Many antibiotic resistance genes (ARGs) conferring resistance to a broad range of antibiotics have often been detected in aquatic environments such as untreated and treated wastewater, river and surface water. ARG proliferation in the aquatic environment could depend upon various factors such as geospatial variations, the type of aquatic body, and the type of wastewater (untreated or treated) discharged into these aquatic environments. Likewise, the strong interconnectivity of aquatic systems may accelerate the spread of ARGs through them. Hence a comparative and a holistic study of different aquatic environments is required to appropriately comprehend the problem of antibiotic resistance. Many studies approach this issue using molecular techniques such as metagenomic sequencing and metagenomic data analysis. Such analyses compare the broad spectrum of ARGs in water and wastewater samples, but these studies use comparisons which are limited to similarity/dissimilarity analyses. However, in such analyses, the discriminatory ARGs (associated ARGs driving such similarity/ dissimilarity measures) may not be identified. Consequentially, the reason which drives the dissimilarities among the samples would not be identified and the reason for antibiotic resistance proliferation may not be clearly understood. In this study, an effective methodology, using Extremely Randomized Trees (ET) Algorithm, was formulated and demonstrated to capture such ARG variations and identify discriminatory ARGs among environmentally derived metagenomes. In this study, data were grouped by: geographic location (to understand the spread of ARGs globally), untreated vs. treated wastewater (to see the effectiveness of WWTPs in removing ARGs), and different aquatic habitats (to understand the impact and spread within aquatic habitats). It was observed that there were certain ARGs which were specific to wastewater samples from certain locations suggesting that site-specific factors can have a certain effect in shaping ARG profiles. Comparing untreated and treated wastewater samples from different WWTPs revealed that biological treatments have a definite impact on shaping the ARG profile. While there were several ARGs which got removed after the treatment, there were some ARGs which showed an increase in relative abundance irrespective of location and treatment plant specific variables. On comparing different aquatic environments, the algorithm identified ARGs which were specific to certain environments. The algorithm captured certain ARGs which were specific to hospital discharges when compared with other aquatic environments. It was determined that the proposed method was efficient in identifying the discriminatory ARGs which could classify the samples according to their groups. Further, it was also effective in capturing low-level variations which generally get over-shadowed in the analysis due to highly abundant genes. The results of this study suggest that the proposed method is an effective method for comprehensive analyses and can provide valuable information to better understand antibiotic resistance. / MS

Raw vegetables can sometimes be the source of outbreaks of human illness; however the potential for fresh vegetables to serve as a vehicle for antibiotic -resistant bacteria is poorly understood. Antibiotics and antibiotic-resistant bacteria have been shown to persist in manure of animals administered antibiotics, and in compost generated from this manure, where there is the potential for their transfer to produce. The purpose of this study was to determine the survival of antibiotic-resistant bacteria on raw, peeled, carrots after washing with commonly used chemical sanitizers. Multi-drug resistant E. coli O157:H7 and Pseudomonas aeruginosa were inoculated into a compost slurry of composted manure from dairy cattle, with and without prior administration of antibiotics, and used to inoculate carrot surfaces prior to the washing studies. This approach provided defined model antibiotic-resistant pathogens present within a background microbial community simulating potential carry over from manure-derived fertilizer. Carrots (n=3, 25g) were air-dried and stored at 4 °C until washing with tap water, XY-12 (sodium hypochlorite, 50 ppm free chlorine) or Tsunami 100 (peroxyacetic acid/hydrogen peroxide, 40 ppm free paracetic acid), according to manufacturer's directions. A second batch of carrots representing each inoculation x wash condition (n=3) were individually packaged for storage at 2 °C for 1,7, and 14 days, or 10 °C for 7 days and enumerated on those day intervals to recover bacteria from the surfaces of washed carrots. The resulting previously washed and stored carrots were subject to serial dilution and plated onto corresponding agar to enumerate total aerobic bacteria (R2A), aerobic bacteria tolerant or resistant to antibiotics (antibiotic-supplemented R2A), E. coli (Eosin Methylene Blue), and Pseudomonas spp. (Pseudomonas Isolation Agar). In addition, the tetA gene was quantified from the carrot samples as a measure of the effect of sanitizers and storage on an antibiotic resistance gene known to be carried by the inoculated bacteria.Inclusion of sanitizer in the wash water significantly reduced the absolute numbers of inoculated bacteria (E.coli and Pseudomonas) as well as populations of bacteria capable of growth on the R2A media containing cefotaxime (10μg/mL), sulfamethoxazole (100μg/mL), or tetracycline (3μg/mL). Comparable reductions in the inoculated P. aeruginosa resistant to tetracycline (PIA T, 4μg/mL), bacteria resistant to cefotaxime (10μg/mL) and tetracycline (3μg/mL) occurred after washing with XY-12 or Tsunami 100. The sanitizer effectiveness may be bacterial dependent, as evident by larger absolute reductions of the inoculated E. coli (EMB) and bacteria grown on sulfamethoxazole (100μg/mL)-amended plates after washing with Tsunami 100 compared to washing with tap water or XY-12. Re-growth of both the inoculated and native compost-associated bacteria was inhibited by storage at 2 °C, as there were no significant differences in the log CFU/g values on the various media (total aerobic bacteria, bacteria on antibiotic-amended plates, E. coli inoculum, P. aeruginosa inoculum) during the 14-day storage period. However, temperature abuse at 10 °C resulted in significant re-growth of native Pseudomonas, compared to storage at 2 °C. A sanitizer-associated interaction between re-growth and temperature was also observed for bacteria resistant to clindamycin (25μg/mL) and cefotaxime (10μg/mL), with substantial re-growth occurring only on carrots washed with Tsunami 100. There was no significant re-growth of the inoculated E. coli O157:H7 at either temperature. Results indicate that some bacterial populations are reduced by post-harvest washes and that temperature abuse of fresh produce may result in increases in antibiotic-resistant bacterial populations. / Master of Science in Life Sciences

Anthropogenically generated antibiotic resistance genes (ARGs) are considered emerging contaminants, as they are associated with a critical human health challenge, are persist independent of a bacterial host, are subject to transfer between bacteria, and are present at amplified levels in human-impacted environments. Given the gravity of the problem, there is growing interest in advancing water treatment processes capable of limiting ARG dissemination. This study examined the potential for membrane treatment of microconstituent ARGs, and the effect of wastewater colloids on their removal. Native and spiked extracellular vanA (vancomycin resistance) and blaTEM (β-lactam resistance) ARGs were tracked by quantitative polymerase chain reaction through a cascade of membrane filtration steps. To gain insight into potential associations occurring between ARGs and colloidal material, the wastewater colloids were characterized by scanning electron microscopy, as well as in their protein, polysaccharide, and total organic carbon content. The results suggest that extracellular DNA (eDNA) containing ARGs interacts with wastewater colloids, and can both be protected against degradation and be removed more efficiently in the presence of wastewater colloidal material. Thus, ARG removal may be achievable in sustainable water reuse scenarios using lower cost membranes than would have been selected based on molecular size alone. As membranes are likely to play a vital role in water sustainability, the results of this study enable consideration of ARG removal as part of a comprehensive strategy to manage emerging contaminants and to minimize overall public health risks. / Master of Science

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Harvey Morgan Scott / A randomized controlled field trial was designed to evaluate the effects of two treatment strategies on ceftiofur and tetracycline resistances in feedlot cattle. The two strategies consisted of administering ceftiofur crystalline-free acid administration (CCFA) at either one or else all of the steers within a pen, and subsequent feeding/not feeding of therapeutic doses of chlortetracycline. Both strategies were hypothesized to reduce ceftiofur resistance. The effects of treatment strategies were evaluated via metagenome-based and culture-based assays. In this 26-day study, 176 steers were allocated to 16 pens of 11 steers each. The two strategies were randomly assigned to the pens in a two-way full-factorial manner resulting in four treatment groups. The blaCMY-2, blaCTX-M, tet(A), tet(B), and 16S rRNA gene copies/g feces were quantified using qRT-PCR from fecal community DNA. Antimicrobial susceptibility profiles were determined using microbroth dilution technique from the non-type-specific (NTS) E. coli isolates (n=1,050). The NTS E. coli DNA was screened for the presence of blaCMY-2, tet(A), and tet(B) genes. Pens in which all the steers received CCFA treatment showed an increase in blaCMY-2 and blaCTX-M log10 gene copies/g feces and in the proportion of ceftiofur-resistant and blaCMY-2 positive NTS E. coli. This was in contrast to the pens where only one animal received CCFA treatment. There was a significant decrease in quantities of tetracycline genes in community DNA in pens where all animals received CCFA treatment. In contrast to metagenome-based assay results, culture-based assays indicated an increase in the proportion of tetracycline resistant NTS E. coli upon CCFA treatment. Thereafter, chlortetracycline administration led to rapid expansion both of ceftiofur (blaCMY-2, blaCTX-M) and tetracycline [tet(A) and tet(B)] log10 gene copies/g feces. Chlortetracycline treatment delayed the return of the ceftiofur resistance prevalence to baseline among NTS E. coli and thus did not lead to the hypothesized decrease in ceftiofur resistance. Our data suggest that chlortetracycline use is contraindicated when attempting to avoid expansion of resistance to critically important 3rd generation cephalosporins in feedlot cattle. Further studies are required to better establish the animal-level effects of co-housing antimicrobial-treated and non-treated animals together at varying ratios on the levels of antimicrobial resistance.

