Made available in DSpace on 2016-08-10T10:55:37Z (GMT). No. of bitstreams: 1 ALINE CRISTINA BATISTA RESENDE.pdf: 921510 bytes, checksum: b4208f02be180566c75ce748e7f29585 (MD5) Previous issue date: 2009-03-27 / Introduction: The emergence of antimicrobial-resistant genes is becoming an increasingly important ecological issue. The selective pressure of antimicrobials is a significant factor in the selection and dissemination of resistance genes in the environment. Objective: The objective of this present study was to detect, isolate and identify Pseudomonas aeruginosa, Acinetobacter spp., Klebsiella pneumoniae and Escherichia coli in the sewage effluents of 10 hospitals located in Goiânia and to assess the susceptibility profile of the isolated microorganisms. Methodology: The samples collected in the hospital effluents and from the Goiânia sewage treatment station were identified using biochemical tests, Gram-staining, microscopy, motility-indole-ornithine (MIO) medium, urea, catalase, oxidase, TAF, API 20E, API STAPH (Bio Merieux) and bile-esculin agar. Following identification, the profile of susceptibility to the antimicrobials was evaluated using the agar diffusion disk susceptibility test, later classified according to the NCCLS (2002). Next, the E. coli strains were analyzed regards the possibility extended spectrum β-lactamase production (ESBL), using phenotypic tests. Results: A total of 73 microorganisms gram-negative were identified including 10 strains of E. coli (14,92 por cent), 10 strains of K. pneumoniae (14,92 por cent), 3 of P. aeruginosa (4.47 ´por cent) and 1 of A. baumannii (1.49 por cent). The E. coli strains presented 100 por cent total resistance to aztreonam, 40 por cent to ampicillin, 30 por cent to piperacillin, 20% to ciprofloxacin and 10 por cent to gentamicin. None of the aztreonam-resistant E. coli strains were identified as being extended spectrum Beta-lactamase producers. P. aeruginosa presented 100 por cent resistance to ampicillin-sulbactam and 100 por cent intermediary resistance to gentamicin. Strains of K. pneumoniae were resistant to ampicillin (70 por cent) and to piperacillin (20 por cent). Additionally, 50 por cent showed intermediate resistance to piperacillin. No cases of total resistance were detected in the sample of A. baumannii, which presented only intermediary resistance to aztreonam and ceftriaxone. Conclusion: The E. coli, P. aeruginosa, K. pneumoniae and A. baumannii isolates showed low resistance rates. None of the bacterial strains produced ESBL or carbapenems. The samples of A. baumannii had the highest susceptibility profile of all the microorganisms isolated. / INTRODUÇÃO: A emergência de genes de resistência antimicrobiana está cada vez mais se tornando um problema ecológico. A pressão seletiva dos antimicrobianos é um importante fator de seleção e disseminação dos genes de resistência no meio ambiente. OBJETIVO: O presente trabalho teve como objetivo a detecção, o isolamento e identificação de Pseudomonas aeruginosa, Acinetobacter spp., klebsiella pneumoniae e Escherichia coli de efluentes de esgoto de 10 hospitais localizados em Goiânia e a caracterização do perfil de susceptibilidade dos microrganismos isolados. METODOLOGIA: As amostras coletadas nos efluentes hospitalares e da Estação de Tratamento de Esgoto de Goiânia (ETE) foram identificadas e analisadas quanto ao perfil de susceptibilidade aos antimicrobianos, por provas bioquimicas, com a coloração de Gram, bacterioscopia, teste de motilidade (MIO), uréia, catalase, oxidase, TAF, API 20E, API STAPH (Bio Merieux) e teste de bilesculina. Após a identificação, a avaliação do perfil de susceptibilidade aos antimicrobianos foi realizada através do método de antibiograma em placa, e depois classificada de acordo com o NCCLS (2002). Posteriormente, as cepas de E.coli foram analisadas quanto a possível produção de Beta-lactamase de amplo espectro (ESBL), utilizando-se testes fenotípicos. RESULTADOS: Foram identificados 73 microrganismos gramnegativos, dentre estes 10(14,92 por cento) E.coli, 10(14,92 por cento) K.pneumoniae, 3(4,47 por cento) P.aeruginosa e 1(1,49 por cento) A.baumannii. As cepas de E.coli apresentaram 100 por cento de resistência total ao aztreonam, 40 por cento à ampicilina, 30 por cento à piperacilina, 20 por cento à ciprofloxacina, 10 por cento à gentamicina. Nenhuma cepa de E.coli resistente ao aztreonam foi identificada como produtora de β-lactamase de espectro ampliado (ESBL). P.aeruginosa apresentou 100% de resistência à ampicilina-sulbactam e 100 por cento apresentaram resistência intermediária à gentamicina. 70 por cento das cepas de K.pneumoniae apresentaram resitência à ampicilina e 50 por cento apresentaram resistência intermediária a este mesmo antimicrobiano. Nenhuma resistência total foi detectada na amostra de A.baumannii apresentando apenas resistência intermediária ao aztreonam e a ceftriaxona. CONCLUSÃO: As amostras de E.coli, P.aeruginosa, K.pneumoniae e A.baumannii apresentaram baixas taxas de resistência aos antimicrobianos, em nenhum dos isolados foi detectada a produção de Beta-lactamase de espectro ampliado e carbapenemase e as amostras de A.baumannii apresentaram o maior perfil de susceptibilidade entre todos os microrganismos isolados.

