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The genus Gluconobacter is industrially important due to the ability to accomplish unusual and almost complete oxidation reactions (bioconversions) and to contaminate high sugar content products. Following preliminary evidence that some strains of Gluconobacter were resistant to cadmium, and realizing that cadmium resistance among gram-negative organisms is often encoded by an operon which also encodes cobalt and zinc resistance via an efflux mechanism, 10 strains of Gluconobacter were tested for heavy-metal resistance. Three of the 10 representative strains appeared to be resistant to cadmium chloride, and two were also resistant to cobalt- and zinc chloride. These strains, as well as two cadmium-sensitive strains were analyzed using PCR and sequencing to establish gene homology with Ralstonia eutropha, the most frequently studied Gram-negative bacterium exhibiting cadmium resistance. Amplification of two genes from the czc operon, known to encode cadmium, cobalt and zinc resistance in Ralstonia, was attempted in the three resistant and two sensitive strains of Gluconobacter. The gene, czcA, thought to encode the main pump protein of the efflux mechanism, was found in all Gluconobacter strains tested. However, amplification of a regulatory gene czcD, thought to sense the extracellular metal ion concentration, was not possible in the Gluconobacter strains tested. The PCR products were sequenced and analyzed for homology to the czc operon in Ralstonia. From the data gathered, it appears as though some strains of Gluconobacter contain at least a portion of the czc operon , encoding cadmium, cobalt and zinc resistance in Ralstonia eutropha. / Master of Science

Made available in DSpace on 2014-12-17T14:20:09Z (GMT). No. of bitstreams: 1 JessicaSM_DISSERT.pdf: 4464945 bytes, checksum: 1436f4eaf0ddca5a64460da0b3822df7 (MD5) Previous issue date: 2013-03-15 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Our research intends to comprehend the configuration of the resistance (Foucault) as the stylization of life in the contemporary world, taking Heavy Metal as the specific object of study. We believe that Heavy Metal is an ethopoietical device which admits practices of freedom withstanding the reified moral habits since the beginning of the socialization. This is reflected, mainly, in the creation of new individual and communal ways to stylize the life. We also suggest an expansion of Foucault s concept of resistance, considering the idea of consumer society described by Zygmunt Bauman. Our hypothesis understands that the contact with the underground of Heavy Metal provides new ethical manners (Foucault), where the individual take the Heavy Metal as a way of life. At this point, the consumption becomes a key-word since the participation in the underground of Heavy Metal is a way of consumption out of the rules of marketing a practice of freedom, a way of particular existence , being different in both mode and duration / Nossa pesquisa visa compreender a configura??o da resist?ncia (Foucault) enquanto estiliza??o da vida na contemporaneidade, tendo o Heavy Metal enquanto objeto de estudo espec?fico. Acreditamos que o Metal se configure em um dispositivo ethopoi?tico possibilitador de pr?ticas de liberdade frente aos h?bitos morais reificados desde os prim?rdios da socializa??o do sujeito. Isso se reflete, principalmente, na cria??o de novas maneiras de estilizar a vida que s?o individuais e grupais, ao mesmo tempo. Sugerimos tamb?m uma amplia??o do pensamento sobre o tema da resist?ncia, em Foucault, tendo em vista a sociedade de consumo descrita por Zygmunt Bauman. Nossa hip?tese ? a de que o contato com o mundo underground do Heavy Metal ? o possibilitador de novas formas ?ticas (Foucault), onde h? a ades?o e o comprometimento do sujeito com o Heavy Metal enquanto um modo de vida. A partir da?, o consumo se torna uma palavra chave, na medida em que, participar do underground do Heavy Metal - enquanto uma pr?tica de liberdade, um modo de exist?ncia particular constitui tamb?m uma forma de consumo que foge ?s regras gerais do mercado, sendo um consumo diferenciado tanto na forma quanto na sua dura??o

Any metal in excess can be toxic; therefore, metal homeostasis is critical to bacterial survival. Bacteria have developed specialized metal import and export systems for this purpose. For broadly toxic metals such as copper, bacteria have evolved only export systems. The copper export system (cop operon) usually consists of the operon repressor, the copper chaperone, and the copper exporter. In Streptococcus pneumoniae, the causative agent of pneumonia, otitis media, sepsis, and meningitis, little is known about operon regulation. This is partly due to the S. pneumoniae repressor, CopY, and copper chaperone, CupA, sharing limited homology to proteins of putative related function and confirmed established systems. In this study, we examined CopY metal crosstalk, CopY interactions with CupA, and how CupA can control the oxidation state of copper. We found that CopY bound zinc and increased the DNA-binding affinity of CopY by roughly an order of magnitude over that of the apo form of CopY. Once copper displaced zinc in CopY, resulting in operon activation, CupA chelated copper from CopY. After copper was acquired from CopY or other sources, if needed, CupA facilitated the reduction of Cu2+ to Cu1+, which is the exported copper state. Taken together, these data show novel mechanisms for copper processing in S. pneumoniae. IMPORTANCE As mechanisms of copper toxicity are emerging, bacterial processing of intracellular copper, specifically inside Streptococcus pneumoniae, remains unclear. In this study, we investigated two proteins encoded by the copper export operon: the repressor, CopY, and the copper chaperone, CupA. Zinc suppressed transcription of the copper export operon by increasing the affinity of CopY for DNA. Furthermore, CupA was able to chelate copper from CopY not bound to DNA and reduce it from Cu2+ to Cu1+. This reduced copper state is essential for bacterial copper export via CopA. In view of the fact that innate immune cells use copper to kill pathogenic bacteria, understanding the mechanisms of copper export could expose new small-molecule therapeutic targets that could work synergistically with copper against pathogenic bacteria.

