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Opioids have been shown to modulate immune function in a variety of assays and animal models. In a more limited number of studies, opioids have been shown to sensitize to infection. Heroin, the prototypical opioid drug of abuse, is rapidly metabolized to morphine in the body. Morphine has been used as an analgesic for hundreds of years, and continues to be a drug of choice for treating pain in ICU and trauma patients. The continued use of these opioid compounds in humans warrants further investigation of their effect on immune responses against, and progression of, common bacterial infections. Two infections were investigated in this thesis using murine models, Acinetobacter baumannii and Salmonella typhimurium. A recent increase in the prevalence of A. baumannii infections among healthy, but wounded, military personnel, lead to the hypothesis that analgesic morphine might sensitize to infection with this multiply-drug resistant bacterium. A systemic, intraperitoneal A. baumannii infection model was established in mice that resulted in rapid, disseminated disease where animals became septic as organisms replicated in the blood, lungs, and other organs. This model was used to investigate the role of various parameters of innate immune defenses to Acinetobacter. Neutralization of neutrophils by antibody depletion greatly sensitized to this infection. Infection resulted in a rapid, biphasic induction of both IL-17 and the chemokine, KC/CXCL1, a major chemotactic factor for neutrophils, that continued to rise through 18h after bacterial inoculation. However, depletion of either IL-17 or KC/CXCL1 using monoclonal antibodies failed to sensitize to Acinetobacter infection. Further, IL-17 receptor KO mice were not sensitized to this infection. Collectively, these results suggest that there must be other chemotactic factors for neutrophils that can compensate for the absence of IL-17 and KC. Morphine, delivered by extended release pellet, sensitized two strains of mice to two strains of Acinetobacter, as measured by mortality to a sublethal challenge dose, and this effect was blocked by administration of the opioid-receptor antagonist, naltrexone. . Morphine increased Acinetobacter burdens in the organs and blood of infected mice, and increased the levels of pro-inflammatory cytokines. Evidence for an effect of morphine on neutrophil infiltration was obtained. Morphine decreased the total numbers of cells, as well as the total numbers of neutrophils and macrophages infiltrating into the peritoneal cavity. This inhibition of neutrophil accumulation correlated with suppression of levels of both IL-17 and KC/CXCL1. The evidence supports the conclusion that morphine sensitizes to Acinetobacter infection by suppressing the response of neutrophils, potentially via depression of neutrophil chemotactic factors IL-17 and KC. However, taken together with the data above there are probably additional factors in addition to IL-17 and KC that are sensitizing the animals to infection in the presence of morphine. In addition to these studies, the opioid-receptor dependency of morphine-mediated sensitization to Salmonella enteric serovar Typhimurium was examined. Previous experiments had determined that extended release morphine pellets sensitized mice to a sublethal dose of Salmonella, as determined by survival and bacterial burdens in the organs of infected mice, but naltrexone resulted in only incomplete reversal of the morphine-mediated effects. To further characterize the receptor dependency of the observed phenomenon, mu-opioid receptor knockout (MORKO) mice were used. MORKO mice were found to be completely resistant to the lethal effects of morphine plus infection observed in wild-type (WT) mice. In addition, MORKO mice showed greatly reduced bacterial burdens and pro-inflammatory cytokine levels when treated with morphine and challenged with a sublethal challenge dose of Salmonella, in comparison to WT mice. In summary, the studies presented in this thesis explored basic mechanisms of innate immunity to A. baumannii using a systemic model of infection. The work provides additional evidence that morphine sensitizes to infection, using models of Acinetobacter and Salmonella in mice. An implication of this work is use of caution in the administration of opioids in patients that are susceptible to opportunistic infections. / Microbiology and Immunology

