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Seaman, Paul F. "Development and horizontal gene transfer of triclosan resistance in Staphylococcus aureus." Thesis, Cardiff University, 2007. http://orca.cf.ac.uk/54591/.
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Serfiotis-Mitsa, Dimitra. "Biophysical and structural studies of the antirestriction proteins ArdA and KlcA." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4358.
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Gene orf18, which is situated in the conjugative transposon Tn916 from the bacterial pathogen Enterococcus faecalis, encodes a putative ArdA (alleviation of restriction of DNA) protein. ArdA from Tn916 may be responsible for the apparent immunity of the transposon to DNA restriction and modification (R/M) systems and for ensuring that the transposon has a broad host range. The orf18 gene was engineered for overexpression in Escherichia coli and the recombinant ArdA protein was purified to homogeneity. Biophysical characterisation of ArdA demonstrated tight association between ArdA and the M.EcoKI. Also, ArdA was shown to efficiently inhibit restriction and modification by all four major classes of Type I R/M enzymes in vivo. Thus, ArdA can overcome the restriction barrier following conjugation and so helps to increase the spread of antibiotic resistance genes by horizontal gene transfer. The amino acid sequence of KlcA, from the incompatibility plasmid pBP136 from Bordetella pertussis, showed a high degree of similarity with the antirestriction protein ArdB from the IncN plasmid pKM101. In this study the solution structure of KlcA was solved with high-resolution NMR and its antirestriction function demonstrated. The structure of KlcA showed a rigid globular molecule with a novel fold. No antimodification function was observed for KlcA in vivo and the antirestriction function of KlcA has been successfully shown in vivo but not in vitro. Because no direct binding of KlcA to EcoKI was observed in vitro, the mechanism of the endonuclease blocking was assumed to be different from that of ArdA. Preliminary experiments including coimmunoprecipitation assays were conducted in order to elucidate the antirestriction mechanism of KlcA.
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Byrne-Bailey, Kathryne Greta. "Bacterial antibiotic resistance and horizontal gene transfer in slurries and slurry amended agricultural soils." Thesis, University of Warwick, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.439746.
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Carrera, Sandra Garcés. "Virulence of Mayetiola destructor (Say) field populations in the Great Plains and levanase/inulase-like genes in the Hessian fly genome." Diss., Kansas State University, 2013. http://hdl.handle.net/2097/16873.
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Doctor of PhilosophyDepartment of Entomology
Ming-Shun Chen
C. Michael Smith
The Hessian fly, Mayetiola destructor (Say), is a major pest of wheat, and is controlled mainly through deploying fly-resistant wheat cultivars. This study investigated five M. destructor populations collected from Texas, Louisiana, and Oklahoma, where infestation by Hessian fly has been high in recent years. Eight resistance genes including H12, H13, H17, H18, H22, H25, H26, and Hdic, were found to be highly effective against all tested M. destructor populations in this region, conferring resistance to 80% or more of plants containing one of these resistant genes. The frequency of biotypes virulent to resistant genes ranged from 0 to 45%. A logistic regression model was established to predict biotype frequencies based on the correlation between the percentages of susceptible plants obtained in a virulence test. In addition to the virulence test, the log-odds of virulent biotype frequencies were determined by a traditional approach to predict the logistic regression model. Characterization of a bacterial artificial chromosome (BAC) clone identified a gene encoding a protein with sequence similarity to bacterial levanases. Blast searching with the levanase-like protein identified 14 levanase/inulase-like genes or gene fragments. In this study, we determined the expression levels of these genes in different developmental stages and different tissues of 3-d old larvae of M. destructor. Sequence analysis revealed that six genes encode full length proteins, three were truncated at the 5’ end, and five truncated at the 3’ end. Sequences of putative proteins showed approximately 42% similarities to bacterial levanases or inulases, and 36% similarity to fungal levanases or inulases. No sequence similarities were found with any known animal or plant proteins. Comparative analysis of sequences among 14 levanase/inulase-like genes revealed that positions for intron/exon boundaries are conserved among different genes even though the length of each intron and exon varied among different genes. The expression patterns of the levanase/inulase-like genes were different among developmental stages and larval tissues of M. destructor. Interestingly, three genes presented alternative splicing bands in different developmental stages, and two genes exhibited splicing bands in different tissues of 3 d old M. destructor. This study would be useful for future studies of the characterization and function of levanase/inulase-like genes of these enzymes in plant-insect interactions.
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Tolba, Sahar T. M. "Distribution of streptomycin resistance and biosynthesis genes in streptomycetes recovered from different soil sites and the role of horizontal gene transfer in their dissemination." Thesis, University of Warwick, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408239.
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Woloszczuk, Kyra. "pCF10 MEDIATES INTERSPECIES DISSEMINATION OF ANTIBIOTIC RESISTANCE DETERMINANTS IN MIXED SPECIES BIOFILMS." Master's thesis, Temple University Libraries, 2016. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/390993.
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Biomedical SciencesM.S.
Enterococcus faecalis is a commensal bacterium, which upon acquisition of virulence factors on mobile genetic elements can cause sepsis, urinary tract infections and endocarditis. E. faecalis isolates can be multi-drug resistant and have been implicated in the dissemination of antibiotic resistance genes to other genera. Although the host range of pheromone inducible conjugative plasmids is restricted to Enterococci, they often carry transposons, which are capable of transposing into the chromosome of other genera. The plasmid pCF10 contains the antibiotic resistance gene tetM on a conjugative transposon Tn925. Tn925 is a Tn916-like plasmid and is capable of pCF10-independent conjugative transfer to multiple bacterial species at low levels. Biofilms are communities of bacteria growing within a matrix. In biofilms, bacteria are more difficult to kill because of their lower susceptibility to antibiotics. In hospital settings, biofilms can grow on medically implanted devices, catheters or even human tissue. In mixed species biofilms, antibiotic resistances are able to be transferred through horizontal gene transfer from E. faecalis to other bacterial species. In mixed species biofilms, it has been show that Tn925 can transpose into S. aureus at rates of 10-8 by Ella Massie Schuh. Using static mixed species biofilms, the transfer of tetM from E. faecalis to S. aureus was studied, hoping to better understand the underlying mechanisms. The goal of these studies was to determine if residence on pCF10 increased the transfer frequency of Tn925 in mixed species biofilms. Mixed species biofilms containing E. faecalis (pCF10) and S. aureus (pALC2073aPSM) were established and pCF10 conjugation was induced with pheromone cCF10. Transfer of Tn925::tetM to S. aureus was detected at a rate of approx. 10-8. No transfer was detected when Tn925 was present in the E. faecalis chromosome (lower limit of approx. 10-10). The increased transfer frequency was dependent on induction with cCF10. These results suggest that pCF10 can disseminate Tn925::tetM to S. aureus and the presence of the conjugative transposon on the plasmid increases its transfer rate. Previous observations in the laboratory show that in some circumstances, E. faecalis would be erythromycin resistant. To understand how this resistance was occurring, we investigated whether retrotransfer was occurring in mixed species biofilms. Retrotransfer is the transfer of genes from the recipient cell, back into the donor cell. For this experimental design, mixed species biofilms were forms erythromycin resistant E. faecalis transconjugants were selected for. While we successfully selected erythromycin resistant E. faecalis, upon gene sequencing it was shown that retrotransfer of the ermC gene was not occurring. Instead, these erythromycin resistant E. faecalis were spontaneous mutants. While transfer was not detected, this model leads to the hypothesis that induction of conjugation may increase the rates of spontaneous mutations of E. faecalis in biofilms.
Temple University--Theses
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Ray, Melissa D. "CHARACTERIZATION OF TRANSFER OF THE MOBILE GENOMIC ISLAND ENCODING METHICILLIN RESISTANCE AMONG STAPHYLOCOCCI." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3946.
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The gene encoding methicillin resistance in Staphylococcus aureus (MRSA) is carried in the chromosome on a large genomic island called SCCmec and is always inserted at the att site within orfX. SCCmec has been designated a mobile genetic element but a mechanism by which it moves among different strains and species of staphylococci has never been demonstrated. This work shows that bacteriophage 80α is capable of transducing SCCmec into a recipient cell, after which it can integrate into the bacterial chromosome via homologous recombination. More importantly, this work characterizes a conjugative mechanism of SCCmec transfer. Results demonstrate the capture of a 30.8 kb SCCmec element on a conjugative plasmid for the first time, its transfer into both S. aureus and S. epidermidis recipients, and its excision from the plasmid with insertion in the orfX att site in recipients. The element was integrated into the plasmid by recombination between IS elements invariably present on all SCCmec types and pGO1/pSK41-like conjugative plasmids. These data explain the movement of SCCmec from reservoirs in commensal coagulase-negative staphylococci into different Staphylococcus aureus lineages using a ubiquitous conjugative plasmid that can transfer among staphylococci of different species and, thus, describes a mechanism for the environmental dissemination of methicillin resistance in nature.
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Albaaj, Mohammed. "Diversity of β-Lactamase Genes in Gram-Negative Soil Bacteria from Northwest Ohio." Bowling Green State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1566553116919146.
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Faucher, Marion. "Le transfert horizontal de gènes chez les mycoplasmes : de l'acquisition de l'antibiorésistance à la dynamique des génomes." Thesis, Toulouse, INPT, 2018. http://www.theses.fr/2018INPT0117/document.
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Les mycoplasmes sont des bactéries atypiques, dépourvues de paroi et souvent considérées comme des cellules minimales du fait de la taille réduite de leur génome. De nombreuses espèces sont pathogènes et ont un impact économique important dans les filières d'élevage, notamment pour les ruminants. Les mycoplasmes n'échappent pas au phénomène mondial de la résistance aux antibiotiques. Contrairement à la plupart des autres bactéries, les mycoplasmes ne contiennent pas de plasmides conjugatifs souvent incriminés dans la dissémination horizontale de gènes de résistance, la base moléculaire principalement décrite étant la mutation chromosomique des gènes cibles. De façon générale, le transfert horizontal de gènes (HGT) chez les mycoplasmes a longtemps été sous-estimé. Récemment, deux mécanismes de HGT ont été décrits chez Mycoplasma agalactiae : le transfert d'élément conjugatif et intégratif (ICE), et le transfert non-conventionnel de régions chromosomiques par conjugaison appelé MCT (Mycoplasma Chromosomal Transfer). Nos travaux se sont attachés à explorer ce dernier mécanisme et à évaluer son impact sur l'acquisition de la résistance aux antibiotiques. Une analyse de génomique comparative a été conduite à partir du séquençage de nombreux mutants spontanément résistants et de transconjugants générés par des expériences de mating et sélectionnés pour leur résistance. Nos résultats montrent que le MCT conduit de façon distributive au transfert simultané de nombreux fragments. En une seule étape de conjugaison impliquant deux souches, ce phénomène génère une population variée de génomes hautement mosaïques. Il accélère la dissémination de l'antibiorésistance, permettant l'acquisition de plusieurs mutations distantes associées à la résistance en un seul événement. De par les multiples possibilités de réassemblage génomique qu'il produit, le MCT pourrait avoir des conséquences importantes sur d'autres processus adaptatifs comme la virulence ou la spécificité d'hôte. Enfin, les modalités distributives et l’ampleur du MCT expliquent l'origine des transferts de gènes précédemment détectés in silico entre de nombreux mycoplasmes. Ce phénomène pourrait donc avoir eu des répercussions importantes sur l'évolution de ces bactéries minimales et être un facteur clé de leur persistance et virulence actuellesMycoplasmas are wall-less bacteria often portrayed as minimal cells because of their reduced genomes. Several species are pathogenic and have a significant economic impact on livestock production, especially for ruminants. Mycoplasmas are also concerned with the worldwide increase in antibiotic resistance. In contrast to the majority of bacteria, these simple bacteria are deprived of conjugative plasmids that are frequently implicated in the horizontal dissemination of resistance genes: in mycoplasmas antibiotic resistance mainly relies on chromosomal mutations in target genes. In Mycoplasmas, the horizontal gene transfer (HGT) has long been underestimated. Recently, two conjugative mechanisms of HGT were described in Mycoplasma agalactiae: the transfer of an integrative and conjugative element (ICE), and the unconventional transfer of chromosomal DNA further designed by “MCT” for Mycoplasma Chromosomal Transfer. Our current study focused on exploring MCT mechanisms and on estimating its impact on antibiotic resistance dissemination. Comparative genomic analyses were performed from the sequencing (i) of spontaneous resistant mutants and (ii) of transconjugants selected by mating experiments and selected based on their resistance. Data revealed that MCT generated the simultaneous transfer of multiple, unrelated donor-fragments following a distributive process. In one conjugative step involving two strains, MCT generated a variety of highly mosaic genomes. This phenomenon was also shown to accelerate the dissemination of antibiotic resistance, by allowing in one step the acquisition of multiple and dispersed mutations associated with resistance. Due to the limitless ability of this phenomenon in reshuffling genomes, MCT may offer a valuable contribution in other adaptive processes such as virulence or host specificity. Finally, the distributive nature and the extent of MCT explain the origin of genes transfers detected in silico in several mycoplasma species. MCT is certainly a major player in the evolution of these minimal bacteria and a key factor of their persistence and virulence
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Balsalobre, Livia Carminato. "Resistência a tetraciclinas em isolados clínicos e ambientais de Escherichia coli, Klebsiella pneumoniae e Aeromonas spp.: identificação e mapeamento do ambiente genético de genes tet." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/6/6135/tde-11102014-091855/.