Orientador: Carla Raquel Fontana / Resumo: A doença periodontal é um processo patológico infecto-inflamatório, que pode causar eventual perda do elemento dental. O principal tratamento realizado é a raspagem e alisamento radicular (RAR), que embora seja efetiva, tem como fator limitante a dificuldade técnica de intervir em biofilmes de áreas de difícil acesso. As associações com antibióticos podem ocasionar efeitos colaterais e em bactérias resistentes podem ser ineficazes. Desta forma, a terapia fotodinâmica antimicrobiana (TFDa) surge como uma opção adjuvante, para o tratamento das doenças periodontais. Este trabalho investigou, in vitro, o potencial de redução e desenvolvimento de resistência bacteriana, em um pool clínico resistente ao metronidazol, frente a múltiplos ciclos de TFDa, em fase planctônica e biofilme, utilizando azul de metileno, clorina-e6 e curcumina, como fotossensibilizadores (FS). Para realização dos ciclos de indução de resistência, foi necessário encontrar as condições sub ótimas da TFDa, através da quantificação das colônias formadas por mililitro de amostra (UFC/mL). Os seguintes parâmetros foram encontrados em fase planctônica: 50 µg/mL de azul de metileno e dose de luz de 120 J/cm2 com redução de 1,67 log10, clorina-e6 e curcumina obtiveram reduções de 1,91 log10 e 1,76 log10, respectivamente, com dose de luz de 25 J/cm2 e 100 µg/mL de FS. Posteriormente o pool de bactérias foi sujeito a múltiplas exposições de TFDa, seguido de um período de recuperação, em que células bacterianas sobreviv... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Periodontal disease is an infectious-inflammatory pathological process, which can cause eventual loss of the dental element. The main treatment is scaling and root planing (RAR), which, although effective, has as a limiting factor the technical difficulty of intervening in biofilms of difficult access areas. Associations with antibiotics can cause side effects and in resistant bacteria they may be ineffective. In this way, antimicrobial photodynamic therapy (aPDT) appears as an adjuvant option for the treatment of periodontal diseases. This study investigated, in vitro, the potential for reduction and development of bacterial resistance in a clinical pool, resistant to metronidazole, against multiple cycles of aPDT in planktonic phase and biofilm using methylene blue, chlorine-e6 and curcumin as photosensitizers (PS). For resistance induction cycles, it was necessary to find the sub optimal conditions of the aPDT, by quantifying the colonies formed per milliliter of sample (CFU/mL). The following parameters were found in the planktonic phase: 50 μg/mL methylene blue and light dose of 120 J/cm2 with a reduction of 1.67 log10, chlorine-e6 and curcumina obtained reductions of 1.91 log10 and 1.76 log10, respectively, with light dose of 25 J/cm2 and 100 μg/mL PS. Subsequently, the pool of bacteria was subjected to multiple exposures of aPDT, followed by a recovery period, in which surviving bacterial cells were resuspended in the same treatment, and so on until resistance developm... (Complete abstract click electronic access below) / Doutor

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-08T12:45:24Z No. of bitstreams: 1 Elane Rafaela Cordeiro Nunes.pdf: 921731 bytes, checksum: eddcacad472e8bebb52d60738ec901b1 (MD5) / Made available in DSpace on 2017-02-08T12:45:24Z (GMT). No. of bitstreams: 1 Elane Rafaela Cordeiro Nunes.pdf: 921731 bytes, checksum: eddcacad472e8bebb52d60738ec901b1 (MD5) Previous issue date: 2013-02-08 / The widespread use of antimicrobials for veterinary and human medicine is the main cause of the selection of resistant bacteria. Monitoring antimicrobial use the occurrence of disease and profile of bacterial resistance to antimicrobial assists in policy formulation and solution of public events related to ecological and microbial diseases. The use of these drugs for milking animals may result in residues in milk and favors the selection of bacteria. In the Southeast and South of Brazil there is more research on antimicrobial residues in raw milk. Thus, the objective of this study was to evaluate the frequency of antibiotic residues in