Made available in DSpace on 2014-06-11T19:22:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-08-15Bitstream added on 2014-06-13T18:48:56Z : No. of bitstreams: 1 rodrigues_lo_me_arafcf.pdf: 364674 bytes, checksum: 25ad80987f7d2eb4d8bf537154a09922 (MD5) / Universidade Estadual Paulista (UNESP) / 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. / 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.

Throughout this thesis, we studied the biochemical and structural impact of the essential residues on the activity of class D beta-lactamases. The production of these enzymes plays a major role in the bacterial resistance. Our work is subdivided in two parts : the study of the post-translational modification of lysine 70 and the screening of new potential inhibitors for the class D β-lactamases. The first part concerns the impact of the residues tryptophan 154 and valine 117 located in the hydrophobic core. Our data indicate that the mutation of tryptophan 154 in alanine or glycine lead to a large decrease of the catalytic efficiencies of the beta-lactamase. The apo-enzyme structures of these mutants show that the lysine 70 is not carboxylated. This absence of carboxylate group induces a modification of the hydrogen network of the active site. The analysis of the complex structure of W154A-benzylpenicillin demonstrates that the deacylation step is clearly the most affected by the mutation. The mutation of tryptophan 154 in histidine leads to a slight decrease of catalytic efficiencies because the imidazol group of histidine mimics the indole group of tryptophan 154. The apo-enzyme structure reveals that lysine 70 is partially carboxylated and stabilized by an hydrogen bond between the carboxylate group and the imidazol group. In the case of the V117T mutant, a strong increase of the catalytic constant values is observed at 50 mM in NaHCO3. The structure of this mutant at pH 8.0 shows that the lysine 70 is partially carboxylated in the monomer A. The determination of individual rate constants of acylation and deacylation steps indicates that the deacylation is the limiting step for the class D beta-lactamase. The k2/k3 ratio is similar between the V117T mutant and the wild-type enzyme. The mutation of lysine 70 in alanine or cysteine leads to a large decrease of the deacylation constants inducing a poorly efficient enzyme. The obtaining of the K70C-Ampicillin complex by X-ray cristallography and the trapping of acyl-enzyme by reaction with fluorescent ampicillin are supplemental proofs that the deacylation step is the limiting rate. By crystallographic and kinetic studies, we demonstrate that the chloride inhibition of the class D beta-lactamases is due to a competition between the carboxylate group of lysine 70 and the chloride ions. At high concentration in bicarbonate, this inhibition is abolished for the wild-type enzyme. The second part of this work concerns the screening of the citrate and aminophosphonate derivated molecules for the class D beta-lactamases. In the case of OXA-10, a citrate molecule is strongly stabilized by hydrogen bonds in the active site. The benzyl esters derivatives of citrate inhibits OXA-10(KI = 20 µM) but the hydrophobic substituents are necessary to obtain a good inhibition.

