Trafficking of GPCRs is a dynamic process that is tightly regulated and sometimes defective in human diseases. Therefore it is important to develop new methods to allow simple and quantitative measurement of surface expression of membrane proteins. Here we describe the development and validation of a new assay for quantification of cell surface expression of GPCRs using β-lactamase as a reporter. For this assay we N-terminally fused β-lactamase (βlac) to the β2-adrenergic receptor (β2AR) and GABA b R1 (GBR1). The results obtained by the βlac assay are quantitatively and qualitatively similar to well established ELISA when measuring agonist induced internalization of β2AR. We also show that measurement of GBR1 surface expression with GBR2 co-expression is quantitatively identical between the βlac and ELISA. In conclusion, our results show that our newly developed βlac assay is quantitatively similar while being less expensive, more robust and higher throughput compared to an ELISA.

Trafficking of GPCRs is a dynamic process that is tightly regulated and sometimes defective in human diseases. Therefore it is important to develop new methods to allow simple and quantitative measurement of surface expression of membrane proteins. Here we describe the development and validation of a new assay for quantification of cell surface expression of GPCRs using β-lactamase as a reporter. For this assay we N-terminally fused β-lactamase (βlac) to the β2-adrenergic receptor (β2AR) and GABA b R1 (GBR1). The results obtained by the βlac assay are quantitatively and qualitatively similar to well established ELISA when measuring agonist induced internalization of β2AR. We also show that measurement of GBR1 surface expression with GBR2 co-expression is quantitatively identical between the βlac and ELISA. In conclusion, our results show that our newly developed βlac assay is quantitatively similar while being less expensive, more robust and higher throughput compared to an ELISA.

In this study, the effects of oxygen transfer (OT) on beta-lactamase production and on intracellular reaction rates were investigated with Bacillus licheniformis ATCC 2597. In order to clarify the oxygen transfer effects on the production of beta-lactamase, firstly a glucose based defined medium was designed and using this medium, the effects of bioreactor operation parameters, i.e., pH and temperature, on beta-lactamase activity and cell formation were investigated in laboratory scale batch-bioreactors using shake bioreactors having V=33 ml working volumes. Among the investigated bioprocess conditions, the highest beta-lactamase activity was obtained as A=115 U cm-3, in the medium with 7.0 kg m-3 glucose, 7.1 kg m-3 (NH4)2HPO4 and the salt solution, at pH0=7.5, T=37C, N=200 min-1. At the optimum conditions found in laboratory scale the effects of OT on cell generation, substrate consumption, product (beta-lactamase) and by-products formations were investigated at three different air inlet (Q0/ VR = 0.2, 0.5 and 1 vvm) and at three agitation rates (N=250, 500, 750 min-1) in V = 3.0 dm3 batch bioreactors consisting of temperature, pH, foam, stirring rate and dissolved oxygen controls. Along with the fermentation, cell, substrate and by-product concentrations, beta-lactamase activity, yield coefficients, specific rates, oxygen uptake rates and the liquid phase mass transfer coefficient values were determined. The highest beta-lactamase activity was obtained at 0.5 vvm 500 min-1 and at 0.2 vvm 500 min-1 conditions as ca. A=90 U cm-3 while the highest cell concentration was obtained as Cx=0.67 kg m-3 at 0.5 vvm 750 min-1 and at 0.2 vvm 750 min-1 conditions. KLa, increased with the increase in the agitation and aeration rates and its values varied between 0.007-0.044 s-1 and oxygen uptake rate varied between 0.4-1.6 mol m-3 s-1. Finally, the influence of OT conditions on the intracellular reaction rates was investigated using metabolic flux analysis to evaluate the effects of oxygen on the metabolism. Keywords: beta-lactamase, production, Bacillus, oxygen transfer, metabolic flux analysis