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Introdução. A resistência bacteriana a antibióticos é aceita como um dos maiores problemas de saúde pública. As tetraciclinas são antibióticos de amplo espectro, e após seu uso indiscriminado observou-se o surgimento de bactérias resistentes, levando médicos e veterinários a diminuírem seu uso. Objetivos. Verificar o perfil de sensibilidade a tetraciclinas em isolados clínicos e ambientais de Escherichia coli, Klebsiella pneumoniae e Aeromonas spp., bem como pesquisar os principais genes tet associados à resistência a esta classe de antibióticos e determinar a potencial forma de disseminação destes genes através da caracterização de seu ambiente genético. Material e Métodos. Os perfis de sensibilidade à tetraciclina (TET), doxiciclina (DOX), minociclina (MIN) e tigeciclina (TGC) de 572 isolados foram obtidos através das técnicas de Disco-Difusão e Concentração Inibitória Mínima. Os isolados não-sensíveis à tetraciclina foram submetidos a reações de PCR para pesquisa de grupos Inc, genes tet e para a caracterização de seu ambiente genético pela pesquisa das integrases de classes 1, 2, 3 e 4, e dos elementos genéticos móveis Tn1721, IS26, Tn10 e ISAS5. Perfis de similaridade genética dos isolados foram obtidos através das técnicas de ERIC-PCR e PFGE. Após análise destes resultados 33 cepas foram selecionadas para as técnicas de S1-PFGE e transformação. Resultados. A partir dos 572 isolados 18,5 por cento foram resistentes à TET, 13,5 por cento à DOX, 8 por cento à MIN e nenhum à TGC. Vinte e dois por cento dos isolados clínicos e 16,3 por cento ambientais foram resistentes à TET. Os genes codificadores de bomba de efluxo tet(A), tet(B), tet(C), tet(D) e tet(E), foram observados em 25,5 por cento , 33 por cento , 6,5 por cento , 18,9 por cento e 23,5 por cento dos isolados, respectivamente. Noventa e cinco por cento, 100 por cento , 100 por cento e 4,5 por cento das cepas carreando o gene tet(A), tet(B), tet(D) e tet(E), foram não-sensíveis à DOX, nesta ordem. Resistência à MIN foi observada em 4,2 por cento , 78,8 por cento e 100 por cento dos isolados carreando tet(A), tet(B) e tet(D), respectivamente. O gene tet(A) estava associado a Tn1721, tet(B) à Tn10 e tet(C) e tet(D) à IS26. Nenhuma das integrases pesquisadas estavam associadas aos genes tet detectados. Os grupos IncF, IncFIB e IncA/C foram observados em 54,8 por cento , 41,1 por cento e 28,7 por cento dos isolados, respectivamente. Uma cepa de Aeromonas spp. carreava um plasmídio do grupo IncP. Através dos perfis de similaridade genética foi observado que dentre os isolados hospitalares de K. pneumoniae houve a ocorrência de perfis genéticos idênticos, no entanto nos demais isolados do estudo os perfis genéticos observados eram distintos. Das 33 cepas selecionadas para os experimentos de linearização plasmidial e de transformação, 8 foram transformadas com sucesso, nas quais foi observada a presença dos genes tet em plasmídios. Conclusões. Uma baixa porcentagem de resistência à TET foi detectada. Verificou-se que a TGC foi a tetraciclina mais ativa, seguida da MIN. Os genes tet(A) e tet(B) foram os mais prevalentes. Todas as cepas carreando tet(B) e tet(D) foram não-sensíveis a DOX e MIN. Plasmídios dos grupos IncF, FIB e A/C foram os mais detectados neste estudo. Os resultados sugerem que os genes tet(A), (B), (C) e (D) são disseminados por meio de plasmídios e estão associados aos transposons Tn1721, IS10 e IS26. Estudos adicionais com isolados mais recentes e outros gêneros bacterianos são necessários, para contribuir com informações da resistência bacteriana a tetraciclinas.Introduction. The antibiotic resistance is accepted as one of the major problems for public health. Tetracyclines are broad spectrum antibiotics, and its indiscriminate use promoted the emergence of resistant bacteria, leading physicians and veterinarians to decrease its use. Objectives. Verify the susceptibility of clinical and environmental isolates of Escherichia coli, Klebsiella pneumoniae and Aeromonas spp. to tetracyclines, and also search for the main tet genes associated with resistance to these antibiotics and determine the potential mechanism of tet genes dissemination by characterizing their genetic context. Material and Methods. Disk-Diffusion and Minimum Inhibitory Concentration tests were carried out in 572 isolates using tetracycline (TET), doxycycline (DOX), minocycline (MIN) and tigecycline (TGC). PCR was carried out in TET non-susceptible isolates for the detection of Inc groups, tet genes and its genetic context determination through the search of classes 1, 2, 3, and 4 integrases, and Tn1721, Tn10, IS26 and ISAS5 mobile genetic elements. Genetic similarities patterns were determined by ERIC-PCR and PFGE techniques. After analyzing the results 33 strains were selected for the S1-PFGE and transformation experiments. Results. From 572 isolates, 18.5 per cent were TET-resistant, 13.5 per cent DOX-resistant, 8 per cent MIN-resistant and none resistant to TGC. Twenty-two per cent and 16.3 per cent of clinical and environmental isolates were TET-resistant, in that order. Genes tet(A), tet(B), tet(C), tet(D) and tet(E), coding for efflux pump mechanism, were found in 25.5 per cent , 33 per cent , 6.5 per cent , 18.9 per cent and 23.5 per cent of the isolates, respectively. Ninety-five per cent, 100 per cent , 100 per cent and 4.5 per cent of the isolates carrying tet(A), tet(B), tet(D) and tet(E) were non-susceptible to DOX, respectively. Resistance to MIN was observed in 4.2 per cent , 78.8 per cent and 100 per cent of isolates carrying tet(A), tet(B) and tet(D), in that order. The gene tet(A) was associated with Tn1721, tet(B) with Tn10, and tet(C) and (D) with IS26. None of the searched integrases were associated with the tet genes detected. Groups IncF, IncFIB and IncA/C were respectively observed in 54.8 per cent , 41.1 per cent and 28.7 per cent of the isolates. One Aeromonas spp. was carrying an IncP plasmid. The genetic similarities patterns demonstrated that there were identical genetic patterns among the hospital K. pneumoniae isolates, however all the remaining isolates possessed distinct genetic patterns. Of the 33 strains selected for plasmid linearization and transformation experiments, 8 were successfully transformed, in which the presence of tet genes in plasmids were observed. Conclusions. A low level of tetracycline resistance was detected. TGC was the most active tested antibiotic, followed by MIN. Genes tet(A) and tet(B) were the most prevalent among the isolates. All strains carrying tet(B) and tet(D) were non-susceptible to DOX and MIN. Groups IncF, IncFIB and IncA/C were the most detected in this study. The results suggest that tet(A), (B), (C) and (D) are disseminated by plasmids and are associated with Tn1721, Tn10 and IS26. Additional studies assembling recent isolates and other genera are necessary in order to contribute with information about the bacteria resistance to tetracyclines.
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Dropa, Milena. "Disseminação da resistência a antimicrobianos em cepas clínicas e ambientais de Enterobacteriaceae: identificação e mapeamento do ambiente genético de genes codificadores de ESBL." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/6/6135/tde-17062014-111143/.
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Introdução. A resistência bacteriana é facilitada pela pressão seletiva do uso de antimicrobianos na clínica e em outras atividades, como a agricultura e pecuária, além de poder ser disseminada para a natureza por meio do lançamento inadequado do esgoto ou pela aplicação do lodo de esgoto na agricultura. As -lactamases de espectro estendido (ESBL) são uma das formas mais prevalentes de resistência em Gram negativos no mundo, e seus genes codificadores são disseminados por meio de diversos elementos genéticos, principalmente transposons e integrons mobilizados para plasmídios. Objetivo. Identificar e caracterizar genes codificadores de ESBL, bem como suas prováveis formas de mobilização, em enterobactérias isoladas de fontes ambientais e clínicas. Material e Métodos. Quarenta e cinco cepas isoladas de um hospital público em 2004 e 2005, responsáveis por infecções hospitalares (14), infecções comunitárias (7) e colonizações (24), e 7 isoladas de estações de tratamento de esgoto (ETE) em 2009, em São Paulo, geneticamente distintas e produtoras de ESBL da família Enterobacteriaceae, foram estudadas. A técnica de PCR seguida de sequenciamento foi utilizada para a identificação dos genes blaESBL, triagem de elementos móveis e mapeamento do ambiente genético de blaESBL. A identificação dos grupos de incompatibilidade plasmidial (Inc) foi realizada pela técnica de PBRT, e a determinação dos tamanhos dos plasmídios pela técnica de S1-PFGE. Resultados. Os genes blaESBL identificados foram: amostras clínicas - blaTEM-15, blaTEM-197, blaSHV-5, blaSHV-12, blaSHV-27, blaSHV-28, blaSHV-45, blaSHV-55, blaSHV-110, blaCTX-M-2, blaCTX-M-59 e blaCTX-M-131; amostras ambientais blaSHV-28, blaCTX-M-15 e blaCTX-M-8. Os genes blaTEM- 15 e blaTEM-197 estavam associados aos elementos Tn2* e Tn3, respectivamente. Os genes blaSHV-5 e blaSHV-12 estavam associados à IS26, e não foi possível determinar o ambiente genético dos demais genes blaSHV. Os genes blaCTX-M-2, blaCTX-M-59 e blaCTXM- 131 estavam inseridos em integrons de classe 1 complexos, blaCTX-M-15 estava associado à ISEcp1 interrompida pela IS26, e blaCTX-M-8 estava associado à IS10, também interrompida pela IS26. Os principais grupos Inc detectados foram IncA/C (37 por cento ) e IncF (30,4 por cento ). Exceto por 7 cepas clínicas, todas apresentavam plasmídios de alto peso molecular, entre 48,5kb e 388kb. Conclusões. Este estudo detectou 15 genes blaESBL diferentes, dos quais dois são genes novos (blaTEM-197 e blaCTX-M-131) e três são inéditos no Brasil (blaTEM-15, blaSHV-55 e blaSHV-110). A maioria das cepas deste estudo possuía genes blaESBL associados a elementos mobilizáveis, bem como continham plasmídios de grupos Inc envolvidos na disseminação da resistência antimicrobiana. Além disso, carreavam plasmídios provavelmente conjugativos. Os resultados deste estudo mostram genes de resistência associados a elementos mobilizáveis em cepas contendo elementos transferíveis. As cepas foram isoladas tanto em uma instituição de saúde como nas ETEs da Grande São Paulo, mostrando o potencial de disseminação da resistência da clínica para o ambiente em nossa região.Introduction. Bacterial resistance is facilitated by selective pressure of antimicrobial use in clinical and other activities, as agriculture and livestock, and can be spread to nature through the inadequate discharge of sewage or by the use of sludge in agriculture. Extended-spectrum -lactamases (ESBL) are the most prevalente forms of resistance in Gram-negative bacteria in the world, and their encoding genes are disseminated through several genetic elements, especially transposons and integrons mobilized to plasmids. Objective. To identify and characterize ESBL-encoding genes, as well as their probable mobilization pathways, in enterobacteria isolated from clinical and environmental sources. Material and Methods. Forty-five strains isolated from a public hospital in 2004 and 2005, responsible for hospital infections (14), community-acquired infections (7) and colonizations (24), and 7 isolated from sewage treatment plants (ETE) in 2009, in São Paulo, genetically distinct and ESBL producers from Enterobacteriaceae family, were studied. PCR technique followed by sequencing was used for blaESBL genes identification, mobile elements screening and blaESBL genetic environment mapping. Plasmid incompatibility groups (Inc) were identified by PBRT technique, and plasmid sizes were determined by S1-PFGE technique. Results. The blaESBL genes identified were: clinical samples - blaTEM-15, blaTEM-197, blaSHV-5, blaSHV-12, blaSHV-27, blaSHV-28, blaSHV-45, blaSHV-55, blaSHV-110, blaCTX-M-2, blaCTX-M-59 and blaCTX-M-131; environmental samples blaSHV-28, blaCTX-M-15 and blaCTX-M-8. Genes blaTEM-15 and blaTEM-197 were associated to the elements Tn2* and Tn3, respectivelly. Genes blaSHV-5 and blaSHV-12 were associated to IS26, and it was not possible to detect the genetic environment of the other blaSHV genes. Genes blaCTX-M-2, blaCTX-M-59 and blaCTX-M-131 were inserted in complex class 1 integrons, blaCTX-M-15 was associated to ISEcp1 interrupted by IS26, and blaCTX-M-8 was associated to IS10, also interrupted by IS26. The most common Inc grups detected were IncA/C (37 per cent ) and IncF (30,4 per cent ). Except for 7 clinical strains, all isolates showed high molecular weight plasmids, rangng from 48,5kb to 388kb. Conclusions. This study detected 15 different blaESBL genes, from which 2 are new genes (blaTEM-197 e blaCTX-M-131) and 3 are still unpublished in Brazil (blaTEM-15, blaSHV-55 and blaSHV-110). Most of the strains from this study had blaESBL genes associated to mobile elements, as well as they had plasmids from Inc groups involved in the spread of antimicrobial resistance. Moreover, the strains probably carried conjugative plasmids. Results from the present work show resistance genes associated to mobile elements in strains carrying transferable elements. The strains were isolated either from a healthcare institution or from ETEs in São Paulo, which shows the spread potential of resistance from the clinic to the environment in our region.
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Oh, Seung Dae. "Metagenomic and metatranscriptomic investigation of microorganisms exposed to benzalkonium chloride disinfectants." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/52928.
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Benzalkonium chlorides (BACs) are widely used, broad-spectrum disinfectants and frequently detected in the environment, even at toxic levels for life. Since such disinfectants can induce broad resistance capabilities, BACs may fuel the emergence of antibiotic resistance in the environment. A substantial body of literature has reported that exposure to BACs causes antibiotic resistance; yet, other studies suggest that the resistance linkage is rare, unsystematic, and/or clinically insignificant. Accordingly, whether or not disinfectant exposure mediates antibiotic resistance and, if so, what molecular mechanisms underlie the resistance link remains to be clearly elucidated. Further, understanding how microbial communities degrade BACs is important not only for alleviating the possible occurrence of antibiotic resistance but also reducing the potential risks to environmental and public health.
