Emergent multidrug resistant Escherichia coli increase clinical challenges. This thesis describes the resistance patterns, molecular epidemiology and mechanisms, for 315 E. coli from patients in the Vancouver Coastal Health Region for 2006/2007. Automated susceptibility testing was confirmed via E-test® for AmpC and/or ESBL production. PFGE, RFLP and PCR were used to assess genotypic relationships, and plasmid character. AmpC production was facilitated mainly by promoter mutations (54.5%). The principal ESBL detected was CTX-M-15 (49.5%). An unidentified ESBL-producer, with a pI near 8.3, was detected. A plasmid displayed variant resistance phenotypes dependent on selective growth media. A positive correlation between ST131 with CTX-M-15 and CIPR indicated the dissemination of companion phenotypes. Ciprofloxacin resistance resulted mainly (98.0%) from a double gyrA mutation. Overall fluoroquinolone resistance was not assessable due to exclusive selection parameters in this evaluation. Fluoroquinolone resistance factors require further examination to understand what causes resistant phenotypes exclusive of chromosomal mutations. ii

Emergent multidrug resistant Escherichia coli increase clinical challenges. This thesis describes the resistance patterns, molecular epidemiology and mechanisms, for 315 E. coli from patients in the Vancouver Coastal Health Region for 2006/2007. Automated susceptibility testing was confirmed via E-test® for AmpC and/or ESBL production. PFGE, RFLP and PCR were used to assess genotypic relationships, and plasmid character. AmpC production was facilitated mainly by promoter mutations (54.5%). The principal ESBL detected was CTX-M-15 (49.5%). An unidentified ESBL-producer, with a pI near 8.3, was detected. A plasmid displayed variant resistance phenotypes dependent on selective growth media. A positive correlation between ST131 with CTX-M-15 and CIPR indicated the dissemination of companion phenotypes. Ciprofloxacin resistance resulted mainly (98.0%) from a double gyrA mutation. Overall fluoroquinolone resistance was not assessable due to exclusive selection parameters in this evaluation. Fluoroquinolone resistance factors require further examination to understand what causes resistant phenotypes exclusive of chromosomal mutations. ii

Stuttgart, Univ., Diss., 2005.

Thesis (M.S.)--Miami University, Dept. of Chemistry and Biochemistry, 2008. / Title from first page of PDF document. Non-Latin script record Includes bibliographical references.

Thèse (M.Sc.) -- Université Laval, 1998. / Bibliogr.: f. 72-84. Publié aussi en version électronique.