As populações de aves antárticas têm sido estudadas e consideradas indicadoras da qualidade do ecossistema marinho, especialmente dos oceanos do sul, ao longo dos últimos 50 anos. Existem cerca de 40 espécies de aves marinhas que se reproduzem em áreas descobertas de gelo. Dentre as aves marinhas antárticas, os pinguins são os que têm a maior representatividade ecológica e são considerados espécies sentinelas para o estudo das mudanças ambientais nesse continente. Esses animais representam 90% da biomassa de aves nos oceanos do sul, e suas colônias estão distribuídas nas ilhas antárticas e subantárticas bem como sobre o Continente Antártico. Há três espécies de pigoscelídeos mais representativos desta biomassa, são eles: Pygoscelis papua (Pinguim gentoo), Pygoscelis adeliae (Pinguim-de-adélia) e Pygoscelis antarcticus (Pinguim-de-barbicha). Os pinguins antárticos estão entre as aves de menor contato com humanos, o que os torna possíveis indicadores da presença natural de genes de resistência a antimicrobianos na microbiota intestinal de aves e no ambiente. O objetivo desta dissertação foi avaliar a presença de resistência a antimicrobianos na microbiota intestinal de P. antarcticus e de P. papua, e compará-las à microbiota de Spheniscus magellanicus (pinguins-de-magalhães). Os S. magellanicus habitam a Argentina, Chile e Ilhas Malvinas, locais em que há variadas atividades humanas. Foram coletadas amostras de fezes aparentemente frescas de 46 pinguins P.antarcticus e de 12 pinguins P. papua, nas suas respectivas colônias na Ilha Elefante, em dezembro de 2014. De S. magellanicus foram coletadas, com auxílio de suabes cloacais, amostras de 19 indivíduos, encontrados na costa norte do Rio Grande do Sul, de Quintão a Torres, durante os meses de inverno de 2015 e de 2016. As amostras de microbiota dos pinguins foram cultivadas em ágar LB e os isolados bacterianos foram triados para os seguintes antimicrobianos: eritromicina (≥ 8μg/mL), estreptomicina (≥ 2.000 μg/mL), tetraciclina (≥ 16 μg/mL) e vancomicina (≥ 32 μg/mL). Em 10 amostras de P. antarcticus e em 15 amostras de S. magellanicus foram identificadas bactérias resistentes a pelo menos um dos antimicrobianos testados. Todas as amostras de P. papua foram sensíveis a esses antimicrobianos. As espécies dos isolados resistentes foram identificadas pelo sequenciamento do rRNA 16S, que revelou sete gêneros, sendo os mais recorrentes Enterococcus sp. e Staphylococcus sp. Esses isolados resistentes também foram triados para a presença de genes de resistência aos antimicrobianos. O tet(M) foi mais abundante em S. magellanicus (5) do que em P. antarcticus (3), ao passo que o int e van(B) foram identificados somente em P. antarcticus (três e um, respectivamente). O gene erm(B) não foi encontrado em nenhum dos isolados. Uma vez que a fração não cultivável das fezes também pode apresentar genes de resistência, foi realizada a extração do DNA das fezes de pinguins antárticos para obtermos DNA de todos os micro-organismos presentes. Os genes mais recorrentes nas amostras de DNA total das fezes de P. antarcticus e P. papua foram, respectivamente, int (5 e 7), seguido de tet(M) (1 e 5). O van(B) foi encontrado em amostras das duas espécies de pinguins, enquanto que o erm(B) foi encontrado somente nas amostras de P. papua. De acordo com esses resultados, houve mais resistência a antimicrobianos na fração cultivável da microbiota de pinguins-de-magalhães do que em pinguins antárticos. Na fração não-cultivável, foram encontrados mais genes de resistência nas amostras de P. papua do que de P. antarcticus. / Antarctic seabird populations have been studied as bioindicators of the nature variability in the Southern Ocean marine ecosystems over the last 50 years. Among the Antarctic seabirds, the most representative species are penguins; they represent 90% of total biomass of birds in the Southern Ocean, and are considered sentinels for environmental changes in the Antarctic region. Pygoscelis antarcticus and P. papua are the most prevalent species in Antarctida. Because they remain among the wild birds with least contact with humans, their microbiota may serve as indicators of antimicrobial resistance in the environment. The aim of this work was to evaluate the antimicrobial resistance present in the microbiota of P. antarcticus and P. papua, and compare it with the microbiota of Spheniscus magellanicus (Magellanicus penguins). Magellanicus penguins inhabit Argentina, Chile and Falkland Islands, and therefore have