Natural products are biologically active metabolites produced by fungi, bacteria, and plants that have an extended application in pharmaceutical and chemical industries. Because of their chemical versatility, flavoenzymes are commonly involved in natural product biosynthetic pathways. This has given rise to the identification of flavoenzymes that are promising candidates for biomedical and biotechnical applications. This dissertation discusses the characterization of three flavoenzymes involved in natural product biosynthesis. The class B flavin-dependent monooxygenases S-monoooxygenase from Allium sativum (AsFMO) and N-hydroxylating monooxygenase from Streptomyces sp. XY332 (FzmM) were studied. Both enzymes perform heteroatom oxidation as part of allicin or fosfazinomycin biosynthesis respectively. AsFMO was predicted to oxidize S-allyl-L-cysteine (SAC) to alliin in allicin biosynthesis. Surprisingly, AsFMO exhibited negligible activity with SAC, and instead was highly active with allyl mercaptan and NADPH. This contradicted the initial proposal and suggested that AsFMO is involved in an alternative path producing allicin directly from allyl mercaptan. FzmM was identified to perform multiple N-oxidations which lead to the formation of a nitro group. FzmM performed a highly coupled and specific reaction with L-aspartate and NADPH to produce nitrosuccinate. Both AsFMO and FzmM followed a kinetic mechanism representative of class B flavin-dependent monooxygenases with a rapid pro-R stereospecific reduction and the formation of a C(4a)-hydroperoxyflavin intermediate during oxidation. In addition, the AsFMO structure was obtained and consisted of two domains for FAD and NADPH binding signature of class B monooxygenases. The biochemical and structural study of the Acinetobacter baumannii siderophore interacting protein (BauF) was also accomplished. This enzyme is essential in acinetobactin mediated iron assimilation and is important for virulence. The characterization of the binding and reduction of acinetobactin-ferric iron complex revealed that BauF is specific for this substrate and does not utilize NAD(P)H as an electron donor. The unique activity and structure of BauF can aid future drug design. / Doctor of Philosophy / Plants, fungi, and bacteria synthesize and excrete unique chemicals called secondary metabolites or natural products. These compounds are used for many applications including dyes, flavorings, fragrances, and medicine. To make natural products, organisms use enzymes to perform complex reactions. Studying the enzymes that are involved in natural product pathways is important for understanding how secondary metabolites are made. Additionally, these enzymes can be engineered to perform reactions relevant to biotechnical applications. Our lab specializes in the study of flavoenzymes which use flavin chemistry for catalysis. Flavin is a yellow coenzyme that contributes to wide array of reactions by performing 1 or 2 electron transfers. This dissertation discussed the characterization of three flavoenzymes. The first enzyme is a S- monooxygenase from Allium sativum (garlic) called AsFMO. Reported here is the kinetic and structural characterization of AsFMO. We demonstrated that AsFMO was cabable of performing an unexpected reaction with allyl mercaptan likely converting it into allicin, the main flavor ingredient of garlic. Secondly, we reported the kinetic characterization of a nitro- forming enzyme termed FzmM. Nitro- formation is a valuable process as nitro- compounds are used in industrial organic synthesis. It was shown that FzmM performs nitro- formation with high efficiency and is specific for the substrate L-aspartate. Lastly, this work described the characterization of the the siderophore-interacting protein from Acinetobacter baumannii, BauF, which was predicted to be involved in iron acqusition. A. baumannii is a serious human pathogen with multidrug resistance, and inhibiting iron acquisition has been shown to prevent its survival. The characterization of the enzymes involved in this pathway is essential for developing new treatments for A. baumannii infection. We report the structure and function of BauF confirming its role in A. baumannii iron uptake and providing information that will aid in future drug design.

