Introdução - A crescente resistência antimicrobiana em bactérias responsáveis por infecções hospitalares é um grande desafio à Saúde Pública. as B-lactamases de espectro estendido (ESBL), que hidrolisam a maioria dos compostos B-lactâmicos, são reconhecidas mundialmente como um grande problema para pacientes hospitalizados, devido à localização de seus genes em elementos transferíveis, facilitando sua disseminação. Objetivo - Caracterizar geneticamente cepas de Enterobactérias produtoras de ESBL isoladas de pacientes de um hospital público da cidade de São Paulo. Material e métodos - Todas as cepas de enterobactérias produtoras de ESBL isoladas em um ano foram submetidas a análises moleculares pela PCR, com iniciadores específicos para oito genes bla, e as cepas de Klebsiella pneumoniae ESBL positivas (ESBL-Kp) identificadas nesse período foram comparadas pela técnica de PFGE.Resultados - Os genes, bla(tem), bla(shv), bla(ctx-m), bla(per-2) bla(veb) and bla(ges) foram identificados em 9 espécies: Klebsiella pneumoniae (71,5 por cento), Escherichia coli (13,5 por cento), Morganella morganii (6 por cento), Proteus mirabilis (3 por cento), Klebsiella oxytoca (1,5 por cento), Providencia rettgeri (1,5 por cento), Providencia stuartii (1,5 por cento), Enterobacter aerogenes (0,75 por cento). Os genes bla(per-1) e bla(oxa) não foram detectados. O PFGE revelou 8 perfis moleculares principais em 68,4 por cento das ESBL-Kp, e 31,6 por cento das cepas não estavam relacionadas. Conclusões - Os resultados de PCR revelaram uma grande variedade de grupos de ESBL, e aparentemente este é o primeiro relato de grupos GES e VEB em enterobactérias no Brasil. / Introduction - The increasing antimicrobial resistance in pathogenic bacteria causing nosocomial infections is a major public health challenge. The extended-spectrum β-lactamases (ESBL), which hydrolyze most of β-lactams, are recognized worldwide as a great problem to hospitalized patients, due to the transferable location of their genes, which facilitates their spreading. Objective - Genetically characterize ESBL-producing Enterobacteriaceae strains isolated from patients of a Public Hospital in the city of São Paulo. Material and Methods - All Enterobacteriaceae ESBL-producing strains isolated in an 1-year period were submitted to molecular analysis by PCR with specific primers for eight bla genes, and all ESBL Klebsiella pneumoniae (ESBL-Kp) identified in this period were compared by the PFGE technique. Results - Genes blaTEM, blaSHV, blaCTX-M, blaPER-2, blaVEB and blaGES were identified in 9 species: Klebsiella pneumoniae (71,5%), Escherichia coli (13,5%), Morganella morganii (6%), Proteus mirabilis (3%), Klebsiella oxytoca (1,5%), Providencia rettgeri (1,5%), Providencia stuartii (1,5%), Enterobacter aerogenes (0,75%) and Enterobacter cloacae (0,75%). Genes blaPER-1 and blaOXA were not detected in any strain. PFGE revealed 8 distinct main molecular patterns in 68,4% of ESBL-Kp, and 31,6% of the strains were totally unrelated. Conclusions - PCR results showed a great variety of ESBL groups in the institution, and apparently this is the first report of GES- and VEB-ESBL groups in enterobacteria in Brazil. The results suggest the spread of resistance genes in different strains of ESBL-Kp in some hospital wards, and also that some strongly related clones of these bacteria colonized patients from a neonatal ward in a 3-month period.