raw milk produced in farms located in the microregion of Garanhuns of the state Pernambuco. Like, make a survey of active antimicrobials sold in pharmacies veterinary and human that region. We analyzed 84 samples of milk collected in expansion tanks or drums, coming from 84 herds in 19 counties. In each farm received a questionnaire on milk production, health management and use antimicrobial. The survey of antimicrobials in milk was performed with kit Charm Cowside II®. The results showed that 14.29% of fresh milk were positive for the presence of antimicrobial residues in milk. The only variable significantly associated with the presence of antimicrobial residues in milk was the type of milking (p=0.038) with a predominance of positive results in the technique of milking mechanics the foot. The active principle antimicrobials often used in dairy cattle herd are tetracyclines, aminoglycosides anda penicillins. The presence of antimicrobial residues in fresh milk was detected in over 50% of the municipalities of the region of Garanhuns, PE. The milking technique as well as the hygienic milking may favor the presence of antimicrobial residues in fresh milk. There were differences in the classes of antimicrobials used in the dairy herd in 47.3% of the municipalities. The diagnosis of antimicrobials showed some of the health and economic risks associated with the use of these drugs in dairy cattle herd in the region. And hence the need to implement intervention strategies in order to control the irrational use of these drugs in animals, and the development of research related to antimicrobial efficacy in human and veterinary medicine. / O amplo uso de antimicrobianos na medicina veterinária e humana é a principal causa da seleção de bactérias resistentes a antimicrobianos. Monitorar o uso de antimicrobianos, a ocorrência de doenças e o perfil de resistência bacteriana a estes fármacos auxilia na formulação de política públicas e na elucidação de fatos relacionados a ecologica microbiana e das doenças. No rebanho leiteiro, a utilização desses fármacos pode resultar na presença de resíduos no leite, o que favorece a seleção de bactérias. As pesquisas elaboradas no intuito de detectar resíduos de antimicrobiano em leite in natura estão concentradas nas regiões Sudeste e Sul do país. Desse modo, o objetivo do presente trabalho foi pesquisar resíduos de resíduos de antimicrobianos em leite in natura produzido em propriedades rurais localizadas na microrregião de Garanhuns do Estado de Pernambuco. Foram analisadas 84 amostras de leite coletadas em tanques de expansão ou latões, procedentes de 84 propriedades localizadas em 19 municípios. Em cada propriedade foi aplicado um questionário sobre produção leiteira e manejo sanitário e uso de antimicrobiano. A pesquisa de antimicrobianos no leite foi realizada com o Kit Charm Cowside II®. Os resultados evidenciaram que 14,29% do leite in natura foram positivos para presença de resíduos de antimicrobianos no leite. A única variável de manejo que teve associação significativa com a presença de resíduos de antimicrobianos no leite foi o tipo de ordenha (p=0,038) com predominância de resultados positivos na técnica de ordenha mecânica ao pé. Os princípios ativos de antimicrobianos frequentemente utilizados no rebanho bovino leiteiro da microrregião pertencem à classe das tetraciclinas, aminoglicosídeos e penicilinas. A presença de resíduos de antimicrobianos em leite in natura foi detectada em mais de 50% dos municípios da microrregião de Garanhuns, PE. A técnica de ordenha, assim como o manejo higiênico durante a ordenha pode favorecer a presença de resíduos de antimicrobianos em leite in natura. Houve diferença nas classes de antimicrobianos utilizadas no rebanho leiteiro em 47,3% dos municípios. O diagnóstico sobre os antimicrobianos evidenciou alguns dos riscos sanitários e econômicos associados ao uso destes fármacos no rebanho bovino leiteiro da região. E consequentemente, a necessidade da implantação de estratégias de intervenção, visando controlar o uso irracional desses fármacos nos animais, e a elaboração de pesquisas relacionada à eficácia antimicrobiana na medicina humana e veterinária.