As infecções relacionadas à assistência à saúde (IRAS) são um grave problema de saúde pública cujo prognóstico tem sido desfavorecido pela emergência e endemicidade de bactérias multirresistentes (MRs). Neste cenário, seguindo uma tendência mundial, no Brasil, infecções por cepas de Acinetobacter baumannii MRs produtoras de carbapenemases do tipo OXA são atualmente consideradas uma emergência clínica e epidemiológica. Na falta de alternativas terapêuticas efetivas para infecções relacionadas, este trabalho objetivou avaliar efeitos sinérgicos (utilizando checkerboard e time-kill) decorrentes da combinação de 10 antimicrobianos de diferentes classes, contra 8 cepas MRs de A. baumannii, clonalmente não relacionadas, produtoras de carbapenemases do tipo OXA-23, OXA-72, OXA-58 e OXA-143, representativas de diferentes centros hospitalares do Brasil. Como resultado, a combinação amicacina/tigeciclina apresentou atividade sinérgica (S= ΣCIF ≤ 0,5) e parcialmente sinérgica (PS= &#931CIF ;0,5-0,75) contra 4 (50%) cepas produtoras de OXA-143 ou OXA-72, e 2 cepas (25%) produtoras de OXA-23, respectivamente. Por outro lado, a combinação polimixina B/imipenem apresentou atividade S e PS contra 3 (37,5%) isolados OXA-143, OXA-23 ou OXA-72 positivos, e 1 (12,5%) isolado produtor de OXA-58, respectivamente. Já, a combinação amicacina/ampicilina-sulbactam foi S contra 2 (25%) A. baumannii OXA-143 ou OXA-23 positivos, sendo PS contra dois (25%) A. baumannii OXA-58 ou OXA-143/23 positivos. De interesse, foi o efeito S da combinação polimixina B/vancomicina, contra 2 cepas (25%) produtoras de OXA-72 ou OXA-23. Por outro lado, a combinação ampicilina-sulbactam/rifampicina apresentou atividade PS contra 6 (75%) cepas produtoras das variantes OXA-23, OXA-143, OXA-72 ou OXA-58. Da mesma forma, rifampicina combinada com polimixina B foi sinérgica para uma cepa OXA-23 (12,5%) e PS para 5/8 (62,5%) cepas produtoras de OXA-72, OXA-58, OXA-23/-OXA143 ou OXA-143. O efeito sinérgico da combinação polimixina B/imipenem foi confirmado, in vivo, no modelo murino de infecção, tanto por avaliação histopatológica como por redução das UFC/g pulmão ou baço (p ≤ 0,05). Finalmente, foi avaliada a atividade, in vitro, do lípide catiônico brometo de dioctadecildimetilamônio (DDA), individualmente e em combinação com tigeciclina. DDA possui efeito bactericida, e potencializou sinergicamente a tigeciclina contra 2 (25%) cepas OXA-143 ou OXA-23 positivas. A atividade do DDA, assim como a atividade da sua combinação com tigeciclina foram efetivas já na segunda hora de interação, como avaliado pelas curvas de morte. Em resumo, o efeito sinérgico decorrente do uso combinado de amicacina, tigeciclina, polimixina B, imipenem, rifampicina ou ampicilina/sulbactam, pode constituir uma alternativa terapêutica para o tratamento de infecções produzidas por cepas de A. baumannii MRs produtoras de oxacilinases, sendo que nanofragmentos catiônicos de bicamada do lipídeo sintético de DDA tem potencial para consolidar um produto de aplicação clínica. / Healthcare-associated infections (HAIs) are a serious public health issue, which have been related with an unfavorable prognosis due to the emergence and endemicity of multidrug-resistant (MDR) bacteria. In this scenario, following a worldwide trend, in Brazil, infections produced by MDR OXA-type carbapenemase-producing Acinetobacter baumannii are currently considered a clinical and epidemiological urgency. In the absence of effective therapeutic alternatives for related infections, this study aimed to evaluate synergistic effects (by using time-kill and checkerboard assays) achieved by the combination of 10 different classes of antimicrobial against 8 strains of MDR, clonally unrelated, A. baumannii strains producing OXA-23, OXA-72, OXA-58 and OXA-143 carbapenemases, being representatives of different medical centers in Brazil. As a result, the combination of amikacin / tigecycline showed synergistic (S = ΣFIC ≤ 0.5) and partially synergistic (PS = 0.5 to 0.75 ΣFIC) activity against 4 (50%) OXA-72 or OXA-143 producing A. baumannii strains, and two strains (25%) producing OXA-23, respectively. Moreover, the combination of polymyxin B / imipenem showed S and PS activity against 3 (37.5%) OXA-143, OXA-23 and OXA-72 positive isolates, and 1 (12.5%) OXA-58 producer, respectively. On the other hand, the combination amikacin / ampicillin-sulbactam was S against 2 (25%) OXA-143 and OXA-23 positive strains, being PS against two (25%) OXA-58- and OXA-143/23-producing A. baumannii. Of interest was the synergistic effect achieved by polymyxin B plus vancomycin against two strains (25%) producing OXA-72 and OXA-23, respectively. Furthermore, the ampicilina-sulbactam / rifampicin combination displayed a PS activity against six (75%) strains producing OXA-23, OXA-143, OXA-72 or OXA-58-type enzymes. Likewise, rifampicin combined with polymyxin B was S against 1 (25%) OXA-23-positive A. baumannii being PS to 5/8 (62.5%) strains producing OXA-72, OXA-58, OXA-23/-OXA143 or OXA-143. The synergistic effect of the combination polymyxin B / imipenem was confirmed, in vivo, in the murine model of infection, by using both histopathological studies and bacterial clearance from the lungs and spleen (CFU/g, p≤ 0.05). Finally, we evaluated the in vitro activity of the cationic lipid dioctadecyldimethylammonium bromide (DDA), alone and in combination with tigecycline. DDA display a bactericidal effect, enhancing synergistically the activity of tigecycline against 2 (25%) OXA-143 and OXA-23 positive strains, respectively. DDA activity alone and in combination with tigecycline was effective on the second hour of interaction, as evaluated by time-kill assays. In summary, the synergistic effect resulting from the combined use of amikacin, tigecycline, polymyxin B, imipenem, rifampicin or ampicillin / sulbactam, could be an alternative therapy for the treatment of infections caused by MDR A. baumannii strains producing oxacilinases. On the other hand, cationic bilayer nanofragments of DDA has potential for consolidating a product for medical application.

