Orientadores: Carlos Arturo Levi D'Ancona, Nelson Rodrigues Netto Júnior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T11:58:20Z (GMT). No. of bitstreams: 1 Zani_EmersonLuis_D.pdf: 2437204 bytes, checksum: a73fbb0add8a13de68169511bc8b989a (MD5) Previous issue date: 2011 / Resumo: A biópsia prostática transretal (BPTR) é um procedimento bem estabelecido utilizado para a obtenção de tecido para o diagnóstico histológico do carcinoma da próstata. Apesar do fato de a BPTR ser geralmente considerada um procedimento seguro, ela pode ser acompanhada por complicações infecciosas e traumáticas. Embora as complicações infecciosas após a BPTR sejam bem conhecidas, há incerteza sobre a necessidade e a eficácia do antibiótico profilático de rotina e uma clara falta de padronização na profilaxia antibiótica para BPTR. O objetivo foi realizar uma revisão sistemática de ensaios clínicos randomizados sobre a profilaxia antibiótica em BPTR para avaliar a eficácia e os efeitos adversos do tratamento antibiótico profilático nessa situação. A pesquisa abrangeu as principais bases eletrônicas: MEDLINE, EMBASE, LILACS e Cochrane Central Register of Controlled Trials (CENTRAL). Especialistas foram consultados e referências de artigos relevantes foram obtidas. Todos os estudos randomizados e controlados (ERCs) de homens que se submeteram à BPTR e receberam antibióticos profiláticos ou placebo / nenhum tratamento foram selecionados, e também todos os ERCs avaliando um tipo de antibiótico contra outro, incluindo doses, vias de administração, freqüência de administração e duração do tratamento antibiótico. A revisão sistemática foi conduzida na Colaboração Cochrane. No geral, mais de 3500 referências foram analisadas e dezenove artigos originais com um total de 3599 pacientes foram incluídos. Nove estudos analisaram antibióticos versus placebo / nenhum tratamento, com todos os resultados favorecendo significativamente o uso de antibióticos (P <0,05 (I2=0%)), incluindo bacteriúria (risco relativo (RR) 0,25 (intervalo de confiança (IC) de 95% de 0,15 a 0,42), bacteremia (RR 0,67, IC 95% 0,49-0,92), febre (RR 0,39, IC 95% 0,23-0,64), infecção do trato urinário (RR 0,37 (IC 95% 0,22-0,62) e hospitalização (RR 0,13 (IC 95% 0,03-0,55)). Diversas classes de antibióticos foram efetivas profilaticamente para a BPTR, e a classe das quinolonas foi a melhor classe analisada, com o maior número de estudos (cinco) e de pacientes (1188). Na comparação "antibiótico versus enema", foram analisados quatro estudos com um número limitado de pacientes. As diferenças entre os grupos não foram significativas. Para "antibiótico versus antibiótico + enema", apenas o risco de bacteremia (RR 0,25, IC 95% 0,08-0,75, P = 0,01) foi reduzido no grupo "antibiótico + enema". Sete estudos relataram os efeitos de antibiótico de curta duração (um dia) versus curso de longa duração (três dias). O uso de antibióticos por longo curso foi significativamente melhor do que o tratamento de curta duração apenas para bacteriúria (RR 2,09, IC 95% 1,17-3,73, P = 0,01 (I2=34%)). Para "dose única versus múltiplas doses", houve maior risco de bacteriúria com dose única (RR 1,98, IC 95% 1,18-3,33, P <0,05 (I2%=7)). Comparando-se a administração oral versus sistêmica - injeção intramuscular (IM) ou intravenosa (IV) - dos antibióticos, não houve diferenças significativas entre os grupos para bacteriúria, febre, ITU e hospitalização. A profilaxia antibiótica é eficaz na prevenção de complicações infecciosas após BPTR. Diversas classes de antibióticos são eficazes profilaticamente para a biópsia da próstata e a classe das quinolonas foi a classe melhor analisada, com o maior número de estudos e de pacientes. Não há dados definitivos para confirmar que os cursos antibióticos de longa duração (três dias) sejam superiores aos tratamentos de curta duração (um dia), ou que o tratamento com doses múltiplas seja superior ao de uma dose única / Abstract: Transrectal prostate biopsy (TRPB) is a well established procedure used to obtain tissue for the histological diagnosis of carcinoma of the prostate. Despite the fact that TRPB is generally considered a safe procedure, it may be accompanied by traumatic and infective complications. Although infective complications after TRPB are well known, there is uncertainty about the necessity and effectiveness of routine prophylactic antibiotics and a clear lack of standardization in antibiotic prophylaxis for TRPB. The objective was to conduct a systematic review of randomized controlled trials on antibiotic prophylaxis in TRPB to evaluate the effectiveness and adverse effects of prophylactic antibiotic treatment in this situation. The search covered the principal electronic databases: MEDLINE, EMBASE, LILACS and the Cochrane Central Register of Controlled Trials (CENTRAL). Experts were consulted and references from the relevant articles were scanned. All randomized, controlled trials (RCTs) of men who underwent TRPB and received prophylactic antibiotics or placebo/no treatment were selected, and all RCTs looking at one type of antibiotic versus another, including comparable dosages, routes of administration, frequency of administration, and duration of antibiotic treatment. The systematic review was conducted in Cochrane Collaboration. Overall, more than 3500 references were considered and nineteen original reports with a total of 3599 patients were included. There were nine trials analyzing antibiotics versus placebo/no treatment, with all outcomes significantly favoring antibiotic use (P < 0.05 (I2 = 0%)), including bacteriuria (relative risk (RR) 0.25 (95% confidence interval (CI) 0.15 to 0.42), bacteremia (RR 0.67, 95% CI 0.49 to 0.92), fever (RR 0.39, 95% CI 0.23 to 0.64), urinary tract infection (RR 0.37 (95% CI 0.22 to 0.62), and hospitalization (RR 0.13 (95% CI 0.03 to 0.55)). Several classes of antibiotics were effective prophylactically for TRPB, and the quinolones were the best analyzed class, with a higher number of studies (five) and patients (1188). For 'antibiotic versus enema', we analyzed four studies with a limited number of patients. The differences between groups were not significant. For "antibiotic versus antibiotic + enema", only the risk of bacteremia (RR 0.25, 95% CI 0.08 to 0.75, P = 0.01) was diminished in the "antibiotic + enema" group. Seven trials reported the effects of short-course (1 day) versus long-course (3 days) antibiotics. Long course was significantly better than short-course treatment only for bacteriuria (RR 2.09, 95% CI 1.17 to 3.73, P = 0.01 ( I2 = 34%)). For "single versus multiple dose", there was significantly greater risk of bacteriuria for singe-dose treatment (RR 1.98, 95% CI 1.18 to 3.33, P < 0.05 (I2 = 7%)). Comparing oral versus systemic administration - intramuscular injection (IM), or intravenous (IV) - of antibiotics, there were no significant differences in the groups for bacteriuria, fever, UTI and hospitalization. Antibiotic prophylaxis is effective in preventing infectious complications following TRPB. Several classes of antibiotics are effective prophylactically for prostate biopsy and the quinolones were the best analyzed class, with a higher number of studies and patients. There is no definitive data to confirm that antibiotics for long-course (three days) are superior to short-course treatments (one day), or that multiple-dose treatment is superior to single-dose / Doutorado / Fisiopatologia Cirúrgica / Doutor em Ciências

