The rapid spread of carbapenem-resistant lineages of Klebsiella pneumoniae, clustered within the clonal group CG258, is a growing public health problem associated with healthcareassociated infections. The objective of this study was to perform a genomic analysis of KPC-2 and/or CTX-M β-lactamase-producing strains of K. pneumoniae belonging to CG258 (ST11, ST258, ST340, ST437) circulating at the human-animal-environment interface, in Brazil and South America. The analysis was conducted to characterize the antimicrobial resistome, virulome, genetic elements of transfer and mobilization associated with the dissemination of the blaKPC-2 gene, and to perform a detailed comparative genomic analysis of the CG258; with subsequent pathogenicity evaluation in an invertebrate (Galleria mellonella) model of infection, aiming to identify biomarkers of virulence. The main results are presented in the format of six manuscripts. Manuscript I: New draft genome sequence of a Klebsiella pneumoniae strain 1194/11, belonging to ST340, showing a wide resisto-me. Manuscript II: The first draft genome sequence of a Klebsiella pneumoniae 606B ST340 carrying blaCTX-M-15 in food-producing animal isolated in Brazil. Manuscript III: The first draft genome sequence of a Klebsiella pneumoniae strain Kp171, recovered from a water sample collected in an urban river in Brazil, demonstrating that anthropogenic activities, including the release of wastewater and sewage from hospitals, may have contributed to the contamination of aquatic environments, raising a concern to public health. Manuscript IV: Identification and complete sequence analysis of an IncX3 plasmid carrying a non-Tn4401 genetic element (NTEKPC-Ic), originating from a hospital associated lineage of K. pneumoniae ST340, showing the spread of blaKPC-2 in new Incompatibility group. Manuscript V: Dissemination of blaKPC-2 in novel non-Tn4401 Element (NTEKPC-IId) carry by new small IncQ1 and Col-Like plasmids in lineages of Klebsiella pneumoniae ST11 and ST340. Manuscript VI: Yersiniabactin, colibactin and wider resistome contribute to enhanced virulence and persistence of KPC-2-producing K. pneumoniae CG258 in South America. The results obtained in the present study allow us to obtain a first genomic landscape of K. pneumoniae lineages of the CG258, circulating at the human-animal-environment interface, in Brazil and South America. In this regard, most likely the interplay of yersiniabactin and/or colibactin, and resistance to clinically significant antibiotics (as carbapenems and polymyxins) are contributing to the emergence of highly virulent and MDR lineages that pose great risk to human health. On the other hand, the wide antimicrobial resistome (antibiotics, disinfectants and heavy metals) could be contributing to adaptation of KPC-2- and/or CTX-M-producing K. pneumoniae CG258 in the human-animal-environment interface, highlighting the urgent need for enhanced control efforts. In conclusion, these findings could contribute to the development of strategies for prevention, diagnosis and treatment of K. pneumoniae infections. / A rápida disseminação de linhagens de Klebsiella pneumoniae resistentes aos carbapenêmicos, agrupadas dentro do grupo clonal GC258, e um crescente problema de saúde pública associado com infecções relacionadas a assistência a saúde. O objetivo deste estudo foi realizar uma análise genômica de cepas de K. pneumoniae produtoras de β-lactamases KPC-2 e/ou CTX-M, pertencentes ao GC258 (ST11, ST258, ST340, ST437), circulando na interface humana-ambiente-animal, no Brasil e na América do Sul. A análise foi direcionada para caracterizar o resistoma e viruloma, elementos genéticos de transferência e mobilização associados com a disseminação de genes blaKPC-2, e realizar uma análise de genômica comparativa detalhada do GC258, com posterior avaliação da patogenicidade em modelo invertebrado (Galleria mellonella) de infecção, visando identificar biomarcadores de virulência. Os principais resultados são apresentados na forma de seis manuscritos. Manuscrito I: Nova sequência \"draft\" do genoma de K. pneumoniae 1194/11isolado de amostra clínica, pertencente ao ST340, mostrando um amplo resistoma. Manuscrito II: O reporte da primeira sequência \"draft\" do genoma de K. pneumoniae 606B (ST340), contendo blaCTX-M-15 em animais de produção isolados no Brasil. Manuscrito III: O primeiro esboço da sequência do genoma de K. pneumoniae Kp171, recuperado de uma amostra de água coletada em um rio urbano no Brasil, demonstrando que atividades antrópicas, incluindo a liberação de esgoto e esgoto de hospitais, podem ter contribuído para a contaminação ambientes aquáticos, levantando