<p>Escherichia coli i Klebsiella pneumoniae su među najznačajnijim uzročnicima infekcija kod ljudi. Problem predstavljaju multirezistentni sojevi koji se javljaju ne samo u bolničkom nego i u vanbolničkom okruženju. Karbapenemi, beta-laktami sa naj&scaron;irim spektrom delovanja, spadaju u lekove poslednje linije odbrane. Rezistencija na karbapeneme među enterobakterijama je u porastu &scaron;irom sveta. Može nastati usled prisustva karbapenemaza, enzima koji degradiraju karbapeneme, ili usled hiperprodukcije AmpC cefalosporinaza ili beta-laktamaza pro&scaron;irenog spektra uz gubitak porina. Geni koji kodiraju karbapenemaze se nalaze na mobilnim genetičkim elementima koji im omogućavaju brz prenos. Najče&scaron;će karbapenemaze su KPC, NDM, VIM, IMP i OXA-48 enzimi. Detekcija sojeva koji produkuju karbapenemaze nije moguća samo na osnovu profila rezistencije izolata, s obzirom da minimalne inhibitorne koncentracije karbapenema mogu biti u referentnom opsegu. Svaki izolat sa smanjenom osetljivo&scaron;ću na karbapeneme bi trebalo ispitati kako bi se sprečilo njihovo &scaron;irenje. Detekcija karbapenemaza može da se zasniva na fenotipskim i genotipskim metodama. Ciljevi istraživanja su bili da se utvrdi postojanje rezistencije na karbapeneme kod multirezistentnih izolata Escherichia coli i Klebsiella pneumoniae iz kliničkih uzoraka, da se dokaže produkcija karbapenemaza kori&scaron;ćenjem fenotipskih i genotipskih testova, kao i da se analizira osetljivost izolata Escherichia coli i Klebsiella pneumoniae sa molekularno dokazanim karbapenemazama. Istraživanje je sprovedeno kao prospektivna studija u periodu 01.11.2013. do 01.11.2014. godine u Centru za mikrobiologiju Instituta za javno zdravlje Vojvodine u Novom Sadu. U istraživanje je bilo uključeno 300 multirezistentnih izolata Escherichia coli i Klebsiella pneumoniae konsekutivno izolovanih iz kliničkih uzoraka (krv, punktat, sekret iz donjeg respiratornog trakta, urin i sekret rana) hospitalizovanih pacijenata. Identifikacija do nivoa vrste je vr&scaron;ena klasičnim bakteriolo&scaron;kim metodama. Za ispitivanje osetljivosti kori&scaron;ćeni su disk difuziona metoda i gradijent testovi. Vrednosti minimalnih inhibitornih koncentracija su ispitane automatizovanim Vitek 2 sistemom (BioM&eacute;rieux, Francuska), a interpretacija izvr&scaron;ena u skladu sa preporukama CLSI (Clinical Laboratory Standards Institute). Za fenotipsko testiranje prisustva betalaktamaza pro&scaron;irenog spektra kori&scaron;ćen je kombinovani disk test. Za fenotipsko testiranje prisustva karbapenemaza kod sojeva rezistentnih na karbapeneme kori&scaron;ćen je kombinovani disk test i test sinergizma sa dva diska. Detekcija gena za beta-laktamaze blaCTXM, gena za karbapenemaze blaKPC, blaVIM, blaNDM, blaIMP i blaOXA-48-like izvr&scaron;ena je metodom lančane reakcije polimeraze. Genotipizacija odabranih izolata Klebsiella pneumoniae izvr&scaron;ena pomoću repetitivne lančane reakcije polimeraze kori&scaron;ćenjem DiversiLab sistema (BioM&eacute;rieux, Francuska). Od 300 multirezistetntnih izolata, bilo je 242 (80,7%) Klebsiella pneumoniae i 58 (19,3%) Escherichia coli izolovanih iz kliničkih uzoraka. Smanjenu osetljivost na bar jedan karbapenem (imipenem, meropenem, ertapenem) pokazalo je 179 (59,7%) izolata. Fenotipski test za dokazivanje produkcije betalaktamaza pro&scaron;irenog spektra bio je pozitivan kod 87/171 (50,9%) izolata. Gen blaCTX-M je dokazan kod 111/121 (91,7%) izolata. Fenotipski test za dokazivanje karbapenemaza bio je pozitivan kod 65/179 (36,3%) izolata, kod 63 (96,9%) je ukazivao na prisustvo metalo-beta laktamaza, a kod 2 (3,1%) na prisustvo karbapenemaza iz grupe A. Senzitivnost fenotipskog testa za dokazivanje karbapenemaza klase A i B iznosila je 100,0%, specifičnost 96,6%, a ukupna tačnost 97,6%. Karbapenemaze su nađene kod 79/179 (44,1%) izolata rezistentnih na karbapeneme. Gen blaNDM nađen je kod 58 (32,4%) izolata, blaOXA- 48-like kod 11 (6,1%), a blaKPC kod 2 (1,1%) izolata. Geni blaVIM i blaIMP nisu detektovani. Kod 8 (4,5%) izolata nađena su 2 gena koja kodiraju karbapenemaze, blaNDM i blaOXA-48-like. Određivanjem osetljivosti disk difuzionom metodom i automatizovanim Vitek 2 