Les intégrons sont des supports génétiques bactériens de capture, d’expression et de dissémination de gènes de résistance aux antibiotiques sous forme de cassettes. Ils sont majoritairement décrits chez les bactéries à Gram négatif chez qui ils confèrent généralement un phénotype de multirésistance. Les intégrons de résistance (IR) jouent un rôle majeur dans l’acquisition de la résistance dans le monde bactérien. Il existe 3 principales classes d’IR ; les IR de classe 1, les IR de classe 2 et les IR de classe 3 (IR3). Contrairement aux 2 premières classes, les IR3 représentent la classe d’intégrons de résistance la moins étudiée. Très peu de travaux s’intéressent à leur étude et on dénombre actuellement moins de 10 IR3 entièrement caractérisés. Les objectifs de ce travail de thèse étaient (i) d’effectuer une étude épidémiologique des IR3 en France et au Cameroun et (ii) d’étudier les modalités d’expression de l’intégrase et des cassettes de ces intégrons. Nos travaux ont permis d’isoler puis de décrire 3 nouveaux IR3 présents au sein de bactéries environnementales appartenant aux genres Aeromonas, Acinetobacter et Citrobacter. Les cassettes de ces IR3 codent des résistances aux bétalactamines, aminosides et ammoniums quaternaires. De plus, nous avons caractérisé des IR3 dans 3 souches de Delftia spp. (2 D.acidovorans et 1 D. tsuruhatensis) isolées en Afrique ; les cassettes de ces intégrons ne codent pas de résistance aux antibiotiques. L’axe plus fondamental de ce travail de thèse a permis de montrer que le PintI3(1) est le promoteur impliqué dans l’expression du gène intI3. De plus, nous avons montré que les variants du promoteur Pc, ainsi que les variants du promoteur PintI3(1) sont fonctionnels et de force différente. Il ressort de nos travaux que l’environnement constituerait un réservoir d’intégrons de classe 3 et que ces supports génétiques pourraient jouer un rôle important dans la dissémination de la résistance au sein de cet écosystème. / Integrons are bacterial genetic elements able to capture and express genes embedded within gene cassettes. They are widely described among Gram-negative bacteria and generally confer a multidrug resistance phenotype. Resistance integrons (RI) play an important role in the acquisition of antibiotic resistance. There are 3 main classes of RI. Class 3 RI has been poorly studied class with less than ten fully IR3 characterized. Objectives of this thesis were (i) to conduct an epidemiological study of class 3 RI in France and Cameroon and (ii) to better understand the modes of expression of the integrase and cassettes of IR3. We described 3 new class 3 RI isolated from environmental bacteria belonging to genus Aeromonas, Acinetobacter and Citrobacter. Gene cassettes encoded resistance to betalactams, aminoglycosides and quaternary ammonium compounds. We also described IR3 from three Delftia strains (2 D.acidovorans and 1 D.tsuruhatensis) in Africa containing cassettes that do not encode antibiotic resistance. The fundamental part of the work showed that the PintI3(1) promoter is involved in the expression of the intI3 gene. Furthermore, we demonstrated that variants of the Pc promoter and variants of the PintI3(1) promoter are functional with different strengths. These results showed that the environment may constitute a reservoir of class 3 integrons and that these genetic elements could play an important role in the spread of the resistance in this ecosystem.

Entre as ß-lactamases, as enzimas CTX-M têm despertado atenção especial pela alta incidência e grande capacidade de propagação. Eventos como recombinação gênica, transferência plasmideal e multirresistência podem ser a razão da manutenção e da ampla disseminação dos genes blaCTX-M. Este é um trabalho retrospectivo que teve como objetivo caracterizar genes blaCTX-M presentes em Klebsiella spp. Foram estudadas 27 linhagens de Klebsiella pneumoniae e 8 linhagens de Klebsiella oxytoca, produtoras de ?