Os psitacídeos estão entre as espécies de aves mais apreendidas e encaminhadas aos centros de triagem animal em São Paulo. Também são comumente mantidos em ambiente doméstico como aves de estimação. A manutenção destas aves em cativeiro pode representar um risco zoonótico e contribuir para a propagação das estirpes de enterobactérias multirresistentes, como Klebsiella spp. produtora de beta-lactamase de espectro estendido (ESBLs), que podem interferir no tratamento de infecções nosocomiais em humanos. O objetivo deste estudo foi identificar e caracterizar estirpes de Klebsiella spp. isoladas de secreções respiratórias de 46 psitácideos doentes, determinando a virulência e o perfil de resistência a 15 antimicrobianos. Dentre as 19 estirpes de Klebsiella spp. isoladas, 16 (16/19) foram identificadas como Klebsiella pneumoniae, e três (3/19) foram identificadas como Klebsiella oxytoca. O perfil de suscetibilidade aos antimicrobianos demonstrou alta resistência para ampicilina (89,5%), e o perfil de virulência demonstrou uma alta prevalência dos genes fimH (94,7%), kpn (89,4%), uge (84,2%) e irp-2 (78,9%). Três estirpes de K. pneumoniae foram positivas para produção de beta-lactamase de espectro estendido. Estas estirpes foram classificadas nos sequence types (STs) ST15, ST147 e ST307. Esses três grupos clonais representam os principais responsáveis por surtos de infecções hospitalares por K. pneumoniae no mundo. No entanto, esse é o primeiro relato desses clones como causadores de doença em aves. Esses dados indicam a ocorrência de K. pneumoniae produtora de CTX-M-15 e CTX-M-8 em psitacídeos cativos e confirmam o potencial zoonótico e antropozoonótico do agente, destacando a relevância clínica para humanos e animais. / Psittacine birds are among the most seized bird species that are sent to animal sorting centers in São Paulo. They are also commonly kept in the domestic environment like pet birds. The maintenance of these birds in captivity may represent a zoonotic risk and contribute to the propagation of strains of multiresistant enterobacteria, such as Klebsiella spp. beta-lactamase extended-spectrum (ESBLs), which may interfere in the treatment of nosocomial infections in humans. The objective of this study was to identify and characterize strains of Klebsiella spp. isolated from respiratory secretions of 46 diseased psittacines, determining virulence and resistance profile to 15 antimicrobials. Among the 19 strains of Klebsiella spp. isolated, 16 (16/19) were identified as Klebsiella pneumoniae, and three (3/19) were identified as Klebsiella oxytoca. The antimicrobial susceptibility profile demonstrated high resistance to ampicillin (89.5%), and the virulence profile demonstrated a high prevalence of fimH (94.7%), kpn (89.4%), uge (84.2% %) and irp-2 (78.9%). Three strains of K. pneumoniae were positive for extended-spectrum beta-lactamase production. These strains were classified in sequence types (STs) ST15, ST147 and ST307. These three clonal groups represent the main responders for outbreaks of K. pneumoniae nosocomial infections worldwide. However, this is the first account of these clones as causing disease in birds. These data indicate the occurrence of K. pneumoniae producing CTX-M-15 and CTX-M-8 in captive parrots and confirm the zoonotic and anthropozoonotic potential of the agent, highlighting the clinical relevance for humans and animals.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-04-05T19:32:12Z No. of bitstreams: 1 2012 - Gabriela Viana da Silva.pdf: 1385561 bytes, checksum: ebedf6cb2382daee2a3bf246edc9848f (MD5) / Made available in DSpace on 2017-04-05T19:32:12Z (GMT). No. of bitstreams: 1 2012 - Gabriela Viana da Silva.pdf: 1385561 bytes, checksum: ebedf6cb2382daee2a3bf246edc9848f (MD5) Previous issue date: 2012-03-27 / CAPES / The sheep milk production in Brazil is a relatively new activity, whose production is directed towards the manufacture of cheeses, yogurts and other products. Therewith a deficit of scientific papers related to this activity. Considered an excellent substrate, many pathogens can be transmitted to humans through consumption of milk and its derivatives, including Staphylococcus spp., one of the main agents involved in outbreaks of food poisoning. Moreover, the increasing of antimicrobial resistance is becoming a global public health problem. Within this problematic, this study aimed to identify the species of Staphylococcus and its antimicrobial susceptibility profile of current use in human and veterinary medicine, isolated from milk of sheep origin from rural properties in the Meridional Agreste of Pernambuco. We identified 13 different species, three coagulase-positive and 10 coagulase-negative and two strains identified only as SCN, especially Staphylococcus aureus (29) followed by S. chromogenes (15) and S. intermedius (9) by its frequency. The determination of antimicrobial susceptibility by disk diffusion revealed high rates of resistance to penicillin (53.2%) and ampicillin (45.6%). The susceptibility to oxacillin was evaluated by the disk diffusion methods for oxacillin and cefoxitin, Screen Agar and determination of Minimum Inhibitory Concentration through tests of broth microdilution and agar, the latter two detected resistance in 24% and 6% of isolates, respectively. All isolates were negative for the presence of blaZ and mecA genes and was not observed a correlation between phenotypic and genotypic testing for resistance to beta-lactams. The results show that this species is a source of infection, through its products, Staphylococcus potentially pathogenic to humans. / A