uma preocupação para a saúde pública. Manuscripto IV: Identificação e análise de sequencia completa de um plasmídeo IncX3 portador de um elemento genético não Tn4401 (NTEKPC-Ic), originado de uma linhagem hospitalar associada a K. pneumoniae ST340, mostrando a disseminação de blaKPC-2 no novo grupo Incompatibilidade. Manuscrito V: Disseminação de blaKPC-2 no novo elemento non-Tn4401 (NTEKPC-IId) portado por novos pequenos plasmídeos IncQ1 e Col-Like em linhagens de K. pneumoniae ST11 e ST340. Manuscrito VI: Os resultados obtidos no presente estudo permitem gerar um panorama genômico das linhagens de K. pneumoniae do GC258, circulando na interface humana-animal-ambiente, no Brasil e na América do Sul. De principal interesse, a convergência da virulência associada com genes codificando yersiniabactina e/ou a colibactina e a resistência a antibióticos clinicamente significativos (como carbapenemicos e polimixinas), estão contribuindo para o aparecimento de linhagens altamente virulentas e multirresistentes que apresentam um grande risco a saúde humana. Por outro lado, a ampla resistência aos antimicrobiana (antibióticos, desinfetantes e metais pesados) poderia estar contribuindo para a adaptação de estirpes de K. pneumoniae do GC258, produtoras de KPC-2- e/ou CTX-M, na interface humana-ambiente-animal, destacando a necessidade urgente de medidas para o controle de disseminação. Em conclusão, esses achados poderiam contribuir para o desenvolvimento de estratégias de prevenção, diagnóstico e tratamento das infecções por K. pneumoniae.

Disertacijos tema: Potencialių hospitalinės pneumonijos sukėlėjų Pseudomonas aeruginosa ir Klebsiella pneumoniae patogeniškumo veiksniai bei jų įtaka ligos eigai Darbo tikslas -ištirti Pseudomonas aeruginosa ir Klebsiella pneumoniae padermių, kolonizavusių apatinius kvėpavimo takus ar sukėlusių hospitalinę pneumoniją, patogeniškumo veiksnius ir jų įtaką hospitalinės pneumonijos eigai. Uždaviniai: • Ištirti hospitalinę pneumoniją sukėlusių ar apatinius kvėpavimo takus kolonizavusių Pseudomonas aeruginosa padermių patogeniškumo veiksnius - atsparumą serumo baktericidiniam poveikiui, gebėjimą įsiskverbti į kvėpavimo takų epitelio ląsteles, atsparumą antibiotikams ir O serogrupinę priklausomybę. • Įvertinti Pseudomonas aeruginosa padermių patogeniškumo veiksnių tarpusavio sąsajas. • Ištirti Klebsiella pneumoniae padermių, sukėlusių hospitalinę pneumoniją ar kolonizavusių apatinius kvėpavimo takus, gebėjimą gaminti plataus spektro beta laktamazes bei atsparumą antibiotikams. • Įvertinti Pseudomonas aeruginosa ir Klebsiella pneumoniae padermių patogeniškumo veiksnių įtaką hospitalinės pneumonijos eigai. Darbas yra pirmas Lietuvoje, kurio metu ne tik nustatytas Pseudomonas aeruginosa patogeniškumo veiksnys – atsparumas serumo baktericidiniam poveikiui, bet ir įvertinta galima šį patogeniškumo veiksnį įgijusių Pseudomonas aeruginosa padermių įtaka hospitalinės pneumonijos vystytis bei ligos eigai. Pirmą kartą apskritai vertintas Pseudomonas aeruginosa padermių gebėjimas įsiskverbti į... [toliau žr. visą tekstą] / The aim of the study: To examine pathogenicity factors of Pseudomonas aeruginosa and Klebsiella pneumoniae strains, colonizing lower respiratory tract or causing hospital-acquired pneumonia, and to evaluate their influence on the course of hospital-acquired pneumonia. Objectives of the sudy: 1. To examine pathogenicity factors – resistance to serum bactericidal activity, ability to penetrate epithelial cells of the respiratory tract, dependence of O serogroup, and resistance to antibiotics – of Pseudomonas aeruginosa strains, colonizing lower respiratory tract or causing hospital-acquired pneumonia. 2. To evaluate the relationship between pathogenicity factors of Pseudomonas aeruginosa strains. 3. To examine the ability of Klebsiella pneumoniae strains, colonizing lower respiratory tract or causing hospital-acquired pneumonia, to produce extended-spectrum beta-lactamases and resistance of these pathogen to antibiotics. 4. To evaluate the influence of pathogenicity factors of Pseudomonas aeruginosa and Klebsiella pneumoniae strains on the course of hospital-acquired pneumonia. Such work is first in Lithuania, because we determined not only pathogenicity factors of Pseudomonas aeruginosa – i.e., resistance to bactericidal activity of serum, but also evaluated possible influence of Pseudomonas aeruginosa strains, having this pathogenicity factor, on hospital-acquired pneumonia development and outcome. Therefore, the ability of Pseudomonas aeruginosa strains to invasive into... [to full text]

Thesis (Ph. D.)--University of Oklahoma. / Vita. Bibliography: leaves 173-205.