-lactamase de espectro estendido, isoladas de pacientes hospitalizados no período de janeiro a junho de 2000. A detecção e identificação dos genes blaCTX-M, assim como dos elementos relacionados com a mobilização destes genes, foi realizada por PCR e seqüenciamento. A localização genética e a mobilidade dos genes blaCTX-M foram pesquisadas por análise plasmideal e hibridação e por conjugação. Os perfis de sensibilidade das linhagens estudadas e das linhagens transconjugantes foram comparados pela determinação da concentração inibitória mínima de antibióticos das classes das cefalosporinas, cefamicinas, aminoglicosídeos e quinolonas. Foram encontrados genes blaCTX-M em plasmídeos conjugativos em 13 (37%) linhagens estudadas: blaCTX-M-9 em 4 K. oxytoca, e blaCTX-M-2 em 9 K. pneumoniae. Os genes blaCTX-M-9 estavam associados ao elemento de inserção ISEcp1, enquanto os genes blaCTX-M-2 estavam associados a integrons de classe I contendo ISCR1. O genes blaCTX-M-2, carreado por plasmídeo, pode estar relacionado com disseminação horizontal entre vários clones de K. pneumoniae, enquanto o gene blaCTX-M-9 foi encontrado sendo carreado por um único clone de K. oxytoca. Este estudo determinou a incidência e a diversidade de enzimas CTX-M no período estudado, além de fornecer dados epidemiológicos que podem explicar a sua prevalência no mundo e contribuir para o entendimento e controle da disseminação deste tipo de resistência. / CTX-M enzymes, the world\'s most prevalent ß-lactamases disseminate very easily. Genetic recombination, plasmid transference and multiresistance could be responsible for the wide spread of blaCTM-X genes. This retrospective study aims to characterize blaCTX-M genes found in Klebsiella spp. The strains were isolated in hospital patients from January to June 2000 and consisted of 27 ESBL-producing Klebsiella pneumoniae and 8 ESBL-producing Klebsiella oxytoca. PCR and sequencing were used in the detection and identification of blaCTX-M genes and genetic elements associated with their mobilization. Determination of genetic localization and mobility of blaCTX-M genes was by plasmid analyses, hybridization and transfer assays. The minimal inhibitory concentrations (MICs) of cephalosporins, cefamicins, aminoglycosides and quinolone antimicrobials evaluated the antibiotic susceptibility profile of transconjugants and strains in the study. The blaCTX-M genes were found in 13 strains (37%): blaCTX-M-9 in 4 K. oxytoca and blaCTX-M-2 in 9 K. pneumoniae. The insertion sequence ISEcp1 was associated with blaCTX-M-9 and blaCTX-M-2 was found in a class I integron bearing ISCR1. Plasmid blaCTX-M-2 genes dissemination was due to horizontal transfer among many K. pneumoniae clones, while blaCTX-M-9 dissemination was associated with a particular clone of K. oxytoca. The study characterized incidence and diversity of CTX-M enzymes during the period studied. Moreover it showed epidemiological data, which may explain CTX-M prevalence worldwide and contribute for the understanding and control of the resistance spread.

No description available.

Global threats to treatment of bacterial infections due to antibiotic resistance (AR) have been on the rise in recent years. Current diagnostic tests identify bacteria by using blood culture, which takes more than 24 hours. This study focuses on the fluorescent labeling of DNA derived from bacterial AR genes (KPC & VIM) and other model DNAs using oligreen dye (OG) and molecular beacons (MB). A NanoDrop 3300 fluorospectrometer was used to take fluorescence measurements. Linear dynamic range and labeling efficiency were dependent on the following optimized conditions: dilution factor of OG (200 fold), buffer (20 mM Tris HCl, pH 8), and heat treatment of 95 °C for 15 min.Fluorescence analysis of a target DNA with a designed MB showed signal-to-background of 10 with our buffer only and 20 with our buffer and 25% ethanol. I also demonstrated a simple microfluidic device capable of detecting AR genes using model DNAs, magnetic beads, and designed MBs for assays of µ50 L volume. This study provides a first step towards detecting MB-DNA complexes by a simple, low cost, and fast non-amplified method, which may be used to detect AR genes in clinical samples in the future.