more contact with humans. We have collected samples of apparently fresh feces from P. antarcticus (n = 46) and from P. papua (n = 12) in their respective colonies located in the Elephant Island in December 2014. From S. magellanicus, we have collected cloacal swabs (n = 19) from specimens found in the northern coast of Rio Grande do Sul, from Quintão to Torres, during the winter months of 2015 and 2016. All samples were evaluated for the presence of resistant bacteria to the following antimicrobials: erythromycin (≥ 8μg/mL), streptomycin (≥ 2.000 μg/mL), tetracycline ( ≥ 16 μg/mL) and vancomycin (32 μg/mL). We have isolated resistant bacteria from 10 samples of P. antarcticus and from 15 samples of S. magellanicus; there was no bacterial growth in the presence of any of these antimicrobials from samples of P. papua feces. The species of resistant bacteria were identified by 16S rRNA sequencing: among the 7 genera identified, the most frequent were Enterococcus sp. and Staphylococcus sp. Resistant bacteria were screened for the resistance genes ermB, tet(M), int and van(B). tet(M) was more frequent in S. magellanicus (5) than in P. antarcticus (3), while int and van(B) were identified only in P. antarcticus (3 and 1, respectively). The erm(B) gene was not identified in any isolate. Considering that the non-cultivable fraction from feces can also harbor resistance genes, we extracted DNA from feces of the Antarctic penguins in a attempt to obtain DNA from all micro-organisms of their microbiota. The most abundant genes present in the microbiota of P. antarcticus and P. papua were, respectively: int (5 and 7) and tet (M) (1 and 5). The van(B) gene was found in one sample of each species, while erm(B) was found in only one sample of P. papua. According to our results, antimicrobial resistance is more frequent in the cultivable microbiota of S. magellanicus than of P. antarcticus. In the non-cultivable fraction, resistance genes were more frequent in samples from P. papua than from P. antarcticus.

Cana-de-açúcar é uma planta agrícola com grande importância econômica para o Brasil. Os cultivares modernos de cana-de-açúcar apresentam uma alta complexidade genômica, devido ao seu genoma ser poliploide e com número cromossômico variando entre 100-120. As plantas de cana-de-açúcar estão sujeitas ao ataque de diversos tipos de patógenos, e sua tolerância depende da ação de genes de resistência. Estes podem desencadear uma cascata de reações ou interagir diretamente com uma molécula efetora, resultando em tolerância por parte da planta à presença do patógeno. O genoma dos cultivares moderno apresentam inúmeros elementos de transposição posicionados em diferentes regiões do genoma. Devido a essa abundância, os elementos de transposição estão sendo usados como marcadores moleculares, por exemplo, RBIP (Retrotransposon-Based Insertion Polymorphism), que identifica polimorfismos baseado na inserção de elementos do tipo retrotransposons. Esta técnica é capaz de diferenciar a presença e a ausência de um elemento de transposição em uma determinada região do genoma. Para a aplicação em organismos poliploides foi desenvolvida a técnica RBIP-qPCR, permitindo dosar a presença e ausência de um elemento de transposição em um organismo poliploide. Sendo assim este trabalho tem como objetivo aplicar a técnica de RBIP-qPCR em elementos de transposição alocados adjacentes a genes de resistência a fim de, posteriormente, verificar a existência de associação entre genes de resistência e elementos de transposição. Para isso foram analisadas três famílias de genes de resistência, I2C-2, Xa21 e Pti1. Foram realizadas análises de composição genômica de cada uma das regiões, genômica comparativa entre as variedades de R570 e SP80-3280 para a região genômica que contém o gene I2C-2, além de uma análise de expressão de genes e elementos de transposição vizinhos neste loco. A partir destes estudos foi possível selecionar um elemento de transposição, scDEL 5.1, para aplicar a técnica de RBIP-qPCR. Os resultados das análises de reconhecimento do ambiente genômico determinou a escolha de um elemento para aplicação da técnica. Verificamos que na ausência de scDEL 5.1 a metodologia RBIPqPCR foi eficaz em todos os genomas testados. As análises de presença de scDEL 5.1 no loco revelaram que elementos com elevado número de cópias no genoma não são adequados para a aplicação da metodologia. / Sugarcane is an agricultural plant with great economic importance for Brazil. The modern cultivars