In this study, the effects of pH and different feeding modes on beta-lactamase production and cell metabolism were investigated with Bacillus licheniformis (ATCC 25972). For this purpose, first, the effects of pH on beta-lactamase activity, cell formation, substrate consumption, as well as intracellular sodium, potassium, ammonium ion, amino acid and organic acid concentrations were investigated in V= 3.0 dm3 batch bioreactors consisting of temperature, pH, foam, stirring rate and dissolved oxygen controls. Among the investigated uncontrolled pH operation with pH0=7.5 and controlled pH operations, pHc=6.75 yielded the highest cell concentration and beta-lactamase activity as Cx=0.60 kg/m3 and A= 54 U/cm3, respectively. Next, the production medium was redesigned in terms of initial glucose and phosphate ion concentrations in order to increase the enzyme activity and cell growth rate, and to determine the feeding strategy in laboratory scale batch-bioreactors using shake bioreactors having V=33 ml working volumes. The medium containing (kg/m3), glucose 2.5 (7.0) / Na2HPO4, 1.0 / K2HPO4 1.0 / (NH4)2HPO4, 7.1 and salt solution at pHc=6.75 was accepted as optimized medium for fed-batch (batch) processes. Using this optimized medium the feeding strategy was investigated for linear and exponential feeding profiles and compared with batch operation. Throughout the fermentation, cell, substrate and intracellular and extracellular by-product, sodium, potassium, ammonium ion concentrations, beta-lactamase activity, yield coefficients, specific rates, oxygen uptake rates and liquid phase mass transfer coefficient values were determined. The highest beta-lactamase activity was obtained at fed-batch operation with exponential feeding (FB1) condition as A= 108 U/cm3, which is ca. 1.7-fold higher than that of the batch operation with optimized medium. Finally, to invesitigate the physiological state of the culture media, viability of the cells was monitored throughout the cultivation time for repeated FB1, pHc=6,75, and pHuc=7.5 experiments. About 9% of the cells were found to be dead through the end of FB1 and pHuc=7.5 operations.

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2008. / Title from second page of PDF document. Includes bibliographical references.

Resumo: A produção de beta-lactamase de espectro estendido (ESBL) em membros da família Enterobacteriaceae pode conferir resistência a cefalosporinas de amploespectro, aztreonam e penicilinas. Devido a esse fenômeno, a detecção exata dos produtores de ESBL é essencial para a seleção apropriada da antibioticoterapia. Para detectar a produção de beta-lactamase de espectro estendido (ESBL) em bacilos Gram-negativos, foi usado um teste de triagem com os discos de aztreonam (ATM), ceftazidima (CAZ), cefotaxima (CTX) e ceftriaxona (CRO) sobre 300 cepas, das quais trinta e cinco eram suspeitas da presença de ESBL. A produção de ESBL foi demonstrada por três métodos fenotípicos confirmatórios de fácil utilização. Os três testes fenotípicos para confirmar a produção de ESBL incluíram o teste do sinergismo (double disk), E-test? ESBL e disco combinado. Os discos utilizados no teste do sinergismo e do disco combinado foram: aztreonam (30?g-ATM), cefotaxima (30?g-CTX), ceftazidima (30?g-CAZ), cefpodoxima (10?g-CPD) ceftriaxone (30?g-CRO) e amoxicilina+ácido clavulânico(30?g-AMC), cefotaxima+ácido clavulânico (30?g-10?g), ceftazidima+ácido clavulânico (30?g- 10?g), cefpodoxima+ácido clavulânico (10?g-1? g). Para E-test foram utilizadas fitas contendo as cefalosporinas: ceftazidima versus ceftazidima/ácido clavulânico; cefotaxima versus cefotaxima/ácido clavulânico. Os testes fenotípicos confirmaram a presença de ESBL em cinco cepas de enterobactérias (1,66%). Todos os métodos são de fácil execução, contudo o método do Etest requer experiência para interpretar os resultados. Os três testes oferecem uma solução viável para confirmar a produção de ESBL no laboratório clínico. / Abstract: The production of extended spectrum beta-lactamase (ESBL) in the members of the family Enterobacteriaceae can check resistance to cephalosporins of extended-spectrum, aztreonam and penicilins. Due to this phenomenon, the exact detection of the producers of ESBL are essential for the appropriate selection of antimicrobial therapy. To detect the production of extended spectrum beta-lactamase (ESBL) in Gram-negative bacilli, a test of screening was used with the discs of aztreonam (ATM), ceftazidime (CAZ), cefotaxime (CTX) e ceftriaxone (CRO) in 300 strains, of which thirty-five were suspicious of the presence of ESBL. The production of ESBL was demonstrated by three phenotypic methods confirmed of easy utilization. The three phenotypic tests to confirm the production of ESBL included the test of sinergy (double disk), E-test? ESBL and combination disk. The disks used on the test sinergy and the