Made available in DSpace on 2015-07-22T20:49:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-28 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Objetivo: O objetivo principal deste estudo foi avaliar a presença de carbapenemases entre amostras de enterobactérias isoladas no Complexo Hospital São Paulo (UNIFESP) entre junho e julho de 2008. Métodos: A detecção dos genes codificadores de MBL e KPC foi realizada pela técnica de PCR. O teste de triagem foi realizado para detectar a presença de enterobactérias com sensibilidade reduzida aos carbapenens, as quais foram submetidas ao teste de sensibilidade aos antimicrobianos pela técnica de ágar diluição. As amostras que confirmaram a sensibilidade reduzida a pelo menos um dos carbapenens foram submetidas ao teste de Hodge modificado, ao teste de hidrólise de β-lactâmicos e à pesquisa fenotípica e genotípica da produção de ESBL e AmpC plasmidial. A avaliação do perfil de proteínas de membrana externa foi realizada através da amplificação e seqüenciamento dos genes codificadores das porinas OmpK35 e OmpK36. A avaliação da relação genética entre as amostras com redução da sensibilidade aos carbapenens foi realizada pelo método de ribotipagem automatizada. A determinação dos grupos de incompatibilidade plasmidial foi realizada conforme descrito por Carattoli et al., 2005 e Götz et al., 1996, para 10% das amostras incluídas no estudo e para as amostras controles produtoras de MBL. Resultados: 450 amostras clínicas de enterobactérias foram estudadas. Os genes codificadores das carbapenemases do tipo MBL e KPC não foram detectados em nenhuma amostra. A taxa de sensibilidade ao meropenem, imipenem e ertapenem entre estas amostras foi de 99,3%, 98,4% e 98%, respectivamente. Os isolados com sensibilidade reduzida a pelo menos um dos carbapenens totalizaram 2,9% das amostras. Destes, somente 45,5%, (5 amostras de Klebsiella pneumoniae) confirmaram este fenótipo pela ágar diluição. O teste de Hodge e o teste de hidrólise não detectaram a produção de carbapenemases nestas amostras. Os mecanismos de resistência responsáveis pela redução de sensibilidade aos carbapenens nas amostras de K. pneumoniae foram a produção de ESBL (blaCTX-M, blaSHV e/ou blaTEM) associada a alteração nas proteínas de membrana externa (n=4), ou somente a alteração das proteínas de membrana externa (n=1). Destas amostras, três K. pneumoniae foram classificadas como pertencentes ao mesmo ribogrupo. A determinação dos grupos de incompatibilidade plasmidial foi realizada para verificar se as amostras não adquiriam os genes codificadores de MBL devido à incompatibilidade entre os plasmídeos carreadores de tais genes e os plasmídeos presentes nas amostras de enterobactérias. Não houve amplificação dos genes relacionados aos grupos de incompatibilidade plasmidial em 58,5% das amostras. Os grupos de incompatibilidade plasmidial encontrados nas demais amostras foram I1, FIA, FIB, FIC, FrepB, FIIs, P, K/B, N, L/M, A/C e Q. Somente para duas das sete amostras controles produtoras de MBL foi possível realizar a tipagem plasmidial, S. marcescens produtora de IMP-1 (IncQ) e E. cloacae produtor de IMP-1 (IncQ e IncA/C). Conclusões: Apesar da grande prevalência de P. aeruginosa e Acinetobacter spp. produtores de MBL no Hospital São Paulo, a produção de carbapenemases por enterobactérias com diminuição da sensibilidade aos carbapenens não foi detectada. A sensibilidade reduzida aos carbapenens ocorre devido à associação de β-lactamases com alteração das proteínas da membrana externa. A hipótese da não transferência dos genes codificadores de MBL devido à incompatibilidade dos plasmídeos carreadores de tais genes e os plasmídeos naturalmente presentes nas enterobactérias não pode ser confirmada porque as amostras de enterobactérias apresentavam plasmídeos não tipavéis pelas técnicas utilizadas. / Objective: The aim of this study was to evaluate the presence of carbapenemases in Enterobacteriaceae isolated in Hospital São Paulo Complex (UNIFESP) between June and July 2008. Methods: The presence of MBL- and KPC-encoding genes was investigated by PCR. A screening test was conducted to detect isolates non-susceptible to at least one carbapenem. The antimicrobial susceptibility profile was determined by the CLSI agar dilution method for all isolates non-susceptible to carbapenens. Modified Hodge test and the detection of β-lactam hydrolysis, carried out by spectrophotometer assays, were conducted for the isolates that confirmed to be non-susceptible to at least one carbapenem. The production of ESBL or plasmid-mediated AmpC β-lactamases was investigated by phenotypic tests and their respective encoding genes were investigated by PCR. Amplifications of ompK35 and ompK36 genes were performed to evaluate whether outer membrane proteins (OMPs)-encoding genes were disrupted or missing. Clonality among isolates non-susceptible to carbapenens was assessed by automated ribotyping. The incompatibility groups of plasmids were determined by PCR-based replicon typing as previously described by Carattoli et al., 2005 and Götz et al., 1996, for 10% of the isolates included in this study and of 7 MBL-producing control strains. Results: 450 Enterobacteriaceae clinical isolates were investigated. The MBL and KPC-encoding genes were not detected in any isolate. The susceptibility rate to meropenem, imipenem and ertapenem were 99.3%, 98.4% and 98%, respectively. Overall, 2.9% of the isolates were classified as nonsusceptible to carbapenens. Of those, only 45.5% (5 K. pneumoniae isolates) confirmed to be non-susceptible to at least one carbapenem by the agar dilution technique. The modified Hodge test and the β-lactam hydrolysis by spectrophotometer assays did not detect carbapenemase production in these isolates. The mechanisms conferring reduced susceptibility to carbapenems among these isolates are the production of ESBL (blaCTX-M, blaSHV and/or blaTEM) associated with altered OMPs (n=4), or only altered OMPs (n=1). Three K. pneumoniae isolates non-susceptible to carbapenens were clustered in one ribogroup. The determination of plasmids’ incompatibility group was carried out to verify if transmission of MBL-encoding genes was prevented due to incompatibility between plasmids occurring in the Enterobacteriaceae clinical isolates studied and those carrying MBL-encoding genes. The incompatibility group of 58.5% of isolates could not be determined due to lack of amplification. Among the remaining isolates the incompatibility groups I1, FIA, FIB, FIC, FrepB, FIIs, P, K/B, N, L/M, A/C and Q were found. Among the MBL producers, the incompatibility group could be determined only for two IMP-1 producers, S. marcescens (IncQ) and E. cloacae (IncQ and IncA/C). Conclusions: While MBL-production is highly prevalent among P. aeruginosa and Acinetobacter spp. clinical isolates from Hospital São Paulo, Enterobacteriaceae isolates nonsusceptible to carbapenens due to carbapenemase production were not detected. In contrast, reduced susceptibility to carbapenems occurred due to the association of β-lactamase production with altered OMPs. The hypothesis that incompatibility between plasmids could have prevented transmission of MBL-encoding genes from non-fermenter rods to Enterobacteriaceae could not be confirmed since most strains presented non-typable plasmid content. / TEDE / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2015-07-22T20:49:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-01-30. Added 1 bitstream(s) on 2015-08-11T03:25:45Z : No. of bitstreams: 1 Publico-10789.pdf: 714723 bytes, checksum: 18fda82488f54ec67849c5ef6e1425d6 (MD5) / Pseudomonas aeruginosa é um importante patógeno oportunista em pacientes hospitalizados, particularmente em pacientes oncológicos e que apresentam neutropenia. Uma das principais características desta bactéria é o desenvolvimento de resistência a múltiplos antimicrobianos, incluindo os antibióticos carbapenêmicos. A primeira amostra produtora da metalo-betalactamase (MBL) blaSPM-1 reportada na literatura foi isolada de paciente hospitalizado no Instituto de Oncologia Pediátrica (IOP/GRAACC/UNIFESP) no ano 2000. Objetivo: Avaliar a prevalência de P. aeruginosa resistente aos carbapenêmicos e produtoras de metalo-beta-lactamase, isoladas de hemoculturas coletadas no período de 2000 à 2005 de pacientes admitidos no IOP; caracterizar essas MBLs e avaliar sua disseminação através de tipagem molecular e caracteristicas clínico/epidemiológicas dos pacientes. Métodos: Foram testadas 56 amostras de P. aeruginosa isoladas de 49 pacientes contra 10 antimicrobianos diferentes por disco difusão. As amostras que foram resistentes aos carbapenêmicos foram submetidas a testes fenotípicos (discoaproximação) e genotípicos (PCR) para a confirmação da presença de MBLs. As amostras produtoras de MBL foram submetidas a tipagem molecular através da técnica de PFGE. A análise clínica/epidemiológica foi realizada a partir de dados obtidos dos prontuários dos pacientes. Resultados: Durante o período entre 2000 e 2005, 32 das 56 amostras de P. aeruginosa, foram classificadas como resistentes aos carbapenêmicos pela técnica de disco difusão. Dezoito das 32 (56,2%) amostras avaliadas carreavam o gene blaSPM-1. Sendo que oito (44,4%) das 18 amostras produtoras de blaSPM-1, apresentaram o mesmo perfil genético. Não foi observada a presença de outras metalo enzimas blaIMP-1, blaVIM-1 e blaVIM-2 nas amostras de P. aeruginosa resistentes a carbapenêmicos. A terapêutica antimicrobiana foi adequada em apenas 27,7% dos pacientes com bacteremia por P. aeruginosa carreando o gene blaSPM-1. Foi observada uma maior letalidade, no período de até 30 dias após a bacteremia, em pacientes com infecção causada por P. aeruginosa carreando o gene blaSPM-1 em relação aos demais pacientes. Conclusões: Evidenciamos a presença de um clone de P. aeruginosa resistente aos carbapenêmicos carreando o gene blaSPM-1 que persistiu no período entre novembro/2000 a dezembro/2005 em hemoculturas de pacientes do IOP-GRAACC. Observamos uma maior letalidade dos pacientes com bacteremia por P. aeruginosa com essa característica de resistência a antimicrobianos e uma indaequação inicial dos esquemas antibióticos utilizados justificando uma vigilância epidemiológica rigorosa e uma readequação dos esquemas terapêuticos. / Pseudomonas aeruginosa is an important opportunist pathogen, particularly for oncologic and neutropenic hospitalized patients. One of the main characterisitic of this bacteria is the development of multiple antibiotic resistance, including the carbapenems. The first metallo-beta-lactamase (MBL) encoding the gene blaSPM-1 reported in the literature was isolated in the year 2000 from a patient admitted to the Instituto de Oncologia Pediatrica (IOP/GRAAC - UNIFESP). Objective: To evalute the prevalence of P. aeruginosa carbapenem resistant and MBL producers isolated from bloodstream samples collected from patients admitted to the IOP in the period 2000- 2005; to describe the dissemination of these enzymes by molecular typing and clinical/epidemiological data. Methods: 56 P. aeruginosa isolates from 49 patients were tested against 10 different antibiotics by disc diffusion. The isolates resistant to carbapenems were tested by disc-approximation method and submitted to PCR reaction for detection of MBLs genes. The isolates producers of blaSPM-1 were molecular typed by PFGE. The clinical and epidemiological data were obtained from the patiens charts. Results: From 2000 to 2005, 32 out of 56 P. aeruginosa isolates were classified as carbepenem reistant by disc diffusion. Eighteen out of 32 (56,2%) isolates carried blaSPM-1 and revealed the same molecular typing profile. We did not detected other MBL blaIMP-1, blaVIM-1 and blaVIM-2. The antibiotic therapy was considered adequate in only 17,7% of the patients with P. aeruginosa bacteremia encoding the gene blaSPM-1. We observed a higher letality rate, in the period of 30 days after bacteremia, in these patients compared with the letality of patients with P. aeruginosa bacteremia resistant to carbapenems but not carring MBL. Conclusion: We detected the presence of a P. aeruginosa clone resistant to carbapenems and carrying the gene blaSPM-1 that persisted in blood stream isolates of patients admitted to the IOP from 2000 to 2005. A high letality rate and inadequacy of initial antibiotic treatment was observed justifying a strict epidemiologic surveillance and re-evalutation of antibiotic treatment protocol. / TEDE