of sugarcane present a high genomic complexity, due to their genome being polyploid and with chromosome number varying between 100-120. Sugarcane plants are targets of several types of pathogens, and their tolerance depends on the action of resistance genes. These can trigger a cascade of reactions or interact directly with an effector molecule, resulting in tolerance by the plant to the presence of the pathogen. The genome of modern cultivars presents several transposable elements allocated in different regions of the genome. Due to this abundance, transposable elements are being used as molecular markers, such as RBIP (Retrotransposon-Based Insertion Polymorphism), which identifies polymorphisms based on the insertion of retrotransposon. This technique is able to differentiate the presence and absence of a transposable element in a particular region of the genome. For the application in polyploid organisms the RBIP-qPCR technique was developed, allowing measuring the presence and absence of a transposable element in a polyploid organism. Therefore, the objective of this work is to apply the RBIP-qPCR technique to on a transposable element localized adjacent to resistance genes in order to, latterly, verify the existence of association between resistance genes and transposable elements. For this, three families of resistance genes, I2C-2, Xa21 and Pti1 were analyzed. Genomic composition analyzes of each region, comparative genomics between the R570 and SP80-3280 varieties for the genomic region containing the I2C-2 gene, as well as an analysis of genes and elements expression neighbors at this locus were performed. From these studies it was possible to select a transposition element, scDEL 5.1, to apply the RBIP-qPCR technique. The results of the genomic environment recognition analysis determined the choice the transposable element for the application of the technique. We verified that in the absence of scDEL 5.1 the RBIP-qPCR methodology was effective in all genomes tested. Analysis of the presence of scDEL 5.1 in the locus revealed that elements with high copy number in the genome are not suitable for the application of the methodology.

As populações de aves antárticas têm sido estudadas e consideradas indicadoras da qualidade do ecossistema marinho, especialmente dos oceanos do sul, ao longo dos últimos 50 anos. Existem cerca de 40 espécies de aves marinhas que se reproduzem em áreas descobertas de gelo. Dentre as aves marinhas antárticas, os pinguins são os que têm a maior representatividade ecológica e são considerados espécies sentinelas para o estudo das mudanças ambientais nesse continente. Esses animais representam 90% da biomassa de aves nos oceanos do sul, e suas colônias estão distribuídas nas ilhas antárticas e subantárticas bem como sobre o Continente Antártico. Há três espécies de pigoscelídeos mais representativos desta biomassa, são eles: Pygoscelis papua (Pinguim gentoo), Pygoscelis adeliae (Pinguim-de-adélia) e Pygoscelis antarcticus (Pinguim-de-barbicha). Os pinguins antárticos estão entre as aves de menor contato com humanos, o que os torna possíveis indicadores da presença natural de genes de resistência a antimicrobianos na microbiota intestinal de aves e no ambiente. O objetivo desta dissertação foi avaliar a presença de resistência a antimicrobianos na microbiota intestinal de P. antarcticus e de P. papua, e compará-las à microbiota de Spheniscus magellanicus (pinguins-de-magalhães). Os S. magellanicus habitam a Argentina, Chile e Ilhas Malvinas, locais em que há variadas atividades humanas. Foram coletadas amostras de fezes aparentemente frescas de 46 pinguins P.antarcticus e de 12 pinguins P. papua, nas suas respectivas colônias na Ilha Elefante, em dezembro de 2014. De S. magellanicus foram coletadas, com auxílio de suabes cloacais, amostras de 19 indivíduos, encontrados na costa norte do Rio Grande do Sul, de Quintão a Torres, durante os meses de inverno de 2015 e de 2016. As amostras de microbiota dos pinguins foram cultivadas em ágar LB e os isolados bacterianos foram triados para os seguintes antimicrobianos: eritromicina (≥ 8μg/mL), estreptomicina (≥ 2.000 μg/mL), tetraciclina (≥ 16 μg/mL) e vancomicina (≥ 32 μg/mL). Em 10 amostras de P. antarcticus e em 15 amostras de S. magellanicus foram identificadas bactérias resistentes a pelo menos um dos antimicrobianos testados. Todas as amostras de P. papua foram sensíveis a esses antimicrobianos. As espécies dos isolados resistentes foram identificadas pelo sequenciamento