produ??o de leite ovino no Brasil ? uma atividade relativamente nova, cuja produ??o est? direcionada para a confec??o de queijos, iogurtes e outros derivados. Com isso h? uma defict de trabalhos cient?ficos ligados a esta atividade. Considerado um excelente substrato, muitos micro-organismos patog?nicos podem ser veiculados ao homem atrav?s do consumo de leite e seus derivados, entre eles Staphylococcus spp., um dos principais agentes envolvidos em surtos de intoxica??es alimentares. Al?m disso, o crescente aumento da resist?ncia aos antimicrobianos vem se constituindo um problema de sa?de p?blica global. Dentro desta problem?tica, o presente estudo objetivou identificar as esp?cies de Staphylococcus e seu perfil de suscetibilidade aos antimicrobianos de uso corrente em medicina humana e veterin?ria, isoladas de leite de origem ovina provenientes de propriedades rurais no Agreste Meridional de Pernambuco. Foram identificadas 13 esp?cies diferentes, sendo tr?s do grupo Staphylococcus coagulase-positivo e 10 de Staphylococcus coagulase-negativo e duas cepas identificadas apenas como SCN, destacando-se por sua frequ?ncia Staphylococcus aureus (29) seguida pelo S. chromogenes (15) e S. intermedius (9). A determina??o da suscetibilidade aos antimicrobianos pelo teste disco difus?o revelou elevados percentuais de resist?ncia ? penicilina (53,2%) e ampicilina (45,6%). A suscetibilidade a oxacilina foi avaliada atrav?s dos m?todos de disco-difus?o para oxacilina e cefoxitina, Screen Agar e determina??o da Concentra??o Inibit?ria M?nima atrav?s dos testes de Microdilui??o em Caldo e em Agar, tendo estes dois ?ltimos detectado resist?ncia em 24% e 6% dos isolados, respectivamente. Todos os isolados foram negativos para a presen?a dos genes blaZ e mecA, n?o tendo sido observada uma correla??o entre os testes fenot?picos e genot?picos para a resist?ncia aos beta-lact?micos. Os resultados obtidos evidenciam que esta esp?cie animal ? mais uma fonte de infec??o e veiculadora, atrav?s de seus produtos, de Staphylococcus potencialmente patog?nicos para o homem.

Fluoroquinolones are a class of antibiotics used clinically to treat a wide array of bacterial infections. These therapeutics act by targeting a bacterial enzyme required for cell viability, bacterial type-II topoisomerases. Fluoroquinolones act by forming a ternary complex with bacterial type II topoisomerases and cleaved DNA; religation of DNA is subsequently blocked, therefore leading to bacterial cell death. In ternary complex the keto-acid moiety of the fluoroquinolone is complexed with a divalent magnesium ion, forming a drug-magnesium-water bridge to a serine and an aspartate (or glutamate) residue on helix-4 of the topoisomerase enzyme. A major issue with fluoroquinolones is the rise in bacterial resistance. Resistance arises through substitutions of the serine or aspartate/glutamate residue, therefore preventing formation of the magnesium-water bridge and dramatically diminishing the overall antibiotic activity of the fluoroquinolone. Quinazoline-2,4-diones are structurally similar to fluoroquinolones; diones also form a ternary complex similar to fluoroquinolones, however, these complexes are less active due to lack of a potent magnesium-water bridge interaction in helix-4. While quinazoline-2,4-diones are therefore less potent antibiotics, their non-reliance on the magnesium water bridge generally affords equipotent activity with wild-type and fluoroquinolone-resistant strains of bacteria. The first objective of this work was to probe the helix-4 interaction of the bacterial type-II topoisomerase by quinazoline-2,4-dione modification, specifically at the N3 and C4 positions of the quinazoline-2,4-dione scaffold to afford potentially new binding contacts. These modified quinazoline-2,4-diones will provide deeper understanding of the helix-4 interaction and potentially afford potent novel quinazoline-2,4-dione scaffolds, against both wild-type and resistant bacteria, for iterative drug design. Metabolism is one of the primary sources of detoxification, inactivation, and clearance of drugs from the body and is a critical consideration for all early stage therapeutic development. Clinically used fluoroquinolones, i.e. Moxifloxacin and Ciprofloxacin, historically are metabolically stable, and are not known to be metabolized by Phase I and/or Phase II drug metabolizing enzymes. However, major modifications to the Moxifloxacin and Ciprofloxacin scaffolds, due to the development of next generation antibiotics, may display different metabolic stability profiles. Moreover, metabolism of quinazoline-2,4-diones, developed for fluoroquinolone-resistant bacteria, is not extensively studied and may be subject to different metabolic liabilities that may render the quinazoline-2,4-dione an ineffective potential antibiotic. The second objective of this work was to determine the in vitro Phase I and Phase II metabolic stabilities of fluoroquinolone and quinazoline-2,4-dione scaffolds to determine any structural features that render the potential therapeutic a metabolic liability. The results from these two objectives have led to the discovery of a novel bacterial type-II topoisomerase catalytic inhibitor and the acquisition of initial metabolic stability data of fluoroquinolone and quinazoline-2,4-dione scaffolds. These findings further promote research into quinazoline-2,4-diones as bacterial topoisomerase targets, and provide metabolic considerations for both fluoroquinolone and quinazoline-2,4-dione therapeutic development, which is severely underrepresented in the field of quinolone antibiotics.