sistemom, izolati koji produkuju karbapenemaze pokazivali su smanjenu osetljivost na sve testirane beta-laktame i gentamicin, odnosno tobramicin. Visok procenat rezistenicije izolati su pokazali u odnosu na ciprofloksacin, levofloksacin i trimetoprim/sulfametoksazol. Najefikasniji antibiotski lekovi su bili amikacin, tigeciklin, fosfomicin i kolistin. Poređenjem minimalnih inhibitornih koncentracija izolata koji produkuju i izolata koji ne produkuju karbapenemaze utvrđena je statistički značajna razlika za meropenem, imipenem, ertapenem, amikacin, gentamicin. Genotipizacijom odabranih izolata Klebsiella pneumoniae kori&scaron;ćenjem DiversiLab sistema klonalno &scaron;irenje je dokazano među izolatima koji produkuju NDM i OXA-48-like karbapenemaze u okviru iste zdravstvene institucije, ali i među različitim zdravstvenim ustanovama. Među izolatima rezistentnim na karbapeneme Klebsiella pneumoniae se če&scaron;će izoluje od Escherichia coli. Kod izolata koji su pokazali smanjenu osetljivost prema bar jednom karbapenemu, karbapenemaze su detektovane u manje od polovine izolata. Kod ostalih izolata dokazane su beta-laktamaze pro&scaron;irenog spektra koje uz gubitak porina mogu uzrokovati rezistenciju na karbapeneme. Kod izolata Klebsiella pneumoniae sa dokazanim genima koji kodiraju karbapenemaze detektovani su pojedinačni blaKPC, blaNDM i blaOXA-48-like geni, kao i kombinacija gena blaNDM i blaOXA-48-like. Kod izolata Escherichia coli nađeni su samo blaNDM geni. Najefikasniji antibiotski lekovi za izolate koji produkuju karbapenemaze su amikacin, tigeciklin, fosfomicin i kolistin. Izolati sa dokazanim karbapenemazama pokazuju rezistenciju na veći broj antibiotika u odnosu na izolate koji ne produkuju karbapenemaze. Dokazano je klonalno &scaron;irenje izolata Klebsiella pneumoniae koji produkuju karbapenemaze. Testove za fenotipsku detekciju karbapenemaza bi trebalo koristiti i u rutinskim mikrobiolo&scaron;kim laboratorijama u skladu sa EUCAST (European Committee on Antimicrobial Susceptibility Testing) preporukama, a konačnu potvrdu treba izvr&scaron;iti molekularnim metodama u referentnoj laboratoriji.</p> / <p>Escherichia coli and Klebsiella pneumoniae are among the most common human pathogens. Multiresistant strains are emerging not only in hospital settings, but also in the community representing a major concern. Carbapenems, beta-lactams with the broadest spectrum of activity are considered to be antibiotics of last resort. Resistance to carbapenems among enterobacteria is spreading worldwide. It is mainly caused by carbapenemases, enzymes capable of degrading carbapenems or by hyperproduction/overexpression of AmpC betalactamases or extended spectrum betalactamases with porin loss. Carbapenemaseencoding genes are usually located on mobile genetic elements providing their fast transfer. The most common carbapenemases are KPC, NDM, VIM, IMP and OXA-48. The detection of carbapenemase-producer cannot rely only on the resistance profile as their minimal inhibitory concentration values may sometimes lay within the susceptibility range. Therefore, every multidrug-resistant isolates with lower susceptibility to carbapenems should be tested for the presence of carbapenemases in order to prevent further spreading. The detection of carbapenemases is based on phenotypic and genotypic methods. The aims of the study were to determine the occurrence of carbapenem resistance in multidrug-resistant Escherichia coli and Klebsiella pneumoniae isolated from clinical samples, to detect carbapenemase production using both phenotypic and genotypic methods and to analyze the susceptibility of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae. The study was conducted from 1st November 2013 to 1st November 2014 at the Center for Microbiology in the Institute for Public Health of Vojvodina, Novi Sad, Serbia. The study included 300 nonrepetitive multidrug-resistant strains of Escherichia coli and Klebsiella pneumoniae isolated from clinical specimen (blood, aspirates, lower respiratory tract secretions, urine and wound secretion) of hospitalized patients. Identification of isolated strains was done using conventional bacteriological methods. Antimicrobial