Type IV pili (T4P) are protein filaments found on the surface of a variety of bacterial species and mediate biofilm formation, adhesion, and flagellum-independent twitching motility. The biogenesis of T4P is dependent on a cell envelope-spanning, multiprotein complex that localizes to the poles in rod-shaped cells. How these proteins localize and cross the peptidoglycan (PG) layer in the absence of dedicated PG-hydrolyzing enzymes is unknown. In P. aeruginosa, PilMNOP interact to form the alignment subcomplex, connected via PilP to PilQ, which forms the outer membrane secretin. We hypothesized that polar localization and integration of the T4P machinery was driven by ordered recruitment to future sites of cell division, placing assembly system components at division septa in the correct position before daughter-cell separation. To determine which T4P components are essential for localization of the complex, we fused the T4P inner membrane assembly protein PilO to the fluorescent protein mCherry to monitor its localization. mCherry-PilO localized to the cell poles and midcell in wild type bacteria. However, it was delocalized in a strain lacking PilQ. A PilQ-mCherry fusion localized to the cell poles, likely through its putative septal PG binding AmiN domains, suggesting that PilQ binds PG and thus localizes its partners to future sites of cell division. In the absence of the associated pilotin protein (PilF), which is required for PilQ multimerization in the OM, T4P components were polarly localized, implying that localization is not dependent on secretin formation. The results of this research support a pre-installation mechanism for integration of protein complexes in the gram negative cell envelope without PG hydrolysis, which may be applicable to other systems. / Thesis / Master of Science (MSc)

This thesis consists of two chapters, each of which is a unique research project. Chapter 1 is focused on the synthesis and biological evaluation of novel sideromycin antibiotics. Sideromycins are bifunctional “Trojan Horse” molecules that have an iron chelator “siderophore” moiety covalently bound to an antibiotic. Such molecules exploit existing bacterial mechanisms for obtaining iron from their environment. Antibiotics that would typically not pass Gram-negative membranes are allowed access via siderophore transporter proteins. This project utilized a siderophore that has not previously been used in this capacity. The synthesis and biological evaluation of multiple sideromycin conjugates is reported. Chapter 2 describes the chemical synthesis of coumarin natural products using a synthetic process recently developed in the Magolan laboratory that enables the efficient prenylation of phenols. These natural products are molecules of biological interest in various capacities but are rare and difficult to isolate from their plant sources. They have also previously been cumbersome to make via chemical synthesis. The chemistry described herein constitutes an inexpensive and efficient process to produce these compounds that is superior to previously known methods. / Thesis / Master of Science (MSc) / This thesis is divided into two chapters. The first chapter is focused on the development of new sideromycin antibiotics. Sideromycins are “Trojan Horse”-like antibiotics that exploit the mechanisms of Gram-negative bacteria for obtaining iron, an essential nutrient, to enable antibiotic entry. This chapter details the synthesis of molecules that attach functionalities called “siderophores” to antibiotics, enabling them to be “smuggled” into Gram-negative microbes. This project uses a siderophore not previously utilized in sideromycin research. The second chapter is focused on the chemical synthesis of rare natural products that are phenols with prenyl substituents. Many such compounds are plant-derived and have potential for biomedical use. However, difficulty in isolating them makes them prohibitively expensive in the purity and quantity required for research. They are also challenging to make synthetically. This chapter details the application of a recently discovered process in the Magolan laboratory to synthesize coumarin-containing prenylated phenolic natural products.

No description available.