Made available in DSpace on 2016-01-07T13:39:49Z (GMT). No. of bitstreams: 2 nilceia_ramos_ioc_2014.pdf: 985413 bytes, checksum: d50610d797598fa42cb546cf07dafa32 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-04-14 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / O gênero Klebsiella compreende bactérias em forma de bastonete, gram-negativas e imóveis que pertencem à família Enterobacteriaceae. As espécies Klebsiella pneumoniae e Klebsiella oxytoca, patógenos oportunistas, são as mais importantes, sob o ponto de vista da clínica. Estas espécies têm sido relacionadas a um número crescente de infecções hospitalares multirresistentes. K. pneumoniae é classificada em três grupos filogenéticos: KpI, KpII e KpIII e os genes que codificam as \03B2-lactamases de classe A cromossômicas SHV, OKP e LEN, estão relacionadas à estes grupos, respectivamente. Da mesma forma, o gene blaOXY codifica a \03B2\2013lactamase de classe A cromossômica que caracteriza a espécie K. oxytoca. blaSHV é um gene cromossômico e plasmidial que codifica \03B2\2013lactamases de espectro restrito (NSBLs) e estendido (ESBL). Até o momento, 183 alelos de blaSHV foram identificados, mas o espectro de atividade de vários destes alelos ainda não foi determinado. As \03B2-lactamases LEN são codificadas em cromossomos e são NSBLs. Recentemente a espécie K. variicola foi identificada. Esta espécie se caracteriza pela fixação de nitrogênio e nenhum gene constitutivo de \03B2-lactamase foi associado à esta espécie. Este estudo objetivou determinar o espectro de resistência de alelos de blaSHV identificados em K. pneumoniae e também identificar, em nível de espécie, isolados carreadores do gene blaLEN. Por PCR e sequenciamento, alelos de blaSHV e blaLEN foram definidos comparando suas sequências com bancos de dados usando as ferramentas blastN e blastP Estes genes foram clonados e expressos em sistema heterólogo, e o espectro de resistência dos transformantes foi avaliado. A taxonomia genômica dos isolados deste gênero foi realizada com base em sequências de genes recuperados de genomas de espécies de Klebsiella e aplicou-se as abordagens MLSA, rMLST e cgMLST. Identificamos blaSHV-28 (n=2), blaSHV-110 (n=1) e alelos novos de blaSHV (n=3). Os transformantes para estes genes apresentaram resistência à amoxicilina, ampicilina e piperacilina, o que corresponde a um espectro restrito de resistência. Quanto a blaLEN, foram identificados oito novos alelos em nove isolados. Dois deles eram idênticos aos encontrados no genoma de K. variicola AT-22 e todos estes alelos codificam NSBLs. A taxonomia genômica de Klebsiella definiu três grupos: K. variicola, K. pneumoniae e K.oxytoca. Curiosamente, alguns isolados de K. pneumoniae agruparam com o K. variicola AT-22, sugerindo pertencerem à esta espécie. Todos os isolados do grupo K. variicola albergavam o gene blaLEN indicando que esta seria a \03B2-lactamase constitutiva de K. variicola, e, por conseguinte, que o grupo KpIII é composto por isolados de K. variicola. Aqui nós determinamos que alelos de blaSHV cromossômicos, incluindo blaSHV-28 e blaSHV-110, previamente identificados como ESBLs, são NSBLs e concluímos que K. variicola pode estar sendo subestimada em infecções humanas / The genus Klebsiella comprises non - motile, g ra m - negative, rod - shaped bacteria, presenting a polysaccharide - based capsule, belonging to the Enterobacteriaceae family. The two most clinically i mport ant species from the genus are the opportunistic pathogens Klebsiella pneumoniae and Klebsiella oxytoca . These species have been related to an increasing number of multiresistant hospital - acquired infections. K. pneumoniae is classified into three phylogen etic groups: KpI, KpII and KpIII. Three chromosomal class A β - lactamase genes were recognized in K pneumoniae : bla SHV , bla OKP and bla LEN , which are related to KpI, KpII and KpIII groups, respectively. Similarly, the bla OXY gene is the chromosomal class A β - lactamase that characterizes the K. oxytoca species. The bla SHV is a chromosomal - and plasmid - borne gene encoding narrow (NSBLs) and extended spectrum β - lactamases (ESBLs). So far, 183 bla SHV