do rRNA 16S, que revelou sete gêneros, sendo os mais recorrentes Enterococcus sp. e Staphylococcus sp. Esses isolados resistentes também foram triados para a presença de genes de resistência aos antimicrobianos. O tet(M) foi mais abundante em S. magellanicus (5) do que em P. antarcticus (3), ao passo que o int e van(B) foram identificados somente em P. antarcticus (três e um, respectivamente). O gene erm(B) não foi encontrado em nenhum dos isolados. Uma vez que a fração não cultivável das fezes também pode apresentar genes de resistência, foi realizada a extração do DNA das fezes de pinguins antárticos para obtermos DNA de todos os micro-organismos presentes. Os genes mais recorrentes nas amostras de DNA total das fezes de P. antarcticus e P. papua foram, respectivamente, int (5 e 7), seguido de tet(M) (1 e 5). O van(B) foi encontrado em amostras das duas espécies de pinguins, enquanto que o erm(B) foi encontrado somente nas amostras de P. papua. De acordo com esses resultados, houve mais resistência a antimicrobianos na fração cultivável da microbiota de pinguins-de-magalhães do que em pinguins antárticos. Na fração não-cultivável, foram encontrados mais genes de resistência nas amostras de P. papua do que de P. antarcticus. / Antarctic seabird populations have been studied as bioindicators of the nature variability in the Southern Ocean marine ecosystems over the last 50 years. Among the Antarctic seabirds, the most representative species are penguins; they represent 90% of total biomass of birds in the Southern Ocean, and are considered sentinels for environmental changes in the Antarctic region. Pygoscelis antarcticus and P. papua are the most prevalent species in Antarctida. Because they remain among the wild birds with least contact with humans, their microbiota may serve as indicators of antimicrobial resistance in the environment. The aim of this work was to evaluate the antimicrobial resistance present in the microbiota of P. antarcticus and P. papua, and compare it with the microbiota of Spheniscus magellanicus (Magellanicus penguins). Magellanicus penguins inhabit Argentina, Chile and Falkland Islands, and therefore have more contact with humans. We have collected samples of apparently fresh feces from P. antarcticus (n = 46) and from P. papua (n = 12) in their respective colonies located in the Elephant Island in December 2014. From S. magellanicus, we have collected cloacal swabs (n = 19) from specimens found in the northern coast of Rio Grande do Sul, from Quintão to Torres, during the winter months of 2015 and 2016. All samples were evaluated for the presence of resistant bacteria to the following antimicrobials: erythromycin (≥ 8μg/mL), streptomycin (≥ 2.000 μg/mL), tetracycline ( ≥ 16 μg/mL) and vancomycin (32 μg/mL). We have isolated resistant bacteria from 10 samples of P. antarcticus and from 15 samples of S. magellanicus; there was no bacterial growth in the presence of any of these antimicrobials from samples of P. papua feces. The species of resistant bacteria were identified by 16S rRNA sequencing: among the 7 genera identified, the most frequent were Enterococcus sp. and Staphylococcus sp. Resistant bacteria were screened for the resistance genes ermB, tet(M), int and van(B). tet(M) was more frequent in S. magellanicus (5) than in P. antarcticus (3), while int and van(B) were identified only in P. antarcticus (3 and 1, respectively). The erm(B) gene was not identified in any isolate. Considering that the non-cultivable fraction from feces can also harbor resistance genes, we extracted DNA from feces of the Antarctic penguins in a attempt to obtain DNA from all micro-organisms of their microbiota. The most abundant genes present in the microbiota of P. antarcticus and P. papua were, respectively: int (5 and 7) and tet (M) (1 and 5). The van(B) gene was found in one sample of each species, while erm(B) was found in only one sample of P. papua. According to our results, antimicrobial resistance is more frequent in the cultivable microbiota of S. magellanicus than of P. antarcticus. In the non-cultivable fraction, resistance genes were more frequent in samples from P. papua than from P. antarcticus.

This master thesis is focused on the role of horizontal gene transfer in disseminating tetracycline resistance among bacteria in farm soils. In the experimental part, plasmids carrying antibiotic resistance, were exogenously isolated in biparental matings with cattle manure and Escherichia coli K-12 CV601 gfp recipients.

