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

Os carbapenêmicos são os antimicrobianos mais amplamente utilizados no tratamento empírico de infecções graves por bacilos Gram-negativos. A pressão seletiva gerada pelo uso desses antimicrobianos ao longo das últimas três décadas contribuiu para a disseminação de enterobactérias e Gram-negativos não fermentadores produtores de carbapenemases, particularmente as do tipo KPC e NDM. Os genes que codificam essas enzimas usualmente estão localizados em plasmídeos e/ou transpósons. A hipótese atualmente mais aceita é que o gene blaNDM-1 seja uma quimera criada em Acinetobacter baumannii. A NDM-1 foi descrita em paciente proveniente da Índia e subsequentemente evidenciou-se sua ampla disseminação nesse país. A epidemiologia que tem sido observada nos casos detectados na Europa e Estados Unidos tem sido viagem à Índia, ou seja, sem casos autóctones. No Brasil, os primeiros casos foram identificados no Rio Grande do Sul, e a seguir no Rio de Janeiro e em São Paulo. Diferentemente dos casos da Europa e América do Norte, os casos do Brasil não tem relação epidemiológica com a Índia. O sequenciamento integral dos plasmídeos e cromossomos albergando o gene blaNDM permitirá entender como ocorre a disseminação desse mecanismo de resistência no Brasil. Para isso, foi avaliado o perfil de susceptibilidade dos isolados, bem como a capacidade conjugativa e clonalidade. Das vinte e oito amostras utilizadas neste trabalho, treze delas pertencem à espécie Enterobacter hormaechei, uma à espécie Citrobacter freundii, sete à espécie Escherichia coli, quatro à Klebsiella pneumoniae e três ao gênero Acinetobacter spp. Os primeiros isolados incluídos neste estudo (Escherichia coli e Enterobacter hormaechei produzindo NDM-1) foram isolados em agosto de 2013, de uma mesma amostra de swab retal de um paciente do Rio de Janeiro que nunca viajou para o exterior. O sequenciamento completo do DNA plasmidial utilizando a plataforma Illumina e a anotação de ambos os plasmídeos albergando o gene blaNDM-1 revelou que estes pertencem a grupos de incompatibilidade diferentes, IncFIIK (E. hormaechei) e IncX3 (E. coli), e abrigam um novo transpóson composto designado Tn3000. A comparação da sequência nucleotídica do Tn3000 com aquelas disponíveis no GenBank evidencia que a mesma estrutura está presente em plasmídeos de isolados da cidade de Porto Alegre e também em diferentes continentes. As espécies de Acinetobacter (A. radioresistens, A. ursingii e A. guillouiae) isoladas em São Paulo e Porto Alegre, possuem o gene blaNDM-1 albergados em um mesmo plasmídeo não tipável de 41.087 pb. A avaliação da clonalidade dos isolados de Enterobacter hormaechei \"subsp. oharae\" mostrou dois perfis diferentes através da técnica de PFGE, sendo que todos os microrganismos foram isolados de um surto no mesmo hospital no Rio de Janeiro. Isolados de Klebsiella pneumoniae de uma mesma paciente internada em hospital em Salvador, de sítios distintos - swab retal, hemocultura e urina, em ordem cronológica - obtiveram o mesmo perfil clonal pela técnica de PFGE. O mesmo ocorreu com três isolados de Escherichia coli, de um mesmo paciente do Rio de Janeiro, em amostras de swab retal. Os achados deste estudo evidenciam que no Brasil, Nepal, Marrocos e Índia há uma disseminação do gene blaNDM-1 mediada por um novo elemento móvel designado Tn3000 em enterobactérias. A detecção de um mesmo plasmídeo em diferentes espécies de Acinetobacter evidencia que neste gênero bacteriano, no Brasil, a disseminação do gene blaNDM-1 ocorre por conjugação. / Carbapenems are the antimicrobials most widely used in the empirical treatment of severe infections caused by Gram-negative bacilli. The selective pressure generated by the use of these antibiotics over the last three decades has contributed to the spread of enterobacteria and Gram-negative non-fermenting producing carbapenemases, mainly KPC and NDM. Genes encoding these enzymes are usually located in plasmids and/or transposons. Currently the most accepted hypothesis is that the blaNDM-1 gene is a chimera created in Acinetobacter baumannii. The NDM-1 was described in a patient from India and subsequently was reported to be broadly disseminate in this country. The epidemiology that has been observed in cases detected in Europe and United States is traveling to India, but no autochthonous cases. In Brazil, the first cases were identified in Rio Grande do Sul, and then in Rio de Janeiro and São Paulo. Differently from the cases described in Europe and North America, the cases from