<div>Antibiotics have been widely used to treat bacterial diseases since the 1940s. However, the benefits offered by antibiotics have gradually faded due to the increased occurrence and frequency of antibiotic resistance. The widespread use of antibiotics has driven selection for resistance in bacteria and is becoming a global problem for human health and the environment. Antibiotic resistance is exacerbated by the ability of bacteria to share their antibiotic resistance genes (ARGs) with other bacteria via horizontal gene transfer (HGT). Many existing studies on HGT of ARGs focused on antibiotic concentrations at or above the minimal inhibitory concentration (MIC), which is the lowest concentration of an antibiotic that prevents visible growth of a bacteria culture. However, knowledge on the development of antibiotic resistance under different stressors at sub-MIC levels is still limited. In addition, carbon nanotubes (CNTs) have been widely studied in environmental, agricultural and biomedical areas due to their unique physical and chemical characteristics, but limited studies have been done to evaluate the effects of CNTs on the spread of ARGs. Electrochemical filtration has been shown to be a cost-effective technique to remove recalcitrant compounds and reduce antibiotic resistance, but limited studies have been done to evaluate the effectiveness of removal of ARGs with electrochemical filtration. Therefore, there is a critical need to evaluate the effects of trace levels of antibiotics and CNTs on the development of antibiotic resistance and electrochemical removal of ARGs. </div><div><br></div><div>The specific research objectives of this study were to evaluate: (1) selective pressure of sub-inhibitory concentrations of antibiotics on the development of antibiotic resistance and HGT, (2) development of antibiotic resistance and HGT under exposure to CNTs and antibiotics, and (3) effectiveness of using an electrochemical MWCNT filter to remove ARGs. </div><div><br></div><div>To evaluate the development of antibiotic resistance exposed to sub-MIC of erythromycin, HGT between environmental donor (<i>E. coli)</i> and pathogenic bacterial recipient (<i>B. cereus</i>) was quantified. The results indicated that extremely low concentration (0.4 ng/L to 4 µg/L) of erythromycin promoted HGT of <i>erm</i>80 gene, which is an erythromycin resistance gene. In addition to traditional culture-based method and quantitative real-time PCR (qPCR), a fluorescence <i>in situ</i> hybridization (FISH) approach was used to detect the occurrence and development of ARGs even the bacteria were in the viable but nonculturable (VBNC) state after treatment of sub-lethal level of erythromycin. Multi-walled carbon nanotubes (MWCNT) was selected as a representative stressor to evaluate the effects on HGT. The results showed that MWCNT enhanced HGT above 1 × MIC, which is the lethal level of erythromycin to recipients, and transfer frequencies of erm80 genes increased up to 101-fold under exposure to 1 × MIC erythromycin and MWCNT as compared to no MWCNT control. However, transfer efficiency of <i>erm</i>80 gene under exposure to sub-MIC of erythromycin was inhibited by MWCNTs. Moreover, transfer of antibiotic resistance plasmids was affected by antibiotics and MWCNTs. Although the concentration of individual stressor was not enough to confer antibiotic selection, effects of both antibiotics above 1 × MIC and MWCNTs could add up and select for antibiotic resistance. The results suggested that CNTs might create additional selective pressure for the spread of ARGs and their effects on HGT should be further investigated. Finally, an electrochemical MWCNT filtration was evaluated to remove genomic DNA and ARGs under the effects of operating conditions, such as pH, phosphate, and NOM. The results showed that the electrochemical MWCNT filtration reactor achieved 79% removal efficiency for genomic DNA and 91% removal efficiency for <i>erm</i>80 genes. The study suggested that electrochemical MWCNT filtration could be a promising technology for the removal of DNA and ARGs.</div><div><br></div><div>Overall, the results improved our understanding of the development of antibiotic resistance and ARGs under various selective pressures. Trace levels of antibiotics promoted the development and spread of ARGs. Conjugative transfer of resistance genes exposed to sub-MIC levels of erythromycin and MWCNTs also contributed to the spread and propagation of ARGs. As antibiotic concentrations detected in natural environment are often in trace levels, the results of this study may improve the understanding of health risks of trace levels of antibiotics and help develop effective mitigation strategies to control the spread of antibiotic resistance. Effective removal of ARGs with electrochemical MWCNT filtration may help the development of cost-effective treatment systems to remove ARGs to protect human health and the environment.</div><div><br></div>

Antibiotic resistance is a worldwide health threat, as bacteria continue to evade antibiotic treatment. In order to survive, bacteria utilize a number of resistance mechanisms, including efflux pumps, which efficiently export antibiotics outside of the cell to reduce intracellular damage. While such mechanisms are well known, there remains a significant gap in knowledge regarding how different environmental dynamics, such as the rate of antibiotic introduction or the diversity within a microbial community, play a role in resistance. In this work, we used the AcrAB-TolC efflux pump as a case study to explore how such complex dynamics promote antibiotic resistance and its evolution. First, through a combined effort using experiments and mathematical modeling, we discovered that the rate of antibiotic introduction impacts the fraction of resistant bacteria in a population. We then explored the impact of mixed populations on survival following antibiotic treatment. In mixed microcolonies, we found that resistant cells can harm their susceptible neighbors by exporting antibiotics to increase the local concentrations of these drugs. Next, we aimed to understand how these environmental effects may impact longer-term survival of an antibiotic treatment, focusing on the evolution of resistance over ~72 hours. Through a series of adaptive evolution experiments, we identified that near-MIC treatments were the most likely to promote antibiotic resistance, regardless of whether the strains contained the AcrAB-TolC pump at wild type or overexpressed levels, or whether the strains lacked the pump altogether. In studying antibiotic introduction rates on evolution, we found that slower introduction rates facilitated the evolution of high levels of resistance with a minimal fitness cost. Meanwhile, mixed populations demonstrated limited evolvability after rapid antibiotic introductions. This work provides important insights into the impacts of environmental factors, such as the rate of antibiotic introduction and the homogeneity of populations, on the promotion and evolution of antibiotic resistance. These lessons may help inform future policies on antibiotic use and mitigate the continued pattern of resistance evolution.