An integrated strategy that combines metagenomics, metatranscriptomics, genetics, and traditional culture-dependent approaches was employed to provide novel insights into these issues. The integrative approach showed that a microbial community exposed to BACs can acquire antibiotic resistance through two mechanisms: i) horizontal transfer of previously uncharacterized efflux pump genes conferring resistance to BACs and antibiotics, which were encoded on a conjugative plasmid and co-selected together upon BACs and ii) selective enrichment of intrinsically multi-drug resistant organisms. Further, a microbial community adapts to BAC exposure via a variety of mechanisms, including selective enrichment of BAC-degrading species and amino acid substitutions and horizontal transfer of genes related to BAC resistance and degradation. The metatranscriptomic data suggests that the BAC-adapted microbial community metabolized BACs by cooperative interactions among its members. More specifically, Pseudomonas nitroreducens cleaved (i.e., dealkylated) BACs, metabolized the alkyl chain (the dealkylated product of BACs), and released benzyldimethylamine (the other product of BACs), which was further metabolized by other community members (e.g., Pseudomonas putida).
Collectively, this study demonstrates the role of BACs in promoting antibiotic resistance and advances current understanding of a microbial community degrading BACs. The results of this work have important implications for (appropriate) usage of disinfectants and for assessing, predicting, and optimizing biological engineering processes treating BAC-bearing waste streams.
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Massie-Schuh, Ella. "The Processing of Replication Initiation Protein PrgW in Enterococcus faecalis is Necessary for Activity and Stable Maintenance of pCF10." Master's thesis, Temple University Libraries, 2013. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/216595.
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Microbiology and ImmunologyM.S.
Enterococcus faecalis are Gram-positive bacteria that colonize the gastrointestinal tracts of mammals, birds and invertebrates and are also found in sewage, soil, food and water. In addition to being commensal organisms, Enterococci can also cause nosocomial infections in humans including urinary tract infections, septicemia and endocarditis. Hospital-acquired infections often present a challenge in treatment due to the emergence of multi-drug resistant strains. Enterococcal plasmids may act as extremely stable reservoirs for resistance genes and other virulence factors. Pheromone responsive plasmids such as pCF10 mediate efficient transfer of genetic material within the species E. faecalis but may also be capable of transferring resistance genes across species and genus boundaries. Polymicrobial environments often found in nosocomial infections may expose plasmid-harboring enterococci to pathogenic species, poising cells for this type of promiscuous horizontal gene transfer of resistance determinants. Previous studies showed that prgW, which encodes the pCF10 replication initiation protein PrgW, is the minimal origin of replication for this plasmid. The replicon, which is usually limited to Enterococcal spp., can replicate in Lactococcus lactis if it is engineered to produce pre-cCF10. Three conserved cysteines (C78/C275/C307) are important for plasmid stability and allow for replication of the pCF10 replicon in L. lactis in the absence of pre-cCF10. PrgW has a predicted molecular weight of 38,635. Four polyclonal antibodies targeting PrgW at the N-terminus (aa 1-20), C-terminus (aa 314-333) and two internal regions (aa 64-80 and aa 250-271) were used in current experiments and retrospective studies. When PrgW was overexpressed in E. faecalis, four different apparent approximate molecular weights were detected by Western blotting (p40*, p36*, p24* and p18*), suggestive of processing. In Enterococci where the replicon is active, p36* was consistently detected by all four antisera; when PrgW was overexpressed in Streptococcus mutans where the replicon is non-functional, p49* and p40* were detected but p36* was not observed. PrgW p24* was detected by a mixture of the internally targeting antibodies as well as the C-terminal targeting antibody, but not the N-terminal targeting antibody, suggesting that the N-terminal domain of PrgW has been cleaved off in p24*. The p24* form may play a role in pCF10 stability. Mutations to three cysteines in PrgW (C78/C275/C307), which reduce the stability of pCF10, result in the loss of p24*. Enterococcal conjugative plasmids have been previously implicated in the transfer of antibiotic resistance genes. The pCF10 plasmid contains the conjugative transposon Tn925, which possesses the tetM tetracycline resistance gene. Proximity of donor and recipient cells is a key part of pheromone-responsive conjugation. Aggregation substance allows for formation of clumps of E. faecalis in liquid mating experiments. E. faecalis forms biofilms; in contrast to filter mating experiments, polymicrobial biofilms provide an in vitro model of a natural scenario during which horizontal gene transfer may occur. Rates of cross-genus genetic transfer of tetM between E. faecalis OG1RF(pCF10) donor cells and Staphylococcus aureus recipient cells growing on glass coverslips as mixed-species biofilm populations were determined to be 10-8 after pheromone induction of pCF10 conjugation. This biofilm transfer model also holds potential to test the efficacy of synthetic peptides in the reduction or even prevention of pCF10 transfer, and the consequential dissemination of antibiotic resistance determinants throughout the genus Enterococcus and beyond.
Temple University--Theses
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Card, Galen Edward. "The Diversity Found Among Carbapenem-Resistant Bacteria." BYU ScholarsArchive, 2018. https://scholarsarchive.byu.edu/etd/6949.
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This work will look at two factors that add to the diversity of carbapenem resistant bacteria. First, it focuses on the diversity of carbapenemase resistance plasmids. 446 plasmids were characterized by size, gene content and replicon groups. We identified that on average, over 30% of the encoded proteins on each plasmid have an unknown function. Plasmid sizes ranged from 1.6kb to 500kb, with an average of around 100kb and median of 80kb. Additionally, six replicon groups account for 80% of all the carbapenemase resistance plasmids. We also highlight the lack of data available for carbapenemase carrying plasmids from bacterial genera other than Escherichia and Klebsiella, and plasmids that carry the New Delhi metallo-β- lactamase or the Verona-integron encoded metallo-β-lactamase. Second, we characterized the β-lactamase diversity of a single carbapenemase resistant Klebsiella pneumoniae. This isolate encodes six distinct β-lactamases, all of which are functional, and three of which are redundant. Additionally, we determined that the CTX-M-15 cephalosporinase imparts a greater fitness when grown in aztreonam (a monobactam) than ceftazidime (a cephalosporin). Finally, we show that individually, these β-lactamases do not account for the elevated levels of resistance seen in the parent strain, indicating that the passive resistance mechanisms (i.e. efflux pumps, altered membrane porins) may play a larger role than originally thought.
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Gluck, Thaler Emile. "Computational, Evolutionary and Functional Genetic Characterization of Fungal Gene Clusters Adapted to Degrade Plant Defense Chemicals." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1555406081422532.
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Bonot, Sébastien. "Persistance et dissémination du plasmide pB10, vecteur de gènes de résistance aux antibiotiques, dans des biomasses issues de stations d'épuration d'eaux usées urbaines." Thesis, Nancy 1, 2010. http://www.theses.fr/2010NAN10050/document.
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L’utilisation massive des antibiotiques, depuis les années 50, génère une libération importante de ces molécules dans l’environnement (excrétion via les urines et les fèces) que l’on peut retrouver à des concentrations allant de 1 à 100 ng/L dans les eaux usées urbaines. Parce qu’elle réunit microorganismes résistants et antibiotiques, la station d’épuration d’eaux usées urbaines pourrait être une zone propice au transfert des gènes de résistance. Cependant, avec sa position stratégique à l’interface entre les activités humaines et l’environnement, la station d’épuration pourrait constituer un « rempart » contribuant à limiter leur dissémination dans l’environnement.Les paramètres qui influencent ces transferts dans les stations d’épuration sont encore mal connus, en particulier du fait de limitations méthodologiques. Aussi l’objectif de notre travail était de déterminer les facteurs environnementaux influant sur la stabilité et le transfert d’un élément génétique mobile modèle, le plasmide pB10, dans des communautés bactériennes (biomasses de stations d’épuration et sédiments de rivière) maintenues en microcosmes. Jusqu’à présent, les transferts de gènes de résistance ont été principalement étudiés avec des méthodes reposant sur la culture de microorganismes sur milieux sélectifs, dont nous savons aujourd’hui qu’elles sous-estiment les phénomènes observés. Aussi, nous avons élaboré une approche basée sur la PCR quantitative pour détecter la dissémination d’un ADN mobile modèle amené via une bactérie hôte E. coli DH5α. Les couples amorces/sondes très spécifiques ont pu être élaborés en tirant profit de la structure mosaïque du génome bactérien. L’approche proposée repose sur des mesures comparées du nombre de plasmide pB10 et de son hôte bactérien DH5α au cours du temps, où une augmentation du rapport (pB10/DH5α) implique une dissémination du plasmide vers les bactéries indigènes. Outre l’intérêt du développement méthodologique proposé, cette méthode a permis d’évaluer l’incidence de quelques paramètres environnementaux sur la dissémination d’un ADN au sein de communautés microbiennes complexes. Deux groupes de facteurs ont pu être distingués selon qu’ils influencent la persistance du plasmide pB10 dans les communautés dans son hôte initial (oxygénation/brassage, ajout d’antibiotiques en concentrations sub-inhibitrices comme l’amoxicilline et le sulfaméthoxazole fréquemment retrouvés en station d’épuration) ou/et qu’ils favorisent sa dissémination dans les communautés bactériennes (biofilms, sédiments). Sans induire de transferts génétiques, les antibiotiques testés, même en concentrations sub-létales, pourraient participer à la dissémination de gènes de résistance en favorisant leur persistanceThe widespread use of antibiotics since the 50s, generates a significant release of these molecules in the environment (excretion via urine and feces) which can be found at concentrations ranging from 1-100 ng/L in wastewater. Due to the high microbial biomass and the abundance of nutrients, wastewater treatment plants (WWTP) represent a suitable habitat for horizontal gene transfer. Because they occupy a key position between human activities and the environment, WWTP may play a major role in limiting the dissemination of antibiotic resistance genes, therefore contributing to the preservation The parameters which influence these transfers in wastewater treatment plants are still poorly known, especially because of methodological limitations. Therefore the aim of our study was to identify environmental factors affecting the stability and transfer of a mobile genetic element model, the plasmid pB10 in bacterial communities (biomass from wastewater treatment plants and river sediments) maintained in microcosms. So far, the transfer of resistance genes have been studied mainly with methods based on the cultivation of microorganisms on selective media that we know now they underestimate the observed phenomena. Also, an approach based on quantitative PCR was developed for detecting the release of a mobile DNA template from the host bacterium E. coli DH5α. Couples of designed primers/probes were very specific and have been developed by taking advantage of the mosaic structure of the bacterial genome. The proposed approach is based on the over time measurements of the number of plasmids pB10 and its bacterial host DH5α, where an increased ratio (pB10/DH5α) implies a release of the plasmid to the indigenous bacteria. This method was used to assess the impact of some environmental parameters on the release of DNA in complex microbial communities. Two groups of factors could be distinguished according to whether they influence the persistence of plasmid pB10 in communities in microcosms (oxygenation / mixing, addition of antibiotics at sub-inhibitory concentrations as amoxicillin and sulfamethoxazole frequently found in treatment plant) and / or they favor his release in bacterial communities (biofilms, sediments). Without inducing genes transfers, the antibiotics tested, even at sub-lethal concentrations, could participate in the dissemination of resistance genes by facilitating their persistence
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Akhtar, Mastura. "Public health aspects of the house fly, Musca domestica L. (Diptera: muscidae) - Enterococcus spp. association." Diss., Manhattan, Kan. : Kansas State University, 2008. http://hdl.handle.net/2097/769.
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GUGLIELMETTI, ELENA. "Antibiotico resistenza in batteri lattici: basi molecolari e trasferibilità." Doctoral thesis, Università Cattolica del Sacro Cuore, 2009. http://hdl.handle.net/10280/404.
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La scoperta e il successivo uso di antibiotici hanno reso resistenti molte specie batteriche sia di origine animale sia umana. I geni di resistenza agli antibiotici possono essere trasferiti tramite la catena alimentare, a partire dagli animali e alimenti, fino al tratto gastrointestinale degli esseri umani.
Il presente studio descrive la proprietà coniugativa di alcuni nuovi plasmidi, in particolare di uno identificato in un ceppo di Lactococcus lactis spp. lactis, isolato dall'intestino di pesce, e di altri plasmidi individuati in ceppi di Lactobacillus brevis, Lb. plantarum e Lb. reuteri, isolati da salame. La trasferibilità dei plasmidi che portano i geni di resistenza per l’eritromicina o tetraciclina è stata valutata con metodi di elettroporazione e coniugazione in vitro. Nello specifico è riportato il trasferimento di tali plasmidi a specie batteriche patogene per l’uomo come Listeria monocytogenes e Staphylococcus spp. e a un agente responsabile di Lactococcosi nei pesci come Lc. garvieae. Dopo lo studio sulle proprietà coniugative si è proceduto alla caratterizzazione di questi elementi extracromosomici con esperimenti di comobilizzazione e stabilità. I dati ottenuti suggeriscono come i LAB possano essere un serbatoio di diffusione dei geni per l’antibiotico resistenza, con gravi rischi per l’allevamento di prodotti ittici e salute umana.The discovery and subsequent widespread use of antibiotics have rendered many bacterial species of human and animal origin resistant to some antibiotics. Antibiotic resistance gene may be transferred via food chain, from animals into fermented and other food or in the human gastrointestinal tract. The transferability of some plasmids that harbor the tetracycline or erythromycin resistance genes to animal and human pathogens was assessed using electrotrasformation and conjugation. The present study describes the proprieties of some new plasmids, originally isolated from fish intestinal Lactococcus lactis ssp. lactis and from fermented sausage Lactobacillus brevis, Lb. plantarum and Lb. reuteri. In particular, here I report the potentially of transferable antibiotic resistance determinants to human pathogenic bacterial like Listeria monocytogenes and Staphylococcus spp. and to an etiologic agent of Lactococcus infection like Lc. garvieae. The possibility of transferring natural Lactococcus and Lactobacillus plasmids into pathogenic bacterial strains involved the characterization of these elements, like comobilization and plasmid stability. These data suggest that lactic acid bacteria (LAB) might be reservoir organism for acquired resistance genes that can be spread both to fish and human pathogens, posing a risk to aquaculture and human health.
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Richard, Damien. "Microévolution et adaptation à une pression de sélection anthropique chez Xanthomonas citri pv. citri, une bactérie pathogène des agrumes : dynamique du compartiment plasmidique." Thesis, La Réunion, 2019. http://www.theses.fr/2019LARE0001.