Made available in DSpace on 2014-07-29T15:30:34Z (GMT). No. of bitstreams: 1 Dissertacao Diana Christina P S Goncalves.pdf: 438422 bytes, checksum: 43edf5906fb5547aa85bb981cb94aaf2 (MD5) Previous issue date: 2009-02-18 / P. aeruginosa is frequently isolated in hospitals and the clinical importance has been increased due to gravity of infections. The metallo-beta-lactamase (MBL) production is an emergent mechanism of resistance in P. aeruginosa. The study aimed to determine the antimicrobial susceptibility profile of P. aeruginosa isolated of patients admitted in a hospital in Goiânia, to verify the MBL production by diffusion test and detect MBL genes by PCR technique. A total of 75 samples were evaluated, isolated of various clinical samples, in the period of January/2005 to January/2007. The biochemical identification was performed by automation technique system (API 20E ®) and antimicrobial susceptibility profile by Kirby- Bauer method. The 75 P. aeruginosa presented multi-drug resistance and, the resistance profile was: 90.7% to ceftazidime: 30.7% to aztreonam, 97.3% to ciprofloxacin; 48.0% of resistance to piperacilin/tazobactam, 88.0% to cefepime; amicacin, gentamicin and tobramicina whit resistance profile of 78.7%, 84.0% and 77.4%, respectively. The MBL production by difusion disc method was 46.7% (35/75). The gene blaSPM-1 was detected in 39 (52.0%) and gene blaIMP-1 in three (4.0%) isolates. The high frequency of P. aeruginosa resistant and MBL production alert to necessity of control the dissemination of bacteria multi-drug resistant in hospital, as well as the adoption of preventive actions and explanation of the health workers about rational use of antibiotics. / P. aeruginosa é frequentemente isolada em ambientes hospitalares e sua importância clínica têm aumentado devido à gravidade das infecções. A produção de metalo-beta-lactamase (MBL) é um mecanismo de resistência emergente entre P. aeruginosa. O estudo teve como objetivo determinar o perfil de suscetibilidade antimicrobiana de P. aeruginosa isoladas de pacientes internados em um hospital de Goiânia, realizar a triagem fenotípica para verificar a produção de MBL e detectar genes que codificam MBL pela técnica de PCR. Foram avaliadas 75 amostras, isoladas de diversos sítios, no período de janeiro de 2005 a janeiro de 2007. A identificação bioquímica foi realizada pelo sistema API 20E e o antibiograma pelo método de Kirby-Bauer. Todos os 75 isolados de P. aeruginosa apresentaram multirresistência, 82,7% foram resistentes ao imipenem; ceftazidima 90,7%; aztreonam 30,7%; ciprofloxacina 97,3%; 48,0% de resistência a piperacilina/tazobactam, 88,0% ao cefepime; amicacina, gentamicina e tobramicina, com resistência de 78,7%, 84,0% e 77,4%, respectivamente. A produção de MBL pelo método de disco aproximação foi detectada em 46,7% (35/75). O gene blaSPM-1 foi detectado em 39 (52,0%) e o blaIMP-1 em três (4,0%) amostras através da técnica de PCR. A frequência elevada de P. aeruginosa multirresistentes e produtoras de MBL alerta para necessidade de controle da disseminação de resistência no ambiente hospitalar, bem como a adoção de medidas preventivas e esclarecimento das equipes de saúde sobre uso racional dos antimicrobianos.

