emrCAB foi obtido, mas a ausência desta bomba de efluxo não tornou C. violaceum mais susceptível ao ácido nalidíxico, sugerindo que ela é importante somente em condições nas quais é induzida. Estas condições indutoras talvez incluam estresse oxidativo, uma vez que enzimas antioxidantes são parte do regulon de EmrR e a proteína EmrR formou dímeros covalentes na presença de agentes oxidantes in vitro. Portanto, nossos dados revelam que mutações pontuais ou moléculas como salicilato abolem a atividade repressora do fator de transcrição EmrR sobre o operon emrCAB, levando a superexpressão da bomba de efluxo EmrCAB e aumentando a resistência ao ácido nalidíxico em C. violaceum. / Antibiotic resistance is a global public health problem with serious consequences for the treatment of various bacterial infections. MarR family transcription factors have been described controlling antibiotic resistance and several other processes in bacteria. In this work, we studied mechanisms of antibiotic resistance in Chromobacterium violaceum, an environmental Gramnegative bacterium that can act as a human opportunistic pathogen. The strategy involved a screening of an available collection of thirteen C. violaceum mutant strains of MarR family transcription factors, by susceptibility testing for 24 antibiotics. These assays revealed that only the ?emrR mutant showed increased resistance to the antibiotic nalidixic acid in relation to the wild-type strain. This increased resistance of the ?emrR mutant to nalidixic acid was reversed in a complemented strain of this mutant, as verified by viability, disk diffusion, and minimal inhibitory concentration (MIC) assays. The phenotype of decreased violacein production of this mutant, observed in a liquid medium, was also complemented. In addition, it was performed the isolation of spontaneous mutants of C. violaceum resistant to nalidixic acid with a point mutation in emrR. DNA microarray assays showed that EmrR represses a few dozen of genes, including the emrCAB operon, which encodes the EmrCAB efflux pump. Northern blot assays confirmed that EmrR represses the emrCAB operon and that the expression of this pump is induced by salicylate, but not other compounds, such as nalidixic acid or ethidium bromide. Electrophoretic mobility shift assays (EMSA) showed that the purified EmrR protein binds directly to the promoter regions of emrR, emrCAB and several other genes of the EmrR regulon, to exert a direct negative regulation of these genes. A ?emrCAB null mutant strain was obtained, but the absence of this efflux pump did not make C. violaceum more susceptible to nalidixic acid, suggesting that it is important only under conditions in which it is induced. These inducing conditions may include 10 oxidative stress since antioxidant enzymes are part of the EmrR regulon and the EmrR protein has formed covalent dimers in the presence of oxidizing agents in vitro. Therefore, our data reveal that point mutations or molecules such as salicylate abolish the repressive activity of the EmrR transcription factor on the emrCAB operon, causing overexpression of the EmrCAB efflux pump and increasing the resistance to nalidixic acid in C. violaceum.