<p>This thesis describes the crystal structures of two proteins in the context of combatting bacterial infections. The GA module is a bacterial albumin-binding domain from a surface protein expressed by pathogenic strains of the human commensal bacterium <i>Finegoldia magna</i>. The structure of the GA module in complex with human serum albumin (HSA) provides insights into bacterial immune evasion, where pathogenicity is acquired by the bacterial cell through the ability to coat (and disguise) itself with serum proteins. The structure shows binding of the GA module to HSA in the presence of fatty acids, and reveals interactions responsible for the host range specificity of the invading bacterium. The complex resulting from binding of the GA module to HSA readily forms stable crystals that permit structural studies of drug binding to HSA. This was exploited to study the specific binding of the drug naproxen to the albumin molecule.</p><p>Antibiotics play a major role in controlling infections by attacking invading bacteria. The enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of the beta-lactam antibiotic cephalosporin C, one of the clinically most important antibiotics in current use. The enzyme uses acetyl coenzyme A as cofactor to acetylate a biosynthetic intermediate. Structures of DAC-AT in complexes with reaction intermediates have been determined. The structures suggest that the acetyl transfer reaction proceeds through a double displacement mechanism, with acetylation of a catalytic serine by the cofactor through a suggested tetrahedral transition state, followed by acetyl transfer to the intermediate through a second suggested tetrahedral transition state. The structure of DAC-AT yields valuable information for the continued study of cephalosporin biosynthesis in the context of developing new beta-lactam compounds.</p>

This thesis describes the crystal structures of two proteins in the context of combatting bacterial infections. The GA module is a bacterial albumin-binding domain from a surface protein expressed by pathogenic strains of the human commensal bacterium Finegoldia magna. The structure of the GA module in complex with human serum albumin (HSA) provides insights into bacterial immune evasion, where pathogenicity is acquired by the bacterial cell through the ability to coat (and disguise) itself with serum proteins. The structure shows binding of the GA module to HSA in the presence of fatty acids, and reveals interactions responsible for the host range specificity of the invading bacterium. The complex resulting from binding of the GA module to HSA readily forms stable crystals that permit structural studies of drug binding to HSA. This was exploited to study the specific binding of the drug naproxen to the albumin molecule. Antibiotics play a major role in controlling infections by attacking invading bacteria. The enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of the beta-lactam antibiotic cephalosporin C, one of the clinically most important antibiotics in current use. The enzyme uses acetyl coenzyme A as cofactor to acetylate a biosynthetic intermediate. Structures of DAC-AT in complexes with reaction intermediates have been determined. The structures suggest that the acetyl transfer reaction proceeds through a double displacement mechanism, with acetylation of a catalytic serine by the cofactor through a suggested tetrahedral transition state, followed by acetyl transfer to the intermediate through a second suggested tetrahedral transition state. The structure of DAC-AT yields valuable information for the continued study of cephalosporin biosynthesis in the context of developing new beta-lactam compounds.