alleles were identified but the activity spectrum of dozen of a lleles remain to be determined. LEN enzymes are chromosomal encoded with restrict activity against β - lactams and confers resistance only to penicillins. More recently, K. variicola species were identified and this species has the ability of fixing nitrogen . There are few studies concerning this species and no constitutive β - lactamase gene has been described in this species. This study aimed to determine the resistance spectrum of bla SHV alleles identified in K. pneumoniae strains and also to identify, at sp ecies level, strains carrying bla LEN chromosomal class A β - lactamase gene. PCR and sequencing were performed and bla SHV , and bla LEN alleles were determined by comparing their sequences with the database using blastN and blastP tools. These genes were clone d and expressed in a heterologous system, and the antibiotic resistance spectrum of the transformants was evaluated by susceptibility test. The genomic taxonomy of strains from this genus were performed based on genes from draft and complete genome sequenc es from Klebsiella species using MLSA, rMLST and cgMLST approaches. Strains harboring bla SHV - 28 (n=2), bla SHV - 110 (n=1) and new bla SHV alleles (n=3) were identified. All the transformants showed resistance to amoxicillin, ampicillin, piperacill in, ticarcil lin , which correspond to a restricted spectrum of resistance. Concerning bla LEN alleles, here were identified eight new alleles in nine strains. Two of them were identical to the one found in K. variicola AT - 22 genome. The antibiotic resistance profile pre sented by the transformants indicated that all these bla LEN alleles encode for NSBLs. The Klebsiella genomic taxonomy revealed three groups corresponding to K. variicola , K. pneumoniae and K. oxytoca species. Curiously, some K. pneumoniae grouped with the K. variicola AT - 22, suggesting that those strains, in fact, belong to K. variicola species and had been misidentified as K. pneumoniae . Moreover, it was observed that all strains from K. variicola cluster harbored the bla LEN gene. This finding indicated th at the bla LEN gene is a constitutive K. variicola β - lactamase, and therefore, the KpIII group is composed by K. variicola strains. Here we determined that different chromosomal bla SHV alleles, including bla SHV - 28 e bla SHV - 110 , previously labeled as ESBLs, and the new ones, are NSBLs. Also, we concluded th at K. variicola can play an underestimated role in human infections .

L'optimisation des procédés utilisant un substrat bon marché et abondant est considéré comme un facteur affectant le prix de production du 2,3-BD. Les valeurs optimales du pH, du taux d'aération, de la vitesse d'agitation, et de la concentration en substrat (maltodextrine) pour la production de 2,3-BD à partir de maltodextrines par la souche génétiquement modifiée Klebsiella oxytoca KMS005 ont été déterminées par une méthode conventionnellefacteur par facteur ainsi que par l’utilisation de la méthode des surfaces de réponse avec un plan d’expériences de Box-Behnken. Les résultats ont montré que les valeurs optimales de pH, taux d'aération, vitesse d'agitation, et concentration en substrat (maltodextrine) ont été respectivement de 6,0, 0,8 wm, 400tr/min et 150 g/L. Les surfaces de réponses ont permis de montrer que la vitesse d'agitation était le paramètre le plus influent pour la production de 2,3-BD. La mise en oeuvre d’un procédé fed-batch a permis d’obtenir en 78 h une concentration en 2,3-BD de 88,1±0,2 g/L avec un rendement de 0,412±0,001 g/g et une productivité de 1,13±0,01 g/L/h. L’influence des conditions de micro-aération sur la croissance des microorganismes et sur la production de 2,3-BD a été étudiée. En bioréacteurs batch, le transfert d’oxygène a été caractérisé via la mesure kLa en faisant varier le débit d'air et la vitesse d'agitation. La quantité optimale d'oxygène fournie aété évaluée à 9,5 g correspondant à un kLa de 25,2 h-1. Ensuite, un Ensuite, un procédé fed-batch a été étudié avec différentes stratégies de débit d'alimentation en glucose. Les meilleurs résultats ont été obtenus pour un débit