A leptospirose é uma zoonose causada por bactérias do gênero Leptospira. A patogênese da doença em humanos é observada principalmente no pulmão, fígado e rins. Neste trabalho, foi avaliado o papel da resposta imune inata na proteção contra a leptospirose usando camundongos como modelo experimental. Os animais foram infectados com L. interrogans e o desenvolvimento da doença foi acompanhado, observando-se a morte de animais C3H/HeJ, enquanto C3H/HePas apresentou icterícia e BALB/c não apresentou sintomas. O perfil de mRNA foi medido por qPCR nas amostras de rim, fígado e pulmão e as concentrações de proteinas TNF-<font face=\"Symbol\">&#945;, TGF-<font face=\"Symbol\">b, MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 e IL-8 foram analisadas por ELISA em extratos dos tecidos e no soro. Os resultados demonstraram que L. interrogans estimula a expressão prematura de TNF-<font face=\"Symbol\">&#945;, TGF-<font face=\"Symbol\">b, MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 e IL-8 na linhagem BALB/c resistente à infecção. A análise histológica indica que estes mediadores podem estar relacionados com o influxo de diferentes células do sistema imune desempenhando importantes funções na proteção contra leptospirose. / Leptospirosis is a worldwide zoonosis caused by Leptospira. The pathogenesis in humans is mainly observed in lungs, livers and kidneys. In this work the role of innate immune response in protection against leptospirosis is being studied using different mice models. The animals were infected intraperitoneally with virulent cells of L. interrogans serovar Copenhageni and the development of the disease was followed, being observed mortality of C3H/HeJ mice, whereas C3H/HePas presented jaundice and BALB/c mice remained asymptomatic. Samples of liver, kidney, lungs and sera were analyzed following the profiles of mRNA and protein of the cytokines TNF-<font face=\"Symbol\">&#945; and TGF-<font face=\"Symbol\">b and chemokine MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 and CXCL1/IL-8. We showed that Leptospira infection stimulates early expression of cytokine TNF-<font face=\"Symbol\">&#945; and TGF-<font face=\"Symbol\">b and chemokine MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 and IL-8 in the resistant mice strain BALB/c. Histological analysis indicates that the expression of those molecules can be related to the influx of distinct immune cells, which play a role in the naturally acquired protective immunity.

A deficiência da proteína C5 do sistema complemento humano é rara com 38 casos relatados na literatura e freqüentemente associada a severas infecções provocadas por bactérias Neisseria. O objetivo do trabalho é caracterizar imunológica e geneticamente esta deficiência encontrada pela primeira vez em brasileiros. Por imunodifusão dupla obtivemos níveis expressivos de C3, C4, C6, C7, C8, C9, Fator B, Fator H e Fator I em todos os membros desta família, a proteína C5 não foi detectada no soro de três irmãos: II:9, II:4 e II:5. Por ELISA a concentração de C5 nestes indivíduos foi (0,9; 1,0; 1,3 µg/ml, 45- 190 µg/ml). Soros destes probandos não apresentam atividade hemolítica mediada pelo sistema complemento. O cDNA de C5 dos indivíduos I:1, I:2, II:4 e II:9 apresenta a deleção do éxon 30. Causada pela substituição de GAG4028 por GAA4028 no último nucleotídeo deste éxon que leva a um erro no splicing. Este defeito provavelmente produz uma proteína incompleta e destinada à degradação. / The deficiency of the C5 component of the complement system is rare with 38 described cases in the literature. This deficiency is frequently associated with severe infections, especially caused by Neisseria. Our objective is to characterize immunologically and genetically this deficiency, the first of its type described in the Brazilian population.We noted that C3, C4, C6, C7, C8, C9, Factor B, Factor H and Factor I have expressive levels in all the individuals sera of this family. C5 was absent in individuals II:4, II:5 and II:9. By ELISA a C5 concentration in this individuals were 0,9; 1,0; 1,3 µg/ml (normal: 45 - 190 µg/ml). Their serum doesn´t present hemolytic activity mediated by complement system. The C5 cDNA from individuals I:1, I:2, II:4 and II:9 has éxon 30 deleted. Caused by the substitution of GAG4028 for a GAA4028 in the last codon of exon 30. This defect was responsible for the deficiency of C5 in this family and this deletion would probably produce an unstable protein destined for degradation.