Conventional wastewater treatment plants are able to reduce contaminant loads within regulations but do not take into account emerging contaminants. Antibiotic resistance genes and antibiotic resistant bacteria have been shown to survive wastewater treatment and remain detectable in effluents. The safety of treated wastewaters is crucial, otherwise unregulated and unmitigated emerging contaminants pose risks to public health and impede wastewater reuse. This dissertation aimed to further understanding of emerging microbial threats, and tested two natural and low-cost tools for their mitigation: sunlight, and bacteriophages. A wastewater bacterial isolate, named E. coli PI-7, which is highly antibiotic resistant, carries the novel antibiotic resistance gene New Delhi metallo-beta-lactamase NDM-1 gene, and displays pathogenic traits, was chosen to model responses to the treatments. Results found that solar irradiation was able to achieve a 5-log reduction in E. coli PI-7 numbers within 12 hours of exposure. However, the results also emphasized the risks from emerging microbial contaminants since E. coli PI-7, when compared with a non-pathogenic strain E. coli DSM1103 that has less antibiotic resistance, showed longer survival under solar irradiation. In certain instances, E. coli PI-7 persisted for over 6 hours before starting to inactivate, exhibited complex stress resistance gene responses, and activated many of its concerning pathogenicity and antibiotic resistance traits. However, upon solar irradiation, gene expression results obtained from both E. coli strains also showed increased susceptibility to bacteriophages. Hence, bacteriophages were coupled with solar irradiation as an additional mitigation strategy. Results using the coupled treatment found reduced cell-wall and extracellular matrix production in E. coli PI-7. DNA repair and other cellular defense functions like oxidative stress responses were also impeded, rendering E. coli PI-7 more susceptible to both stressors and successfully hastening the onset of its inactivation. Overall, the dissertation is built upon the need to develop strategies to further mitigate risks associated with emerging microbial contaminants. Solar irradiation and bacteriophages demonstrate potential as natural and low-cost mitigation strategies. Sunlight was able to achieve significant log-reductions in tested E. coli numbers within a day’s exposure. Bacteriophages were able to overwhelm E. coli PI-7’s capacity to resist solar inactivation while not affecting the indigenous microbiota.

Background The antimicrobial stewardship is developed to provide a guide on the responsible use of antimicrobial drugs. Thus, slowing down the development of antimicrobial resistance. However, the nurse's role in antimicrobial stewardship is not clarified. Failure toinclude the nurse within the antimicrobial stewardship guidelines may result in poor execution of antimicrobial stewardship.Aim To explore the role of nurses in antimicrobial stewardship and how it can be practically implemented within the medical field.Method This is a literature review where seven qualitative studies, two quantitative studies and a mix-methods study examines the nurse's role in antimicrobial stewardship.Results Two main themes and five sub-themes were created. The two main themes were clinical role and collaboration. The clinical role described the nurse's role as a patient advocate and the nurse's contribution to antimicrobial stewardship through monitoring and evaluation of the patient and treatment, as well as through safe sampling, drug administration and hygiene. The collaboration showed and identified the nurse's role as a communicator and educator. Conclusion Conclusions that can be drawn from the literature review are that the potential roles the nurse may have in antimicrobial stewardship are many and those we have identified are already included in the nurse's daily work.

No description available.