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Le cuivre, souvent utilisé pour gérer les bactérioses en agriculture, est largement utilisé dans la lutte contre Xanthomonas citri pv. citri (Xcc), agent responsable du chancre asiatique des agrumes. La récente détection d’un phénotype résistant au cuivre (CuR) chez Xcc dans deux territoires ultramarins français a motivé une étude génomique qui a révélé, dans les génomes de souches CuR, la présence d’un plasmide conjugatif portant un transposon adaptatif de type Tn3. Sa conservation chez plusieurs espèces de Xanthomonas phytopathogènes suggère le rôle des transferts horizontaux (HGT) dans l’adaptation de Xcc. Nous avons donc analysé, dans l’océan Indien, les relations phylogénétiques de souches sensibles et CuR en prenant en compte à la fois les SNP et les variations de contenu en gènes. La datation de la phylogénie a permis de formuler des scenarii d’introduction de la bactérie dans la région. La phylogénie a montré une structure géographique forte à l’échelle de l’océan Indien, qui s’estompe à l’échelle de la Réunion et disparaît à l’échelle du verger. Au sein des vergers, l’admixture est un élément favorable aux HGT entre souches génétiquement différentes. Ils sont pourtant peu caractérisés chez les bactéries du genre Xanthomonas. Nous avons ainsi analysé la dispersion de l’ensemble des gènes plasmidiques connus de la famille des Xanthomonadaceae dans l’ensemble des génomes bactériens de NCBI, mettant en évidence à la fois la forte prévalence des gènes plasmidiques au sein des Xanthomonadaceae mais aussi la limite taxonomique forte à leur échange par conjugaison. L’importance du mosaïsme plasmidique, en partie lié aux éléments mobiles a aussi été illustrée. L’ensemble de nos résultats souligne l’importance des HGT dans l’évolution des bactéries du genre Xanthomonas, et la nécessité de caractériser finement le contenu et le fonctionnement du génome environnemental des Xanthomonadaceae pour appréhender au mieux l’adaptation de ces bactéries phytopathogènesCopper, frequently used in agriculture to control bacterial diseases, is commonly used against Xanthomonas citri pv. citri (Xcc), the bacterial agent of Asiatic citrus canker. The recent detection of a copper-resistant phenotype in two French overseas regions motivated a genomic study which revealed, in copper-resistant (CuR) strains, a conjugative plasmid encoding an adaptive transposon of the Tn3 family. Its conservation in several Xanthomonas species suggested the role of horizontal gene transfer (HGT) in Xcc adaptation. We therefore analyzed the evolutionary history of susceptible and CuR Xcc strains in the Indian Ocean using both SNP and gene content variations. The dating of the obtained phylogeny allowed us to hypothesize the history of Xcc introduction into the region. The phylogeny showed a strong geographic structure among islands of the Indian Ocean region, which faded at the Réunion scale and disappeared at the grove scale. Among the groves, admixture is a factor favoring HGT between genetically distinct strains. This form of evolution is however largely uncharacterized in the Xanthomonas genus. To fill this gap, we searched genetic homology between the whole known plasmid gene content of the Xanthomonadaceae family and the complete set of genomes hosted in NCBI databases. We highlighted both the ubiquity of plasmid genes in the Xanthomonadaceae family and the taxonomical barrier of their sharing by conjugation. The small fraction of genes that were exchanged through the complete sharing of plasmids also revealed the importance of plasmid mosaicism, partly due to mobile genetic elements. Taken together, our results highlight the importance of bacterial communities in the evolution of phytopathogenic bacteria of the Xanthomonas genus, and the need for a precise characterization of the content and the functioning of the Xanthomonadaceae environmental genome in order to fully apprehend the adaptation of these phytopathogenic bacteria
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Li, Xiaojing. "Prevalence and Characteristics of Antibiotic Resistant Bacteria in Selected Ready-to-Consume Deli and Restaurant Foods." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1259770996.
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Ong, Han Chuan. "Intracellular and horizontal transfer of mitochondrial genes in grass evolution pseudogenes, retroprocessing and chimeric genes /." [Bloomington, Ind.] : Indiana University, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3232559.
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Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2006."Title from dissertation home page (viewed July 11, 2007)." Source: Dissertation Abstracts International, Volume: 67-08, Section: B, page: 4258. Adviser: Jeffrey D. Palmer.
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Laskey, Alexander. "Horizontal Transfer of β-Lactam Resistance in the Mouse Gut Microbiota Under Antibiotic Treatment." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/41404.
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The rise of β-lactam-resistant bacteria from agricultural settings, including food-producing animals and their related food products has become a significant public health concern. Consumption of food contaminated by such bacteria may cause infection as well as the transmission of resistance genes. Here we used a mouse model to assess the impact of different antibiotic treatments on the composition of the gut microbiota and any impact on the transfer of β-lactam resistance genes between donor and recipient bacteria. Mice were inoculated with β-lactam resistant Escherichia coli and an antibiotic-susceptible Salmonella Heidelberg strain. The mice were treated with either streptomycin, ampicillin or both antibiotics. Mouse feces were collected at regular intervals and processed using selective culture techniques to capture potential transfer of resistance genes. Gene transfer was confirmed by whole genome sequencing. DNA extracted from the feces was used for monitoring changes in microbial profiles by 16S rDNA sequencing. In the absence of antibiotic treatment, the inoculated bacteria were only transiently detected and no transconjugants were recovered from the mouse feces. In comparison, antibiotic treatment changed microbial profiles in the mouse gut, enhanced colonization of the bacterial isolates, and facilitated the transfer of the resistance genes into both S. Heidelberg and commensal E. coli recipient strains. The results of this study indicated that the use of multiple antibiotics may enhance infection of opportunistic β-lactam resistant bacterial pathogens relative to single antibiotics and pose a greater risk in terms of antibiotic resistance gene transfer. Such process might occur in clinical settings where patients are under prolonged antibiotic treatments. Information gained through this study together with future work will inform the development of new policies guiding the prudent use of antibiotics.
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Barnard, Danielle. "Conjugative Transfer Pathways of High-Level Mupirocin Resistance and Conjugative Transfer Genes in Staphylococcus." Digital Commons @ East Tennessee State University, 2006. https://dc.etsu.edu/etd/2188.
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To combat widespread infections caused by Staphylococcus aureus, mupirocin was introduced at the Veterans Affairs Medical Center, Mountain Home, Tennessee. Soon after introduction, high-level mupirocin-resistance emerged. The rapid emergence was hypothesized to be due to conjugative transfer of the mupA resistance gene from S. epidermidis to S. aureus. Results have shown that transfer of high-level mupirocin-resistance from S. aureus donors commonly occurs. However, transfer from naturally-occurring S. epidermidis donors was not attainable. Staphylococcus epidermidis transconjugants, however, were capable of serving as donors. Further examination of non-transmissibility included PCR analysis of conjugative transfer genes (tra genes) in capable and non-capable donors. Results confirmed that capable donors possess full-length copies of selected transfer genes. Non-capable donors varied in the presence/absence of full-length copies of transfer genes, but none had all three genes. The genetic differences among non-capable donors suggest that non-transmissibility has arisen independently in different strains via gene deletions and recombinations.
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Ludidi, Ndomelele Ndiko. "Characterization of two Arabidopsis thaliana genes with roles in plant homeostasis." Thesis, University of the Western Cape, 2004. http://hdl.handle.net/11394/4602.
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Philosophiae Doctor - PhDPlants are continuously exposed to varying conditions in their environment, to which they have to adapt by manipulating various cellular processes. Environmental (abiotic) and pathogen (biotic) stress are challenges against which plants have to defend themselves. Many plant responses to stress stimuli are a result of cellular processes that can be divided into three sequential steps; namely signal perception, signal transduction m1d execution of a response. Stress signal perception is, in most of these cases, facilitated by cell surface or intracellular receptors that act to recognize molecules presented to the cell. In several cases, hormones are synthesized in response to stress signals and in turn these hormones are perceived by cellular receptors that trigger signal transduction cascades. Propagation of signal transduction cascades is a complex process that results from activation of various signaling molecules within the cell. Second messengers like calcium (Ca2+) and guanosine 3', 5'-cyclic monophosphate (cGMP) play a vital role in mediating many signal transduction processes. The result of these signal transduction cascades is, in most instances, expression of genes that contribute to the plant's ability to cope with the challenges presented to it. Plant natriuretic peptides (PNPs) are novel plant hormones that regulate water and salt homeostasis via cGMP-dependent signaling pathways that involve deployment of Ca2+. The aim of this study is to partially characterize a PNP and a guanylyl cyclase, both from Arabidopsis thaliana. Guanylyl cyclases synthesize cGMP from the hydrolysis of guanosine 5' -triphosphate (GTP) in the cell. The study also aims to investigate the effect of drought and salinity on cGMP levels in plants, using sorbitol to mimic the osmolarity/dehydration effect of drought and NaCl as a source of salinity stress and thus link NaCl and sorbitol responses to both AtPNP-A and cGMP up-regulation.
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Rangel, Luiz Thibério Lira Diniz. "O papel de transferência horizontal de genes na história evolutiva de duas classes de genes em bactérias." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/95/95131/tde-06122017-114559/.
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A Transferência Horizontal de Genes (THG) é um dos principais mecanismos de evolução bacterianos, impactando a evolução de praticamente todas famílias gênicas. Neste trabalho identificamos e avaliamos padrões de possíveis transferências horizontais de genes pertencentes a duas classes funcionais de dois níveis taxonômicos distintos. Caracterizamos a ocorrência e evolução de 45 genes importantes para a fixação de N2 em 479 genomas de Proteobacteria. Identificamos cinco potenciais aquisições de genes ligados a fixação de N2 por linhagens de Proteobacteria, as quais foram identificadas consistentemente em 36 dos genes analisados. Realizamos predições de transferências horizontais dos 45 entre todos os 479 genomas de Proteobacteria e identificamos possíveis enriquecimentos de THG, provavelmente ligados à sinais filogenéticos e ecológicos. Desenvolvemos um pipeline para identificação semi-automática de efetores do Sistema Secretor do Tipo III em Aeromonas, o qual reportou 21 famílias de potenciais efetores presentes em 105 genomas. Entre os 21 efetores identificados 17 foram descritos pela 1º vez em Aeromonas, corroborando a sensibilidade de nosso pipeline. Com o auxílio de nossos colaboradores foram realizados testes de citotoxidade para efetores identificados in silico, e apenas quatro não inibiram o crescimento de Saccharomyces cerevisiae. Por fim, desenvolvemos um método para agrupamento de famílias gênicas com histórias evolutivas similares que não requer a reconstrução de árvores filogenéticas, aumentando a eficiência computacional. Aplicamos o método desenvolvido para reconstrução da filogenia de Aeromonas, o qual mostrou-se compatível com dados presentes na literatura.Horizontal Gene Transfer (HGT) is one of main mechanisms of bacterial evolution, affecting virtually all gene families. In this document we identified and assessed putative horizontal transfers of genes from two functional classes from two distinct taxonomic levels. We characterized the distribution and evolution of 45 genes important to N2 fixation among 479 Proteobacteria genomes. We identified five potential distinct acquisitions of such genes by Proteobacteria lineages. The distinct origins are consistently identified in 36 out of the 45 assessed genes. We computed possible horizontal transfers of the 45 genes among the 479 Proteobacteria genomes, and we identified enrichments of HGT, likely related to phylogenetic and ecological signals. We developed a semi-automated pipeline to identify effectors of the Type III Secretion System within Aeromonas, which reported 21 putative effector families distributed among 105 genomes. Among the 21 likely effectors 17 have been described in Aeromonas for the first time, highlighting the sensibility of our pipeline. Our colaborators performed cytotoxicity tests for the 21 likely effector families identified by in silico analysis, and only four did not inhibited Saccharomyces cerevisiae growth. Lastly, we developed a method to cluster gene families according to shared evolutionary history, without the requirement of phylogenetic tree reconstruction, increasing computational efficiency. We applied this proposed method during Aeromonas phylogenetic reconstruction, and it showed up compatible with data available on the literature.
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Cury, Gisele Cristiane Gentile 1980. "Estudo molecular in vitro da transferência horizontal de genes entre as bactérias Haemophilus influenzae e Neisseria meningitidis." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314455.
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Orientador: Marcelo LancellottiTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Doutorado
Bioquimica
Doutora em Biologia Funcional e Molecular
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Kacar, Betül, Eva Garmendia, Nurcan Tuncbag, Dan I. Andersson, and Diarmaid Hughes. "Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs." AMER SOC MICROBIOLOGY, 2017. http://hdl.handle.net/10150/625744.
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Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreign tuf genes are transferable to the E. coli genome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coli genome. However, when the tufB gene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tuf with its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tuf partially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria. IMPORTANCE Horizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacing tuf with ancestral homologs and homologs from distantly related organisms, we investigated the constraints on HGT of a highly conserved gene with multiple interaction partners. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 bya. Only variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth, demonstrating the limited functional interchangeability of E. coli tuf with its homologs. Our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.
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To, Pui-chi Amanda, and 杜佩芝. "Molecular epidemiology of erythromycin resistance in Streptococcus bovis and lancefield group G beta-hemolytic streptococci andhorizontal gene transfer of antibiotic resistance genes." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B29387401.
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Olayat, Sophie. "Utilisation de vecteurs rétroviraux aviaires pour l'étude de l'implication de gènes dans la resistance des oiseaux aux maladies viro-induites." Tours, 1996. http://www.theses.fr/1996TOUR3309.
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Melville, Claire Margaret. "Molecular analysis of novel tetracycline resistance genes and the elements involved in their transfer between diverse gut bacteria." Thesis, University of Aberdeen, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367364.