Made available in DSpace on 2014-06-11T19:32:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-04Bitstream added on 2014-06-13T20:03:25Z : No. of bitstreams: 1 morceli_tgs_dr_jabo.pdf: 699244 bytes, checksum: f1671e3db3a2b611f86d79aaccfe36e0 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A ferrugem asiática da soja, causada pelo fungo Phakopsora pachyrhizi (Sidow & P. Sidow) foi relatada no Brasil ao final da safra 2001 e já nas safras seguintes, ocasionou severas perdas de produtividade. Cinco genes de resistência à ferrugem asiática da soja (Rpp1 a Rpp5) estão descritos na literatura. O objetivo geral deste trabalho foi identificar marcadores moleculares microssatélites ligados ao gene de resistência a P. pachyrhizi presente na linhagem de soja PI 200526. Uma população de plantas F2 derivada do cruzamento entre esta linhagem e a cultivar suscetível Coodetec 208, foi artificialmente inoculada e avaliada quanto à sua reação de resistência à ferrugem. Marcadores microssatélites foram testados nos genitores e em dois bulks contrastantes para possibilitar a identificação de possíveis marcadores ligados. Os três marcadores polimórficos que foram caracterizados como potencialmente associados com a resistência à ferrugem asiática foram, posteriormente, avaliados em toda a população. A resistência comportou-se como governada por um gene com dominância completa. O gene de resistência da PI 200526 foi mapeado no grupo de ligação N da soja, estando próximo ao marcador Sat_166. Existe grande possibilidade de que o gene mapeado neste estudo corresponda ao novo loco de resistência à ferrugem asiática da soja, denominado de Rpp5, recentemente descrito. / The Asian soybean rust caused by the Phakopsora pachyrhizi (Sidow & P. Sidow) fungus was related in Brazil at the end of 2001 crop year, and already in the following seasons, caused severe losses in productivity. Five distinct resistance genes to Asian rust (Rpp1 to Rpp5) are described. The main objective of this work was to identify microsatellite markers linked to a resistance gene to P. pachyrhizi present in the soybean line PI 200526. One population of F2 plants originated from the cross between this resistant line and the suscetible cultivar Coodetec 208 was artificially inoculated and evaluated for the Asian rust resistance. Microsatellite markers were tested on parents and in the two contrasting bulks to enable the identification of linked markers. The three polymorphic markers that were identified potentially associated with resistance to Asian rust were then evaluated throughout the progeny. The resistance showed to be governed by a gene with complete dominance. The resistance gene of PI 200526 was mapped on the soybean linkage group N, being close to Sat_166 marker. Possibly, the gene mapped on this linkage group is part of the new locus of resistance to Asian soybean rust, called Rpp5, recently described.

As populações de aves antárticas têm sido estudadas e consideradas indicadoras da qualidade do ecossistema marinho, especialmente dos oceanos do sul, ao longo dos últimos 50 anos. Existem cerca de 40 espécies de aves marinhas que se reproduzem em áreas descobertas de gelo. Dentre as aves marinhas antárticas, os pinguins são os que têm a maior representatividade ecológica e são considerados espécies sentinelas para o estudo das mudanças ambientais nesse continente. Esses animais representam 90% da biomassa de aves nos oceanos do sul, e suas colônias estão distribuídas nas ilhas antárticas e subantárticas bem como sobre o Continente Antártico. Há três espécies de pigoscelídeos mais representativos desta biomassa, são eles: Pygoscelis papua (Pinguim gentoo), Pygoscelis adeliae (Pinguim-de-adélia) e Pygoscelis antarcticus (Pinguim-de-barbicha). Os pinguins antárticos estão entre as aves de menor contato com humanos, o que os torna possíveis indicadores da presença natural de genes de resistência a antimicrobianos na microbiota intestinal de aves e no ambiente. O objetivo desta dissertação foi avaliar a presença de resistência a antimicrobianos na microbiota intestinal de P. antarcticus e de P. papua, e compará-las à microbiota de Spheniscus magellanicus (pinguins-de-magalhães). Os S. magellanicus habitam a Argentina, Chile e Ilhas Malvinas, locais em que há variadas atividades humanas. Foram coletadas amostras de fezes aparentemente frescas de 46 pinguins P.antarcticus e de 12 pinguins P. papua, nas suas respectivas colônias na Ilha Elefante, em dezembro de 2014. De S. magellanicus foram coletadas, com auxílio de suabes cloacais, amostras de 19 indivíduos, encontrados na costa norte do Rio Grande do Sul, de Quintão a Torres, durante os meses de inverno de 2015 e de 2016. As amostras de