Pseudomonas aeruginosa is a dreaded opportunistic pathogen that causes severe and often intractable infections in immunocompromised and critically ill patients. This bacterium is also the primary cause of fatal lung infections in patients with cystic fibrosis and a leading nosocomial pathogen responsible for nearly 10% of all hospital-acquired infections. P. aeruginosa is intrinsically recalcitrant to most classes of antibiotics and has the ability to acquire additional resistance during treatment. In particular, resistance to the widely used β-lactam antibiotics is frequently mediated by the expression of AmpC, a chromosomally encoded β-lactamase that is ubiquitously found in P. aeruginosa strains. This dissertation delved into the role of a recently reported chromosomal β-lactamase in P. aeruginosa called PoxB. To date, no detailed studies have addressed the regulation of poxB expression and its contribution to β-lactam resistance in P. aeruginosa. In an effort to better understand the role of this β-lactamase, poxB was deleted from the chromosome and expressed in trans from an IPTG-inducible promoter. The loss of poxB did not affect susceptibility. However, expression in trans in the absence of ampC rendered strains more resistant to the carbapenem β-lactams. The carbapenem-hydrolyzing phenotype was enhanced, reaching intermediate and resistant clinical breakpoints, in the absence of the carbapenem-specific outer membrane porin OprD. As observed for most class D β-lactamases, PoxB was only weakly inhibited by the currently available β-lactamase inhibitors. Moreover, poxB was shown to form an operon with the upstream located poxA, whose expression in trans decreased pox promoter (Ppox) activity suggesting autoregulation. The transcriptional regulator AmpR negatively controlled Ppox activity, however no direct interaction could be demonstrated. A mariner transposon library identified genes involved in the transport of polyamines as potential regulators of pox expression. Unexpectedly, polyamines themselves were able induce resistance to carbapenems. In summary, P. aeruginosa carries a chromosomal-encoded β-lactamase PoxB that can provide resistance against the clinically relevant carbapenems despite its narrow spectrum of hydrolysis and whose activity in vivo may be regulated by polyamines.