A deficiência da proteína C5 do sistema complemento humano é rara com 38 casos relatados na literatura e freqüentemente associada a severas infecções provocadas por bactérias Neisseria. O objetivo do trabalho é caracterizar imunológica e geneticamente esta deficiência encontrada pela primeira vez em brasileiros. Por imunodifusão dupla obtivemos níveis expressivos de C3, C4, C6, C7, C8, C9, Fator B, Fator H e Fator I em todos os membros desta família, a proteína C5 não foi detectada no soro de três irmãos: II:9, II:4 e II:5. Por ELISA a concentração de C5 nestes indivíduos foi (0,9; 1,0; 1,3 µg/ml, 45- 190 µg/ml). Soros destes probandos não apresentam atividade hemolítica mediada pelo sistema complemento. O cDNA de C5 dos indivíduos I:1, I:2, II:4 e II:9 apresenta a deleção do éxon 30. Causada pela substituição de GAG4028 por GAA4028 no último nucleotídeo deste éxon que leva a um erro no splicing. Este defeito provavelmente produz uma proteína incompleta e destinada à degradação. / The deficiency of the C5 component of the complement system is rare with 38 described cases in the literature. This deficiency is frequently associated with severe infections, especially caused by Neisseria. Our objective is to characterize immunologically and genetically this deficiency, the first of its type described in the Brazilian population.We noted that C3, C4, C6, C7, C8, C9, Factor B, Factor H and Factor I have expressive levels in all the individuals sera of this family. C5 was absent in individuals II:4, II:5 and II:9. By ELISA a C5 concentration in this individuals were 0,9; 1,0; 1,3 µg/ml (normal: 45 - 190 µg/ml). Their serum doesn´t present hemolytic activity mediated by complement system. The C5 cDNA from individuals I:1, I:2, II:4 and II:9 has éxon 30 deleted. Caused by the substitution of GAG4028 for a GAA4028 in the last codon of exon 30. This defect was responsible for the deficiency of C5 in this family and this deletion would probably produce an unstable protein destined for degradation.

Helicobacter pylori (H. pylori) is a microaerophilic, Gram-negative motile curved rod that inhabits the gastric mucosa of the human stomach. The organism chronically infects billions of people worldwide and is one of the most genetically diverse of bacterial species. Infection with the organism potentially induces chronic gastritis and peptic ulcer disease. In addition, H. pylori plays a role in the etiology of gastric cancer and gastric MALT lymphoma. The risk of infection is increased in those living in the developing world, which has been ascribed to precarious hygiene standards, crowded households, and deficient sanitation common in this part of the world. Thus, the aim of this study was to identify the risk factors in the transmission of H. pylori in our environment, i.e. in Nkonkobe Municipality in the Eastern Cape Province, South Africa. Faecal samples were collected from 356 apparently healthy subjects, consisting of 168 males and 188 females aged from 3 months to 60 years (Mean = 31 years). A standardized questionnaire was applied, it described demographic characteristics including age, sex, household hygiene, socioeconomic status, area of residence, duration of stay in the area, sharing bath water, sharing tooth brush, habit of sucking thumb, medication currently being taken or medication taken within the past three months, source of water, type of toilet used, education and occupation. A sandwich-type enzyme immunoassay amplification technology (Amplified IDEIA TM Hp StAR TM , Oxoid, UK) was used to analyze the faecal samples for the detection of H. pylori antigens using monoclonal antibodies specific for H. pylori antigens. To assess the possibility of faecal oral route with tap water as an intermediary link, PCR targeting the ureC (glmM), a highly conserved gene in H. pylori ii was carried out to detect H. pylori DNA in faecal samples of already positive samples by HpSA test as well as in direct tap water used by the H. pylori positive subjects. QIAamp DNA stool mini kit was used to extract DNA from faecal samples. Tap water samples were then obtained using sterile bottles from areas inhabited by H. pylori positive subjects as determined by HpSA test and PCR. DNA extraction from water samples was done using UltraCleanTM Water DNA Isolation Kit (0.22μm) according to the