combination disk were: aztreonam (30?g-ATM), cefotaxime (30?g-CTX), ceftazidime (30?g-CAZ), cefpodoxime (10?g-CPD) ceftriaxone (30?g- CRO) e amoxicillin+clavulanic acid (30?g-AMC), cefotaxime+clavulanic acid (30?g- 10? g), ceftazidime+clavulanic acid (30?g-10? g), cefpodoxime+clavulanic acid (10?g- 1? g). For E-test, were utilized strips containing the cephalosporins: ceftazidime and ceftazidime/clavulanic acid; cefotaxime and cefotaxime/clavulanic acid. The phenotypic tests confirmed the presence of ESBL in five strains Enterobacteriaceae (1,66%). All of the methods are of easy execution; however, the method of Etest requires experiment to interpret the results. The three tests offer a viable solution to confirm the production of ESBL on a clinic laboratory. / Orientador: Elisabeth Loshchagin Pizzolitto / Coorientador: Antonio Carlos Pizzolitto / Banca: Wilton Rogério Lustri / Banca: Izabel Yoko Ito / Mestre

Conselho Nacional de Desenvolvimento Científico e Tecnológico / In recent years, the isolation of bacteria producing β-lactamases has caused concern around the world, due to the fact these enzymes hydrolysis the ring β-lactam antimicrobials used in the main clinic. This aim of this study was asses the prevalence metallo-β-lactamases (MbL) in isolates of Pseudomonas aeruginosa and Acinetobacter baumannii obtained from patients admitted at the University Hospital of Santa Maria (HUSM). The profile of susceptibility for all isolates was evaluated by the disk diffusion method standardized by CLSI. The antimicrobial disks were distributed in a way that allows the identification of strains producers of AmpC and ESBL. For the identification of the producers of MbL the test of disk approximation with EDTA 0.1 M, EDTA 0,5M and acid 2-mercaptopropionic were performed. Isolates that did not have any of the mechanisms of resistance search were classified as multiresistant (MDR). The minimum inhibitory concentration (MIC) for ceftazidima, imipenem and polymyxin B was assessed by broth method microdilution for all isolated, according to CLSI. From January to June 2006, were obtained 32 isolates the P.aeruginosa and 41 the A. baumannii, the those 17 (23.29%) were β-lactamase AmpC-type producers, 11 (15.07%) were MbL producers, and 45 (61,64%) were classified as MDR. All strains producing MbL were Pseudomonas aeruginosa. The sensitivity of the isolates according to the CIM for antimicrobial evaluated were: 90,28% for polymyxin B, 36,11% for imipenem and 18% for ceftazidima. There was a high prevalence of MDR isolates and producers of β-lactamase-type AmpC and MbL in HUSM, this is extremely worrying once there is limiting therapy available. This situation becomes even more worrying with the find of isolates resistant the polymyxin B, witch is one of the last options of treatment for MDR isolates and producers of MbL. The detection of microorganisms is extremely important for the committees of infection hospital with the goal of preventing outbreaks, as well as guide the medical team on the conduct therapy, since there are few effective antimicrobial clinically for these pathogens and no prospects for development the new antimicrobial in the near future. / Nos últimos anos, o isolamento de bactérias produtoras de β-lactamases tem causado preocupação em todo o mundo, devido ao fato dessas enzimas hidrolisarem o anel β- lactâmico dos principais antimicrobianos utilizados na clínica. Este trabalho teve por objetivo avaliar a prevalência de metalo-β-lactamases (MbL) em isolados de Pseudomonas aeruginosa e Acinetobacter baumannii obtidos de pacientes atendidos no Hospital Universitário de Santa Maria (HUSM). O perfil de sensibilidade para todos os isolados foi avaliado pelo método de disco difusão padronizado pelo CLSI. Os discos de antimicrobianos utilizados foram distribuídos de forma que permitisse a identificação dos isolados produtores de AmpC e ESBL. Para a identificação dos produtores de MbL utilizou-se o teste de disco aproximação com os seguintes agentes quelantes: EDTA 0,1M, EDTA 0,5 M e ácido 2-mercaptopropiônico. Os isolados que não possuíam nenhum dos mecanismos de resistência pesquisados foram classificados como multirresistentes (MDR). A concentração inibitória mínima (CIM) para ceftazidima, imipenem e polimixina B foi avaliada pelo método de microdiluição em caldo para todos os isolados, de acordo com o CLSI. Durante o período de janeiro a junho de 2006 foram obtidos 32 isolados de P.aeruginosa e 41 de A. baumannii, destes 