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Pseudomonas aeruginosa et Stenotrophomonas maltophilia, pathogènes opportunistes majeurs, pourraient acquérir leur résistance aux antibiotiques dans l’environnement, sous la pression exercée par les métaux lourds par co-sélection de résistance. Nous avons tout d’abord évalué la distribution et l’abondance de ces espèces dans un large panel de sols d’origine géographique différente (France et Afrique) et évalué l’influence d’activités anthropiques susceptibles d’exposer les sols en éléments métalliques sur cette distribution. Alors que la présence de P. aeruginosa est sporadique et plutôt liée à un apport exogène, S. maltophilia est présente dans tous les sols étudiés, suggérant son endémicité. L’évaluation des résistances des souches isolées de ces sols a également montré des différences entre les deux espèces. Les souches environnementales de P. aeruginosa sont pour la plupart caractérisées par un phénotype sauvage alors que celles de S. maltophilia présentent une grande diversité de phénotypes en fonction des sites, parfois similaires à ceux de souches cliniques. Cette diversité peut être attribuée à l’adaptation aux conditions environnementales très différentes rencontrées mais il est difficile d‘attribuer précisément aux métaux un rôle dans la co-sélection de ces résistances. L’étude menée sur la communauté bactérienne d’un sol contaminé a également permis de mettre en évidence une forte proportion de bactéries résistantes à différents antibiotiques représentée par des espèces qualifiées de pathogènes opportunistes ainsi que la présence du gène blaIMP, permettant la résistance à l’imipénème, utilisé en milieu clinique pour le traitement de clones multi-résistants. / Pseudomonas aeruginosa and Stenotrophomonas maltophilia, two major opportunistic pathogens, could acquire antibiotic resistance in the environment under heavy metal pressure that co-selects both resistances. We first investigated the distribution and abundance of these species in a wide range of soils of different geographical origin (France and Africa) and evaluated the influence of human activities that may expose soils to metallic elements on this distribution. While the presence of P. aeruginosa is rather sporadic and could be linked to exogenous intake, S. maltophilia is present in all studied soils, that suggests its endemicity. Evaluating resistance capacities of strains isolated from these soils also showed differences between the two species. Environmental strains of P. aeruginosa are mostly characterized by a wild type phenotype, whereas those of S. maltophilia present a wide diversity of phenotypes depending on the site, sometimes similar to those of clinical strains. This diversity could be attributed to a deep adaptation to the very different environmental conditions encountered in the original niche but it is difficult to attribute specifically to metals a role in coselection of resistance. The study conducted on the bacterial community present in a contaminated soil has also highlighted a high proportion of bacteria resistant to different antibiotics represented by species qualified as opportunistic pathogens and the presence of the gene blaIMP, enabling resistance to imipenem, used in the hospital to treat infections due to multidrug-resistant clones.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Sanjeevkumar Narayanan / Antimicrobials are included in finishing cattle diets for growth promotion, feed efficiency, and protection against liver abscesses. The inclusion of in-feed antimicrobials at or below therapeutic concentrations may provide a selective pressure for antimicrobial resistant microorganisms. Additionally, heavy metals such as copper and zinc may be included in cattle diets because of growth-promoting effects. Heavy metal resistance genes are on transferable plasmids that also contain antimicrobial resistance genes. The objectives of this research were to 1) determine the prevalence of food-borne pathogens, Salmonella and E. coli O157, in cattle fed diets with or without monensin and tylosin and 0 or 25% wet corn distiller's grains (WDGS), 2) determine the prevalence of food-borne pathogens in cattle fed elevated concentrations of copper and zinc 3) evaluate the effect of antimicrobials on antimicrobial susceptibility of food-borne pathogens and commensal fecal bacteria, and 4) determine a possible association between in-feed antimicrobials and the concentration of antimicrobial resistance genes in the feces of cattle. Inclusion of 25% WDGS was associated with a higher prevalence of E. coli O157 on one of two sample collection days; however, there was no association between the use of monensin and tylosin, or copper and zinc on the prevalence of food-borne pathogens. Including monensin and tylosin in cattle diets was associated with an increased resistance of enterococci to macrolides, but was not related to concentration of the common macrolide resistance gene, ermB. In cattle fed diets with copper and/or zinc, no differences were observed in antimicrobial susceptibility or the concentration of antimicrobial resistance genes. In conclusion, results indicate that including growth-promoting antimicrobials in cattle diets at below therapeutic concentrations only limitedly impacted antimicrobial susceptibility and concentration of fecal antimicrobial resistance genes; however, this research encompassed only a select number of microorganisms. The positive association between WDGS and E. coli O157 prevalence in cattle has important implications for food safety, and warrants further investigation.