susceptibility was tested using the disk diffusion method and MIC test strips. Minimal inhibitory concentrations were determined using Vitek 2 Compact automated system (BioM&eacute;rieux, France), interpreted according to the CLSI (Clinical and Laboratory Standards Institute) recommendations. Phenotypic testing of extended-spectrum beta-lactamases production was done using combined disk test. Phenotypic testing of carbapenemase production was done by combined disk test and double-disk synergy test. Detection of blaCTX-M, gene encoding extended-spectrum beta-lactamases and blaKPC, blaVIM, blaNDM, blaIMP i blaOXA-48-like, genes encoding carbapenemases was done using PCR. Genotyping of selected Klebsiella pneumoniae isolates was done by repPCR using DiversiLab system (BioM&eacute;rieux, France). From the total of 300 multiresistant isolates, 242 (80.7%) were Klebsiella pneumoniae and 58 (19.3%) were Escherichia coli obtained from clinical samples. Reduced susceptibility to at least one carbapenem (imipenem, meropenem, ertapenem) was found in 179 (59.7%) isolates. Phenotypic test for extended-spectrum betalactamases production was positive in 87/171 (50.9%) isolates. A total of 111/121 (91.7%) isolates harbored blaCTX-M. Phenotypic test for carbapenemase production was positive in 65/179 (36.3%) isolates, 63 (96.9%) indicating the presence of metallo-beta-lactamases and 2 (3.1%) indicating the presence of class A carbapenemases. Sensitivity of the phenotypic test for carbapenemase production of class A and B was 100.0%, specificity 96.6% and overall accuracy 97.6%. Carbapenemases were detected in 79/179 (44.1%) carbapenemresistant isolates. Gene blaNDM was found in 58 (32.4%) isolates, blaOXA-48-like in 11 (6.1%) and blaKPC in 2 (1.1%) isolates. Genes blaVIM and blaIMP were not detected. In 8 (4.5%) isolates 2 genes encoding carbapenemases were found, blaNDM and blaOXA-48-like. Using both disk diffusion method and Vitek 2 automated system for antimicrobial susceptibility testing carbapenemase-producing isolates were resistant to all beta-lactams and also to gentamicin and tobramicin respectively. Resistance rates were high for ciprofloxacin, levofloxacin and cotrimoxazole. Good activity maintained for amikacin, tigecycline, fosfomycin and colistin. Comparing minimal inhibitory concentrations of carbapenemaseproducing isolates and non-carbapenemase producers, significant difference was found for meropenem, imipenem, ertapenem, amikacin and gentamicin. Genotyping of selected Klebsiella pneumoniae isolates using DiversiLab system, revealed the clonal spread of NDM- and OXA-48-like-producers not only within one healthcare-setting, but also between different healthcare centers. Among carbapenem-resistant isolates, Klebsiella pneumoniae was found more often than Escherichia coli. Carbapenemases were detected in less than 50% of isolates resistant to at least one carbapenem. In other carbapenem resistant isolates extended-spectrum betalactamases were confirmed most likely causing carbapenem-resistance with porin deficiency or porin loss. Among carbapenemase-producing Klebsiella pneumoniae blaKPC, blaNDM and blaOXA-48-like genes were detected, as well as combination of 2 genes blaNDM and blaOXA-48-like. In carbapenemase-producing Escherichia coli only blaNDM was found. The most efficient antimicrobial drugs among tested carbapenemase-producing isolates were amikacin, tigecycline, fosfomycin and colistin. Carbapenemase-producing isolates were resistant to more antimicrobial agents compared to non-carbapenemase producers. Clonal dissemination of carbapenemase-producing Klebsiella pneumoniae was confirmed. Phenotypic detection of carbapenemase production should be done in routine microbiology laboratories according to EUCAST (European Committee on Antimicrobial Susceptibility Testing) recommendations. Final confirmation should be done by molecular methods in the reference laboratory.</p>