As infecções relacionadas à assistência à saúde (IRAS) são um grave problema de saúde pública cujo prognóstico tem sido desfavorecido pela emergência e endemicidade de bactérias multirresistentes (MRs). Neste cenário, seguindo uma tendência mundial, no Brasil, infecções por cepas de Acinetobacter baumannii MRs produtoras de carbapenemases do tipo OXA são atualmente consideradas uma emergência clínica e epidemiológica. Na falta de alternativas terapêuticas efetivas para infecções relacionadas, este trabalho objetivou avaliar efeitos sinérgicos (utilizando checkerboard e time-kill) decorrentes da combinação de 10 antimicrobianos de diferentes classes, contra 8 cepas MRs de A. baumannii, clonalmente não relacionadas, produtoras de carbapenemases do tipo OXA-23, OXA-72, OXA-58 e OXA-143, representativas de diferentes centros hospitalares do Brasil. Como resultado, a combinação amicacina/tigeciclina apresentou atividade sinérgica (S= ΣCIF ≤ 0,5) e parcialmente sinérgica (PS= &#931CIF ;0,5-0,75) contra 4 (50%) cepas produtoras de OXA-143 ou OXA-72, e 2 cepas (25%) produtoras de OXA-23, respectivamente. Por outro lado, a combinação polimixina B/imipenem apresentou atividade S e PS contra 3 (37,5%) isolados OXA-143, OXA-23 ou OXA-72 positivos, e 1 (12,5%) isolado produtor de OXA-58, respectivamente. Já, a combinação amicacina/ampicilina-sulbactam foi S contra 2 (25%) A. baumannii OXA-143 ou OXA-23 positivos, sendo PS contra dois (25%) A. baumannii OXA-58 ou OXA-143/23 positivos. De interesse, foi o efeito S da combinação polimixina B/vancomicina, contra 2 cepas (25%) produtoras de OXA-72 ou OXA-23. Por outro lado, a combinação ampicilina-sulbactam/rifampicina apresentou atividade PS contra 6 (75%) cepas produtoras das variantes OXA-23, OXA-143, OXA-72 ou OXA-58. Da mesma forma, rifampicina combinada com polimixina B foi sinérgica para uma cepa OXA-23 (12,5%) e PS para 5/8 (62,5%) cepas produtoras de OXA-72, OXA-58, OXA-23/-OXA143 ou OXA-143. O efeito sinérgico da combinação polimixina B/imipenem foi confirmado, in vivo, no modelo murino de infecção, tanto por avaliação histopatológica como por redução das UFC/g pulmão ou baço (p ≤ 0,05). Finalmente, foi avaliada a atividade, in vitro, do lípide catiônico brometo de dioctadecildimetilamônio (DDA), individualmente e em combinação com tigeciclina. DDA possui efeito bactericida, e potencializou sinergicamente a tigeciclina contra 2 (25%) cepas OXA-143 ou OXA-23 positivas. A atividade do DDA, assim como a atividade da sua combinação com tigeciclina foram efetivas já na segunda hora de interação, como avaliado pelas curvas de morte. Em resumo, o efeito sinérgico decorrente do uso combinado de amicacina, tigeciclina, polimixina B, imipenem, rifampicina ou ampicilina/sulbactam, pode constituir uma alternativa terapêutica para o tratamento de infecções produzidas por cepas de A. baumannii MRs produtoras de oxacilinases, sendo que nanofragmentos catiônicos de bicamada do lipídeo sintético de DDA tem potencial para consolidar um produto de aplicação clínica. / Healthcare-associated infections (HAIs) are a serious public health issue, which have been related with an unfavorable prognosis due to the emergence and endemicity of multidrug-resistant (MDR) bacteria. In this scenario, following a worldwide trend, in Brazil, infections produced by MDR OXA-type carbapenemase-producing Acinetobacter baumannii are currently considered a clinical and epidemiological urgency. In the absence of effective therapeutic alternatives for related infections, this study aimed to evaluate synergistic effects (by using time-kill and checkerboard assays) achieved by the combination of 10 different classes of antimicrobial against 8 