Brazil have no epidemiological link with India. The complete sequencing of plasmids and chromosomes harboring blaNDM gene will understanding how the dissemination of this resistance mechanism in Brazil occurs. In this work we will be evaluate the susceptibility profile of the isolates, and their conjugal capacity and clonality. Of the twenty-eight samples used in this study, thirteen of them belong to the species Enterobacter hormaechei, one to Citrobacter freundii, seven to Escherichia coli, four to Klebsiella pneumoniae and three to the genus Acinetobacter sp. The first two isolates included in this study (Escherichia coli and Enterobacter hormaechei) were isolated in August 2013, from the same rectal swab sample from a patient from Rio de Janeiro that never traveled abroad. Complete sequencing of plasmid DNA using Illumina platform and annotation of both plasmids harboring the blaNDM-1 gene revealed that they belong to different incompatibility groups, IncFIIK (E. hormaechei) and IncX3 (E. coli), and are harbor to a new transposon designated Tn3000. The comparison of the Tn3000 nucleotide sequence with those available at GenBank shows that the same structure is present in plasmids from other Porto Alegre and also in different continents. The Acinetobacter species (A. radioresistens, A. ursingii and A. guillouiae) isolated in São Paulo and Porto Alegre, have the blaNDM-1 gene harbored in a single non-typing plasmid of 41,087 bp. The evaluation of clonal relationship of Enterobacter hormaechei \"subsp. oharae\" showed two different profiles by PFGE technique; of note all microorganisms were isolated from an outbreak in the same hospital in Rio de Janeiro. Isolates of Klebsiella pneumoniae from a single patient hospitalized in Salvador, from different anatomical sites - rectal swab, blood culture and urine, in chronological order - obtained the same clonal profile by the PFGE technique. The same occurred with three Escherichia coli isolates, from the same patient from Rio de Janeiro, in swab rectal strains. Our findings suggest that in Brazil, Nepal, Morocco and India there is a spread of blaNDM-1 gene mediated by Tn3000 in enterobacteria. The detection of a same plasmid in different species of Acinetobacter shows that in this bacterial genus, in Brazil, the dissemination of the blaNDM-1 gene occurs by conjugation.

Made available in DSpace on 2014-08-20T14:37:54Z (GMT). No. of bitstreams: 1 tese_clairton_marcolongo_pereira_resumo.pdf: 10803 bytes, checksum: 3f7507f184a7adb5ceb1296595a35eca (MD5) Previous issue date: 2014-02-21 / This thesis is a study developed in period of 34 years about equine diseases diagnosed through out 34 municipalities of Southern Rio Grande do Sul, which includes de influence area of the Laboratório Regional de Diagnóstico of the Veterinary School of the Federal University of Pelotas. There is a general paper about the diseases diagnosed in this specie related by etiologic agent. This paper establishes the importance of dermatological diseases, including equine sarcoid and squamous cell carcinoma as a cause of economic losses in equines. It also demonstrates that leucoencephalomalacia, rabies, thromboembolism by Strongylus vulgaris and equine monocytic ehrlichiosis are the leading causes of death in horses in the region. Other three papers are presented: two related to the causes of abortion and the one about the epidemiology of pythiosis. These conditions are important causes of economic losses in the breeding of horses in the region. / Esta tese trata-se de um estudo das enfermidades de equinos diagnosticadas em 34 municípios da região Sul do Rio Grande do Sul, que compreende a área de influência do Laboratório Regional de Diagnóstico da Faculdade de Veterinária da Universidade Federal de Pelotas, em um período de 34 anos. É apresentado um trabalho geral das enfermidades diagnosticadas nesta espécie animal, relacionadas por agente etiológico tendo sido estabelecida a importância das doenças dermatológicas, dentre elas o sarcoide equino e o carcinoma de células escamosas como causas de prejuízos econômicos nesta espécie animal. Com este estudo foi possível, ainda, demonstrar que a leucoencefalomalacia, a raiva, o tromboembolismo por Strongylus vulgaris e a erliquiose monocítica são as principais causas de morte de equinos na região. São apresentados, também, dois trabalhos científicos referentes às causas de aborto e um à epidemiologia da pitiose, respectivamente, que são importantes causas de prejuízos econômicos na criação de equinos na região, identificadas neste estudo.