Bacterial resistance to non-antibiotic agents is being increasingly studied. Plasmid-mediated resistance to cationic agents, which are important biocides, has been described in antibiotic-resistant Staphylococcus aureus. Multi-resistant Staphylococcus aureus (MRSA) are often found to express resistance to a range of cationic biocides including quaternary ammonium compounds (QACs), biguanides, diamidino compounds, cationic dyes and nuclear stains. Three resistance determinants, qacA, qacB and smr genes, have been identified that confer resistance to cationic biocides in staphylococci. These genes encode multi-drug efflux pumps that remove the cationic biocides from the cytoplasm using a membrane bound pumping mechanism dependent on the cell's proton-motive force (PMF). This prevents the build up of lethal concentrations of cationic compounds within the cytoplasm avoiding cell death.This research project has focused on the S. aureus strain WBG4364, a transcipient strain carrying the cationic biocide resistant plasmid pWBG1773. The plasmid encodes resistance to several QACs, including benzalkonium chloride and CTAB, and cationic dyes rhodamine 6G, crystal violet and safranin O but not to the dye ethidium bromide and therefore differing from other cationic biocide resistant plasmids previously identified in staphylococci (Emslie et al. 1986). This unique phenotype was further classified in this study alongside those strains carrying the qac gene families, qacA/B and smr.Plasmid pWBG1773 was cloned, sequenced and analysed to reveal a unique plasmid of 2,916 bp in length. Plasmid pWBG1773 was placed with the pC194 family of rolling-circle replicating plasmids. This family appear to be largely composed of interchangeable cassette structures.The plasmid was found to carry three ORFs, designated ORF1, ORF2 and ORF3. ORF1 was homologous to rep genes of small staphylococcal ++ / plasmids belonging to the pC194 rolling-circle replication family and has been redefined as repWBG1773. ORF2 was found to have no similarity to any proteins of known function in the GenBank database whereas ORF3 was found to have homology to the marR gene, a regulator of the multiple antibiotic resistance (mar) operon of Gram-negative organisms. MIC analysis of these ORFs found both ORF2 and ORF3 were essential for expression of resistance to cationic biocides. The exact ORF2 sequence required for resistance to be expressed was reduced to only 141 nt in size. This translated to a 47 aa sequence that contained a hydrophobic C-terminus indicating ORF2 to be a membrane-bound protein. The aa sequence of ORF3 contained a helix-turn-helix motif characteristic of the DNA binding domains of MarR-like proteins. Further analysis of pWBG1773 identified a putative 'marbox', a binding site for the homologous transcriptional activators of mar, within the ORF2 sequence. This indicated that ORF3 was binding to the 'marbox' sequence and activating transcription. Induction studies have not been able to ascertain any compounds capable of interacting with the ORF3 regulatory protein resulting in induction of cationic biocide resistance. Each ORF when analysed alone had no effect on the expression of cationic biocide resistance and it is thought that a efflux pump was not involved. This is further corroborated by the CCCP efflux experiments performed in an attempt to determine the mechanism of resistance. The unique ORFs of plasmid pWBG1773 appears to encode a novel cationic biocide resistance phenotype and mechanism.MRSA strains from all around the world were analysed to determine if they possessed sequences homologous to ORF2 and ORF3. Sequences sharing a high degree of homology to ORF2 and/or ORF3 were detected in several MRSA strains including strains sensitive to all cationic ++ / biocides tested. These findings suggest that the appearance of ORF2 and ORF3 sequences in MRSAs was not an isolated event and the fact that some MRSAs do not carry both ORF2 and ORF3 sequences simultaneously indicates that these genes have another role that does not involve expression of resistance to cationic biocides.Bacteria are noteworthy for their remarkable ability to adapt to changes in their environments and possess an impressive set of tools with which to adjust the blueprint of the cell to this change. The acquisition of a single system that may decrease a potential pathogenic organisms susceptibility to a wide range of cationic biocides, such as seen in pWBG1773, poses a clinical threat, one that needs to be thoroughly investigated.