17 (23,29%) foram produtores de β-lactamase do tipo AmpC, 11 (15,07%) foram produtores de MbL e 45 (61,64%) foram classificados como MDR. Todas as cepas produtoras de MbL foram de Pseudomonas aeruginosa. A sensibilidade dos isolados de acordo com a CIM para os antimicrobianos avaliados foram as seguintes: 90,28% para polimixina B, 36,11% imipenem e 18% ceftazidima. Observou-se uma alta prevalência de isolados MDR no HUSM, além de isolados produtores de β-lactamase do tipo AmpC e MbL, o que é extremamente preocupante devido limitar a terapia a poucos antimicrobianos. Esta situação torna-se ainda mais preocupante com a detecção de isolados resistentes a polimixina B, a qual é uma das últimas opções de tratamento para infecções causadas por isolados de P. aeruginosa e Acinetobacter baumannii MDR e produtores de MbL. A detecção desses microrganismos é de grande importância para as comissões de controle de infecção hospitalar com o objetivo de prevenir surtos, bem como orientar a equipe médica sobre a conduta terapêutica, uma vez que há poucos antimicrobianos efetivos clinicamente para esses patógenos e as perspectivas para o desenvolvimento de novos antimicrobianos em um futuro próximo são mínimas.

Orientador: Domingos da Silva Leite / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T00:21:34Z (GMT). No. of bitstreams: 1 Domingos_DanielaFerreira_M.pdf: 1627311 bytes, checksum: ba7a6545a75d72a741bfe8d1cda234f4 (MD5) Previous issue date: 2010 / Resumo: O número de animais de companhia tem crescido substancialmente na sociedade atual, com estimativas de 48 milhões de cães e gatos no Brasil. A relação entre animais de companhia e os humanos mudou radicalmente nos últimos anos, esses estão em contato mais próximo com humanos. Como consequência dessas mudanças, agentes antimicrobianos, incluindo antibióticos usados no tratamento de infecções humanas, tem sido utilizados em cães. O objetivo deste trabalho foi investigar fenotipicamente a ocorrência de ß-lactamase de espectro estendido (ESBL) em amostras de Escherichia coli isoladas de cães e identificar os genes de resistência nestas amostras. A presença e variedade de integrons nas amostras de E. coli foi analisada. A classificação das amostras nos grupos filogenéticos A, B1, B2 e D também foram investigadas. Dentre 158 amostras de E. coli isoladas de cães sendo 51 de ITU (infecção do trato urinário), 52 de piometra e 55 de fezes de cães sadios, foram selecionadas 67 amostras que apresentavam pelo menos uma marca de resistência aos ß-lactâmicos. As amostras selecionadas: 41 isoladas de ITU, 20 isoladas de piometra e seis isoladas de fezes de animais sadios, foram submetidas a testes de sensibilidade microbiana pelo método de difusão de disco. A produção de ESBL foi verificada pelo método de aproximação de disco. A identificação dos genes das ß-lactamases foi realizada em ensaios de PCR (Reação em Cadeia da Polimerase) com primers específicos para os genes blaTEM, blaSHV, blaCTX-M, blaGES-1, blaOXA-10, ampC e cmy. A classificação filogenética (chuA, yjaA, TspE4.C2), bem como a detecção de integrons das classes 1 e 2 (intI1 e intI2) também foram realizadas por PCR. Os resultados dos antibiogramas mostraram elevada resistência aos ß-lactâmicos de 1ª geração, ampicilina (82,0%) e cefalexina (62,7%) entre as amostras selecionadas. Resistência aos antimicrobianos ß-lactâmicos de 3ª e 4ª geração e ao monobactam foi observada em 7,5% das amostras. As amostras também apresentaram resistência aos antimicrobianos tetraciclina (82,0%), trimetoprim-sulfametoxazol (62,7%), enrofloxacina (35,8%), florfenicol (34,3%) e ciprofloxacina (32,3%). A expressão fenotípica de ESBL foi observada em duas amostras (3,3%), ambas isoladas de animais sadios. Na análise genotípica, foram identificados os genes blaTEM, (98,5%), ampC (95,5%), blaCTX-M (35,8%), blaSHV (6%) e cmy (2,9%) destacando que os genes blaCTX-M, blaSHV e cmy apresentaram-se associados ao blaTEM e ao ampC. Pelo sequenciamento foi possível identificar as ß-lactamases do tipo TEM-1 e CTX-M-2. Na classificação filogenética as amostras foram agrupadas nos grupos B2 (59,7%), B1 (25,4%) e A (14,9%). Dentre as 67 amostras com marcas de resistência aos ß-lactâmicos, o gene int foi detectado em 26,9% , das quais 71,4% da classe 1 e 28,6% da classe 2. Por outro lado, dentre as 91 amostras sem marcas de resistência a à ß-lactâmicos, somente 3,3% apresentaram integrons, sendo todos da classe 1. As amostras com integrons foram mais comumente alocadas nos