La compréhension de la fonction des protéines et des systèmes biologiques passe par une connaissance fine des mécanismes moléculaires sous-jacents. La cristallographie et la résonance magnétique nucléaire permettent d'appréhender ces mécanismes au niveau atomique en fournissant des informations sur la structure et sur la dynamique des macromolécules biologiques. Nous nous sommes ainsi intéressés à deux protéines, la lipase lip2 de la levure Yarrowia lipolytica et la protéine membranaire OmpA de la bactérie Klebsiella pneumoniae. Nous avons recherché des conditions d'expression de la protéine lip2 marquée uniformément ou spécifiquement sur une boucle (appelée " lid ") afin d'en étudier la dynamique. Des conditions de marquage uniforme à l'azote 15 de lip2 recombinante dans Yarrowia lipolytica ont été mises au point, mais le marquage acide aminé spécifique n'a pu être réalisé à cause de phénomènes de dilution isotopique trop importants dans cette levure. Nous avons résolu par cristallographie aux rayons X la structure du domaine C-terminal de la protéine OmpA et étudié sa dynamique en solution par RMN (techniques de relaxation 15N). Nous avons caractérisé la dynamique de son domaine N-terminal membranaire reconstitué en liposomes par RMN du solide : en utilisant la rotation à l'angle magique à 60kHz et à la détection 1H sur un spectromètre 1 GHz, nous avons pu attribuer une majorité des résonances du tonneau ? et établir un profil de paramètre d'ordre des vecteurs NH. Des expériences de protéolyse ménagée ont révélé par ailleurs un site de coupure unique à la trypsine au sein de la boucle extracellulaire L3. Enfin, une première caractérisation de la protéine complète exprimée dans la membrane externe d'Escherichia coli a été entreprise par RMN du solide sur membranes externes natives. / Understanding the function of proteins and biological systems requires an accurate knowledge of the underlying molecular mechanisms. Crystallography and nuclear magnetic resonance provide a detailed description of these mechanisms, with an atomic resolution, by providing data on both structures and motions. We investigated two proteins, the lip2 lipase from the yeast Yarrowia lipolytica and the membrane protein OmpA from the bacteria Klebsiella pneumoniae. We tried to produce lip2 with uniform and amino-acid specific stable isotope labelling on its functional loop (the lid) for NMR experiments. The homologous recombinant expression in Yarrowia lipolytica turned out to be the most efficient for uniform labelling but failed for specific labelling due to extensive isotope scrambling. We solved the structure of OmpA C-terminal domain by X-ray crystallography, and analyzed its dynamics in solution by NMR (15N relaxation techniques). We characterized its transmembrane N-terminal domain in proteoliposomes by solid state NMR: using state of the art ultra-fast MAS (60 kHz), 1H detection and a 1 GHz spectrometer, we could assign most ?-barrel resonances and establish a NH order parameter profile. In a complementary approach, we used proteolysis to reveal a unique trypsin cleavage site on the extracellular loop 3. Finally, a first characterization of the full-length protein expressed in the outer membrane of Escherichia coli was initiated by solid state NMR on intact outer membranes.