d'alimentation constant en glucose de 2 g/h après une phase batch de croissance de 48 heures, et suivie d'une phase batch finale de 40 heures. Il en est résulté une concentration finale de 2,3-BD de 74,7 g/L avec une productivité de 0,64 g/L/h et peu de sous-produits formés (environ 3 g/L d’acide succinique, acétate et éthanol). D’autre part, les résultats issus des expérimentations en bioréacteur de 2 L ont été mis en oeuvre à l’échelle pilote. La production de 2,3-BD de maltodextrine a été réalisée dans des bioréacteurs de 10, 90 et 300 L pour différentes vitessesd’agitation et un taux d'aération fixé à 0,8 vvm.). Une concentration de 2,3- BD de 53,8 g/L et un rendement de 0,40 g/g de sucre consommé en 48 hont été obtenus avec une vitesse d’agitation constante de 295 tr/min en réacteur de 10 L. Pour le réacteur de 90 L, une concentration en 2,3-BD de52,53 g/L et un rendement de 0,43 g/g de sucre consommé ont été atteints en 72 h pour une vitesse d’agitation constante à 130 tr/min. Les meilleuresconditions d’inoculation ont été obtenues pour les précultures en phase exponentielle de croissance (12 h d’incubation) et une DO550 égale à 4 avant transfert dans le bioréacteur de 90 L. Pour le réacteur de 300 L, la concentration en 2,3-BD était de 45,02 g/L pour un rendement de 0,43 g/g de sucre consommé, obtenusaprès 72 h. / An optimization process with a cheap and abundant substrate is considered one of the factors affecting the price of commercial 2,3-Butanediol (2,3-BD) production. The optimized levels of pH, aeration rate, agitation speed, and substrate concentration (maltodextrin)were optimized by a conventional method and Response Surface Methodology (RSM) with Box-Behnken design in which metabolically engineered Klebsiell oxytoca KMS005 utilized maltodextrin to produce 2,3-BD. Results revealed that pH, aeration rate, agitation speed,and maltodextrin concentration at levels of 6.0, 0.8 vvm, 400 rpm, and 150 g/L, respectively, were the optimal conditions. RSM indicated that the agitation speed was the most influential parameter when either agitation and aeration interaction or agitation and substrate concentrationinteraction played important roles for 2,3-BD production. Under interim fedbatch fermentation, 2,3-BD concentration, yield, and productivitywere obtained at 88.1±0.2 g/L, 0.412±0.001 g/g sugar supplied, and 1.13±0.01 g/L/h, respectively, within 78 h. The influence of micro-aerobicconditions on microbial growth and 2,3-BD production was also studied. In batch bioreactors, air flow rate and agitation rate characterized through kLa measurement were tested. The optimal amount of oxygen supply was evaluated at 9.5 g corresponding to a kLa of 25.2 h-1 for cell growth and 2,3-BD production. Then, a fed-batch process was investigated by different glucose feeding rate strategies. Fedbatch with a glucose feeding rate of 2 g/h starting at the end of the growth phase during 48 h, followed by a final batch phase of 40 h was found satisfactory. It resulted in a final 2,3- BD concentration of 74.7 g/L with a productivity of 0.64 g/L/h but few byproducts formed (about 3 g/L including succinate, acetate and ethanol). Validated information in the 2 L bioreactor was further applied in a larger scale production of 2,3-BD with series of bioreactors from 10, 90 and 300 L vessels. Batch experiments were conducted based on various agitation speeds with the fixedaeration rate at 0.8 vvm. As a result, 2,3-BD concentration, and yield were achieved at 53.8 g/L, and 0.40 g/g sugar supplied within 48 h, respectively, under the constant tip speed at 295 rpm using a 10 L vessel. Its concentration of 52.53 g/L and yield of 0.43 g/g sugar consumedwithin 72 h were attained under the condition of the constant tip speed at 130 rpm using a 90 L fermenter. An appropriate seed inoculum condition was found with an optical cell density (OD550) around 4 at the log phase (12 h incubation) prior to transferring of the inoculum into the 90 L fermenter. Under the constant tip speed at 70 rpm, 2,3-BD concentration and yield were obtained at 45.02 g/L and 0.43 g/g sugar consumed in the pilot scale of 300 L bioreactor after 72 h incubation.