strains of MDR, clonally unrelated, A. baumannii strains producing OXA-23, OXA-72, OXA-58 and OXA-143 carbapenemases, being representatives of different medical centers in Brazil. As a result, the combination of amikacin / tigecycline showed synergistic (S = ΣFIC ≤ 0.5) and partially synergistic (PS = 0.5 to 0.75 ΣFIC) activity against 4 (50%) OXA-72 or OXA-143 producing A. baumannii strains, and two strains (25%) producing OXA-23, respectively. Moreover, the combination of polymyxin B / imipenem showed S and PS activity against 3 (37.5%) OXA-143, OXA-23 and OXA-72 positive isolates, and 1 (12.5%) OXA-58 producer, respectively. On the other hand, the combination amikacin / ampicillin-sulbactam was S against 2 (25%) OXA-143 and OXA-23 positive strains, being PS against two (25%) OXA-58- and OXA-143/23-producing A. baumannii. Of interest was the synergistic effect achieved by polymyxin B plus vancomycin against two strains (25%) producing OXA-72 and OXA-23, respectively. Furthermore, the ampicilina-sulbactam / rifampicin combination displayed a PS activity against six (75%) strains producing OXA-23, OXA-143, OXA-72 or OXA-58-type enzymes. Likewise, rifampicin combined with polymyxin B was S against 1 (25%) OXA-23-positive A. baumannii being PS to 5/8 (62.5%) strains producing OXA-72, OXA-58, OXA-23/-OXA143 or OXA-143. The synergistic effect of the combination polymyxin B / imipenem was confirmed, in vivo, in the murine model of infection, by using both histopathological studies and bacterial clearance from the lungs and spleen (CFU/g, p≤ 0.05). Finally, we evaluated the in vitro activity of the cationic lipid dioctadecyldimethylammonium bromide (DDA), alone and in combination with tigecycline. DDA display a bactericidal effect, enhancing synergistically the activity of tigecycline against 2 (25%) OXA-143 and OXA-23 positive strains, respectively. DDA activity alone and in combination with tigecycline was effective on the second hour of interaction, as evaluated by time-kill assays. In summary, the synergistic effect resulting from the combined use of amikacin, tigecycline, polymyxin B, imipenem, rifampicin or ampicillin / sulbactam, could be an alternative therapy for the treatment of infections caused by MDR A. baumannii strains producing oxacilinases. On the other hand, cationic bilayer nanofragments of DDA has potential for consolidating a product for medical application.

Acinetobacter baumannii is an important nosocomial pathogen characterized by the ability to acquire and develop complex resistance to antimicrobial agents. This capability is caused by eflux systems removing molecules of antibiotics from bacterial intracellular space. AdeABC is an RND-type chromosomal eflux system specific for A. baumannii which has a broad substrate spectrum. In this work, we focused on functional analysis of AdeABC to define its role in the resistance development to aminoglycosides in genetically different strains. We studied a set of 15 epidemiologically and genotypically well characterized strains of A. baumannii which were fully susceptible to aminoglycosides and other antibiotics primarily effective against this species. We determined genotyp of AdeABC for these strains and performed a selection for resistant variants in the presence of netilmicin. Using real-time qRT-PCR we compared the expression of the transporter gene adeB in originally sensitive strains and selected variants. The obtained results confirmed that the increased expression of AdeABC significantly reduces susceptibility to aminoglycosides and other antibiotics. The results also suggest that the efflux system provides a significant selective advantage for nosocomial strains of A. baumannii.