No description available.

β-lactam antibiotics have been the most widely used drug of choice to combat infectious disease caused by bacteria. Unfortunately, the effectiveness of these antibiotics is drastically threatened by bacterial β-lactamases. β-lactamases are currently responsible for the resistance to most β-lactam antibiotic drugs. For decades, β-lactam β-lactamases inhibitors have been used to reduce bacterial resistance, however, in this study, we will employ the use of 1,2,4-oxadiazolidinone derivatives as a non-β-lactam β-lactamases inhibitor against TEM-1 and P99 β-lactamases. The significance of oxadiazolidinone is the prominent five-membered ring in its structure, which is configurationally stable and present in other biologically active compounds such as linezolid and avibactam. Oxadiazolidinones were synthesized in two steps procedure using nitroalkanes and benzaldehyde as starting materials to produce nitrones, which in turn undergo 1,3- dipolar cycloaddition with substituted isocyanates to give the desired 1,2,4-oxadiazolidin analogs (2a, 2b, 2c and 3). Each product was purified and characterized using 1H NMR and 13C NMR, GC-MS, IR, and UV/Vis analysis. Following their successful synthesis and structural elucidation, they were tested with TEM-1 and P99 serine β-lactamase using Nitrocefin as the substrate to ascertain their effectiveness against β-lactamase. 2a, 2b, 2c and 3 showed inhibition ranging from 12-38 %.