During the last decades, K. pneumoniae has emerged as one of the most clinically significant pathogens, especially due to the high prevalence of strains producing extended spectrum β-lactamases (ESBL) and carbapenemases. The therapeutic options to treat these infections are constrained not only by this enzyme production, but also by the regular ability to form biofilms. The development of K. pneumoniae biofilms on the host tissue eventually protect the microorganisms from the action of antimicrobial agents and from the immune response, apart from driving the expression of virulence factors. Thus, the aim of this study was to detect mechanisms of resistance, adhesive properties and ability of multi-resistant K. pneumoniae clinical isolates to form biofilms. In the first stage of the study, 33 strains of K. pneumoniae with reduced susceptibility to carbapenems were isolated. By using phenotypic tests, 10 samples positive for the modified Hodge test were detected, suggesting the production of carbapenemases, and 2 strains manifested positivity for the synergic test with boronic acid, suggesting the production of KPC. When employing molecular techniques, such as polymerase chain reaction, the gene blaKPC was not detected, characterizing failure on the phenotypic detection of KPC. In the second stage of the study, 14 strains of multi-resistant K. pneumoniae were analyzed in order to verify the ability of biofilm formation, as well as adhesive/aggregative properties. Most of the strains analyzed presented low affinity to p-xylene, suggesting hydrophilic character, aside from strong affinity to the basic solvent ethyl acetate indicating acidic surface characteristics. It was also verified that the strains studied manifested high ability of biofilm formation and important adhesion in epithelial cells. The combination of all the characteristics studied may contribute to the survival of K. pneumoniae in the host and in the environment, as the organization of microorganisms in biofilms complicates the pharmacological treatment and favors its spread and multi-resistance. / Durante as últimas décadas, a Klebsiella pneumoniae tem emergido como um dos patógenos mais importantes clinicamente, especialmente em função da alta prevalência de amostras produtoras de beta-lactamases de espectro estendido (ESBL) e carbapenemases. As opções terapêuticas para o tratamento destas infecções são limitadas não somente por esta produção enzimática, mas também pela frequente capacidade de formação de biofilmes. O estabelecimento de biofilmes de K. pneumoniae sobre tecidos do hospedeiro acaba por proteger os microrganismos da ação de agentes antimicrobianos e da resposta imunológica, além de conduzir a expressão de determinantes de virulência. Neste sentido, o objetivo deste trabalho foi detectar mecanismos de resistência, propriedades adesivas e capacidade de formação de biofilmes de isolados clínicos de K. pneumoniae multirresistentes. Na primeira fase deste estudo, foram isoladas 33 isolados clínicos de K. pneumoniae com perfil de sensibilidade reduzida aos carbapenêmicos. Através de testes fenotípicos foram detectadas 10 amostras que apresentaram positividade para o teste modificado de Hodge (MHT) sugerindo produção de carbapenemases e duas amostras que apresentaram positividade para o teste sinérgico com ácido borônico (AB), indicando produção de KPC (Klebsiella pneumoniae carbapenemase). Quando utilizado ferramentas moleculares, como a reação em cadeia da polymerase, não foi detectado o gene blaKPC, caracterizando desta forma uma falha na detecção fenotípica de KPC. Na segunda fase deste estudo, foram analisadas 14 amostras de K. pneumoniae multirresistentes a fim de verificar a capacidade de formação de biofilmes, bem como propriedades adesivas/agregativas. A maioria das isolados clínicos analisadas apresentaram baixa afinidade ao xileno, sugerindo um caráter hidrofílico, além de alta afinidade ao solvente básico acetato de etila indicando características superficiais ácidas. Foi verificado ainda que os isolados clínicos estudados apresentaram alta capacidade de formação de biofilme e importante adesão em células epiteliais. As combinações de todas estas características estudadas podem contribuir para a sobrevivência de K. pneumoniae no hospedeiro e no ambiente, pois a organização dos microrganismos em biofilme dificulta o tratamento farmacológico e favorece a sua disseminação e multirresistência.

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.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2018-01-22T12:47:14Z No. of bitstreams: 1 PATRICIA VOLLÚ SILVA.PDF: 2575547 bytes, checksum: c2489f8abe58d46ea1c2ba8977ba02e1 (MD5) / Made available in DSpace on 2018-01-22T12:47:14Z (GMT). No. of bitstreams: 1 PATRICIA VOLLÚ SILVA.PDF: 2575547 bytes, checksum: c2489f8abe58d46ea1c2ba8977ba02e1 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A ilha de patogenicidade pks de 54 kb é responsável pela produção de colibactin, uma molécula genotóxica, que causa rupturas de fita dupla de DNA (DSB) das células hospedeiras, e que parece estar relacionada ao aumento do risco de desenvolvimento de câncer colorretal. Colibactin foi inicialmente descrita em Escherichia coli, mas também pode ser encontrada em outras enterobactérias, como Klebsiella pneumoniae. A biossíntese de colibactin requer a atividade enzimática da