Les hypersensibilités retardées (HSR) cutanées sont hétérogènes, à la fois par la nature des mécanismes impliqués (allergiques versus non allergiques) mais aussi par les différents degrés de sévérité rencontrés. Seules les HSR allergiques sont dues à la présence de lymphocytes T (LT), mal caractérisés chez l'homme. Le but de ce travail est d'étudier la contribution des LT CD8 cytotoxiques dans le développement et la sévérité des HSR cutanées chez l'homme, à partir de deux pathologies fréquentes : les toxidermies aux béta lactamines et l'eczéma allergique de contact à la para-phénylènediamine (PPD). Tout d'abord, la présence de LT spécifiques de médicament au sein des toxidermies aux béta lactamines a été recherchée in vivo et in vitro. Nous avons montré que les HSR sévères étaient plus souvent d'origine allergique que les HSR bénignes. Nous avons ensuite caractérisé le rôle des LT CD8 dans les HSR allergiques. Dans les toxidermies bénignes à l'amoxicilline, l'étude de la cinétique de recrutement des LT au niveau cutané ainsi que l'analyse des LT spécifiques du sang circulant ont permis de mettre en évidence le rôle essentiel des LT CD8 cytotoxiques dans l'initiation de ces réactions. Ensuite, dans l'eczéma allergique de contact à la PPD, un recrutement épidermique précoce des LT CD8 associés à des marqueurs de cytotoxicité, a été retrouvé, corrélé avec la sévérité des lésions. Ces résultats ont été confortés par ceux obtenus dans un modèle pré-clinique d'HSR allergique à la PPD chez la souris. En conclusion, ce travail montre que les LT CD8 cytotoxiques pourraient être les principales cellules effectrices des HSR cutanées allergiques chez l'homme / Skin delayed hypersensitivity (DHS) are heterogeneous, by the nature of the mechanisms involved (allergic versus non allergic) and also by their different degrees of severity. Only allergic DHS is due to T cells, poorly characterized in humans. The aim of this work is to study the contribution of cytotoxic CD8 T cells in the development and severity of skin DHS in humans, induced by two common diseases: cutaneous adverse drug reactions to beta lactam antibiotics and allergic contact dermatitis to para-phenylenediamine (PPD). First, the presence of drug specific T cells in cutaneous adverse drug reactions to beta lactams was investigated in vivo and in vitro. We showed that severe DHS were more often allergic than benign DHS. Then, we characterized the role of CD8 T cells in allergic DHS. In benign cutaneous adverse drug reactions to amoxicillin, the study of the kinetics of skin T cell recruitment as well as the analysis of circulating specific T cells highlight the essential role of cytotoxic CD8 T cells in the initiation phase of these reactions. In allergic contact dermatitis to PPD, early recruitment of epidermal CD8 T cells associated with cytotoxic markers was found, correlated with the severity of lesions. These results were supported by those obtained in a mouse model of allergic contact dermatitis to PPD. In conclusion, this work showed that cytotoxic CD8 T cells could be the main effector cells of allergic skin DHS in humans

Streptococcus pneumoniae, le pneumocoque, est une bactérie pathogène qui entraîne le décès de plus d'un million et demi de personnes dans le monde chaque année. Les β-lactamines sont très utilisées pour traiter les infections à pneumocoques. Ces antibiotiques inhibent la synthèse du peptidoglycane, une molécule géante constituant un réseau de chaînes glycopeptidiques qui englobe la cellule, lui confère sa forme et lui permet de maintenir son intégrité face à la pression osmotique. Le mécanisme d'action des β-lactamines est bien connud'un point de vue biochimique. En revanche, la réponse physiologique empêchant la multiplication des bactéries traitées est mal connue. Au cours de ma thèse, j'ai étudié les mécanismes moléculaires de la morphogenèse du pneumocoque par des approches de biochimie et de microbiologie. Un modèle de morphogenèse est proposé intégrant mes résultats à la littérature et permettant de formuler des hypothèses sur la réponse physiologique de S. pneumoniae aux β-lactamines. / Streptococcus pneumoniae, the pneumococcus, is a bacterial pathogen that causes more than 1.5 million deaths each year in the world. β-Lactams are widely used to treat patients with pneumococcal infections. These antibiotics inhibit the synthesis of the peptidoglycan, a giant molecule constituting a mesh of aminosugar strands encasing the cell. This main constituent of the cell wall allows cells to maintain their integrity under the turgor pressure, and endows bacteria with their shape. The action of β-lactams is well understood from a biochemical point of view. However, a complete understanding of the physiological response of treated bacteria remains elusive. In this thesis, I investigated the molecular mechanisms of the morphogenesis of S. pneumoniae using methods of biochemistry and microbiology. A morphogenesis model is built based on my results and the literature, which permits to emit hypotheses concerning the response of the pneumococcus to β-lactams.