Background. Carbapenem-resistant Enterobacteriaceae, including Escherichia coli and Klebsiella pneumoniae, are newly emerging pathogens of public health importance. Currently no nationally representative or mandatory surveillance or reporting system exists to examine trends of these important pathogens. Objective. The purpose of the current study was to estimate trends in overall microbial burden and carbapenem resistance in E. coli and K. pneumoniae and to understand the extent to which hospitals which report to voluntary surveillance systems represent all hospitals in the United States. Design. We conducted a descriptive study to compare the hospitals participating in voluntary reporting systems of the University HealthSystem Consortium and the National Healthcare Safety Network with the Healthcare Utilization Project’s Nationwide Inpatient Sample, a nationally representative sample of hospital discharges. Methods. Descriptive analyses examined hospital characteristics (region, bed size, hospital control, teaching status, case mix index) and patient characteristics (age, sex, race/ethnicity, admission source, admission type, discharge status, primary payer) of participant hospitals versus all US hospitals. ICD-9-CM codes identified discharges coded for E. coli and K. pneumoniae diagnoses; linear regression was used to evaluate trends in overall microbial burden of E. coli and K. pneumoniae in all US Hospitals and US Academic Centers. Trends in E. coli and K. pneumoniae resistance to carbapenem were also evaluated in hospitals participating in voluntary surveillance systems (n=13). Results. Between 2002 and 2007, slight increasing trends in burden of both E. coli and K. pneumoniae were observed (E. coli: slope = 0.0537; K. pneumoniae slope = 0.0168). Hospitals participating in voluntary surveillance systems are larger and care for fewer elderly patients than all US hospitals. Conclusions. These results suggest that hospitals that participate in voluntary surveillance systems like the National Healthcare Safety Network and the University HealthSystem Consortium may underrepresent trends in smaller hospitals, as well as those that treat elderly patients. Increasing overall burden of infection due to these isolates only reinforces the importance carbapenem resistance in E. coli and K. pneumoniae. This important public health threat may warrant the creation of a national, mandatory reporting system for these and other antimicrobial resistant organisms.

Thesis (MSc (Biomedical Technology))--Cape Peninsula University of Technology, 2017. / Background: It is well established that Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogenic organism that has been frequently identified as the cause of nosocomial and community acquired infections. Furthermore, studies have shown that over the last few decades strains of the genus Klebsiella have systematically developed resistance to numerous antibiotics. Aims and Methods: The primary aim of this study was to investigate the prevalence of K. pneumoniae in nosocomial and community isolates in the Western Cape province of South Africa. Various identification techniques such as the polymerase chain reaction (PCR) using the API 20 E, the VITEK®2 system, primers specific for the 16S-23S rDNA ITS region and the Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) were compared for the identification of this pathogen. The VITEK 2 system was used to detect antibiotic resistant profiles of the K. pneumoniae isolates and to identify the extended spectrum beta-lactamase (ESBL) phenotypic among these isolates. The PCR was used to detect Beta-lactam genes viz. CTX-M (blaCTX-M), TEM (blaTEM) and SHV (blaSHV) respectively in both the genome and plasmid DNA of K. pneumoniae using gene specific primers. Results: In total 57 agar plate bacterial cultures or glycerol stock bacterial cultures were obtained during 2011. Of the 57 isolates, the API 20 E test identified 47 (82.5%) of the isolates (n = 57) as K. pneumoniae while 10 isolates (17.5%) were identified as Raoultella species. The VITEK 2 method and PCR identified all 57 isolates as K. pneumoniae (100%). Of the isolates, 82.5% (47/57) were positively identified as Klebsiella species, 14% (8/57) were identified as Klebsiella variicola and 3.5% (2/57) were shown as no reliable identification (NRI) when using the MALDI-TOF MS. Examination of the 57 isolates using primers specific for the CTX-M (blaCTX-M), TEM (blaTEM) and SHV (blaSHV) respectively showed the following: PCR amplicons for the TEM gene were produced successfully for 46 (81%) of the 57 isolates included in this project, while 11 (19%) of the samples did not yield any TEM amplicons; PCR amplicons for the blaSHV gene were obtained successfully for 56 (98%) of the 57 DNA samples, while 1 sample (2%) did not yield any SHV amplicons; and PCR amplicons for the blaCTX-M gene were produced successfully by 89% (n = 51) of the DNA samples included in this project, while 11% (n = 6) did not yield any CTX-M amplicon. Extended-spectrum beta-lactamase phenotypes had been confirmed in 84% (n = 48) K. pneumoniae isolates while nine isolates were found to be non-ESBL. Resistance rates for these 48 isolates were high and showed resistance patterns of: Amoxicillin/Ampicillin, Amoxycillin/Clavulanate, Ceftriaxone/Cefotaxime, Cefuroxime/Cefprozil and Ceftazidime (100%, n = 48); Piperacillin/Tazobactam and Cefoxitin (98%, 47/48); Cefepime (96%, 46/48); Aztreonam (94%; 45/48); Tobramycin (81%, 39/48); Gentamycin and Ciprofloxacin (77%, 37/48); Trimethoprim/Sulfamethoxazole (67%, 32/48); and Tigecycline (25% 12/48). Conclusion: For the analysis by all four methods employed, a total agreement of 68.4% was obtained, indicating the positive identification of K. pneumoniae in 39 of the 57 samples analysed. An average agreement of 28.1% was then obtained for the comparison of results generated for three of the methods utilised, while a 3.5% average agreement was obtained for at least two methods. Furthermore, all four methods agreed that 82.5% of the isolates were Klebsiella species while three methods agreed that 17.5% of the isolates were Klebsiella species. Based on the results obtained in the current study, PCR and VITEK 2 were the methods of choice for the identification of K. pneumoniae. The current study also showed, that ESBL-K. pneumoniae strains are present in the Western Cape province, South Africa; with high resistance profiles to numerous antibiotics including the Cephalosporins.