manufacturer’s instructions. PCR with primers specific for H. pylori glmM gene was carried out with both positive and negative controls incorporated. Fisher’s exact test was used to assess the univariate association between H. pylori infection and the possible risk factors. Odds ratio (OR) and the corresponding 95 percent confidence interval (CI) were calculated to measure the strength of association using EPI INFO 3.41 package. P values of < .05 were required for significance. The precision rate of the diagnostic tests used was also determined. H. pylori antigen was detected in 316 of the 356 subjects giving an overall prevalence of 88.8 percent. Prevalence increased with age from 75.9 percent in children < 12 years age to 100 percent in the age group from 13 years to 24 years, also 100 percent prevalence of H. pylori was recorded in young adults aged 25-47 years and subjects aged 60 years (P < .05). H. pylori prevalence was higher in females than in males. Of 188 females who participated in the study, H. pylori antigen was detected in 172 (91.5 percent) versus 144 (85.7 percent) of 168 males (P > .05). Interestingly, H pylori antigen was detected more often (100 percent) in the high socioeconomic group than in those of low socioeconomic group (85.9 percent). Sixteen (66.7 percent) of twenty four faecal samples that had previously tested positive for the organism by HpSA test were confirmed positive by PCR. However none of the treated tap water samples tested positive for the organism by PCR. The present iii study revealed a high prevalence of H. pylori in faecal samples of asymptomatic individuals in the Nkonkobe Municipality, an indication of active infection. The obtained results also revealed that direct treated tap water might not be playing a crucial role in the oral transmission of H. pylori in the studied population.

A leptospirose é uma zoonose causada por bactérias do gênero Leptospira. A patogênese da doença em humanos é observada principalmente no pulmão, fígado e rins. Neste trabalho, foi avaliado o papel da resposta imune inata na proteção contra a leptospirose usando camundongos como modelo experimental. Os animais foram infectados com L. interrogans e o desenvolvimento da doença foi acompanhado, observando-se a morte de animais C3H/HeJ, enquanto C3H/HePas apresentou icterícia e BALB/c não apresentou sintomas. O perfil de mRNA foi medido por qPCR nas amostras de rim, fígado e pulmão e as concentrações de proteinas TNF-<font face=\"Symbol\">&#945;, TGF-<font face=\"Symbol\">b, MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 e IL-8 foram analisadas por ELISA em extratos dos tecidos e no soro. Os resultados demonstraram que L. interrogans estimula a expressão prematura de TNF-<font face=\"Symbol\">&#945;, TGF-<font face=\"Symbol\">b, MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 e IL-8 na linhagem BALB/c resistente à infecção. A análise histológica indica que estes mediadores podem estar relacionados com o influxo de diferentes células do sistema imune desempenhando importantes funções na proteção contra leptospirose. / Leptospirosis is a worldwide zoonosis caused by Leptospira. The pathogenesis in humans is mainly observed in lungs, livers and kidneys. In this work the role of innate immune response in protection against leptospirosis is being studied using different mice models. The animals were infected intraperitoneally with virulent cells of L. interrogans serovar Copenhageni and the development of the disease was followed, being observed mortality of C3H/HeJ mice, whereas C3H/HePas presented jaundice and BALB/c mice remained asymptomatic. Samples of liver, kidney, lungs and sera were analyzed following the profiles of mRNA and protein of the cytokines TNF-<font face=\"Symbol\">&#945; and TGF-<font face=\"Symbol\">b and chemokine MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 and CXCL1/IL-8. We showed that Leptospira infection stimulates early expression of cytokine TNF-<font face=\"Symbol\">&#945; and TGF-<font face=\"Symbol\">b and chemokine MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 and IL-8 in the resistant mice strain BALB/c. Histological analysis indicates that the expression of those molecules can be related to the influx of distinct immune cells, which play a role in the naturally acquired protective immunity.