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2015-06-29T18:23:12Z No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_InvestigacaoGenesBla.pdf: 1111769 bytes, checksum: 0a840604d3d503325d9540739858f390 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2015-07-23T13:49:39Z (GMT) No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_InvestigacaoGenesBla.pdf: 1111769 bytes, checksum: 0a840604d3d503325d9540739858f390 (MD5) / Made available in DSpace on 2015-07-23T13:49:39Z (GMT). No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_InvestigacaoGenesBla.pdf: 1111769 bytes, checksum: 0a840604d3d503325d9540739858f390 (MD5) Previous issue date: 2012 / As beta-lactamases são produzidas por bactérias gram-positivas e gram-negativas. Cerca de 40% a 50% das mulheres desenvolveram um infecção urinária durante a sua vida adulta. O objetivo o presente trabalho foi realizar a caracterização dos genes Bla SHV, Bla TEM e Bla CTX-M produtoras de beta-lactamases de espectro estendido em E. coli e Klebsiella spp isoladas de gestante com infecção do trato urinário atendidas na Unidade Básica de Saúde de Imperatriz - MA no período de maio a agosto de 2012. Participaram do presente estudo 50 de mulheres grávidas maiores de 18 anos, que apresentaram sintomas com caracterização clínica de infecção do trato urinário (ITU), referenciadas no ambulatório na Unidade Básica de Saúde Imperatriz - MA. Foi coletada urina, para realização da urinocultura e antibiograma, posteriormente foi realizado a Reação em Cadeia da Polimerase (PCR) detectar a presença dos genes Bla SHV, Bla TEM e Bla CTX-M produtores das enzimas Beta-lactamases. A média de idade destes pacientes foi de 21 anos, sendo 42% estando na faixa etária de 18 a 25 anos, 70% destas tem residência fixa em Imperatriz e os outros 30% são oriundos de outros municípios. Entre estas, 24% das voluntárias já apresentaram ITU durante a gravidez e fora do estado gravídico e 80% delas afirmaram fazer o uso de antibióticos sem prescrição médica. Das 12 amostras com crescimento microbiológico positivo, 11 foram positivas para Escherichia coli com resultados do antibiograma que apresentaram resistência para o antibiótico cefalotina, em 91,7%. Sendo isolado, uma cepa de Klebsiella ssp, e esta apresentou resistência a todos os antibióticos testados. Pela análise dos resultados da PCR das amostras isoladas, os três genes Bla SHV, Bla TEM e Bla CTX-M, não foram observadas nas amostras P9, P11 e P12, porém nas demais amostras houve a ocorrência de pelo menos um dos genes. Este estudo confirmou a presença dos três tipos de genes (Bla SHV, Bla TEM e Bla CTX-M) entre as amostras estudada. / Beta-lactamases are produced by gram-positive and gram-negative bacteria. About 40% to 50% of women developed a urinary tract infection during their adult life. The aim of the present study was the characterization of genes Bla SHV, Bla TEM e Bla CTX-M beta-lactamase-producing extended-spectrum E. coli and Klebsiella spp isolated from pregnant women with urinary tract infection treated at a Basic Health Unit of Empress - MA from May to August 2012. The study included 50 pregnant women over 18 years who presented with symptoms clinical characterization of urinary tract infection (UTI), referenced in the clinic in the Basic Health Empress - MA. Urine was collected for urine culture and sensitivity of achievement, subsequently was performed Polymerase Chain Reaction (PCR) to detect the presence of genes Bla SHV, Bla TEM e Bla CTX-M, enzymes producing beta-lactamases. The mean age of these patients was 21 years, 42% being aged 18 to 25 years, 70% of these fixed address in Empress and the other 30% come from other municipalities. Among these, 24% of the volunteers already had UTI during pregnancy and pregnancy out of state and 80% of them reported using antibiotics without prescription. Of the 12 samples with positive microbiological growth, 11 were positive for Escherichia coli with the antibiogram results that were resistant to the antibiotic cephalothin at 91.7%. Being isolated, a strain of Klebsiella spp, and this was resistant to all antibiotics tested. By analyzing the results of PCR samples isolated three genes Bla SHV, Bla TEM e Bla CTX-M, were not observed in samples P9, P11 and P12, but in the other samples was the occurrence of at least one of the genes. This study confirmed the presence of three types of genes (Bla SHV, Bla TEM e Bla CTX-M) among the samples studied.