This work will look at two factors that add to the diversity of carbapenem resistant bacteria. First, it focuses on the diversity of carbapenemase resistance plasmids. 446 plasmids were characterized by size, gene content and replicon groups. We identified that on average, over 30% of the encoded proteins on each plasmid have an unknown function. Plasmid sizes ranged from 1.6kb to 500kb, with an average of around 100kb and median of 80kb. Additionally, six replicon groups account for 80% of all the carbapenemase resistance plasmids. We also highlight the lack of data available for carbapenemase carrying plasmids from bacterial genera other than Escherichia and Klebsiella, and plasmids that carry the New Delhi metallo-β- lactamase or the Verona-integron encoded metallo-β-lactamase. Second, we characterized the β-lactamase diversity of a single carbapenemase resistant Klebsiella pneumoniae. This isolate encodes six distinct β-lactamases, all of which are functional, and three of which are redundant. Additionally, we determined that the CTX-M-15 cephalosporinase imparts a greater fitness when grown in aztreonam (a monobactam) than ceftazidime (a cephalosporin). Finally, we show that individually, these β-lactamases do not account for the elevated levels of resistance seen in the parent strain, indicating that the passive resistance mechanisms (i.e. efflux pumps, altered membrane porins) may play a larger role than originally thought.

The treatment options for multidrug resistant bacteria are dwindling and it is an important issue of research in medicine to solve. One of the more problematic bacterial species is Klebsiella pneumoniae, it can cause infections with high morbidity that are difficult to treat. Common antibiotics for treatment of these infections are carbapenems but K. pneumoniae can produce enzymes called carbapenemases that can hydrolyze carbapenems and most other beta-lactam antibiotics. In this study carbapenemase genes were introduced chromosomally to a previously susceptible K. pneumoniae strain using λ-Red recombineering. Further constructs were made with non-functional porins to examine how they affect combination treatment with carbapenems. Antibiotic combination therapy was evaluated against constructed carbapenemase- (KPC-2, NDM-1 and OXA-48) producing K. pneumoniae strains. Screening was done using time-lapse microscopy (oCelloScope), and combinations with better effect than treatment with a single antibiotic were chosen for time-kill assays. The results shows that a triple combination of colistin, meropenem and the beta-lactamase inhibitor avibactam gives an improved effect, up to twice the effect compared to monotherapy and up to 1.8 times increased effect compared to double combination. The synergistic effect was greater when adding colistin to treat the strains with non-functional porins, indicating that colistin can increase the permeability for other antibiotics into the cell. This is an interesting finding that need to be researched further.