Orientadores : Thales Rocha de Mattos Filho, Francisco Carlos Groppo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-06T06:37:26Z (GMT). No. of bitstreams: 1 Junqueira_MarcelodeSouza_D.pdf: 648615 bytes, checksum: 3b46ad75b56af2255d981b942d82fa8f (MD5) Previous issue date: 2006 / Resumo: O uso de antimicrobianos e de antiinflamatórios é prática comum na odontologia, embora haja escassez de trabalhos sobre seu uso simultâneo. Portanto, o objetivo deste trabalho foi avaliar, em ratos, os efeitos do diclofenaco sódico na absorção, concentração sérica e excreção da amoxicilina utilizando a técnica de perfusão tecidual e o método microbiológico. Foram utilizados 108 ratos Wistar machos (12 grupos, n= 9), com peso entre 150 e 200 g. Foram administrados aos animais: amoxicilina 25 mg/Kg (grupos: G1, D1, S1 e R1), diclofenaco sódico 2,5 mg/Kg + amoxicilina 25 mg/Kg (grupos: G2, D2, S2 e R2) e 1,0 mL de solução de cloreto de sódio a 0,9% (grupos: G3, D3, S3 e R3). As drogas foram administradas por injeção intraluminal aos animais dos grupos G1, G2, G3, D1, D2 e D3 e por via oral aos animais dos grupos S1, S2, S3, R1, R2 e R3. Foram avaliadas nos tempos de 15, 30, 45, 60, 120, 180, 240, 300 e 360 minutos as concentrações plasmáticas e urinárias de amoxicilina e a absorção gastrintestinal ¿in vitro¿ do antimicrobiano. O diclofenaco sódico causou uma redução significativa na absorção intestinal e um aumento na excreção renal do antimicrobiano em ratos, com conseqüente diminuição da sua concentração sérica. Portanto, em algumas situações clínicas, a eficácia da amoxicilina poderia ser prejudicada pela sua co-administração com o diclofenaco sódico / Abstract: The prescription of antibiotics associated to anti-inflammatory drugs is common in dentistry; however its effects have not been studied enough. Thus, the aim of this work was to evaluate the influence of sodium diclofenac on absorption, serum concentration and excretion of amoxicillin, in rats, by the microbiologic and tissue perfusion methods. The sample consisted of 108 male Wistar rats (12 groups, 9 rats each), weighing 150¿200 g. The animals were given: amoxicillin 25 mg/Kg (groups G1, D1, S1 and R1), sodium diclofenac 2.5 mg/Kg plus amoxicillin 25 mg/Kg (groups G2, D2, S2 and R2) and 1.0 mL of solution of sodium chloride 0.9% (groups G3, D3, S3 and R3). The animals in the groups G1, G2, G3, D1, D2 and D3 were administered drugs by intra-luminal injection and the animals in the groups S1, S2, S3, R1, R2 and R3 were administered drugs p.o. After 15, 30, 45, 60, 120, 180, 240, 300 and 360 minutes, were evaluated the blood and urine concentrations of amoxicillin and the ¿in vitro¿ absorption of the antibiotic. Sodium diclofenac had decreased the intestinal absorption, increased renal excretion and, consequently, decreased the serum concentration of the amoxicillin. Thus, sodium diclofenac could decrease the efficacy of amoxicillin under some clinical situations / Doutorado / Farmacologia, Anestesiologia e Terapeutica / Doutor em Odontologia

Orientador: Francisco Carlos Groppo / Dissertação (mestrado) Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-12T01:52:09Z (GMT). No. of bitstreams: 1 Simoes_RobertaPessoa_M.pdf: 1951478 bytes, checksum: fa986a3125f61b41fa59a6ea91b73746 (MD5) Previous issue date: 2000 / Resumo: O presente trabalho teve por objetivo observar o efeito da amoxicilina, do diclofenaco sódico e da associação de ambos sobre a infecção estafilocócica induzida em tecidos granulomatosos, em ratos. Também foram avaliadas as concentrações sérica e teciduais da amoxicilina, observando o efeito da associação do diclofenaco sódico sobre as mesmas. Trinta animais receberam implantes de quatro esponjas de poliuretana subcutâneamente no dorso e, após 14 dias, dois dos tecidos resultantes (posicionados caudalmente) receberam 0,5mL de um inóculo de 108 ufcjmL de S. aureus ATCC 25923. Dois dias