microbiota dos pinguins foram cultivadas em ágar LB e os isolados bacterianos foram triados para os seguintes antimicrobianos: eritromicina (≥ 8μg/mL), estreptomicina (≥ 2.000 μg/mL), tetraciclina (≥ 16 μg/mL) e vancomicina (≥ 32 μg/mL). Em 10 amostras de P. antarcticus e em 15 amostras de S. magellanicus foram identificadas bactérias resistentes a pelo menos um dos antimicrobianos testados. Todas as amostras de P. papua foram sensíveis a esses antimicrobianos. As espécies dos isolados resistentes foram identificadas pelo sequenciamento do rRNA 16S, que revelou sete gêneros, sendo os mais recorrentes Enterococcus sp. e Staphylococcus sp. Esses isolados resistentes também foram triados para a presença de genes de resistência aos antimicrobianos. O tet(M) foi mais abundante em S. magellanicus (5) do que em P. antarcticus (3), ao passo que o int e van(B) foram identificados somente em P. antarcticus (três e um, respectivamente). O gene erm(B) não foi encontrado em nenhum dos isolados. Uma vez que a fração não cultivável das fezes também pode apresentar genes de resistência, foi realizada a extração do DNA das fezes de pinguins antárticos para obtermos DNA de todos os micro-organismos presentes. Os genes mais recorrentes nas amostras de DNA total das fezes de P. antarcticus e P. papua foram, respectivamente, int (5 e 7), seguido de tet(M) (1 e 5). O van(B) foi encontrado em amostras das duas espécies de pinguins, enquanto que o erm(B) foi encontrado somente nas amostras de P. papua. De acordo com esses resultados, houve mais resistência a antimicrobianos na fração cultivável da microbiota de pinguins-de-magalhães do que em pinguins antárticos. Na fração não-cultivável, foram encontrados mais genes de resistência nas amostras de P. papua do que de P. antarcticus. / Antarctic seabird populations have been studied as bioindicators of the nature variability in the Southern Ocean marine ecosystems over the last 50 years. Among the Antarctic seabirds, the most representative species are penguins; they represent 90% of total biomass of birds in the Southern Ocean, and are considered sentinels for environmental changes in the Antarctic region. Pygoscelis antarcticus and P. papua are the most prevalent species in Antarctida. Because they remain among the wild birds with least contact with humans, their microbiota may serve as indicators of antimicrobial resistance in the environment. The aim of this work was to evaluate the antimicrobial resistance present in the microbiota of P. antarcticus and P. papua, and compare it with the microbiota of Spheniscus magellanicus (Magellanicus penguins). Magellanicus penguins inhabit Argentina, Chile and Falkland Islands, and therefore have more contact with humans. We have collected samples of apparently fresh feces from P. antarcticus (n = 46) and from P. papua (n = 12) in their respective colonies located in the Elephant Island in December 2014. From S. magellanicus, we have collected cloacal swabs (n = 19) from specimens found in the northern coast of Rio Grande do Sul, from Quintão to Torres, during the winter months of 2015 and 2016. All samples were evaluated for the presence of resistant bacteria to the following antimicrobials: erythromycin (≥ 8μg/mL), streptomycin (≥ 2.000 μg/mL), tetracycline ( ≥ 16 μg/mL) and vancomycin (32 μg/mL). We have isolated resistant bacteria from 10 samples of P. antarcticus and from 15 samples of S. magellanicus; there was no bacterial growth in the presence of any of these antimicrobials from samples of P. papua feces. The species of resistant bacteria were identified by 16S rRNA sequencing: among the 7 genera identified, the most frequent were Enterococcus sp. and Staphylococcus sp. Resistant bacteria were screened for the resistance genes ermB, tet(M), int and van(B). tet(M) was more frequent in S. magellanicus (5) than in P. antarcticus (3), while int and van(B) were identified only in P. antarcticus (3 and 1, respectively). The erm(B) gene was not identified in any isolate. Considering that the non-cultivable fraction from feces can also harbor resistance genes, we extracted DNA from feces of the Antarctic penguins in a attempt to obtain DNA from all micro-organisms of their microbiota. The most abundant genes present in the microbiota of P. antarcticus and P. papua were, respectively: int (5 and 7) and tet (M) (1 and 5). The van(B) gene was found in one sample of each species, while erm(B) was found in only one sample of P. papua. According to our results, antimicrobial resistance is more frequent in the cultivable microbiota of S. magellanicus than of P. antarcticus. In the non-cultivable fraction, resistance genes were more frequent in samples from P. papua than from P. antarcticus.