Ameisen der Gattung Camponotus beherbergen bakterielle Symbionten der Gattung Blochmannia in spezialisierten Zellen des Mitteldarms (Blochmann, 1882; Buchner, 1965; Sauer, 2000; Schröder et al., 1996). Die Genomsequenzierung dieser Symbionten zeigte, dass Blochmannia, ähnlich den Symbionten von Blattläusen, hauptsächlich Gene der Aminosäurebiosynthese beibehalten hat (Degnan et al., 2005; Gil et al., 2003). Die Relevanz dieser nahrungsaufwertenden Funktion konnte experimentell bestätigt werden (Feldhaar et al., 2007). Ein Schwerpunkt der vorliegenden Arbeit war die Aufklärung der dynamischen Interaktion der beiden Partner während des komplexen Lebenszyklus des holometabolen Wirtes. Frühere Studien deuteten darauf hin, dass die Symbiose vor allem während der Larven- und Puppenphasen von Bedeutung sein könnte (Feldhaar et al., 2007; Wolschin et al., 2004; Zientz et al., 2006). Mit fluoreszenter in situ Hybridisierung (FISH) und konfokaler Laserscanning Mikroskopie konnte in der vorliegenden Arbeit die Lokalisierung von B. floridanus während der wichtigsten Entwicklungsstadien aufgeklärt werden. Hierbei konnte gezeigt werden, dass die Symbionten schon im ersten Larvenstadium in spezialisierten Zellen um den Darm angeordnet sind, aber in späteren Stadien nicht, wie bisher angenommen, auf diese Bakteriozyten beschränkt sind, sondern bis zum Schlupf der jungen Arbeiterinnen massiv andere Darmzellen infizieren. Übereinstimmend mit Bestimmungen der Zellzahl in den verschiedenen Wirtsstadien ist die Anzahl der Symbionten gegen Ende der Metamorphose am höchsten. Die Symbiose degeneriert in sehr alten Arbeiterinnen, gut gefüllte Bakteriozyten werden jedoch noch monatelang beibehalten. Mit Macroarray- und qRT- PCR- basierten Transkriptomanalysen wurde die Expression der bakteriellen Gene in charakteristischen Entwicklungsstadien des Wirtes untersucht. Allgemein zeigen vor allem Gene für molekulare Chaperons und bestimmte bakterielle Grundfunktionen eine hohe Expression. Aber auch viele Gene, die möglicherweise wichtige Funktionen in der Symbiose besitzen, wie die Biosynthese essentieller Aminosäuren und das Recycling von Stickstoffverbindungen, zeigen ein hohes absolutes Transkriptlevel. Zudem besteht eine positive Korrelation zwischen dem Expressionsniveau und dem GC- Gehalt der Gene, die in dem höheren Selektionsdruck und damit einer geringeren Mutationsrate der essentiellen Gene begründet liegt (Schaber et al., 2005). Durch Proteinanalysen konnte bestätigt werden, dass die Faktoren mit der höchsten absoluten Transkription die dominanten Proteine der Symbionten darstellen. In den unterschiedlichen Entwicklungsstadien zeigen viele Gene eine deutliche Dynamik, deren Ausmaß aber, verglichen mit freilebenden Bakterien, gering ist. Aus den Expressionsprofilen aufeinanderfolgender Gene lassen sich mögliche Transkriptionseinheiten ableiten, die teilweise auch experimentell bestätigt wurden. Oftmals zeigen auch Gene, die nicht in Transkriptionseinheiten angeordnet sind, aber verwandten Stoffwechselwegen angehören, ähnliche Muster. Dies deutet auf das Vorhandensein grundlegender Genregulations-mechanismen hin, obwohl im Genom von B. floridanus nur noch sehr wenige Transkriptionsfaktoren codiert sind (Gil et al., 2003). Auf übergeordneter Ebene zeigt sich, dass bei Symbionten aus späten Puppenstadien viele symbioserelevante Gene im Vergleich zu Genen des Grundmetabolismus eine erhöhte Expression zeigen. Dies betrifft besonders die Biosynthese aromatischer und verzweigter Aminosäuren, die in diesen Stadien vom Wirt in hoher Menge benötigt werden, während die internen Reserven gleichzeitig zur Neige gehen. Dies äußert sich auch im deutlichen Abfallen der Speicherproteinmenge des Wirts gegen Ende der Puppenphase. Die festgestellte Veränderung der Symbiontenzahl übertrifft das geringe Ausmaß der Genregulation um ein Vielfaches. Die Bakterien liegen in jedem Stadium polyploid mit bis zu 100 Genomkopien vor, dieser Polyploidiegrad bleibt jedoch während der