após, os animais foram divididos em grupos de seis, os quais receberam uma dose única de amoxicilina 50mgjkgjvo (grupo 1), amoxicilina 25 mg/kg/vo (grup02), diclofenaco sódico 2,5mg/kg/im (grupo 3), diclofenaco sódico 2,5mg/kg/im + amoxicilina 50mg/kg/vo (grupo 4) e soro fisiológico 1mLjanimal/vo (controle). Após 6h, dois tecidos granulomatosos (posicionados em direção à cabeça) e 10 _L de soro sangüíneo foram obtidos de cada animal, os quais foram dispostos em diferentes placas com MHA semeado com 108 ufcjmL de S. aureus ATCC 25923. Após incubação, foram medidos os halos de inibição proporcionados pelos tecidos granulomatosos e pelo soro sangüíneo. Dez microlitros, provenientes dos tecidos infectados após dispersão com auxílio de sonicador em tubos de ensaio contendo 10m L de NaCI foram distribuídos em placas contendo ágar salt manitol; permitiram a contagem do número de microrganismos em cada grupo. Os resultados mostraram concentrações teciduais de amoxicilina de 6,6 I-Ig/g para o grupo 1; 2,8I-1g/g para o grupo 2 e 0,8 I-Ig/g para o grupo 4. As concentrações séricas do antimicrobiano encontradas foram 11,6 I-Ig/mL para o grupo 1; 5,4I-1g/mL para o grupo 2 e 1,3I-1g/mL para o grupo 4. Não foram observadas diferenças estatisticamente significantes entre o número de microrganismos dos grupos 1, 2 e 4. Entretanto, estes foram significativamente diferentes do controle e do grupo 3, os quais foram diferentes entre si. Foi concluído que, embora o diclofenaco sódico (grupo 4) tenha diminuído a concentração tecidual da amoxicilina, a concentração resultante foi suficiente para permitir uma redução do número de microrganismos viáveis similar à observada nos grupos 1 e 2 / Abstract: The aim of this work was to observe the effect of the amoxicillin, sodium diclofenac and amoxicillin plus sodium diclofenac against staphylococcal infection in granulomatous tissues. Four polyurethane sponges placed under the back skin of thirty rats induced these tissues. After 14 days two tissues (tall position) received 0.5 mL of 108 ufc of S. aureus ATCC 25923/mL. Two days after, the animais were divided into five groups of six each. Group 1 received an only dose of amoxicillin 50mgjkgjpo, group 2 received amoxicillin 25mgjkgjpo, group 3 received sodium diclofenac 2.5mgjkgjim, group 4 received sodium diclofenac 2.5mg/kg/im plus amoxicillin 50mg/kg/po, and group 5 (control group) received NaCI 1mLjpo. After six hours of drug administration, two tissues of each animal were removed. Blood was collected from cervical plexus and centrifuged. Then, 10j.JL of serum were placed on paper disks. These disks and tissues were placed on dishes with Mueller Hinton agar inoculated with 108 ufc of Staphylococcus aureus/mL The dishes were incubated over 18 hours, and the inhibition zones were measured. The infected granulomatous tissues were placed in 10mL of NaCI, and dispersed by sonic system. Ten microliters of this suspension were spread on salt manitol agar dishes. The resulting microorganisms were counted after the incubation. Results showed tissue concentrations of amoxicillin of 6.6 ug/g for group 1; 2.8ug/g for group 2, and 0.8 ug/g for group 4. Serum concentration of amoxicillin showed 11.6 ug/mL for group 1; 5.4ug/mL for group 2, and 1.3ug/mL for group 4. Statistically significant differences among the number of microorganisms groups 1, 2, and 4 were not observed. However, these previous groups were statistically different from groups 3 and 5, which were statistically different from each other. It was concluded that, although sodium diclofenac (group 4) reduced amoxicillin concentration in the tissue, the resulting concentration was enough to reduce the count of microorganism. Also, the number of microorganisms of group 4 was similar to the one observed in groups 1 and 2 / Mestrado / Farmacologia, Anestesiologia e Terapeutica / Mestre em Odontologia

No description available.