fosfopantoteinil transferase (PPTase) ClbA, que também pode contribuir para a produção dos sideróforos enterobactin e yersiniabactin em E. coli. O objetivo deste trabalho foi avaliar a virulência em amostras de E. coli e de K. pneumoniae, presença da ilha de patogenicidade pks e determinar o papel de colibactin e do sideróforo enterobactin em infecções por K. pneumoniae. Para tal, uma coleção de 218 amostras de E. coli isoladas de pacientes com infecção do trato urinário atendidas no Instituto Nacional do Câncer, Brasil e 258 amostras de K. pneumoniae isoladas de sangue de pacientes internados no Centre Hospitalier Universitaire de Toulouse, França foram avaliadas. A tipificação filogenética e a presença do gene de virulência fyuA em E. coli e do gene clbN em ambas bactérias foram avaliadas por PCR. O fenótipo hipermucoviscoso (HMV) de K. pneumoniae foi determinado pelo teste string. Foi ainda investigado o papel de colibactin e enterobactin em um modelo de pneumonia em camundongos C57BL/6JRJ e em infecções de células HeLa, utilizando a cepa K. pneumoniae SB 4496 e mutantes isogênicos deficientes em clbA, clbN ou entD. Os resultados demonstraram que entre as amostras de E. coli pesquisadas, o grupo filogenético B2 foi o mais prevalente (44,9%), seguido pelos grupos filogenéticos A (13,8%), B1 (22,0%) e D (19,3%). O gene fyuA, relacionado a produção do sideróforo yersiniabactin, foi detectado em todos os grupos filogenéticos, no entanto, a detecção do referido gene foi mais frequente no grupo B2 (P = 0,0228), sendo detectado em 74,5% das amostras deste grupo. O gene clbN, relacionado a produção de colibactin, foi detectado em 14,7% das amostras de E. coli. Vale ressaltar que todas as amostras clbN positivas pertenciam ao grupo filogenético B2, as quais também portavam o gene fyuA. Adicionalmente, três amostras de E. coli apresentaram efeito citopático em células HeLa, independente da produção de colibactin e da presença dos genes hlyC/A, hlyF, cdt e cnf1. O gene clbN foi detectado em 5,8% das amostras de K. pneumoniae. Também foi observado que a detecção de clbN foi estatisticamente significantemente (P < 0,0001) entre as amostras caracterizadas como HMV, este gene foi observado em 35% das amostras HMV analisadas. Além disso, a síntese de colibactin não pôde ser mantida pela PPTase EntD e a produção de sideróforos pela K. pneumoniae SB 4496 foi continuada por outro sistema independente de PPTases EntD e ClbA. Os resultados obtidos neste trabalho parecem indicar que a produção de colibactin não afeta a sobrevivência de K. pneumoniae hipervirulenta (hvKP) nos tecidos pulmonares de camundongos C57BL/6JRJ, nas condições estudadas. No entanto, é importante salientar que a toxina colibactin produzida por hvKP é capaz de induzir genotoxicidade em células HeLa. Ambos os genes clbA e clbN foram necessários para a manutenção da megalocitose e DBS induzida por colibactin em K. pneumoniae / The 54-kb pks pathogenicity island produces a genotoxic molecule named colibactin, which causes double-strand DNA breaks (DSB) in the host cells and enhanced colorectal cancer development. Colibactin was initially described in Escherichia coli, but can be found in other enterobacteria, including Klebsiella pneumoniaE. colibactin biosynthesis requires the enzymatic activity of phosphopantetheinyl transferase (PPTase) ClbA, which may also support the enterobactin and yersiniabactin siderophores synthesis in E. coli. The goal of this work was to evaluate the virulence of E. coli and K. pneumoniae isolates, presence of pks pathogenicity island and to determine the role of colibactin and enterobactin siderophore in K. pneumoniae infections. For this purpose, it was evaluated a collection of 218 E. coli isolates obtained from patients with urinary tract infection assisted at Instituto Nacional do Câncer, Brazil, and 258 K. pneumoniae isolates collected from blood samples from patients admitted to the Centre Hospitalier Universitaire in Toulouse, France. Phylogenetic typing and the detection of fyuA virulence gene in E. coli and clbN gene in both bacteria were assessed by PCR. K. pneumoniae hypermucoviscous (HMV) phenotype was determined by the string test. The role of colibactin and enterobactin in a C57BL/6JRJ mice pneumonia model and in HeLa cells infection was investigated using K. pneumoniae SB 4496 strain and isogenic mutants deficient in clbA, clbN or entD. Among the E. coli isolates, the phylogenetic group B2 was more prevalent (44.9%) followed by phylogroups A (13.8%), B1 (22.0%) and D (19.3%). The fyuA gene, involved in the yersiniabactin production, was detected in all phylogenetic groups studied; however, its presence was more frequently detected among the phylogroup B2 (P = 0.0228), with a high percentage of 74.5%. The clbN gene, associated to colibactin production, was detected in 14.7% of E. coli isolates. It is noteworthy that all clbN positive isolates belong to the phylogroup B2 and carry the fyuA gene. In addition, three E. coli isolates studied showed cytopathic effect in HeLa cells independently of colibactin production and the presence of hlyC/A, hlyF, cdt and cnf1 genes. Regarding K. pneumoniae, clbN gene was detected in 5.8% of the isolates. It was also observed that this detection was statistically significant (P < 0.0001) among the isolates of the HMV phenotype group, being this gene observed in 35% of the HMV isolates analyzed. In addition, it was observed that colibactin synthesis could not be maintained by the PPTase EntD. Indeed, siderophores production by K. pneumoniae SB 4496 was successful continued by another system that does not require the activity of PPTases EntD and ClbA. Results obtained in this work seem to indicate that production of colibactin does not affect the survival of hypervirulent K. pneumoniae (hvKP) in C57BL/6JRJ lung mice. Despite that, it is interesting that colibactin produced by hvKP is capable of inducing genotoxicity in HeLa cells. Both clbA and clbN genes were required for the maintenance of megalocytosis and DBS induced by colibactin in K. pneumoniae