grupos filogenéticos A e B1. A presença de ESBL, de genes de resistência e integrons em E. coli isoladas de cães é um achado importante, uma vez que o contato íntimo entre humanos e cães oferece condições para a transmissão das amostras e ainda, que estes animais podem servir de reservatórios de genes de resistência. Esses resultados reforçam a necessidade de controle no uso de antimicrobianos em animais de companhia e bem como o papel dos animais domésticos como reservatório de genes de resistência bacteriana, precisa ser melhor investigado / Abstract: The number of cats and dogs has substantially increased in modern society, with an estimated population of above 48 million in Brazil. The relationship between companion animals and humans has radically changed throughout the years, and animals have become in closer contact with humans. As a consequence of these changes, antimicrobial agents, including antimicrobial preparations licensed for human use, are frequently used in dogs. The aim of this study was to investigate, in Escherichia coli strains isolated from dogs, the occurrence of ß-extended-spectrum ß-lactamase (ESBL) phenotype and to identify resistance genes in these samples. The presence and diversity of integrons in strains of E. coli was analyzed. The classification of microorganisms in the phylogenetic groups A, B1, B2 and D was also determined. Among 158 E.coli strains isolated from dogs (51 from UTI [urinary tract infection], 52 from piometra and 55 from faeces of healthy dogs) 67 strains were selected that had at least one sign of resistance to ß-lactams. The strains selected: 41 isolated from UTI, 20 isolated from piometra and six isolated from the faeces of healthy dogs, were tested for antibiotic sensitivity by the disk diffusion method. ESBL production was screened by the double-disk synergy method. The identification of ß-lactamases was performed by PCR (Polymerase Chain Reaction) using specific primers for blaTEM, blaSHV, blaCTX-M, blaGES-1, blaOXA-10, ampC and cmy and, subsequently, sequencing of the PCR product of strains showing the ESBL phenotype was performed. The phylogenetic classification (chuA, yjaA, TspE4.C2), as well as the detection of class 1 and class 2 integrons, were also determined by PCR. The results of susceptibility tests showed high resistance to 1st generation ß lactams, ampicillin (82.0%) and cephalexin (62.7%), among the selected strains. Resistance to 3rd and 4th generation ß-lactam antibiotics and monobactam was observed in 7.5% of isolates. The strains also showed resistance to antimicrobial tetracycline (82%), trimethoprim-sulfamethoxazole (62.7%), enrofloxacin (35.8%), florfenicol (34.3%) and ciprofloxacin (32.3%). Phenotypic expression of ESBL was observed in 2 samples (3.3%), both isolated from healthy animals. In the genotypic analysis, were identified the genes, blaTEM, (98.5%), ampC (95.5%), blaCTX-M (35.8%), blaSHV (6%) and cmy (2.9%); the genes blaCTX-M, blaSHV and cmy were shown to be associated with blaTEM and ampC. By sequencing, it was possible to identify the TEM-1 and CTX-M-2 ß-lactamases. In the phylogenetic classification, strains were classified B2 (59.7%), B1 (25.4%) and A (14.9%) groups. Among the 67 strains with resistance markers to ß-lactams, the int gene was detected in 26.9% of the strains (71.4% of class 1 and 28.6% of Class 2). Moreover, among the 91 samples without a trace of resistance to ß-lactams, only 3.3% had integrons, all of class 1. Strains with integrons were more commonly housed in the phylogenetic groups A and B1. The presence of ESBL, resistance genes and integrons in E. coli isolated from dogs is an important finding, due to close contact between humans and dogs provides conditions for the transmission of microorganisms and these animals may also serve as reservoirs of isolates harboring resistance genes. These results emphasize the need to control the use of antimicrobials in companion animals and the role of livestock as a reservoir for genes of resistance should be further investigated / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-04-18T16:42:00Z No. of bitstreams: 1 Fleming, Maria Emília de Castro Kling [Dissertação, 2011].pdf: 3850457 bytes, checksum: 76e7a64373d2898f956190ecd2aa26c3 (MD5) / Made available in DSpace on 2017-04-18T16:42:00Z (GMT). No. of bitstreams: 1 Fleming, Maria Emília de Castro Kling [Dissertação, 2011].pdf: 3850457 bytes, checksum: 76e7a64373d2898f956190ecd2aa26c3 (MD5) / Introdução: As infecções de