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A previously undescribed tetracycline resistance gene, tet(W), was isolated by our group from the rumen anaerobe Butyrivibrio fibrisolvens. tet(W) is less than 65% homologous to tet(M) and in the B. fibrisolvens strain 1.230, is carried on a 50 kb mobile chromosomal element, TnB1230, which is unrelated to known conjugative transposons. This thesis has identified a gene >99% homologous to tet(W) from two groups of human faecal bacteria, a low % G+C Gram Positive human colonic bacteria, Strain K10 and Bifidobacterium longum. In each isolate tet(W) was identified by Southern blot on differently sized chromosomal fragments which are unrelated to TnB1230. Sequence analysis of 13 kb of TnB1230 from a cosmoid library clone identified several open reading frames with identity to transfer functions encoded by conjugative elements from important human pathogens. The DNA% G+C of tet(W) is considerably higher than the flanking orf's in TnB1230 and also the Butyrivibrio host itself, suggesting that it may have originated in a higher % G+C bacterium. The tetracycline resistance (TcR) was transmissible from Strain K10 to the rumen anaerobe B. fibrisolvens 2221R by filter mating, however tet(W) was not detected in the transconjugants by PCR with the tet(W) specific primers. Instead a second novel TcR gene designated tet(32) was shown to be responsible for transmissible resistance in K10. This novel ribosome protection gene has also been sequenced and characterised and is most related to tet(O), expresses a higher level of TcR than other ribosome protection proteins and is abundant in the ovine rumen, human and porcine gut.
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Hadziabdic, Sead [Verfasser]. "Transfer and structural alterations of resistance plasmids carrying carbapenemase-encoding genes in a broiler chicken infection model / Sead Hadziabdic." Berlin : Freie Universität Berlin, 2020. http://d-nb.info/1220691569/34.
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Espinosa, Zuleima Del Carmen Caballero. "Origem, evolução e relações filogenéticas de homólogos de prolina racemase em espécies de Trypanosoma." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-24022015-162835/.
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Os genes de Prolina racemase (PRACs) são enzimas intracelulares ou secretadas de T. cruzi (TcPRAC); essas enzimas estão envolvidas no metabolismo, diferenciação e virulência. Os genes PRAC foram identificados e caracterizados molecularmente em isolados representantes de toda a diversidade interespecífica de T. cruzi, T. cruzi marinkellei, T. dionisii, T. erneyi, T. rangeli, T. conorhini e T. lewisi. Além dessas espécies de tripanossomas restritas a mamíferos; homólogos de PRAC foram encontrados em tripanossomas de cobra (T. serpentis), crocodilo (T. grayi) e anuro (T. sp. 339). Análises filogenéticas e de sintenia entre homólogos de PRAC suportaram uma historia evolutiva totalmente congruente com as relações evolutivas previamente descritas dentro do gênero Trypanosoma.Proline racemaces (PRACs) are intracellular or secreted enzymes of Trypanosoma cruzi (TcPRAC), implicated in metabolism, differentiation, virulence and the induction of nonspecific polyclonal B-lymphocyte in the host. We identified and molecularly characterized PRAC genes from isolates representing all intra-specific diversity (TcI-TcVI and Tcbat), T. cruzi marinkellei, T. dionisii, T. erneyi, T. rangeli (isolates of lineages A-E), T. conorhini, and T. lewisi. In addition to these trypanosome species restricted to mammals, PRAC homologs were found in trypanosomes from snake (T. serpentis), crocodile (T. grayi) and anuran (T. sp 339). Phylogenetic and synteny analysis of PRAC homologs supported an evolutionary history totally congruent with the evolutionary relationships within the genus Trypanosoma.
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Arthur, Michel. "Origine et evolution des genes de resistance a l'erythromycine chez les enterobacteries." Paris 6, 1986. http://www.theses.fr/1986PA066336.
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Silva, Susan Ienne da. "Gene da putativa indolpiruvato descarboxilase de Phytomonas: caracterização, arranjo genômico, marcador molecular e origem filogenética." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-04062008-123820/.
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O gênero Phytomonas está associado a enfermidades devastadoras em plantações de interesse econômico, enquanto que outros vegetais parasitados não apresentam nenhum dano aparente. A seqüência-consenso de um agrupamento de ESTs de P. serpens determinado anteriormente apresentou alta similaridade com indolpiruvato descarboxilases (IPDCs) de fitobactérias e putativas piruvato/indolpiruvato descarboxilases de Leishmania spp. A enzima IPDC está na via de biossíntese do ácido 3-indolil acético, um dos hormônios vegetais mais importantes. Verificamos que o gene IPDC está presente em diversos isolados de Phytomonas, em múltiplas cópias (cerca de 104 cópias em P. serpens e 200 cópias em P. françai), contíguas e concentradas em uma banda cromossômica. Análises filogenéticas e amplificações por PCR com oligonucleotídeos degenerados apontam o clado Phytomonas-Leishmania como grupo-irmão de IPDCs de fitobactérias, sugerindo um processo de transferência horizontal anterior à separação dos tripanossomatídeos do clado que também inclui Leptomonas, Herpetomonas e Crithidia.The genus Phytomonas is associated to devastating diseases in commercially important crops, whereas in other plant species no apparent damage is observed. The consensus sequence of one group of ESTs of P. serpens previously determined showed high similarity with indolepyruvate decarboxylases (IPDCs) from phytobacteria and putative pyruvate/indolepyruvate decarboxylases of Leishmania spp. The enzyme ipdC participates in the biosynthetic pathway of the indole-3-acetic acid, one of the most important plant hormones. We found that the IPDC gene is present in several Phytomonas isolates, in multiple copies (about 104 copies in P. serpens and 200 copies in P. françai, in tandem and concentrated in one chromosomal band. The phylogenetic analyses and data of PCR amplifications with degenerated primers point the clade Phytomonas-Leishmania as a sister group of IPDCs of phytobacteria, suggesting a process of horizontal gene transfer prior to the separation of the trypanosomatid clade that also includes Leptomonas, Phytomonas, Herpetomonas, Leishmania and Crithidia.
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Santos, Viviane Piccin dos [UNESP]. "Filogenia molecular de cianobactérias baseada em sequências do 16S-23S-ITS rDNA e PC-IGS: investigação de transferência lateral do PC." Universidade Estadual Paulista (UNESP), 2011. http://hdl.handle.net/11449/87867.
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As cianobactérias apresentam uma ampla variabilidade fenotípica e ecológica. Porém, esta variabilidade, muitas vezes, não corresponde à sua diversidade genética. Assim, o uso de marcadores moleculares é fundamental para os estudos de filogenia neste grupo. Entretanto, a filogenia molecular enfrenta um desafio na seleção dos marcadores devido à ocorrência relativamente frequente da transferência de genes de forma lateral entre os procariotos. Em cianobactérias os marcadores dos espaçadores dos genes ribossomais (16S-23S-ITS rDNA) e do operon da ficocianina (PC-IGS) estão entre os mais utilizados nestes estudos. Contudo, alguns trabalhos sugerem que o PC-IGS possa ter sito transferido lateralmente em sua história evolutiva. A identificação de morfoespécies dos gêneros Microcystis e Geitlerinema é baseada em caracteres morfológicos que em geral não correspondem à sua variabilidade genética. Com o objetivo de investigar a transferência lateral do operon da ficocianina em Geitlerinema e Microcystis, foram obtidas e comparadas árvores filogenéticas de ambas espécies baseadas nos marcadores PC-IGS e 16S-23S-ITS rDNA. As topologias das árvores obtidas para ambos os marcadores foram muito semelhantes e indicaram que o PC-IGS é estável e indicado para os estudos de taxonomia e filogenia de linhagens de Geitlerinema e Microcystis. Assim, hipótese inicial de transferência lateral foi refutada. Algumas linhagens tiveram seu posicionamento divergente entre um marcador e outro, o que ressalta a importância do uso de mais de um marcador em estudos de filogenia. O marcador PC-IGS apresentou melhor desempenho que 16S-23S-ITS rDNA. As árvores filogenéticas de Geitlerinema baseadas em ambos os marcadores indicaram a ocorrência de espécies crípticas dentre as linhagens estudadas e corroboraram que G. amphibium e G. unigranulatum devem ser consideradas sinonímias...
Cyanobacteria show a wide phenotypic and ecological variability, but frequently this variability does not correspond to their genetic variation. Therefore, the use of molecular markes is critical for phylogenetic studies in this group. At the same time, the selection of molecular markers represents a challenge for the molecular phylogeny due to the horizontal gene transfer, witch is a relatively common process among the prokaryotes. In cyanobacteria, makers for the ribosomal genes spacer (16S-23S-ITS rDNA) and for the phycocyanin operon spacer (PC-IGS) are among of the most used for phylogeny. However, some studies suggest that the PC-IGS marker may have been horizontally transferred during its evolutionary history. The identification of the morphospecies from the genus Microcystis and Geitlerinema is based in their morphological characters, but they generally do not correspond to genetic variability. In order to investigate the possibility of horizontal transfer of the phycocyanin operon in Microcystis and Geitlerinema, phylogenetic trees based on the PC-IGS and 16S- 23S-ITS rDNA were generated and compared. The topologies obtained for both markers were very similar, indicating that the PC-IGS marker is stable and suitable for taxonomical and phylogenetic studies in Microcystis and Geitlerinema. Therefore, the initial hypothesis of horizontal transfer was rejected. Some strains were found to have divergent positions between the trees based on the two molecular markes, witch highlights the importance of using more than one marker in phylogenetic studies. The PC-IGS marker performed better than 16S-23SITS rDNA. The Geitlerinema phylogenetic trees based on both markers indicated the occurrence of cryptic species among the strains and corroborated that G. amphibium and G. unigranulatum should be treated as synonyms. The phylogenetic tree based on PC-IGS formed a monophyletic clade... (Complete abstract click electronic access below)
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Tinjuangjun, Porntip. "Transfer of pest and disease resistance genes to varieties of Thai rice (Oryza sativa L.) : enhancing transformation efficiency using a novel bombardment and selection system." Thesis, University of East Anglia, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323239.
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Pepper, Karen. "Etude de la localisation et de la dispersion des genes de resistance aux antibiotiques chez les streptocoques et les enterocoques." Paris 7, 1987. http://www.theses.fr/1987PA077144.
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Santana, Jhonne Pedro Pedott. "Localização de regiões potenciais para integração do kDNA de Trypanosoma cruzi no genoma humano." Universidade Federal de São Carlos, 2016. https://repositorio.ufscar.br/handle/ufscar/7537.
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Knowledge about horizontal gene transfer has been proposed even before the determination of the molecular structure of DNA. It has been experimentally shown that micro-homologies rich in adenine and cytosine mediates the integration of Trypanosoma cruzi’s kDNA minicircle, in the vertebrate genome. After human genome sequencing, the genome characterization of different organisms has been one of the main driving forces of science, providing a quantity of biological data for modern biomedical research, unprecedented in the history of science. However, even though traditional DNA mapping algorithms are highly accurate, they operate at a much lower rate than that needed for the next generation sequencers to accumulate new data. This great asymmetry between data generation and analysis capability requires the rapid evolution of mapping and reading algorithms so that this large volume of information can be worked through targeted searches. Thus, this work proposes an efficient, fast and easy way to search and locate multiple signatures of indicators that allow exogenous kDNA integration in the human genome, by creating a set of scripts for in silico analysis adapted to large files sequences. Three scripts based in R language were developed: to permute the elements (nucleic acids or amino acids codes); for search, grouping and plotting matches in genome; and for counting total matches and chromosomal window. All adenine and cytosine signatures were properly identified in the human genome, but no point more susceptible to T. cruzi kDNA integration was identified. With the obtained data, a genetic map was created, listing all matchings in each cytogenetic band, but it was not possible to identify which chromosome was more prone to mutations, since the bigger the chromosome is, the higher the quantity of matches are.
Com o sequenciamento do genoma humano e tantas outras espécies, abre-se agora uma nova janela de oportunidades analíticas. Podemos pensar em fazer buscas orientadas dentro dessa massa enorme de dados publicados em bancos de dados biológicos. Tendo isso em foco, buscamos estruturar uma forma automatizada de busca dentro do genoma humano, pela qual pudéssemos inferir sobre os sítios mais prováveis de integração de DNA exógeno. Para isso utilizamos como modelo os trabalhos que indicam que a doença de Chagas é produzida pela introgressão do kDNA de Trypanosoma cruzi no genoma hospedeiro, por meio de herança genética horizontal. Já foi demonstrado experimentalmente que micro-homologias ricas em adenina e citosina medeiam as integrações de minicírculos de kDNA do T. cruzi, no genoma de vertebrados. Deste modo, o presente trabalho propõe uma maneira eficiente, fácil e rápida para a busca e localização de múltiplas assinaturas dos sinalizadores que propiciam a introgressão do kDNA exógeno no genoma humano, através da criação de um conjunto de scripts para análises in silico, adaptados a grandes arquivos de sequências. Foram desenvolvidos três scripts, baseados na linguagem R: para permutação de elementos (ácidos nucleicos ou aminoácidos); para busca, agrupamento e plotagem das correspondências em genoma; e para contagem total de correspondências e contagem por janela cromossômica. Todas as assinaturas compostas por adenina e citosina (motivos CA’s) foram devidamente identificadas no genoma humano, porém não foi identificado nenhum ponto mais suscetível à integração do kDNA de T. cruzi. Com os dados obtidos, um mapa genético foi criado, listando as correspondências em cada banda citogenética, porém não foi possível identificar qual cromossomo possui maior propensão à mutações, já que quanto maior o cromossomo, maior é a quantidade de correspondências presentes.
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Takahashi, Elizabete Keiko. "Transferência do gene atacina A para plantas de maracujá amarelo (Passiflora edulis Sims f. flavicarpa Deg.) por biobalística." Universidade de São Paulo, 2002. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-03122002-083637/.