Escherichia coli and Klebsiella pneumoniae cause a wide range of infections. Multidrug-resistance strains carrying extended-spectrum β-lactamases (ESBLs) has become a growing problem worldwide. The CTX-M type ESBLs has emerged distinctly, especially in Escherichia coli and Klebsiella pneumoniae. CTX-M type has been associated with many outbreaks of infections both in the hospitals and community. CTX-M-15 is now identified as the most predominantly distributed CTX-M enzyme. Clonal outbreaks of CTX-M-15 producing Enterobacteriaceae have been described in many countries including the United Kingdom, and Escherichia coli is the most commonly involved species. A total of 100 isolates were received in 2010 from London St George’s hospital, England, 50 Escherichia coli, 17 Klebsiella spp, 9 Enterobacter spp, 13 Proteus spp, 6 Lactose fermenting coliforms, 2 Pantoea spp, one Serratia marcescens, one Morganella morganii, and one Hafnia alvei. The antimicrobial susceptibility results showed that 5 Escherichia coli and one Klebsiella pneumoniae isolates were found to be resistance to cefotaxime, ceftazidime, ceftriaxone, cefotaxime, ciprofloxacin, and gentamicin, making them multi-drug resistant bacteria. None of the isolates showed resistance to imipenem, ertapenem, or morepenem, thus making carbapenems the drug of choice for the treatment of these infections due to multi-resistant isolates. The overall frequency of CTX-M-15 type ESBL-producers detected in this study was 6 (6%) most of them 5/6 (83%) were from Escherichia coli and one was (17%) Klebsiella pneumoniae isolates. The 6 CTX-M-positive isolates were typed by PFGE, only two strains of Escherichia coli showed more than 85% similarity, owing to clonal homology for both strains. The rest strains showed less than 85% similarity. S1 nuclease plasmid profiles were obtained for ESBL-producers isolates. A total of one to three plasmids per isolate, ranging from approximately 78.0 to 152.0 kb, were observed. The plasmids from most isolates were assigned to be IncFA and IncFB replicons. Analysis of phylogenetic groups showed group A and group B2. The method of phylogenetic classification of exteraintestinal pathogenic Escherichia coli depends on examine and combination of two preserved genes (chuaA and yjaA) and the DNA fragment TSP. Primer walking and PCR experiments were used for the genetic environment studies which showed 5 different genetic constructions for the described blaCTX-M-15 genes. Conjugation studies were used to detect the transferability of the plasmids harbouring the reported blaCTX-M-15 genes. Three isolates were found transferable by conjugation. In conclusion, this study reports the presence of hospital highly resistant blaCTX-M-15 in St George’s hospital. The spread of blaCTX-M-15 is probably due to horizontal gene transfer harbouring ISEcp1 and the conjugative properties of plasmids carrying blaCTX-M-15.

Antibiotic resistance is a serious global public health problem. ESBLs are enzymes that destroy expanded-spectrum beta-lactam antibiotics rendering these drugs ineffective. Infection with ESBL-producing K.pneumoniae are hard to treat and result in longer hospital stay and higher mortality rates. The Clinical Laboratory Standard Institute (CLSI) have standard methods for detection of ESBL producing strains of bacteria in infected patients to guide antibiotic therapy, reduce the risk of mortality and risk of transmission. The presence of K.pneumoniae and E.coli which produce ESBLs have been confirmed in natural environments such as soil and water but no standard methods exist to identify directly and quantify these bacteria to understand the risk of human exposure in these settings. The purpose of this research is to assess the ability of an agar dilution method, using a differential agar Bio-Rad Rapid E.coli 2 agar utilized in environmental water quality studies, to identify correctly ESBL-producing K.pneumoniae. The minimum inhibitory concentration (MIC) of ceftriaxone antibiotic for wild-type ESBL producing K.pneumoniae isolates were compared on Mueller-Hinton broth (MHB) and Bio-Rad Rapid E.coli 2 agar. Using the MIC values, the isolates were classified as susceptible, intermediate or resistant. The MIC of wild-type strains of K.pneumoniae were above 4μg/mL for both methods on all susceptibility tests performed. The results of this research suggest that Bio-Rad Agar dilution method performed well, correctly identifying these strains as resistant to ceftriaxone, an indication of ESBL production. The Bio-Rad agar dilution method can be considered as a viable standard method for direct identification of ESBL-producing K.pneumoniae in natural environments.