The University Hospital of Liège has 955 beds in 8 intensive care units, 15 medical wards, 10 surgical wards and 1 paediatric ward. Approximately 36,000 patients are admitted each year, giving a total of 265,000 patient-days hospitalization. Extended-spectrum beta-lactamase-producing Enterobacteriaceae (E-ESBL) constitute, along with methicillin-resistant Staphylococcus aureus (MRSA), the main multi-resistant bacteria recovered in our hospital. The aims of the present study were to: - evaluate the epidemiology of E-ESBL - evaluate the impact of an infection control programme to reduce the spread of E-ESBL in the University Hospital of Liège. In order to do this, several studies were carried out between 2001 and 2007: 1. Determination of the high risk units in the CHU (2001) The high risk units were determined by comparing the incidence rates of each type of unit. Two types of high risk unit were identified in this way: the Intensive Care Units (ICUs) and the Onco-Haematology Unit. 2. Epidemiology of E-ESBL in the Onco-Haematology unit (2002-2003 and 2005-2006) Digestive tract colonization by E-ESBL was found to be relatively high (7.3%) and this explains the high incidence of E-ESBL in Onco-Haematology in comparison with the rest of the hospital. However, the clinical gravity associated with exposure to the risk factor (digestive tract colonization by E-ESBL) was found to be relatively weak. 3. Importance of digestive tract colonization by E-ESBL in General ICUs (2002-2003) Digestive tract colonization by E-ESBL was found to be relatively common (8.8%) and faecal carriage of E-ESBL was found to be a good marker for infection with E-ESBL at another body site. Even though the number of infected patients was found to be low, the risk of infection due to E-ESBL was multiplied by 14.7 in a group of digestive carriers of E-ESBL with regard to a group of non carriers. Our data also showed that Enterobacter aerogenes is the most frequent species producing extended-spectrum beta-lactamase (ESBL) and that TEM-24 is the most prevalent ESBL produced by E-ESBL species in our ICUs. No CTX-M-type genes were identified. With regard to antibiotic susceptibility, meropenem and cefepime appeared to be the most active agents against the majority of isolates. 4. Impact of an infection control programme to reduce the spread of E-ESBL (2006-2007) A surveillance programme was carried out to evaluate the implementation of infection control procedures including surveillance of ESBL-producing strains, utilization of computer alerts for E-ESBL positive patients and the application of contact precautions for colonized or infected patients. Infection control compliance observations were performed by trained referring nurses. During the 2 years of application, one or more E-ESBL were identified in 500 patients. A total of 2268 internal messages regarding the identification of E-ESBL were sent within the hospital, among which 91.84 % were received (at least 1 for every patient). An alert was associated with 406 patients, who were always hospitalized as the identification of the E-ESBL by the laboratory was obtained. A total of 257 registration forms were filled in by the referring nurses, resulting in a survey compliance of 63%. This survey showed that door signs identifying positive patients, hydro-alcoholic solution and gloves were present in 90% of the cases, but that gowns were only present in 59%. The overall incidence of nosocomial acquisition of E-ESBL between 2006 and 2007 was 0.92/1000 patient-days, more or less the same as in 2002. In relation to this research, several questions remain: - Even though the rates of digestive tract colonization with E-ESBL in the 2 types of high risk unit were found to be more or less the same (7.3 and 8.8%), the impact on infections due to E-ESBL was very different. - Are the infections due either to E-ESBL endogenous infections (owing notably to the use of broad spectrum antibiotics) or to secondary infections (resulting from cross-transmission) or to both? The implementation of an infection control programme to limit the spread of E-ESBL has been based on the limitation of the cross-transmission of these micro-organisms. An enhanced barrier precautions policy has been in place in our institution for 2 years, and we have seen no erosion in compliance. We should not however lose sight of the fact that, whatever the institutional policy for the management of multi-resistant bacteria, the correct application of standard precautions for all patients is the first measure to limit the cross-transmission of all micro-organisms.

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.

ß-hemolytic streptococci persist in 10-25% of patients with acute streptococal tonsillitis (about 10.000-25.000 per year in Sweden) in spite of treatment with a recommended dosage and schedule of Phenoxymethylpenicillin. The aim of the study was to investigate different bacteriological factors involved in treatment failures of streptococcal tonsillitis. Patients included in the study were 33 patients who underwent tonsillectomy, 62 persons included in a tonsillitis epidemic outbreak, 267 tonsillitis patients contacting the ENT-clinic, Sahlgrenska Hospital, Göteborg, and 20 healthy volunteers taking Phenoxymethylpenicillin. It was found that the Steer's steel pin replicator was a useful tool to study interference between a- and ß-hemolytic streptococci and a guantitative differen ce in. the inhibitory capacity of the different a-strains was noted, a-streptococci with a strong inhibitory capacity on ß-streptococci were isolated mainly from individuals seemingly resistant to ß-streptococcal tonsillitis, while from patients with repeated tonsillitis no or low numbers of inhibiting a-streptococci were demonstrated. Patients with clinical treatment failure had less a-streptococci with inhibiting capacity on their own ß-streptococcal strain compared with the healthy carriers. These treatment failures also showed beta-lactamase activity in their saliva pellet significantly more often than patients in the control groups. In volunteers penicillin was released from ordinary sugar coated tablets already in the mouth resulting in a decrease of the a-strep- tococcal flora. A synergistic effect on ß-hemolytic killing by low concentration of penicillin and inhibition of a-streptococci was noted in vitro and in vivo. Penicillin tolerance was registered in most strains from the treatment failure group, but in none of the strains from the group of successfully treated patients. A co-operation between different bacteriological factors (bacterial interference, beta-lactamase production, penicillin tolerance) seems to be important in treatment failures of streptococcal tonsillitis. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1986, härtill 6 uppsatser</p> / digitalisering@umu

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