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O Brasil é o principal produtor de maracujá amarelo. Entretanto, a produtividade é baixa, cerca de 1 0.000 t por hectare. A produção de frutos varia com o cultivar, condições climáticas, manejo e outros fatores, principalmente doenças causadas por bactérias e vírus. Metodologias de transformação genética são alternativas modernas para obter plantas resistentes. A proteína derivada de inseto, atacina A atua como bactericida, e tem sido utilizada para conferir resistência a espécies vegetais. Os objetivos do presente estudo foram (i) obter a regeneração de brotos in vitro, (ii) testar a eficiência de agentes seletivos durante o processo organogênico, (iii) construir o cassete contendo o gene atacína A, e (iv) determinar as condições físicas e biológicas para a transformação genética de plantas de maracujá amarelo utilizando o método de biobaiística. Em relação a estudos in vitro, três recipientes de cultura foram avaliados como também diferentes concentrações de benzylaminopurina (BA) e água de coco que foram adicionadas ao meio basal. Phytagei e agar também foram testados como agentes solidificantes. As culturas foram avaliadas quanto à resposta morfogênica dos discos foliares. O gene atacina A foi sequenciado e clonado para receber o promotor CAMV 35S com um enhancer duplicado e o terminador 35S. Este vetor foi denominado pFFatacina. O cassete de expressão foi cionado nos vetores pcambia 1300 e pcambia 2300 que contêm os genes higromicina (hpt) e canamicina (nptll), respectivamente. Discos foliares, assim como segmentos entrenodais e hipocotiledonares que induzem calos, foram usados nos experimentos de biobaiística. A expressão do gene uida foi avaliada para testar os parâmetros de bombardeamento, pressão de gás Hélio (psi) e a distância da tela de retenção até o tecido alvo (cm). A resposta organogênica dos discos foliares não diferiu quando placas de petrí, tubos ou frascos foram usados, embora os tubos (2,4 x 8,5 cm, 30 mi) mostraram uma resposta ligeiramente melhor. O meio MS (Murashige & Skoog, Physiologia Plantarum, 15, 1962) solidificado com agar (0,6%) e suplementado com 0,5 mg/L BA e 5% de água de coco (w/v) provou ser eficiente na indução de organgênese. Brotos foram obtidos após 30 dias. Segmentos entrenodais e hipocotiledonares produziram 300 estruturas semelhantes a gemas por explante. Microscopia de varredura e análises histológicas demonstraram ser estruturas foliares, as quais evoluíram em brotos após 50 dias em Y2 MS. Higromicina a 5 mg/L provou ser um agente seletivo apropdado, inibindo organogênese em 60% dos explantes. Canamicina a 50 mg/L foi também efetiva. Calos morfogênicos de até 10 dias e discos foliares de 3 dias de cultivo mostraram elevados níveis de expressão transiente sob 80016,5 ou 100019,5 (psi de gás Héliolcm de distância de võo dos microprojéteis). Foram realizados experimentos de co-transformação com pB[426 (8,7 kb) que contém o gene npdi e pFFatacina (5,25 kb), como também utilizando-se um único vetor (pcatacina 1300), Freqüência de transformação estável de 0,85% foi obtida. A integração do transgene foi confirmada por PCR para o gene atacina A. Este é o primeiro trabalho que relata a transferência de um gene de interesse para plantas de maracujá amarelo por biobalística.Brazil is the leading producer of the yellow passion fruit. However, the productivity is fairly low, about 10,000 t per hectare. Fruit yields vary with cultivars, climatic conditions, management and other factors, namely bacterial and virus díseases. Genetic transformation methodologies are modern alternatives to obtain plant resistance. The insectderived protein, attacin A acts as bacterícide, and it has been used to confer resistance to plant species. The objectives of the present study were (i) to obtain in vitro shoot regeneration, (ii) to test the efficiency of certain selective agents during the organogenesis process, (iií) to construct the cassette containing the attacin A gene, and (iv) to determine the physical and biological conditions for genetic transformation of passion fruit plants by using the biolistic approach. Regarding the ín vítro studies, three culture recipients were evaluated as well as different concentrations of benzylaminopurine (BA) and coconut water that suppiemented the basal medium. Phytagei and agar were aiso tested as solidifying agents. Cultures were evaluated with respect to leaf dises morphogenic responses. The attacín A gene was sequenced, and cioned to receive the CAMV 35S promoter with a duplicated enhancer sequence, and the 35S terminator. This vector was denoted pFFatacina. The cassette was cioned in pcambia 1300 and pcambia 2300 vectors that contain the hygromícin (hpt) and kanamycin (nptil) genes, respectively. Leaf discs, as well as internodal segments and hypocotyl-derived sections chosen to índuce calii, were used in the biolistic experiments. The uida gene expression was evaluated for testing bombardment parameters, namely the helium pressure (psi) and the distance from the stopping screen to the target tissue (cm). The organogenic response of the leaf discs did not differ when petri dishes, tubes or culture vesseis were used although the tubes (2,4 x 8,5 cm, 30 ml capacity) showed to be slightly better. The agar (0.6%)-solidifíed MS (Murashige & Skoog, Physíología Plantarum, 15, 1962) medium suppiemented with 0.5 mg/L BA and 5% (wlv) coconut water proved to be efficient to induce organogenesis. Shoots were obtained after 30 days. Internada[ and hypocotyl-derived segments produced 300 bud-like structures per explant. Scanning microscopy and histologícal anaiyses provided evidences that they were leaf structures, which last 50 days in 1/2 MS to evolve into shoots. Hygromicin at 5 mg/L proved to be proper as selective agent, inhibiting organogenesis in 60% of the explants. Kanamycin at 50 mg/L was also effective. Morphogenic calli up to 10 days old and 3 d-leaf discs showed high levels of transient uida gene expression under 80016.5 or 1000/9.5 helium pressureldistance from the stopping screen to the target tissue. Cotransformation experiments with pBI426 (8.7 kb) that contain the nptil gene and pFFatacina (5.25 kb) were carried on as well singre vector transformation tríals (pcatacina 1300). Stable transformation frequency of 0.85% was obtained. Transgene integration was confirmei by PCR for the attacin A gene. This is the first report on agronomicaily useful-gene transfer to yeilow passion fruit plants by biolistics.
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Dangla, Pélissier Gauthier. "Identification et caractérisation des régulateurs du transfert horizontal de l’îlot de pathogénicité PAPI-1 chez Pseudomonas aeruginosa PA14." Thesis, Aix-Marseille, 2020. http://www.theses.fr/2020AIXM0136.
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Le transfert horizontal d’ADN est un des moteurs importants de l’évolution. Par ce biais, les bactéries acquièrent de nouvelles voies métaboliques, résistent à de plus en plus de stress environnementaux et s’adaptent aux stratégies thérapeutiques. Chez P. aeruginosa PA14, l’îlot de pathogénicité majeur acquis par transfert horizontal est nommé PAPI-1. Cet îlot augmente considérablement la virulence de P. aeruginosa PA14. Cet îlot, appartenant à la famille des ICE, est capable de se transmettre de façon autonome au sein de l’espèce P. aeruginosa via une machinerie de conjugaison (SST4-GI) impliquant au moins 55 gènes encodés en son sein. Au cours de ma thèse, j’ai montré que l’H-NS MvaT réprime la biosynthèse du pilus conjugatif et que l’anti-H-NS NdpA2 encodé sur PAPI-1 lève cette inhibition afin que le facteur de régulation transcriptionnelle (FRT) TprA (encodé sur PAPI-1) puisse activer la synthèse du SST4-GI. J’ai montré que TprA régule également un changement de phénotype chez P. aeruginosa PA14 lié à l’induction de la majorité des gènes de l’ICE PAPI-1. Ces travaux ont été également l’occasion de caractériser le domaine régissant la spécificité de fixation des FRT de la famille RHH. En effet, la région N-terminale de ces FRT interagit spécifiquement avec l’ADN et leur confère leur spécificité de fixation. Enfin, à travers un crible de mutagénèse aléatoire, j’ai identifié ce qui semble être les prémices d’une cascade de régulation du transfert horizontal chez P. aeruginosa PA14Horizontal transfer of DNA is one of the major motors of evolutive forces. It allows bacteria to obtain extra biological functions such as new metabolic pathways, resistance factors against environmental stresses and adaption to therapeutic strategies. The opportunistic human pathogen P. aeruginosa PA14 is a gram-negative bacterium with a large genome plasticity partially due to genomic island acquisition. The major genomic acquisition is PAPI-1 which considerably increases the virulence potential of the PA14 strain. Indeed, a strain of P. aeruginosa without PAPI-1 is less infectious against various organisms. This genomic island, belonging to ICE family, is self-transmissible among P. aeruginosa species via a conjugative machinery (T4SS-GI) requiring at least 55 genes encoded within it. During my PhD, I proved that MvaT repress the conjugative pilus biosynthesis and that the anti-H-NS NdpA2 can release this repression to allow the transcriptional regulation factor (TRF) TprA to induce T4SS-GI synthesis. I also proved that TprA controls phenotype changes in P. aeruginosa PA14 through the regulation of the majority of PAPI-1 encoded genes. Through this work I also characterised the domain leading to the specificity of action of RHH family TRF. As a matter of fact, the N-terminal region of these TRF interacts directly with DNA leading their binding specificity. At last, by a random mutagenesis screening, I identified what seems to be a regulation cascade of horizontal transference in P. aeruginosa PA14
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Hermetet, Francois. "La dualité de l'apoptose des cellules du cancer du col de l'utérus ou la face de Janus de l'apoptose : un objectif thérapeutique et une implication dans le transfert horizontal d'oncogènes viraux." Thesis, Besançon, 2015. http://www.theses.fr/2015BESA3009/document.
Full textAbstract:
À l'heure actuelle, une large proportion des recherches vouées à l'identification et au développement de nouvelles thérapies anticancéreuses est basée sur l'apoptose. Dans les dernières décennies, divers composés phytochimiques ont été caractérisés comme des agents pharmacologiques susceptibles d'éliminer les cellules cancéreuses via l'induction de l'apoptose. Parmi ceux-ci, l'isoliquiritigénine (ILG), un flavonoïde naturellement présent dans les racines de réglisse, se distingue des autres par ses nombreuses propriétés thérapeutiques incluant une activité antitumorale. Chez les mammifères, les cellules apoptotiques (CA) peuvent être complètement dégradées via leur capture par des cellules phagocytaires spécialisées ou servir de vecteurs d'ADN dans un processus appelé transfert horizontal de gènes (THG). Ainsi, il a été mis en évidence que des CA dérivées de cancer du col de l'utérus peuvent induire le transfert de séquences d'oncogènes de papillomavirus humains (HPV) à des fibroblastes primaires humains (HPFs) qui acquièrent des propriétés de cellules transformées. Cependant, les mécanismes cellulaires et moléculaires impliqués dans l'internalisation de CA par les fibroblastes, un modèle de phagocyte non-professionnel, n'ont pas encore été clairement identifiés et la caractérisation de ces événements qui précèdent le THG est essentielle à la compréhension de ce processus et de la transformation des cellules receveuses qui peut en découler.Dans ce contexte, les objectifs de cette thèse étaient, (i) d'analyser les effets anticancéreux de l'ILG sur des cellules dérivées de cancer du col de l'utérus, (ii) de caractériser les mécanismes cellulaires et moléculaires impliqués dans la capture des CA par les HPFs, (iii) d'étudier les événements cellulaires qui font suite à ce processus comme la maturation des phagosomes, le THG et l'acquisition de propriétés de transformation et enfin, (iv) de démontrer la preuve de la conservation de la capacité tumorigénique des CA in vivo.Un premier travail a permis de mettre en lumière les propriétés antitumorales de l'ILG via une multiplicité d'actions sur des modèles cellulaires de cancer du col de l'utérus in vitro, incluant des activités anti-proliférative, pro-apoptotique, anti-migratoire. Concernant la lignée cellulaire Ca Ski, p53 sauvage et représentative du carcinome du col utérin le plus fréquent, associé à une infection transformante par HPV16, l'apoptose induite par l'ILG semble dépendante de p53 et de la mitochondrie, mais aussi de la voie des récepteurs de mort, comme attesté par l'augmentation des niveaux de protéines p53 et p21, la dissipation du potentiel membranaire mitochondrial, la libération du cytochrome c et le clivage des caspases-9, -8 et -3. Ces effets pourraient être la conséquence de la diminution de l'expression de l'oncoprotéine virale E6 d'HPV16 induite par l'ILG entraînant la restauration de l'expression de p53. Nos travaux révèlent un fort potentiel de l'ILG contre différents types de cellules malignes et ouvrent le champ à de nouvelles modalités de traitement des cancers associés à HPVMost of the research strategies aiming at improving anticancer therapies currently target apoptosis. Over the last decades, several natural products derived from herbal medicine or food have been identified as pharmacological agents for cancer cell elimination through apoptosis induction. Among them, isoliquiritigenin (ILG) is a chalcone derivative isolated from liquorice and shallots which exhibits a wide variety of biological functions including antitumor properties.In mammals, apoptotic cells (AC) can either be eliminated after their capture by specialized phagocytes or act as vectors of DNA in a process named horizontal gene transfer (HGT). For instance, AC derived from cervical cancer cells can transfer human papillomavirus (HPV) oncogene sequences to human primary fibroblasts (HPFs) which subsequently acquire transformed cell properties. The molecular mechanisms underlying AC uptake by HPFs, a model of non-professional phagocytes, have not been clearly identified. Characterizing these upstream events appears critical to broaden our understanding of HGT and the ultimate transformation of recipient cells which may subsequently occur.The aims of this work were to (i) study the antitumor effects of ILG on cervical cancer cell lines,(ii) characterize the cellular and molecular mechanisms underlying AC uptake by HPF, (iii) study the cellular events which occur in HPFs following AC engulfment such as phagosome maturation, HGT and acquisition of transformed properties, and (iv) evaluate the tumorigenic properties of AC in vivo.In a first part of this PhD project, we found that ILG exhibits multiple antitumor actions on cervical cancer cells in vitro including anti-proliferative, pro-apoptotic and anti-migration properties. Further studies on apoptosis-related events were conducted in Ca Ski cells (p53wt, HPV16 DNA positive), which are representative of the most frequent cervical carcinoma. The treatment of Ca Ski cells with ILG is associated with increased levels of p53 and p21 proteins, loss of mitochondrial membrane potential, cytochrome c release and caspase-9, -8 and -3 cleavage. These features suggest that ILG-induced apoptosis is dependent on p53 and involve both mitochondrial and death receptor- mediated pathways. The effect of ILG in Ca Ski cells may be partly explained by the decrease of HPV16 E6 oncoprotein expression and the associated raise of p53 levels observed after cell treatment. Our work highlights the potential of ILG as an antitumor agent and provides the opportunity of new treatment. ln the second part of this work, we set up a method based on flow cytometry to quantitatively analyze AC uptake. This original method and microscopy analysis allowed us to show that HPFs act as non-professional phagocytes and are able to engulf subcellular fragments rather than dying whole cells, with lower efficiency and rapidity compared to professional phagocytes as macrophages. Uptake of AC by HPFs depends on time, temperature and the presence of bivalent ions. Morphological analysis and fonctional assays using endocytosis inhibitors revealed a mechanism related to phagocytosis and/or macropinocytosis. The recognition of phosphatidylserine exposed on the surface of AC by their receptor BAIl has emerged as a required event for AC uptake by HPFs
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Gaiffe, Emilie. "Les cellules apoptotiques vecteurs d'oncogènes viraux : Une voie alternative de la carcinogenèse associée aux HPV." Thesis, Besançon, 2011. http://www.theses.fr/2011BESA3006/document.