As international travel is rapidly increasing, more people are being exposed to potentially more antibiotic resistant bacteria, a changed infectious disease epidemiology, and an increased risk of accidents and crime. Research-based advice is needed to adequately inform travellers about these risks. We studied travellers who sought advice from the Travel Medicine Clinic at the Department of Infectious Diseases, Umeå University Hospital, as well as university students from Umeå, Stockholm, and Gothenburg travelling abroad for study, research, and clinical exchange programs. From retrospective data at the Travel Medicine Clinic, we found that pre-existing health problems were rare among travellers from Umeå seeking pre- travel health advice and vaccinations. In addition, we found that the travel destination and the sex of the traveller affected vaccination levels. Although hepatitis A is endemic to both Thailand and Turkey, compared to travellers to Thailand few travellers to Turkey visited the clinic for hepatitis A vaccination. The data also revealed that more women than men were vaccinated against Japanese encephalitis despite comparable trips. A prospective survey study showed that travellers felt that the pre-travel health advice they received was helpful. Two-thirds of the travellers followed the advice given although they still fell ill to the same extent as those who were not compliant with the advice. Factors outside the control of travellers likely affect the travel-related morbidity. Compared to older travellers, younger travellers were less compliant with advice, fell ill to a greater extent, and took greater risks during travel. In a prospective survey study, we found that healthcare students had higher illness rates and risk exposure when abroad compared to students from other disciplines. This difference was mainly due to the fact that healthcare students more often travelled to developing regions during their study period abroad. When abroad, half of all students increased their alcohol consumption and this was linked to an increased risk of theft and higher likelihood of meeting a new sex partner. The healthcare students participating in the survey study also submitted stool samples before and after travel. These samples were tested for the presence of antibiotic resistance, both by selective culturing for ESBL-PE (Extended-Spectrum Beta-Lactamase Producing Enterobacteriaceae) as well as by metagenomic sequencing. About one-third (35%) of the students became colonised by ESBL-PE following their study abroad. The strongest risk factor for colonisation was travel destination; for example, 70% of students who had travelled to India became colonised. Antibiotic treatment during travel was also a significant risk factor for colonisation. The stool samples from a subset of study subjects were analysed using metagenomic sequencing. From this we learned that although the majority of resistance genes in the gut microbiome remained unchanged following travel, several clinically important resistance genes increased, most prominently genes encoding resistance to sulphonamide, trimethoprim, and beta-lactams. Overall, taxonomic changes associated with travel were small but the proportion of Proteobacteria, which includes several clinically important bacteria (e.g., Enterobacteriaceae), increased in a majority of the study subjects. Clearly, there are risks associated with international travel and these risks include outside factors as well as the personal behaviour of travellers. We believe our results can be used to develop better pre-travel advice for tourists as well as university students studying abroad resulting in safer travel.

Orientador: Maria Luiza Moretti / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T02:52:37Z (GMT). No. of bitstreams: 1 Kuboyama_RogerioHakio_D.pdf: 1899697 bytes, checksum: 863826cd141f1e5f89eb90d272c0c330 (MD5) Previous issue date: 2009 / Resumo: Os objetivos do presente estudo, conduzido retrospectivamente, utilizando cepas isoladas de espécimens clínicos obtidos de pacientes internados em dois hospitais brasileiros entre fevereiro de 2001 e junho de 2004 foram descrever: a presença de cepas de Klebsiella pneumoniae produtoras de beta-lactamases de espectro estendido (ESBLs), o melhor método e o substrato preferido para os testes de triagem e de confirmação da produção de ESBLs, a relação epidemiológica das cepas obtidas e analisar os fatores de risco para infecção por cepa produtora de ESBLs. A fim de investigar a relação genética das cepas, foram utilizadas as análises do DNA plasmidial e do DNA cromossômico por eletroforese em campo pulsátil (PFGE). Um total de 89 cepas de K. pneumoniae foram coletadas de diversos sítios anatômicos. Os espécimens clínicos mais comuns dos quais foram isoladas cepas produtoras foram urina (12,4%) e sangue (10,1%). Utilizando os critérios estabelecidos pelo CLSI (testes de triagem), 35 (39,3%) cepas de K. pneumoniae foram consideradas possivelmente produtoras de ESBLs, enquanto que o teste de aproximação de discos (DDAT) revelou distorções características nas zonas de inibição produzidas pela