gesamten Wirtsentwicklung weitestgehend konstant. Somit scheint die Kontrolle des Wirts über die bakterielle Vermehrung der entscheidende Faktor dieser Symbiose zu sein. Die verbleibenden regulatorischen Fähigkeiten der Bakterien stellen möglicherweise eine Feinjustierung von optimierten Produktionseinheiten dar, deren Anzahl nach den Bedürfnissen des Wirtes verändert wird. Insgesamt konnten in der vorliegenden Arbeit neue Einblicke in das komplexe Zusammenleben von Blochmannia und Camponotus gewonnen werden, die zu einem besseren Verständnis der biologischen Funktion und der grundlegenden Mechanismen dieser Symbiose führen. Eine der wichtigsten Fragestellungen nach dem Sinn einer nahrungsaufwertenden Symbiose für einen Nahrungsgeneralisten konnte mit starken Hinweisen auf eine stadienabhängige Relevanz der Symbiose beantwortet werden, die den enormen evolutionären Erfolg dieser Ameisengattung erklären könnte.&#8195; / Ants of the genus Camponotus harbor bacterial endosymbionts of the genus Blochmannia in specialized cells of their midgut (Blochmann, 1882; Buchner, 1965; Sauer, 2000; Schröder et al., 1996). The complete sequencing of the symbiont’s genome revealed, that Blochmannia, comparable to the symbionts of aphids, mainly retained genes involved in the biosynthesis of essential amino acids (Degnan et al., 2005; Gil et al., 2003). The biological relevance of a nutritional upgrading by Blochmannia could be confirmed experimentally (Feldhaar et al., 2007). One focus of this thesis was the elucidation of the dynamic interactions between the two partners during the complex life cycle of the holometabolic host animal. Previous studies pointed towards a temporal relevance of this symbiosis especially during larval and pupal development (Feldhaar et al., 2007; Wolschin et al., 2004; Zientz et al., 2006). In this thesis the localization of B. floridanus could be documented throughout all life stages of the host by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy. A layer of densely filled bacteriocytes surrounding the gut could already be identified in first instar larvae. In contrast to previous assumptions, the bacteria are not restricted to these cells in later stages, as until the eclosion of the young adult workers bacteria massively infect other midgut cells. Concordant with previous findings, bacterial load is highest at the end of metamorphosis and symbiont numbers decrease in older workers, yet densely filled bacteriocytes are still visible after several months. The expression of the bacterial genes during characteristic life stages of the C. floridanus was assessed by macroarray and qRT- PCR- based experiments. In general, especially molecular chaperones, central basic metabolism and may putative symbiosis related factors like pathways leading to essential amino acids or nitrogen recycling show highest absolute expression levels. A positive correlation between expression level and GC- content of the genes can be observed, which is caused by a higher selection pressure and lower mutation rate of these essential factors (Schaber et al., 2005). Protein analyses confirmed the correlation between gene expression and translation of the most abundant factors. Many B. floridanus genes exhibit a dynamic expression during the different host stages but the extent of this gene regulation is modest as compared to free living bacteria. Expression profiles of genes located next to each other on the genome allow proposal of local transcription units, which were confirmed experimentally in several cases. Often genes that are not clustered locally but belong to related metabolic functions also exhibit similar expression patterns. This indicates the existence of basic mechanisms of gene regulation despite the low number of transcription factors annotated in the B. floridanus genome (Gil et al., 2003). In late pupal stages symbiosis related genes often show a higher expression compared to basic metabolic functions. This especially includes