Full textAbstract:
Chez les mammifères, les cellules apoptotiques peuvent être complètement dégradées par des cellules phagocytaires spécialisées ou servir de vecteur d'ADN Le transfert d'oncogènes via les cellules apoptotiques aboutit à la transformation des cellules receveuses uniquement si celles-ci sont déficientes en p53. Sachant que Fniicogène E6 des papillomavirus humains (HPV) à haut risque induit la dégradation de p53, il est concevable que son transfert par la cellule apoptotique soit à l'origine d'un mécanisme alternatif de carcinogenèse associée aux HPV. Afin de confirmer cette hypothèse, l'apoptose de cellules dérivées de cancer du col de l'utérus, abriiam ou non des séquences d'HPV, a été induite. En collaboration avec l'équipe de Patrick Sandoz du laboratoire d'optique de FEMTO-ST, nous avons adapté leur inicr;système référencé en position à l'observation automatisée de Finternalisation des cellules apoptotiques. Nous avons aussi déterminé que les cellules apoptotiques son! phagocytées par les fîbroblastes quel que soit leur statut virologique. Seules les cellules apoptotiques dérivées de cellules abritant de l'ADN d'HPV transforment les cellules receveuses. L'expression de l'ADN viral, dont E6, dans les fîbroblastes transformés ainsi que la perte d'expression des protéines p53 et p21 suggère que les oncogènes d'HPV pourraient être à l'origine de la transformation. Les résultats présentés dans ces travaux mettent en évidence un nouveau mécanisme de carcinogenèse associée aux HPV via la phagocytose des cellules apoptotiques, potentiellement impliqué dans la transformation de cellules primaires et la progression des tumeurs associées aux HPVApoptotic cells in mammals may be completely degraded by specialized phagocytic cells or serve as a DNA vector. The oncogene transfer via apoptotic cells leads to the transformation of récipient cells but only when they are p53 deficient. As the E6 oncogene of high-risk human papillomavirus (HPV) leads to p53 dégradation, its transfer from apoptotic cells may be the cause of an alternative mechanism of HPV-associated carcinogenesis. To confirm this hypothesis, we induced apoptosis of cervical cancer cells that may harbor HPV sequences. In collaboration with Patrick Sandoz's team at the FEMTO-ST optical laboratory, we used their position-referenced microsystem for the automated observation of apoptotic cell internalization. We also found that apoptotic cells are phagocytized by fibroblasts regardless of their virological status. Only apoptotic cells from cells harboring HPV DNA transform recipient cells. Viral DNA expression, including E6, in transformed fibroblasts and the loss of p53 and p21 protein expression suggest that HPV oncogènes may cause transformation. These results highlight a new mechanism of HPV-associated carcinogenesis via apoptotic cell phagocytosis. This mechanism may be involved in thé transformation of primary cells and the progression of HPV-associated tumors
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Chen, Ke. "Sequencing and functional analysis of cT-DNAs in Nicotiana." Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAJ007/document.
Full textAbstract:
La bactérie Agrobacterium tumefaciens est bien connue pour son utilisation en génie génétique végétale où elle sert comme vecteur de gènes. A l’origine, cette bactérie ainsi que l’espèce voisine Agrobacterium rhizogenes sont des bactéries phytopathogènes qui induisent respectivement des tumeurs et des racines anormales sur des plantes sensibles telles que la vigne ou des arbres fruitiers. L’action pathogène résulte d’un transfert horizontal de gènes de la bactérie vers l’hôte végétal, à partir d’un plasmide, le pTi (plasmide inducteur de tumeurs) ou pRi (plasmide inducteur de racines). Mon travail de thèse concerne deux aspects particuliers de cette bactérie.1. Sa capacité à transformer durablement des espèces végétales dans la nature, donnant ainsi naissance à des plantes naturellement transformées, notamment dans le genre Nicotiana. Nous avons pu montrer par séquençage à haut débit du génome de N. tomentosiformis et par l’analyse d’autres séquences complètes de Nicotianées publiées récemment l’existence inattendue de 5 séquences venant d’Agrobacterium (cT-DNAs) avec une taille total de 65 kb, dont certaines portent des gènes intacts. Nous avons montré que deux de ces gènes (TB-mas2’ de N. tabacum et TE-6b de N. otophora) ont une activité biologique. Une étude comparative approfondie a permis de mieux comprendre l’évolution de ces cT-DNAs (Chen et al., 2014). Le gène mas2’ est bien connu, il code pour une enzyme qui catalyse la synthèse du désoxyfructosyl-glutamine (DFG) dans des tumeurs ou racines induites par Agrobacterium. Des résultats récents dans notre groupe portant sur le gène TB-mas2’ montrent que ce gène est exprimé de façon très active dans plusieurs cultivars de N. tabacum, et y donne naissance à l’apparition de quantités mesurables de DFG. Ce travail est présenté sous forme d’un manuscrit à soumettre.2. Une deuxième partie de la Thèse concerne les propriétés du gène T-6b, qui fait partie de l’ADN transféré par A. vitis souche Tm4 et provoque une croissance anormale caractérisée par l’apparition d’énations, sans que l’on connaisse son mode d’action. Le gène 6b fait partie de la famille des gènes plast (pour plasticité phénotypique), avec des effets différents et souvent remarquables sur la croissance des plantes. Le gène T-6b a été mis sous contrôle d’un promoteur inductible par le dexaméthasone, et des plantes de tabac transformées par cette construction ont été étudiées en détail, à différents moments après son induction. Un grand nombre de changements a été décrit incluant des analyses anatomiques montrant des modifications encore jamais décrites chez les plantes, comme par exemple l’apparition de méristèmes foliaires ectopiques à la base de trichomes, ou l’apparition de systèmes vasculaires ectopiques parallèles au système vasculaire normal avec un développement régulier menant à des structures complexes ordonnées (Chen and Otten, 2015). Le gène TE-6b de N. otophora a été mis sous contrôle d’un promoteur fort constitutif et introduit dans des plantes de tabac, où il provoque des changements de croissance différents de ce qui a été observé pour le gène T-6b. Ces derniers résultats préliminaires sont présentés en complément des observations sur le gène T-6b. Ils indiquent que le transfert horizontal du gène TE-6b vers l’ancêtre de N. otophora aurait pu contribuer à une modification de la croissance et ainsi à la création d’une nouvelle espèceThe bacterium Agrobacterium tumefaciens is well-known for its utilisation in plant genetic engineering where it serves as a gene vector. This bacterium and the related species Agrobacterium rhizogenes are phytopathogens that induce tumors and hairy roots respectively on susceptible plants like grapevine or fruit trees. Their phytopathogenicity is due to horizontal transfer of bacterial genes to the plant host, from a plasmid called the Ti (tumor-inducing) or Ri (root-inducing) plasmid. The subject of my Thesis concerns two particular aspects of this bacterium.1. Their capacity to stably transform several plant species in nature, thereby yielding naturally transformed plants, especially in the genus Nicotiana. We have shown by deep sequencing of the Nicotiana tomentosiformis genome and by analysis of other recently published Nicotiana sequences the presence of five different Agrobacterium-derived sequences (cT-DNAs), totalling 65 kb, some of which carry intact genes. We have shown that two of them (TB-mas2’ from N. tabacum and TE-6b from N. otophora) have biological activity. A detailed comparative study has allowed us to better understand the evolution of these cT-DNAs (Chen et al., 2014). The mas2’ gene is well-known, it codes for the synthesis of desoxyfructosyl-glutamine (DFG) in tumors or roots induced by Agrobacterium. Recent work in our group has shown that the TB-mas2’ gene is highly expressed in some N. tabacum cultivars and leads to the accumulation of detectable amounts of DFG. This work is presented as a manuscript to be submitted.2. A second part of the Thesis describes new properties of the T-6b gene, which is part of the DNA transferred by A. vitis strain Tm4 and leads to abnormal growth caracterized by the appearance of enations, so far the mode of action of this gene is unknown. The 6b gene is part of the so called plast family (for phenotypic plasticity), with different and often remarkable growth effects on plants. The T-6b gene wasearlier placed under control of a dexamethasone-inducible promoter, and tobacco plants transformed with this construct have now been studied in detail, at different times after the start of induction. A large number of changes was analyzed, both at the morphological and anatomical level, these include various unprecedented morphological changes, like for example the appearance of shoot primordia at the base of trichomes, or the appearance of ectopic vascular strands parallel to the normal strands with a regular development leading to complex but predictable structures (Chen and Otten, 2015). The TE-6b gene from N. otophora was placed under strong and constitutive promoter control and introduced into tobacco, where it was found to cause new types of morphological change, different from those observed for T-6b. The latter results are preliminary and will be presented as a complement to the work on T-6b. They indicate that the introduction of the TE-6b gene in the N. otophora ancestor could have caused a change in growth pattern, and might have favored the appearance of a new species
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BALAZS, ERVIN. "Construction d'un vecteur hybride pour le transfert de genes chez les plantes superieures : utilisation de sequences virales appartenant a l'adn du virus de la mosaique du chou-fleur." Université Louis Pasteur (Strasbourg) (1971-2008), 1986. http://www.theses.fr/1986STR13123.
Full textAbstract:
Confirmation de la carte genetique du camv et identification de 4 produits de transcription. Caracterisation des regions promotrices de type "tata". Construction d'un vecteur hybride par association de sequences virales strategiques a un plasmide bacterien et au gene de resistance a la kanamycine
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Centis, Sonia. "Transfert de fragments de genes d'un caulimovirus dans le colza (brassica napus l. ) : obtention de plantes transgeniques." Toulouse 3, 1988. http://www.theses.fr/1988TOU30198.
Full textAbstract:
Le virus de la mosaique du chou-fleur, representant modele des caulimovirus, comprend une molecule circulaire d'adn double brin d'organisation genetique connue. Des fragments de l'adn viral ont ete introduits dans le genome du colza, grace au vecteur naturel de transformation agrobacterium rhizogenes, afin de faire produire par la cellule vegetale un arn antisens permettant d'interferer avec l'expression d'un gene essentiel dans le cycle vital du virus. Des plantes transgeniques ont ete regenerees a partir des racines transformees
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(6616589), Mian Wang. "DEVELOPMENT AND REMOVAL OF ANTIBIOTIC RESISTANCE GENES." Thesis, 2019.
Find full textAbstract:
Antibiotics have been widely used to treat bacterial diseases since the 1940s. However, the benefits offered by antibiotics have gradually faded due to the increased occurrence and frequency of antibiotic resistance. The widespread use of antibiotics has driven selection for resistance in bacteria and is becoming a global problem for human health and the environment. Antibiotic resistance is exacerbated by the ability of bacteria to share their antibiotic resistance genes (ARGs) with other bacteria via horizontal gene transfer (HGT). Many existing studies on HGT of ARGs focused on antibiotic concentrations at or above the minimal inhibitory concentration (MIC), which is the lowest concentration of an antibiotic that prevents visible growth of a bacteria culture. However, knowledge on the development of antibiotic resistance under different stressors at sub-MIC levels is still limited. In addition, carbon nanotubes (CNTs) have been widely studied in environmental, agricultural and biomedical areas due to their unique physical and chemical characteristics, but limited studies have been done to evaluate the effects of CNTs on the spread of ARGs. Electrochemical filtration has been shown to be a cost-effective technique to remove recalcitrant compounds and reduce antibiotic resistance, but limited studies have been done to evaluate the effectiveness of removal of ARGs with electrochemical filtration. Therefore, there is a critical need to evaluate the effects of trace levels of antibiotics and CNTs on the development of antibiotic resistance and electrochemical removal of ARGs.
The specific research objectives of this study were to evaluate: (1) selective pressure of sub-inhibitory concentrations of antibiotics on the development of antibiotic resistance and HGT, (2) development of antibiotic resistance and HGT under exposure to CNTs and antibiotics, and (3) effectiveness of using an electrochemical MWCNT filter to remove ARGs.
To evaluate the development of antibiotic resistance exposed to sub-MIC of erythromycin, HGT between environmental donor (E. coli) and pathogenic bacterial recipient (B. cereus) was quantified. The results indicated that extremely low concentration (0.4 ng/L to 4 µg/L) of erythromycin promoted HGT of erm80 gene, which is an erythromycin resistance gene. In addition to traditional culture-based method and quantitative real-time PCR (qPCR), a fluorescence in situ hybridization (FISH) approach was used to detect the occurrence and development of ARGs even the bacteria were in the viable but nonculturable (VBNC) state after treatment of sub-lethal level of erythromycin. Multi-walled carbon nanotubes (MWCNT) was selected as a representative stressor to evaluate the effects on HGT. The results showed that MWCNT enhanced HGT above 1 × MIC, which is the lethal level of erythromycin to recipients, and transfer frequencies of erm80 genes increased up to 101-fold under exposure to 1 × MIC erythromycin and MWCNT as compared to no MWCNT control. However, transfer efficiency of erm80 gene under exposure to sub-MIC of erythromycin was inhibited by MWCNTs. Moreover, transfer of antibiotic resistance plasmids was affected by antibiotics and MWCNTs. Although the concentration of individual stressor was not enough to confer antibiotic selection, effects of both antibiotics above 1 × MIC and MWCNTs could add up and select for antibiotic resistance. The results suggested that CNTs might create additional selective pressure for the spread of ARGs and their effects on HGT should be further investigated. Finally, an electrochemical MWCNT filtration was evaluated to remove genomic DNA and ARGs under the effects of operating conditions, such as pH, phosphate, and NOM. The results showed that the electrochemical MWCNT filtration reactor achieved 79% removal efficiency for genomic DNA and 91% removal efficiency for erm80 genes. The study suggested that electrochemical MWCNT filtration could be a promising technology for the removal of DNA and ARGs.