Biosynthetic natural products are invaluable resources that have been gleaned from the environment for generations, and they play an essential role in drug development. Natural product biosynthesis also possesses the latent ability to affect biological systems adversely. This work implements recent advances in genomic, proteomic and microbiological technologies to understand further biosynthetic molecules that may influence progression of human disease. Azinomycin A and B are antitumor metabolites isolated from the terrestrial bacterium Streptomyces sahachiroi. The azinomycins possess an unusual aziridine [1,2-a] pyrrolidine ring that reacts in concert with an epoxide moiety to produce DNA interstrand cross-links. Genomic sequencing of S. sahachiroi revealed a putative azinomycin resistance protein (AziR). Overexpression of AziR in heterologous hosts demonstrated the protein increases cell viability and decreases DNA damage response in the presence of azinomycin. Fluorescence titration indicated AziR functions as an azinomycin binding protein. An understanding of azinomycin resistance is important for future engineering and drug delivery strategies. Additionally, the S. sahachiroi draft genome obtained via 454 pyrosequencing and Illumina sequencing revealed several silent secondary metabolic pathways that may provide new natural products with biomedical application. β-lactoglobulin (BLG), the most abundant whey protein in bovine milk, has been observed to promote the self-condensation of retinal and similar α,β-unsaturated aldehydes. BLG is a possible non-genetic instigator of cycloretinal and A2E accumulation in the macula, a condition associated with age-related macular degeneration. BLG-mediated terpenal condensation has been optimized for in vitro study with the retinal mimic citral. In rabbits fed retinal and BLG or skim milk, cycloretinal formation was detected in the blood by 1H-NMR, and SDS-PAGE analysis indicated BLG was present in blood serum, suggesting the protein survives ingestion and retains catalytic activity. Mass spectrometry and site-directed mutagenesis provided mechanistic insight toward this unusual moonlighting behavior. The experiments described in this dissertation serve to further natural product biosynthesis discovery and elucidation as they relate to consequences for human health. Efforts to solve azinomycin biosynthesis via enzymatic reconstitution, characterize compounds produced by orphan gene clusters within S. sahachiroi, and obtain a clear mechanism for BLG-promoted cycloterpenal formation are immediate goals within the respective projects.

Orientador: Carlos Magno Castelo Branco Fortaleza / Resumo: Atualmente, estamos diante de uma notável presença de isolados de enterobactérias resistentes aos carbapenêmicos em um hospital público do município de Bauru-SP desde outubro de 2012. No entanto, não estão disponíveis estudos relacionando a epidemiologia e os fatores associados à aquisição de tais isolados. Este estudo teve como objetivo identificar fatores de risco para aquisição de Enterobactérias Resistentes aos Carbapenêmicos (CRE) em pacientes internados no Hospital Estadual Bauru, os fatores associados ao desenvolvimento de quadro infeccioso em uma coorte de pacientes colonizados por CRE e os fatores preditores de óbito. Foram incluídos pacientes do local de estudo que apresentaram colonização do trato digestório por CRE, de outubro de 2012 a dezembro de 2016, dos quais foram levantados dados clínicos e demográficos. Os isolados foram identificados por métodos fenotípicos e foram testadas as suscetibilidades por concentração inibitória mínima (MIC). Realizamos um estudo de caso-controle que incluiu 427 casos e igual número de controles. Os fatores de risco observados foram queimadura (HR 3,91; IC95% 2,36-6,46; p=<0,001), índice de Charlson (HR 1,12; IC95% 1,05-1,20; p=<0,001), uso prévio de esteróides (HR 2,79; IC95% 1,94-4,02; p=<0,001) e antimicrobianos como as penicilinas/inibidores de beta-lactamases (HR 2,01; IC95% 1,43-2,82; p=<0,001), cefalosporinas de 3ª. e 4ª. gerações (HR 2,45; IC95% 1,75-3,44; p=<0,001), quinolonas (HR 1,70; IC95% 1,75-2,45; p=0,003) e anaero... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Currently, we are facing a remarkable presence of isolates of carbapenem-resistant enterobacteriacea in Bauru city public hospital, São Paulo state, Brazil, since october 2012. However no studies are available relating the epidemiology and the factors associated with acquisition of such isolates. The purpose this study was to identify risk factors for acquisition of Carbapenem Resistant Enterobacteriaceae in patients hospitalized at the Bauru State Hospital, factors associated with the development of infectious disease in a cohort of patients colonized by CLP and factors predicting death. We included patients from the study site who presented colonization of the digestive tract by CRE, from October 2012 to December 2016, from which clinical and demographic data were