biosynthetic pathways for aromatic and branched amino acids, which are needed by the host at this stage in increased amounts, while internal storages are depleted. This could be demonstrated by the significant decrease in storage proteins of the host at the end of the pupal phase. The observed change in bacterial numbers per host exceeds the extent of bacterial gene regulation by far. The symbionts are polyploid in each host stage with up to 100 genome copies per cell. The degree of polyploidy is largely constant during host development. Thus the control over bacterial reproduction seems to be the decisive factor in this symbiosis. The residual regulatory capacities of the symbionts might represent a mechanism of fine tuning of a production unit that has been streamlined by evolution and whose numbers are adjusted according to the host’s needs. In conclusion, this thesis delivers new insights into the complex symbiosis of Blochmannia and Camponotus leading to a better understanding of its biological function and the underlying mechanisms. One of the central mysteries concerning the need of a symbiont for nutritional upgrading for an omnivorous host could be explained by a temporal, stage- dependent relevance of this symbiosis, possibly being the reason for the enormous evolutionary success of this ant genus.

A multirresistência aos antibióticos observada em bacilos gram-negativos é um grave problema de saúde pública devido a alta morbidade e mortalidade apresentada, especialmente em instituições assistenciais de saúde. Como consequência do intenso uso de antibióticos, a multirresistência a esses fármacos é principalmente mediada por enzimas hidrolisantes, onde destaca-se as enzimas ?-lactamases, principal mecanismo de resistência aos ?-lactâmicos verificado em bacilos gram-negativos. Os esgotos de origem hospitalar e de estações de tratamento de esgoto (ETE) são considerados como reservatórios de bactérias multirresistentes pela presença de antibióticos que as selecionam e por favorecem a transmissão de determinantes de resistência. Nesse sentido, o presente estudo objetivou avaliar a multirresistência a antibióticos e a produção de enzimas ?-lactamases em bacilos gram-negativos isolados de efluente hospitalar e da estação de tratamento de esgoto, na cidade de Ribeirão Preto, SP. No hospital terciário, amostras de esgotos foram coletadas dos ambulatórios, das enfermarias e da junção do esgoto hospitalar. Na ETE, amostras foram coletadas na caixa de entrada do esgoto bruto e após ao tratamento. Dez microlitros foram semeados em ágar MacConkey, SalmonellaShigella, Cetrimide e TCBS e a identificação dos bacilos gram-negativos foi realizada pelo kit Bactray®. O teste de susceptibilidade aos antibióticos foi realizado pelo método de discodifusão em ágar. A detecção fenotípica de bacilos produtores de ESBL foi realizada pelos testes de sinergia de disco-duplo e disco combinado com ácido clavulânico, e para detecção de isolados produtores de carbapenemases foi utilizado os testes de disco combinado com ácido fenilborônico e EDTA e o teste Blue Carba. A PCR foi utilizada para amplificação dos genes codificadores de ESBL e carbapenemases. No total, 45 bacilos gram-negativos foram isolados, sendo as espécies Klebsiella pneumoniae e Pseudomonas aeruginosa as de maiores prevalências. Ampla resistência foi verificada aos antibióticos ?-lactâmicos, sendo a resistência ao aztreonam, a cefepime e a cefotaxima mais expressiva nos isolados do esgoto hospitalar, com diferenças estatisticamente significante (p<0,05). O fenótipo multidroga resistente foi atribuído a 33,3%, nos isolados exclusivamente do esgoto hospitalar, com diferença estatisticamente significante (p = 0,0025) em relação aos isolados do esgoto da ETE. Genes de ?-lactamases foram encontrados em 35,6% das bactérias, sendo o blaKPC e blaTEM os de maiores ocorrências, ambos em 17,8% dos isolados, e os genes blaSHV e blaCTX-M em 13,3% e 8,9%. Somente em um isolado de Enterobacter cloacae no esgoto tratado da ETE foi identificado o gene blaSHV, os demais isolados portadores dos genes de ?