Overall, the results improved our understanding of the development of antibiotic resistance and ARGs under various selective pressures. Trace levels of antibiotics promoted the development and spread of ARGs. Conjugative transfer of resistance genes exposed to sub-MIC levels of erythromycin and MWCNTs also contributed to the spread and propagation of ARGs. As antibiotic concentrations detected in natural environment are often in trace levels, the results of this study may improve the understanding of health risks of trace levels of antibiotics and help develop effective mitigation strategies to control the spread of antibiotic resistance. Effective removal of ARGs with electrochemical MWCNT filtration may help the development of cost-effective treatment systems to remove ARGs to protect human health and the environment.
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Domingues, Sara Margarida dos Santos. "Unravelling novel mechanisms of horizontal transfer of class I integrons in bacteria. Implications for the dissemination of antibiotic resistance." Doctoral thesis, 2016. http://hdl.handle.net/10316/31894.
Full textAbstract:
Horizontal gene transfer (HGT) contributes to the genetic diversity and
evolutionary trajectories of bacterial populations. In particular, HGT of mobile
genetic elements (MGEs) is a major contributor to the emergence,
recombination and dissemination of multidrug resistance among bacterial
pathogens (Nakamura et al., 2004; Thomas and Nielsen, 2005). MGEs are
often shared between bacterial species and separately evolving lineages due to
their capacity to physically move within genomes, and also between host
genomes and cytoplasms. A variety of MGEs have been described so far
(Stokes and Gillings, 2011).
Integrons are genetic elements that contain a site-specific recombination
system able to capture, express and exchange specific DNA elements, called
gene cassettes (Hall and Collis, 1995). The complete integron is not considered
to be a mobile element as such as it lacks functions for self-mobility. In contrast,
the gene cassettes present in integrons are considered mobile within genomes,
although the frequencies and modes of exchange of cassettes are rarely
observed experimentally (Guerin et al., 2009; Baharoglu et al., 2010).
Nevertheless, sequence similar integrons appear to be widespread among
bacterial species and genetic backgrounds, suggesting that they are frequently
exposed to mechanisms that allow them to disseminate horizontally through
bacterial populations (Stokes and Hall, 1989).Foundation for Science and Technology, Portugal by the grant SFRH/BD/49061/2008
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Chidiamassamba, Sekerani Benedito. "IncP-1 plasmids in antibiotic resistant Enterobacteriaceae." Master's thesis, 2020. http://hdl.handle.net/10773/30447.
Full textAbstract:
Antibiotic resistance is increasing worldwide closely linked with antibiotic
misuse and overuse. Antibiotic gene resistance is commonly disseminated
through MGEs (mobile genetic elements), especially plasmids. IncP-1
plasmids are BHR presumably found in several bacterial families and have
been associated with antibiotic resistance and tolerance to metals.
The identification of plasmid groups has been frequently done using the
PBRT (PCR-based replicon typing) approach which assigns plasmids to Inc
groups, including the IncP-1 plasmid group. The efforts to further
characterize these structures, especially when associated with antibiotic
resistant bacteria, has been lacking, and consequently, information is
scarce and dispersed. Therefore, a systematic analysis was carried out to
understand the occurrence, distribution, and genetic traits of IncP-1
plasmids associated with antibiotic resistant bacteria. To do so, a
bibliographic search strategy was followed, where the Scopus platform was
used to look for studies that used the PBRT method developed by Carattoli
et al. (2005) for plasmid identification and that actually detected IncP-1
plasmids structures.
Article collection for a period of 5 years resulted in 96 eligible articles,
which were used to retrieve relevant information about IncP-1 plasmids
occurrence and features. The articles combined reported the identification
of 629 IncP-1 replicons. Altogether, the bacterial hosts of IncP-1 plasmids
were found in 32 countries and were collected from a variety of
environmental sources. Bacterial hosts belonged to 28 species distributed
in 10 genera, of Enterobacteriaceae and Pseudomonadaceae families and
were resistant to 10 different antibiotic classes, harboring 32 different
resistance genes. IncP-1 plasmid-positive bacteria usually harbored at
least 2 different Inc groups. Of all the IncP-1 plasmids identified, 229
(~36%) were further described, their sizes ranging from 35 to 320 kb and
have been associated with medically important resistance genes and
additional genetic elements that could potentiate gene dissemination.
Furthermore, we studied the molecular diversity of previously reported
IncP-1 plasmids occurring in 9 Escherichia coli isolates from an UWTP in
Portugal. This was accomplished via PCR amplification of the 281 bp trfA
gene fragment sequences and transfer to new well-known bacterial hosts
for further characterizations. Amplicon sequencing showed 100% identity in
all trfA fragments suggesting genetic structure conservation association to
similar bacteria and environments. Similarity searching of the trfA fragment
sequence was used to select closely related fully sequenced IncP-1β1
plasmids for comparisons. As a result, 63 closely related replicons were
selected for comparative analysis and found to be usually large genetic
structures, isolated mainly from wastewater and soil, harboring genetic
determinants associated with resistance to aminoglycosides,
sulphonamides, and β-lactams, and with tolerance to Hg. Attempts to
transfer the 9 IncP-1 plasmids to a recipient strain were unsuccessful
probably due to the chosen selective markers. The high prevalence of mer
operon genes in these IncP-1β1 plasmids, suggesting mercury tolerance,
could be used in a future work as a selective marker to transfer these 9
IncP-1 plasmids another bacterial host allowing for proper genomic
characterization of these promiscuous structures.A resistência aos antibióticos está a aumentar em todo o mundo, intimamente associada ao uso incorreto e excessivo de antibióticos. Os genes de resistência a antibióticos são comummente disseminados por meio de EGMs (elementos genéticos móveis), especialmente plasmídeos. Os plasmídeos IncP-1 têm amplo espetro de hospedeiros, são presumivelmente encontrados em várias famílias de bactérias, e têm sido associados à resistência a antibióticos e tolerância a metais. A identificação de grupos de plasmídeos tem sido frequentemente realizada utilizando a abordagem PBRT (PCR-based replicon typing), que atribui plasmídeos a grupos de incompatibilidade, incluindo o grupo IncP-1. Os esforços para caracterizar melhor essas estruturas, principalmente quando associadas a bactérias resistentes a antibióticos, têm falhado e, consequentemente, as informações são escassas e dispersas. Portanto, uma análise sistemática foi realizada para compreender a ocorrência, distribuição e características genéticas de plasmídeos IncP-1 associados a bactérias resistentes a antibióticos. Para tal, a plataforma Scopus foi utilizada para realizar uma pesquisa bibliográfica, para selecionar estudos que identificaram plasmídeos IncP-1 através do método PBRT desenvolvido por Carattoli et al. (2005). A pesquisa abrangeu um período de 5 anos e resultou em 96 artigos elegíveis, os quais foram usados para a obtenção de informações relevantes sobre a ocorrência e características dos plasmídeos IncP-1. Os artigos combinados relataram a identificação de 629 replicões IncP-1. Ao todo, os hospedeiros bacterianos dos plasmídeos IncP-1 foram encontrados em 32 países e foram coletados de uma variedade de fontes ambientais. Os estudos identificaram hospedeiros bacterianos pertencentes a 28 espécies, distribuídas em 10 géneros das famílias Enterobacteriaceae e Pseudomonadaceae, foram identificados como resistentes a 10 classes de antibióticos, possuindo 32 genes de resistência diferentes, e pelo menos apresentando replicões de 2 grupos Inc diferentes. De todos os plasmídeos IncP-1 identificados, 229 (~ 36%) foram descritos posteriormente, e seus tamanhos variam de 35 a 320 kb, foram associados a genes de resistência clinicamente importantes e a elementos genéticos adicionais que poderiam potenciar a disseminação de genes. Além disso, foi estudada a diversidade molecular de plasmídeos IncP-1 identificados anteriormente em 9 isolados de Escherichia coli de uma UWTP em Portugal. Isso foi realizado por meio da amplificação por PCR de um fragmento do gene trfA de 281 bp, e tentativa de transferência para novos hospedeiros bacterianos bem conhecidos para posterior caracterização. A sequenciação dos amplicões mostrou 100% identidade entre fragmentos trfA sugerindo conservação das suas estruturas genéticas e associação com bactérias e ambientes semelhantes. A pesquisa por similaridade da sequência do fragmento trfA foi usada para selecionar e comparar plasmídeos IncP-1β1 totalmente sequenciados. Isso resultou na análise de 63 replicões, geralmente grandes estruturas genéticas, isolados de águas residuais e solos, possuindo determinantes genéticos associados à resistência a aminoglicosídeos, sulfonamidas e βlactâmicos, e à tolerância ao Hg. As tentativas de transferir os 9 plasmídeos IncP-1 para uma estirpe de laboratório não tiveram sucesso, provavelmente devido aos marcadores seletivos escolhidos. A ampla prevalência de genes do operão mer nestes plasmídeos IncP-1β1, sugerindo tolerância ao mercúrio, poderia ser usada em um trabalho futuro como um marcador seletivo para a transferência dos 9 plasmídeos IncP-1 de água residual para outro hospedeiro, permitindo a caracterização genética dessas estruturas.
Mestrado em Microbiologia
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Rosário, Natasha de Fátima Oliveira Esteves. "Internship Reports and Monograph entitled “Natural Transformation in Acinetobacter spp.”." Master's thesis, 2018. http://hdl.handle.net/10316/84369.
Full textAbstract:
Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de FarmáciaA crescente resistência antimicrobiana entre agentes patogénicos bacterianos, responsáveis por infeções, especialmente, as adquiridas em ambiente hospitalar, tem vindo a constituir uma grande ameaça para a saúde pública a nível global. A transferência horizontal de genes desempenha um papel importante na disseminação de genes de resistência antimicrobiana, incluindo a transformação natural, que é frequentemente negligenciada em ambiente clínico. Ainda assim, um grande número de isolados clínicos parece ser competente para a transformação natural, tal como foi recentemente descrito para Acinetobacter spp. Este facto poderá explicar a elevada capacidade de Acinetobacter baumannii adquirir multirresistências. Os objetivos deste estudo foram analisar a capacidade de deteção da captura e integração de ADN livre num vasto grupo de isolados clínicos de Acinetobacter spp., o seu envolvimento na transferência e aquisição de determinantes genéticos de resistência e de alterações relacionadas com o perfil de resistências das células transformantes. Numa primeira fase, foi usado um protocolo de transformação através da mobilidade em meio semi-sólido para detetar competência em 15 isolados clínicos de Acinetobacter spp. obtidos ao longo de 14 anos de cinco hospitais portugueses diferentes. Isolados naturalmente competentes (9 de 15) foram detetados em todos os hospitais, o que sugere que a transformação natural é mais comum em ambiente hospitalar do que o expectável. De seguida, os ensaios de transformação natural foram realizados com dois isolados clínicos de A. baumannii: o multirresistente Ab51 como dador de ADN e o naturalmente competente A118 como recetor. As células de A118 foram transformadas pelo ADN de Ab51. A técnica de PCR foi utilizada para determinar a presença da sequência de inserção ISAba1 no dador, no recipiente e nas células transformantes, bem como o contexto genético da mesma, seguido da sequenciação dos amplicões. Todos os transformantes testados adquiriram ISAba1 adjacente ao gene ampC. Em dois transformantes, a aquisição da ISAba1 ocorreu por transposição e foi inserida entre os habituais genes folE e ampC. Os restantes transformantes adquiriram ISAba1 adjacente a um segundo gene ampC, sendo parte do Tn6168, provavelmente por recombinação homóloga. Globalmente, este estudo revela a elevada prevalência de isolados clínicos de Acinetobacter spp. naturalmente competentes, assim como a contribuição do desenvolvimento de competência para a disseminação da resistência aos antibióticos beta-lactâmicos, sendo que a aquisição de determinantes não-codificadores de resistência pode contribuir para alterações no perfil de suscetibilidade de estirpes de A. baumannii.
The ever-increasing antimicrobial resistance among the bacterial pathogens causing infections, especially those who are healthcare-acquired has posed as a major life-threat for the public health worldwide. Horizontal gene transfer events play a relevant role in the dissemination of antimicrobial resistance genes, including natural transformation, which is often neglected in the clinical environment. Nevertheless, a high number of clinical isolates seems to undergo natural transformation, as has been recently described for Acinetobacter spp. This ability may explain the Acinetobacter baumannii high propensity to acquire multidrug resistance. The aims of this study were to assess to what extent the ability to take up naked DNA can be detected in a broad set of clinical Acinetobacter spp. isolates, its involvement in the transfer and acquisition of resistance genetic determinants and related changes in the resistance profile of transformed cells. First, an established transformation protocol, during motility in semisolid media, was used to detect competence in 15 clinical isolates of Acinetobacter spp. collected over a 14-year time period from five different Portuguese Hospitals. Naturally competent isolates (9 out of 15) were detected from all the hospitals, suggesting that natural transformation is more common in hospital settings than expected. Then, natural transformation assays were performed with two clinical isolates of A. baumannii: the multidrug-resistant Ab51 as a DNA donor source and the naturally competent A118 as a recipient cell. A118 cells were transformed by Ab51 DNA. PCR was used to ascertain the presence of ISAba1 in donor, recipient and transformant cells, as well as the genetic context of the insertion sequence, followed by amplicons sequencing. All tested transformants acquired ISAba1 upstream the ampC gene. In two transformants, the ISAba1’s acquisition was by transposition and its insertion was between the usual folE and ampC genes. The remaining transformants acquired the ISAba1 adjacent to a second ampC gene, as part of Tn6168, likely by homologous recombination. Overall, this study reveals the high prevalence of naturally competent Acinetobacter spp. clinical isolates, as well as the contribution of competence development to the widespread beta-lactams resistance, where the acquisition of non-resistant determinants can lead to changes in the susceptibility profile of A. baumannii strains.
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Burris, Kellie Parks. "Horizontal gene transfer to bacteria of an Arabidopsis thaliana ABC transporter that confers kanamycin resistance in transgenic plants." 2006. http://etd.utk.edu/2006/BurrisKellie.pdf.
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