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

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[ES] Los fármacos pertenecientes a la familia de los ß-lactámicos son los antibióticos más usados en todo el mundo. Estudios recientes revelan que hasta un 10% de la población refiere haber presentado síntomas alérgico, esta porción de la población se considera "alérgica". En el diagnóstico de la alergia a antibióticos ß-lactámicos existen varios tipos de pruebas que sirven para orientar y confirmar la presencia de una alergia, clasificadas como ensayos in vivo e in vitro. En lo que se refiere a los ensayos in vivo, la detección de los procesos alérgicos más comunes se realiza mediante pruebas de exposición oral controladas, y pruebas intraepidérmicas e intradérmicas. No obstante, estas pruebas tienen sus limitaciones como es el riesgo de inducir reacciones alérgicas sistémicas graves. Por todo ello, su práctica requiere personal entrenado. Este requisito limitan la realización de este tipo de pruebas a centros hospitalarios especializados. La aplicación y desarrollo de técnicas de diagnóstico in vitro tiene como objetivo llegar a un diagnóstico de la alergia sin riesgos para el paciente. Entre las distintas pruebas serológicas y celulares que se pueden emplear, la detección de IgE específica es una de las más extendidas debido a que, dada su sensibilidad y especificidad, y mayor seguridad. Los sistemas de diagnóstico in vitro actuales utilizan una instrumentación analítica cara y de gran tamaño, así como materiales desechables caros, manejados por personal cualificado, por lo que este tipo de pruebas se realizan únicamente por servicios de alergia y/o análisis clínicos de grandes centros sanitarios. En consecuencia, hay una necesidad de desarrollar nuevos métodos de diagnóstico que cumplan los requisitos de sensibilidad, rapidez, sencillez, multiplexado y portabilidad, a un precio reducido para su implantación en todo tipo de laboratorios clínicos de los diferentes niveles de atención sanitaria. Por este motivo, en esta tesis doctoral se plantea como objetivo principal la puesta a punto de un inmunoensayo múltiple, utilizando la tecnología de disco compacto para la detección y cuantificación in vitro de niveles de IgE específica a un amplio espectro de antibióticos ß-lactámicos en muestras de suero humano. Las limitaciones más críticas para la determinación de IgE específica multianalito mediante métodos inmunoquímicos son las relacionados con la sensibilidad y selectividad. Por lo tanto, una parte importante de la tesis se ha centrado en la preparación y caracterización de un panel antígenos ß-lactámicos, selección de inmunoreactivos, formato y estudio de diferentes parámetros claves del inmunoensayo. Otra parte importante de la tesis se ha enfocado en abordar la mejora de la reproducibilidad de los resultados. La estrategia ha consistido en estudiar diferentes sistemas de calibración con el objetivo de estandarizar la cuantificación de los métodos in vitro de diagnóstico de este tipo de alergias. Para ello, se han evaluado las prestaciones de la calibración homóloga, heteróloga y el uso de un patrón interno, comparado sus prestaciones analíticas (relación señal ruido, sensibilidad, reproducibilidad, etc.) con el sistema de referencia. Finalmente, la validación del inmunoensayo se realizó con un conjunto de 101 muestras de suero de pacientes y controles. Los resultados obtenidos han sido comparados con los obtenidos mediante las técnicas de referencia, mostrando una mejor sensibilidad, especificidad, precisión, y linealidad. La capacidad múltiplex de la tecnología de disco compacto permitió llevar a cabo paralelamente estudios de reactividad cruzada frente a diferentes antibióticos ß-lactámicos esclareciendo el patrón de reconocimiento de los pacientes. En esta línea de trabajo, el uso de otras estrategias de conjugación de haptenos ha resultado en la mejora de la sensibilidad clínica del ensayo, identificando nuevos epítopos / [CA] Els fàrmacs que pertanyen a la família dels beta-lactams, són els antibiòtics d'us més estès a tot el món. Estudis recents posen de manifest que fins un 10% de la població ha presentat símptomes d'al·lèrgia, essent considerats població al·lèrgica als beta-lactams. En el diagnòstic de l'al·lèrgia front aquest tipus d'antibiòtics, existeixen diferents tipus de proves que serveixen per orientar i confirmar la presència d'una al·lèrgia, classificades com assajos in vivo i in vitro. Pel que fa als assajos in vivo, la detecció dels processos al·lèrgics més comuns es realitza mitjançant proves d'exposició oral controlades, i proves intra-epidèrmiques i intradèrmiques. No obstant això, aquestes proves tenen les seues limitacions com és el risc d'induir reaccions al·lèrgiques sistèmiques greus. Per tot això, la seua pràctica requereix personal entrenat. Aquests requisits limiten la realització d'aquesta mena de proves a centres hospitalaris especialitzats. L'aplicació i desenvolupament de tècniques de diagnòstic in vitro té com a objectiu arribar a un diagnòstic de l'al·lèrgia sense riscos per al pacient. Entre les diferents proves serològiques i cel·lulars que es poden emprar, la detecció d'IgE específica és una de les més esteses donada la seua sensibilitat, especificitat, i major seguretat. Els sistemes de diagnòstic in vitro actuals utilitzen una instrumentació analítica cara i de gran grandària, així com materials d'un sol ús també cars, que requereixen de personal qualificat, per la qual cosa aquest tipus de proves es realitzen únicament per serveis d'al·lèrgia i/o anàlisis clíniques de grans centres sanitaris. En conseqüència, hi ha una necessitat de desenvolupar nous mètodes de diagnòstic que complisquen els requisits de sensibilitat, rapidesa, senzillesa, multiplexatge i portabilitat, a un preu reduït per a la seua implantació en tota mena de laboratoris clínics dels diferents nivells d'atenció sanitària. Per aquest motiu, en aquesta tesi doctoral es planteja com a objectiu principal la posada a punt d'un immunoassaig múltiple, utilitzant la tecnologia de disc compacte per a la detecció i quantificació in vitro de nivells d'IgE específica a un ampli espectre d'antibiòtics ß-lactáms en mostres de sèrum humà. Les limitacions més crítiques per a la determinació d'IgE específica multianàlit mitjançant mètodes inmunoquímics estan directament relacionades amb la sensibilitat i selectivitat. Per tant, una part important de la tesi s'ha centrat en la preparació i caracterització d'un panell antígens ß-lactáms, selecció d'inmunoreactius, del format d'assaig i estudi de diferents paràmetres claus de l'immunoassaig. Una altra part important de la tesi s'ha enfocat a abordar la millora de la reproductibilitat dels resultats. L'estratègia ha consistit a estudiar diferents sistemes de calibratge amb l'objectiu d'estandarditzar la quantificació dels mètodes in vitro de diagnòstic d'aquesta mena d'al·lèrgies. Per a això, s'han avaluat les prestacions del calibratge homòleg, heteròleg i l'ús d'un patró intern, comparant les seues prestacions analítiques (relació senyal soroll, sensibilitat, reproductibilitat, etc.) amb el sistema de referència. Finalment, la validació de l'immunoassaig es va realitzar amb un conjunt de 101 mostres de sèrum de pacients i controls. Els resultats obtinguts han sigut comparats amb els obtinguts mitjançant les tècniques de referència, mostrant una millor sensibilitat, especificitat, precisió, i linealitat. La capacitat múltiplex de la tecnologia de disc compacte va permetre dur a terme paral·lelament estudis de reactivitat creuada enfront de diferents antibiòtics ß-lactáms esclarint el patró de reconeixement dels pacients. En aquesta línia de treball, l'ús d'altres estratègies de conjugació d'haptens ha resultat en la millora de la sensibilitat clínica de l'assaig, identificant nous epítops / [EN] ß-lactam antibiotics are one of the most widely used antimicrobials worldwide. However, up to 10% of the population reports having