corrente sanguínea estão entre as mais graves infecções, principalmente se causadas por microrganismos resistentes a antimicrobianos. O objetivo deste estudo foi avaliar a prevalência e o perfil de resistência de microrganismos isolados em hemoculturas em um hospital público e outro privado localizados em Niterói, Rio de Janeiro, Brasil. MÉTODOS: O estudo foi conduzido em um hospital geral de natureza pública com 227 leitos e um hospital geral, privado contendo 123 leitos, entre Agosto de 2009 a Agosto de 2010. Todas as amostras provenientes de pacientes maiores de 18 anos foram consecutivamente coletadas após identificação por métodos de rotina de cada laboratório de microbiologia. As cepas de E. coli, K. pneumoniae, K. oxytoca e P. mirabilis foram testadas quanto a produção de ESBL (Extended Spectrum Betalactamase) de acordo com as recomendações do CLSI. As enterobactérias foram testadas quanto a produção de carbapenemases pelo teste modificado de Hodge (CLSI). As amostras de P. aeruginosa foram testadas para a produção de MBLs pelo teste fenotípico de disco combinado. A reação em cadeia da polimerase (PCR) foi utilizada para a detecção dos genes (blaIMP, blaVIM, blaSPM), relacionados com a produção de metalo-beta-lactamases (MBLs). A similaridade genética entre as cepas de P. aeruginosa foi avaliada pela técnica de eletroforese em gel de campo pulsado (PFGE). RESULTADOS: Foram coletadas 195 amostras de microrganismos isolados em hemoculturas no hospital público e 123 amostras no hospital privado. Os nãofermentadores foram a maior causa de bacteremias na unidade de terapia intensiva (UTI) da instituição pública. As enterobactérias foram os microrganismos mais prevalentes nas enfermarias da unidade privada. No hospital público foram detectadas amostras produtoras de ESBL, enquanto no hospital privado foram identificadas cepas produtoras de carbapenemases. Dentre as cepas de P. aeruginosa, 40 amostras foram testadas para a produção de MBLs. Treze cepas (32,5%) foram positivas no teste fenotípico e no PCR, todas positivas para o gene blaSPM-1, sendo que apenas uma foi proveniente da instituição pública e 12 do hospital particular. Nenhuma amostra carreadora dos genes blaIMP-1 e blaVIM-2 foram detectadas. Os resultados de PFGE mostraram que todas as cepas carreadoras do gene blaSPM-1, isoladas no hospital privado, foram geneticamente relacionadas (Pulsotipo A), o que pode indicar uma transmissão cruzada entre os pacientes e profissionais de saúde. CONCLUSÕES: As características dos microrganismos isolados em hemoculturas variou entre as unidades de internação e entre os hospitais, demonstrando que os dados locais podem orientar a terapia antimicrobiana e as medidas de controle e prevenção das infecções. Devido ao impacto das infecções de corrente sanguínea e a presença de microrganismos resistentes no ambiente hospitalar, estudos adicionais e medidas de vigilância são necessárias / Introduction: Bloodstream infections are one of the most serious bacterial infections, especially if caused by resistant microorganisms. The purpose of this study was to assess the prevalence and resistance profile of pathogens isolated from blood cultures in a public and a private hospital of Niterói, Rio de Janeiro, Brazil. METHODS: A case-series of patients with blood stream infection was conducted at a 227-bed public general hospital and at a 123-bed private general hospital from August 2009 to August 2010. All isolates were consecutively detected from patients minimum age of 18 years and identified by the routine methodology used at each laboratory. Every E. coli, K. pneumoniae, K. oxytoca and P. mirabilis isolates were tested for ESBL (Extended Spectrum Beta-lactamase) production using the CLSI guidelines. The Enterobacteriaceae was tested for carbapenemase production using the Modified Hodge test (CLSI). Every P. aeruginosa were tested for metallo-betalactamases (MBLs) producing by the phenotypic method of combined disk. The polymerase chain reaction (PCR) was used to detect the MβLs genes (blaIMP, blaVIM, blaSPM). The genetic similarity between the strains was evaluated in samples which were positive for MBLs using the pulsed field gel electrophoresis technique (PFGE). RESULTS: Were collected 195 samples of microorganisms isolated in blood cultures in the public hospital and 123 samples in the private hospital. The non-fermentatives were the major cause of bacteremia in the ICU of public hospital. The Enterobacteriaceae were the most prevalent