Klebsiella pneumoniae is an opportunistic pathogen responsible for lobar pneumoniae, liver abscess, and septicemia. Clinical isolates are found to be extended spectrum beta lactamase positive with differential expression of the two classical porins, OmpK35 and OmpK36. Porin loss is associated with increased minimum inhibitory concentrations of beta lactam, cephalosporin, and carbapenem antibiotics that target the peptidoglycan. However, little is known about how porin loss affects other aspects of the cell envelope. The focus of this study was to characterize clinical isolates exhibiting differential porin expression and determine if the cumulative changes altered the resistance to phagocytosis by macrophages. The results support the hypothesis that porin loss significantly impacts the overall cell envelope composition, which in turn alters interactions with macrophages.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-09-04T12:06:25Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CaracterizacaoMolecularKlebsiella.pdf: 3383189 bytes, checksum: 1b8dc4391b448cb697a3dd6a7612a585 (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-09-04T17:57:15Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CaracterizacaoMolecularKlebsiella.pdf: 3383189 bytes, checksum: 1b8dc4391b448cb697a3dd6a7612a585 (MD5) / Made available in DSpace on 2017-09-04T17:57:15Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CaracterizacaoMolecularKlebsiella.pdf: 3383189 bytes, checksum: 1b8dc4391b448cb697a3dd6a7612a585 (MD5) Previous issue date: 2016-12-13 / A resistência aos antimicrobianos em bactérias da família Enterobacteriaceae está aumentando de forma alarmante no mundo todo. As K. pneumoniae constituem um importante grupo de patógenos humanos, causadores de infecções hospitalares e comunitárias. Nestas bactérias, a produção de beta-lactamases é um dos principais mecanismos de resistência aos antimicrobianos e responsável pela falha da terapia antimicrobiana. Este trabalho teve como objetivo realizar a caracterização molecular de espécimes de K. pneumoniae produtoras de ESBL e KPC quanto a resistência aos antimicrobianos em pacientes hospitalizados em Belém-PA. Foram analisadas 124 espécimes de K. pneumoniae oriundas de um hospital público de Belém-Pará. Foram realizadas nesses espécimes testes de suscetibilidade a antimicrobianos, testes fenotípicos para detecção de betalactamases de espectro ampliado (ESBL) e K. pneumoniae produtora de carbapenemase (KPC). Posteriormente foram realizadas reações de cadeia de polimerase (PCR) e o sequenciamento de DNA para identificar os genes determinantes de resistência aos antimicrobianos. Foi observado que 83% dos isolados apresentaram o gene bla CTX-M, 85,5% o bla SHV, 83% o bla TEM e 5% o gene bla KPC. Quanto aos genes que codificam ESBL o gene bla CTX-M-71 foi isolado com maior freqüência e foi identificado em 60% dos isolados analisados. Os outros genes que codificam ESBL foram bla SHV-38 (5%), bla SHV-100 (5%) and bla SHV-12 (3,5%). O gene bla KPC-2 foi detectado em 100% dos isolados. Estas enterobactérias apresentaram fenótipos de multidroga resistência com elevados níveis para os quinolonas e aminoglicosideos. Foram observadas associações entre os genótipos e à resistência aos antibióticos. A presença de micro-organismos multirresistentes em unidades hospitalares reforça a necessidade de medidas para a rápida contenção de possíveis infecções causadas por esses patógenos. / The antimicrobial resistance in Enterobacteriaceae is increasing worldwide. The K. pneumoniae constitute an important group of human patogen, causing of hospital and communitarian infections. In these bacteria, the production of extended spectrum beta-lactamases (ESBL) is one of the main mechanisms of resistance the antimicrobials, responsible for the imperfection of the therapy against infections for gram-negative bacilli. This work aimed to do the molecular characterization of the K. pneumoniae producing ESBL and KPC about antimicrobial resistence in pacients from Belém-PA. A total of 124 K. pneumoniae isolates were collected from public hospital from Belém-PA and susceptibility test was performed to detect its susceptibility patterns antibiotics. Phenotypic tests for extended-spectrum beta-lactamases (ESBLs) and carbapenemase-producing Klebsiella pneumoniae (KPC) producing strains were performed to detect the resistance phenotype of the isolates. Then PCR amplification and sequencing analysis were performed for the drug resistance determinants genes. The results showed that 83% strains harbored bla CTX-M gene, 85,5% carried bla SHV , 83% carried bla TEM and 5% carried bla KPC. The most frequent gene ESBL detected was bla CTX-M-71, which was observed in 60% of isolates. Other ESBL genes were bla SHV-38 (5% of isolates), bla SHV-100 (5% of isolates) and bla SHV-12 (3,5% of isolates). O gene bla KPC-2 was detected in 100% of isolates.These enterobacterias showed multidrug resistance phenotypes with high levels for quinolones and aminoglycosides. Associations between genotypes and antibiotic resistance were observed.The presence of multidrug resistant micro-organisms in hospitals, reinforces the need for measures for rapid containment of possibles infections caused by these pathogens.