presented allergic symptoms derived from their consumption. Consequently, this portion of the population is considered "allergic". In the diagnosis of allergy to ß-lactam antibiotics there are several types of tests that serve to orient and confirm the presence of an allergy. These diagnostic methods are classified as in vivo and in vitro assays. Regarding in vivo tests, the most standardized tests are the provocation, prick and intradermal test. The aim of these assays is to observe the response produced by the different beta-lactam antibiotics in the patient after oral or cutaneous administration. Despite their wide use, these tests have their limitations, such as the risk of inducing severe systemic allergic reactions. Therefore, their practice requires specialized professionals for their indication, performance and interpretation. This requirement limit the performance of this type of test to specialized hospitals. The use and development of in vitro diagnostic techniques can overcome these disadvantages and allow the diagnosis of allergy without risks. In vitro assays are less invasive and therefore neither pose a risk of adverse reactions. Among the different serological and cellular tests that can be used, the detection of specific IgE is one of the most widespread due to its sensitivity and specificity. Current in vitro diagnostic systems use expensive and bulky analytical instrumentation and high-cost single-use materials, handled by qualified professionals. Therefore, such tests are performed only by allergy and/or clinical analysis services of hospitals or by companies specialized in clinical diagnostics. Consequently, there is a need to develop new diagnostic methods that can be implemented in all types of clinical laboratories at diverse levels of health care, meeting the requirements of sensitivity, speed, simplicity, multiplexing and portability at a reduced price. For this reason, the main objective of this doctoral thesis is the development of a multiplex immunoassay using compact disc technology for the detection and quantification of specific IgE levels to a wide variety of ß-lactam antibiotics in human serum samples. The most crucial limitations for the determination of multianalyte specific IgE by immunochemical methods are those related to sensitivity and selectivity. Hence, an important part of the thesis has been focused on the preparation and characterization of a pool of beta-lactam antigens, selection of immunoreagents, format and optimization of different critical parameters of the immunoassay. Another important aspect of the thesis has been focused on improving the reproducibility of the results. The strategy consisted of studying different calibration systems with the aim of standardizing the quantification of in vitro diagnostic tests for this type of allergy. To achieve that goal, the performance of homologous and heterologous calibration and the use of an internal standard have been evaluated, comparing their analytical performance (signal-to-noise ratio, sensitivity, reproducibility, etc.) with the reference system. Finally, the validation of the immunoassay was performed with a total of 101 serum samples from patients and controls. The results obtained have been compared with those obtained using the reference techniques, showing improved sensitivity, specificity, precision, and linearity. The multiplex capability of the compact disc technology allowed carrying out parallel cross-reactivity studies against different beta-lactam antibiotics both from the penicillin family and from other families, elucidating the recognition profile. Following this working line, the use of other hapten conjugation strategies improved clinical sensitivity of the assay by identifying new epitopes. / Juárez Rodriguez, MJ. (2022). Metodologías bioanalíticas para el diagnóstico in vitro de alergia a antibióticos ß-lactámicos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/184011 / Premios Extraordinarios de tesis doctorales