in the the wards of private hospital. In the public hospital, we found strains producing ESBL and in the private hospital, strains producing carbapenemase. Forty samples of P. aeruginosa were tested for MBL producing. Thirteen strains (32,5%) were positive in phenotypic test and in PCR, every sample were positive for blaSPM-1, and only one was from the public institution and 12 of the particular hospital. No blaIMP-1 and blaVIM gene were detected. The PFGE analysis showed that all blaSPM-1 gene-carrying strains isolated in private hospital were genetically related (Pulsetype A), suggesting a cross transmission between patients and health professionals. CONCLUSIONS: The characteristics of the microorganisms isolated from blood culture varied from hospital to hospital and between inpatient units, showing that local data can help with therapeutic choices and with the prevention and control of infection. Due to the impact of bloodstream infections and the presence of resistant microorganisms in the hospitals, additional studies and monitoring measures are necessary

Extended-spectrum β-lactamases (ESBLs) constitute a major problem given their broad substrate specificity and ability to hydrolyse many of the extended-spectrum third-generation cephalosporins currently in use in hospital settings. Guiana extended-spectrum-type (GES-1 – GES-9) ESBL enzymes have mainly been found in Pseudomonas aeruginosa (P. aeruginosa) and only at a limited number of geographical sites, mainly France, Greece and South Africa. Detection of GES-type ESBL-producing P. aeruginosa isolates in the clinical microbiology laboratory using conventional methods is problematic with molecular methods yielding better results. The aim of this study was to utilise various molecular techniques to determine the prevalence of GES-type ESBLs, characterise their genetic determinants and determine their clonal relatedness. The study further aimed to apply a sequence-selective, competitive PNA-based multiplex PCR in real-time for the identification and differentiation of GES-type enzymes. The prevalence of GES-type ESBLs was determined successfully through DNA sequencing. An increase in GES-2 prevalence since 2000 was noted which emphasised the importance of constant surveillance to monitor antibiotic determinants, their spread and overall prevalence. The knowledge on prevalence could be used in turn to monitor the efficacy of infection control measures and antibiotic regimens. Repeated sequencing confirmed the presence of blaGES-5 in P. aeruginosa isolates. As far as could be established, this study reported the first occurrence of GES-5 in South Africa and was the second description of GES-5 in P. aeruginosa. Application of a sequence-specific, competitive PNA-based multiplex PCR in real-time utilising SYBR Green was not suitable for the identification and differentiation of the blaGES genes. Although the method achieved different melting temperatures for the bla<GES genes tested, these temperatures were not suitable for accurate differentiation. Melting temperatures obtained for the same blaGES gene varied and those for different genes overlapped. An approach exploiting the high temperature shift caused by the PNA-probe rather than its competitive nature might be more successful. Random amplified polymorphic DNA typing has been described as a fast and simple method with high discriminatory power for the typing of P. aeruginosa and was thus used to determine the clonal relatedness of the bla<GES positive P. aeruginosa isolates. The occurrence of identical or similar P. aeruginosa isolates producing ESBLs in a single hospital setting emphasised the importance of constant surveillance. The study further identified identical P. aeruginosa clones that occurred in different hospitals indicating spread from a common external reservoir into these hospitals. The occurrence of highly drug-resistant P. aeruginosa in the environment has serious implications in a country with an ever increasing immune-compromised population. These finding were of concern since they demonstrated that acquired GES ESBLs can rapidly emerge and become a major cause of broad-spectrum β-lactam resistance among nosocomial pathogens. The information obtained in this study should be used to create awareness of the potential ESBL problem threatening current antimicrobial regimens in South Africa. / Dissertation (MSc (Medical Microbiology))--University of Pretoria, 2008. / Medical Microbiology / unrestricted