-lactamases foram encontrados no esgoto hospitalar. Os dados obtidos neste estudo são importantes levando em consideração que no Brasil o esgoto hospitalar pode ser lançado in natura na rede coletora municipal, no entanto, acredita-se que tal permissão favorece a disseminação da multirresistência bacteriana, posto que, os resultados demonstram alta frequência de bactérias portadoras de genes de resistência a antibióticos no esgoto hospitalar estudado. Assim, a implementação do tratamento de efluentes hospitalares, especialmente os de hospitais terciários, e adicionalmente ao tratamento da ETE evitaria a propagação dessas bactérias no ambiente e de impactar negativamente os recursos hídricos / Antibiotic multi-resistance observed in Gram-negative bacilli is a serious public health problem due to high morbidity and mortality, especially in health care institutions. As a consequence of the intense use of antibiotics, multi-resistance to these drugs is mainly mediated by hydrolyzing enzymes, in which ?-lactamases, the main ?-lactam resistance mechanism observed in Gramnegative bacilli, are prominent. Hospital sewage and wastewater treatment plants (WWTP) are considered reservoirs of multiresistant bacteria by the presence of antibiotics that select these bacteria and favor the transmission of resistance determinants. In this sense, the present study aimed to evaluate the antibiotics multi-resistance and the production of ?-lactamase enzymes in Gram-negative bacilli isolated from hospital effluent and the wastewater treatment plants in Ribeirão Preto city, SP. In the tertiary hospital, sewage samples from the outpatient clinics, rooms patients and the hospital sewage junction were collected. In the WWTP, raw and treated sewage were collected. Ten microliters were seeded on MacConkey, Salmonella-Shigella, Cetrimide and TCBS agar and the identification of Gram-negative bacilli was performed by the Bactray® kit. Antibiotic susceptibility test was performed by agar-diffusion method. Phenotypic detection of ESBL-producing bacilli was performed by double-disc and discsynergy tests combined with clavulanic acid, and for the detection of carbapenemase-producing isolates the combined disk tests with phenylboronic acid and EDTA and Blue Carba test were used. PCR amplification of ESBL and carbapenemases-encoding genes was used. In total, 45 Gram-negative bacilli were isolated, and Klebsiella pneumoniae and Pseudomonas aeruginosa being the most prevalent. Extensive resistance was verified to ?-lactam antibiotics and resistance to aztreonam, cefepime and cefotaxime was more pronounced in hospital sewage isolates, with statistically significant differences (p<0.05). Multidrug-resistant phenotype was attributed to 33.3% in isolates exclusively from hospital sewage, with a statistically significant difference (p = 0.0025) in relation to the sewage isolates from the WWTP. ?-lactamase genes were found in 35.6% of the bacteria, with blaKPC and blaTEM having the highest occurrences, both in 17.8% of the isolates, and the blaSHV and blaCTX-M genes in 13.3% and 8, 9%. Only in an isolate of Enterobacter cloacae in the treated sewage from WWTP was the blaSHV gene identified, the other isolates carrying the ?-lactamases genes were found in hospital sewage. The data obtained in this study are important considering that in Brazil the hospital sewage can be released in nature in municipal collection network, however, it is believed that such permission favors the dissemination of bacterial multi-resistance, since, the results show high frequency of bacteria carrying antibiotic resistance genes in the hospital sewer studied. Thus, the implementation of treatment of hospital effluents, especially those in tertiary hospitals, and in addition to the treatment of WWTP would prevent the spread of these bacteria in the environment and negatively impact water resources