collected. Isolates were identified by phenotypic methods and susceptibilities were tested by minimum inhibitory concentration (MIC). We performed a case-control study that included 427 cases and an equal number of controls. The risk factors observed were burn (HR 3.91, 95% CI 2.36-6.46, p = 0.001), Charlson index (HR 1.12, 95% CI 1.05-1.20, p = <0.001), previous use of steroids (HR 2.79, 95% CI 1.94-4.02, p = <0.001) and antimicrobials such as penicillins / beta-lactamase inhibitors (HR 2.01, 95% CI, 43-2.82, p = <0.001), cephalosporins of 3rd. and 4ª. (HR 2.45, IC 95% 1.75-3.44, p = 0.001), quinolones (HR 1.70, IC 95% 1.75-2.45, p = 0.003) and anaerobicides (HR 1, 63, 95% CI 1.04-2.56, p = 0.03). The cohort stud... (Complete abstract click electronic access below) / Doutor

Cancer represents one of the leading causes of death worldwide and efforts to discover more effective anticancer therapies have led to the synthesis of a wide diversity of molecular species. Another major challenge is the anti-infective treatment, because despite the large arsenal of active substances available, many show inefficiency due to the rapid emergence of resistant bacterial strains. As a result, arises the necessity of finding new active substances, more effective and targeted properties in both treatments of neoplasms as microbial resistance. Notably, the Triazenes compounds (TZCs) have been asserting itself as a promising class of metallodrugs with relevant antimicrobial and antiproliferative activity. Moreover, the association of pharmacophoric radical TZC with metal ions, such as gold, leads to a significant increase in biological activity. Because of this wide pharmacological versatility, this study aimed the evaluating in vitro of the biological activity of a compound TZC complexed with ion gold (I). The antibacterial activity was carried out by the conventional method of broth microdilution, through the technique of the minimum inhibitory concentration (MIC), against bacterial strains reference standard American Type Culture Collection (ATCC), clinical isolates with multidrug resistance (MDR) and clinical isolates biofilm producers. Cytotoxicity was evaluated by colorimetric assay based on the reduction of bromide of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) against the standard cell line K562 (Chronic Myeloid Leukemia). The results obtained demonstrate that the compound in study present a narrow spectrum of action, being active only against microorganisms classified as Gram positive, moreover, proved to be active against all isolates producing biofilm when compared to non-producing strains of biofilm. Also showed remarkable cytotoxic activity, with IC50 4.96 μM. Thus, these results demonstrate an alternative to the design of a new class of antibacterial and antitumor metallodrugs with activity. / O câncer representa uma das principais causas de óbito no mundo e esforços para descobrir terapias antineoplásicas mais eficazes têm conduzido à síntese de inúmeras moléculas. Outro grande desafio é o tratamento anti-infeccioso, pois apesar do grande arsenal de substâncias ativas disponíveis, muitas mostram-se ineficazes devido ao rápido aparecimento de estirpes bacterianas resistentes. Em razão disso, surge a necessidade da descoberta de novas substâncias ativas, com propriedades mais eficazes e direcionadas, tanto nos tratamentos de neoplasias como de resistência microbiana. Notoriamente, os compostos Triazenos (TZCs) vêm afirmando-se como uma classe promissora de metalofármacos com relevante atividade antimicrobiana e antiproliferativa. Além disso, a associação do radical farmacofórico TZC com íons metálicos, como o ouro, leva a um aumento significativo na atividade biológica. Em virtude dessa ampla versatilidade farmacológica, este estudo teve como objetivo a avaliação in vitro da atividade biológica de um composto TZC complexados com íon ouro (I). A atividade antibacteriana foi realizada pelo método convencional da microdiluição em caldo, através da técnica da concentração inibitória mínima (CIM), frente às cepas bacterianas padrão de referência American Type Culture Collection (ATCC), isolados clínicos com resistência a múltiplas drogas (RMD) e isolados clínicos produtores de biofilme. A citotoxicidade foi analisada através do ensaio colorimétrico baseado na redução do brometo de 3-(4,5-dimetiltiazol-2-il)-2,5 difeniltetrazólio) (MTT), frente às células da linhagem padrão K562 (Leucemia Mieloide Crônica). Os resultados obtidos demonstram que o composto em estudo apresentou estreito espectro de ação, sendo ativo somente frente aos microrganismos classificados como Gram positivos, além disso, mostrou-se ativo frente a todos os isolados produtores de biofilme, quando comparado às cepas não produtoras de biofilme. Também demonstrou notável atividade citotóxica, tendo a IC50 4.96 μM. Sendo assim, esses resultados demonstram uma alternativa para a concepção de uma nova classe de metalofármacos com atividade antibacteriana e antitumoral.