molécula do clavulanato em 96, 62,5, 50, 18,8 e 12,5% das cepas ao redor dos discos contendo aztreonam, cefotaxima, ceftazidima, ceftriaxona e cefpodoxima, respectivamente. Das 89 cepas, 32 (36%) foram consideradas produtoras de ESBLs baseadas no teste confirmatório pelo método Oxoid de discos-combinados. O disco contendo cefotaxima foi capaz de confirmar 100 % dos produtores de ESBLs enquanto que o disco contendo ceftazidima deixou de confirmar 2 cepas produtoras. Dez e 32 diferentes perfis plasmidiais foram observados dentre as cepas de K. pneumoniae produtoras e não produtoras, respectivamente. A PFGE demonstrou melhor poder discriminatório fornecendo 15 e 55 perfis de DNA cromossômico dentre as cepas ESBLs-positivas e ESBLs-negativas, respectivamente. Da análise univariada, as variáveis significativamente associadas com infecção por cepas de K. pneumoniae produtoras de ESBLs foram: uso de cefalosporinas de quarta geração, de lincosamida, de carbapenêmicos, de glicopeptídeos, cirurgia recente, traqueostomia, idade, dias em uso de lincosamida, dias em uso de glicopeptídeos, número total de antibióticos e duração da terapia antimicrobiana. Ao realizar a análise multivariada, utilizando um modelo de regressão logística que incluía as variáveis estatisticamente significantes da análise univariada (P < 0,05), número total de antibióticos permaneceu como única variável independente para infecção por cepa de K. pneumoniae produtora de ESBLs (OR, 1,60; IC95%, 1,194-2,145.; P = 0,0017). Os dados obtidos revelam: uma taxa relativamente alta da produção de ESBLs em cepas de K. pneumoniae obtidas de pacientes das instituições estudadas; a insuficiência da análise plasmidial na elucidação da relação genética dentre as cepas produtoras de ESBLs; aztreonam como melhor substrato indicador da produção presuntiva de ESBLs e cefotaxima como o melhor substrato no teste confirmatório pelo método Oxoid de discoscombinados / Abstract: The objectives of this study conducted restrospectively using strains isolated from clinical specimens obtained from patients hospitalized in two Brazilian hospitals between February 2001 to June 2004 were to describe the presence of extended-spectrum b-lactamase (ESBL)-producing Klebsiella pneumoniae strains, the best method and the preferred substrate for screening and confirming ESBL production and the epidemiological relatedness of ESBL-producing strains and analyse the risk factors for infection due to ESBL-producing K. pneumoniae. To investigate the genetic relatedness of the strains, plasmid analysis and chromosomal DNA analysis by pulsed-field gel electrophoresis were used. A total of 89 K. pneumoniae were collected from diverse body sites. The most commom specimens yielding ESBL-producing strains were urine (12.4%) and blood (10.1%). Using CLSI criteria (ESBL screening breakpoints), 35 K. pneumoniae (39.3%) had presumptive ESBL phenotype, while using the double-disk approximation test (DDAT) characteristic clavulanate-induced distortions of inhibition zones were found in 96, 62.5, 50, 18.8 and 12.5% of the strains around the disks containing aztreonam, cefotaxime, ceftazidime, ceftriaxone and cefpodoxime, respectively. Of 89 isolates, 32 (36%) produced ESBL based on the confirmatory Oxoid combination disk method. The disk containing cefotaxime was able to confirm 100% of the ESBL producers while the disk containing ceftazidime was not able to confirm 2 ESBL-positive strains. Ten and 32 different plasmid profiles were observed among the ESBL-producing K. pneumoniae and non ESBL producers, respectively. Pulsed-field gel electrophoresis showed the best discriminatory power giving 15 and 55 different chromosomal DNA profiles among the ESBL-positive and ESBL-negative K. pneumoniae, respectively. From univariate analysis, variables significantly associated with infection by an ESBL-producing strain of K. pneumoniae included the following: use of 4st-generation cephalosporins, lincosamide, carbapenems, glycopeptides, recent surgery, tracheostomy, age, days in using of lincosamide, days in using of glycopeptides, total number of antibiotics and duration of the antimicrobial therapy. The only variable that remained independent risk factor for acquiring infection due to ESBL-producing K. pneumoniae after multivariable analysis using a logistic regression model, which included the variables associated with acquiring infection by ESBL-producing K. pneumoniae by univariate analysis (P < 0.05), was total number of antibiotics (OR, 1.60; 95%CI, 1.194-2.145; P = 0.0017). In summary, these data indicate that ESBL-producing K. pneumoniae occur at a relatively high incidence at our institutions and the plasmid analysis is not sufficient to identify relationships between ESBL-producing strains of K. pneumoniae. The ESBL screening breakpoints and DDAT with aztreonam appear to be good indicators in presumptive detection of ESBL-producing strains and the cefotaxime used in the Oxoid combination disk method constituted the best substrate in the confirmatory test / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

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