The rising number of antibiotic resistant bacteria (ARB) may introduce to the post antibiotic era because they cause a loss of the therapeutic potential of antibiotics. For many years the important role of the natural environment as reservoir and dissemination pathway for ARB and responsible genes has been largely overlooked. However, especially aquatic ecosystems provide optimal conditions for the antibiotic resistance (AR) evolution: first, aquatic ecosystems are frequently affected by anthropogenic activities that cause multiple pollutions for example with heavy metals, that potentially cause co-selection of antibiotic- and heavy metal resistance. Second, aquatic ecosystems feature a dissemination pathway between human populations and natural environments via the urban water cycle. Water cycles between human associated environments (e.g. house holds and clinics) via waste water through waste water treatment plants into natural ecosystems (e.g. water bodies) and back as drinking water after purification. Third, ecosystem internal biotic interactions such as competition between bacteria and predation by the natural consumers seem to impact AR evolution sustainably. The present doctoral thesis focuses on the impact of abiotic and biotic factors on the proliferation of AR and responsible genes in natural aquatic environments, with special emphasis on (i) heavy metal driven co-selection of antibiotic and heavy metal resistance and (ii) on the impact of competition and predation on the evolution of AR. In order to quantify the risk of heavy metal driven co-selection for AR spread, I provide a first risk assessment based on literature values of environmental heavy metal loadings and related AR. Additionally, I developed a limit value named minimum co-selective concentration (MCC), which is the lowest concentration of a heavy metal that can potentially cause coselection in nature. It turned out that Cu, Zn, Ni, Hg, and Cd are suspected to be the main co-selecting heavy metals in the aquatic environment. I further investigated heavy metal driven co-selection of AR in a river ecosystem, the Western Bug River (Ukraine). I found indications for co-selection of resistance to five antibiotics (ciprofloxacin, gentamicin, amikacin, tobramycin, and cefepime) and two metals (Ni and Cd) caused by Ni- and Cd-levels. Both metals exceed their MCC for water samples and Cd additionally in sediments. As a second focal point the present work emphasis on ecological interactions effecting AR evolution. Currently three possible effects of ecological interactions on AR spread are discussed. First, environmental antibiotic levels are rather low, however they might favour ARB due to a competitive advantage. The reason is that even sublethal antibiotic levels reduce the growth of sensitive bacteria while resistant cells remain unaffected by the antibiotic action. Second, predation by protozoa is believed to impact conjugation between prey bacteria (and thus the transfer of DNA and potential resistance genes) by keeping bacteria in a growing stage that favours conjugation. Third, in order to escape predation by protozoa, bacteria evolved grazing defence mechanisms such as the formation of inedible biofilms, which can feedback on the evolution of AR. With an ordinary differential equation model, I tested the effect of low antibiotic levels and losses (e.g. due to predation) on the proliferation of ARB in a modelled planktonic system. In case that the model contains the mechanism that conjugation frequencies are highest during exponential growth, I found that (i) (grazing) losses enhance conjugation frequencies between bacteria and that (ii) medium levels of antibiotics and (grazing) losses favour resistant cells in the competition to sensitive bacteria. Biofilms are thought to be \'hot spots\' for conjugation but some plasmids have lower conjugation frequencies in biofilms compared to planktonic systems. As a first step, in order to discover predation effects on plasmid spread in plankton - biofilm systems I investigated grazing resistance of bacteria in grazing experiments. Both plankton and biofilm phenotypes were consumed, when exposed to their specialized grazer (either plankton-feeder or biofilmfeeder), whereas the other phenotype remained grazing-resistant and thus became the dominant prey type. Both predators together effectively control planktonic and biofilm prey. With regards to the spread of AR-genes via conjugation, I speculate that the feeding preference of the present predator can affect the invasion success of resistance plasmids in planktonic - biofilm systems. For dynamic systems, I assume that dynamics of predator and prey traits (plankton vs. biofilm-feeder and biofilm vs. planktonic prey) will lead to dynamics of conjugation frequencies in planktonic or biofilm bacteria. I assume that conjugation events are more frequent in the dominant prey type (plankton or biofilm). However, other factors such as pili-type of the plasmid (short and rigid pili, prefers conjugation in biofilms or long and flexible pili, prefers conjugation in plankton) might additionally influence plasmid invasion success in plankton - biofilm morphotypes.

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