The main MG detoxification pathway in <i>Escherichia coli </i>consists of two enzymes, the glyoxalases I and II, and is dependent on glutathione (GSH). MG readily conjugates with GSH in a non-enzymatic manner.  Two subsequent enzymatic reactions via the glyoxalases complete a cyclic process that recycles GSH and produces the non-toxic compound, D-lactate.  An intermediate compound in the detoxification pathway, S-lactoylglutathione (SLG), activates potassium efflux systems KefB and KefC.  This triggers a second mechanism of protection mediated by cytoplasmic acidification, enhancing chances of survival.  Therefore, it is important to understand how cells regulate the concentration of this important intermediate compound. A deterministic model was proposed that describes the series of chemical reactions in the MG detoxification pathway, allowing one to predict the flux of all compounds produced during detoxification. Through an iterative process involving model formulation, parameter estimation, data fitting and validation against experimental data, different models were analysed and discriminated in this study.  Mathematical simulations predicted that the glyoxalase pathway is not linear because it involves feedforward mechanisms for the control of SLG, the activator of the potassium efflux systems. The activities of the potassium efflux systems were investigated using deterministic models that describe the interactions between protein and ligand.  From this model, it was possible to quantify the dependence of the possible binding states on the kinetic parameters of the system.  Parameter estimation methods were used for the analysis of experimental data on the gating of the efflux systems, which proved useful for the design of new experimental strategies.

The ligand-gated potassium channels KefC and KefB of <i>Escherichia coli </i>are critical components in protecting cells from toxic electrophiles.  Potassium efflux through these channels is coupled to a decrease in cytoplasmic pH which in turn reduces the damage to DNA by electrophiles.  KefC and KefB are both inhibited by cytoplasmic glutathione and activated by glutathione adducts, such as ESG, formed by conjugation of glutathione with electrophilic compounds. Robust membrane purification protocols were developed to isolate both the wild type full-length KefC and KefB and the mutants required for biophysical analysis.  <i>In vivo </i>K⁺ measurements were performed to ensure that all of the constructs used were fully functional.  Structural and functional analysis used electron paramagnetic resonance (EPR) and stead state emission fluorescence measurements <i>in vivo</i>, on wild type and mutated full-length proteins to elucidate the gating mechanism and test the model generated from crystallographic data.  In particular, EPR spectroscopy combined with site-directed spin labelling revealed a substantial conformational change and thus provided the first insight into coupling between sensing and gating.  Steady state fluorescence spectroscopy was used to precisely measure binding affinities for both activating and inhibitory ligands and characterise nucleotide binding to KefC.  Finally, a variety of chemically diverse glutathione adducts was tested on KefC <i>in vitro </i>to elucidate the mechanism by which these ligands initiate K⁺ flux through the associated transmembrane domain.

HdeA and HdeB are stationary phase periplasmic proteins believed to function as acid-induced chaperones to prevent the aggregation of periplasmic proteins at low pH.  The aims of this project were to examine the importance of HdeA and HdeB for <i>E. coli</i> acid resistance, to determine the fate of periplasmic proteins at acid pH and to establish the extent to which HdeA and HdeB aid protein stability during an acid challenge. This study has demonstrated that HdeA and HdeB are important for acid resistance, but to a lesser extent than is currently described in the literature.  The work presented in this thesis shows that <i>hdeAB </i>mutants retained a degree of acid resistance, albeit at a lower level than the wild type strains.  HdeA and HdeB contribute to, but are not essential for acid resistance.  The temperature at which cells were grown and subsequently acid challenged at was also shown to be an important factor in acid resistance. Cytoplasmic and exponential phase periplasmic proteins were found to be acid sensitive and aggregated after treatment at acid pH.  In contrast, stationary phase periplasmic proteins were resistant to aggregation at acid pH.  The absence of HdeA and HdeB from stationary phase periplasmic extracts did not result in protein aggregation.  This demonstrated that HdeA and HdeB are not required for protein stability at acid pH.  An exception to this was the periplasmic protein, FkpA, which was found to precipitate upon acid treatment in the absence of HdeA and HdeB.  This presented a potential role for HdeA and HdeB in stabilising FkpA upon exposure to acid pH.

Conventionally, in Escherichia coli, phylogenetic groups A and B1 are associated with commensal strains while B2 and D are associated with extraintestinal strains. The aim of this study was to evaluate diarrheagenic (DEC) and commensal E. coli phylogeny and its association with antibiotic resistance and clinical characteristics of the diarrheal episode. Phylogenetic groups and antibiotic resistance of 369 E. coli strains (commensal strains and DEC from children with or without diarrhea) isolated from Peruvian children <1 year of age were determined by a Clermont triplex PCR and Kirby-Bauer method, respectively. The distribution of the 369 E. coli strains among the 4 phylogenetic groups was A (40%), D (31%), B1 (21%), and B2 (8%). DEC-control strains were more associated with group A while DEC-diarrhea strains were more associated with group D (𝑃 < 0.05). There was a tendency (𝑃 = 0.06) for higher proportion of persistent diarrhea (≥14 days) among severe groups (B2 and D) in comparison with nonsevere groups (A and B1). Strains belonging to group D presented significantly higher percentages of multidrug resistance than the rest of the groups (𝑃 > 0.01). In summary, DEC-diarrhea strains were more associated with group D than strains from healthy controls.

Objetivos: Determinar la prevalencia de Escherichia coli BLEE en urocultivos del Hospital Central FAP en el periodo Enero-Junio 2016. Material y métodos: Se realizó un estudio observacional, descriptivo, analítico, retrospectivo y transversal. Se revisaron 1772 aislamientos bacterianos en orina (urocultivos) generados desde el laboratorio de microbiología del Hospital en el periodo comprendido entre Enero a Junio del 2016. Resultados: Encontramos 1175 urocultivos positivos de los cuales el 26.5% resultaron ser urocultivos E coli BLEE (+).El 80 % de los urocultivos E coli BLEE (+) fueron de género femenino y el 20% masculino. La media de la edad de los pacientes con E coli BLEE (+) fue de 64+/-22,2 años, siendo la mínima de 1 año y la máxima de 102 años. En la mujer, la prevalencia de Escherichia coli BLEE (+), fue más frecuente en el grupo etáreo de 35 a 64 años (34,7%), y en el hombre fue más frecuente en el grupo etáreo mayor de 80 años (42,6%). Observamos que la media de edad de los BLEE (+) fue mayor en relación a los BLEE negativos (64 vs 60.9 años). Encontramos una mayor frecuencia de sensibilidad antimicrobiana de los aislados de Escherichia coli BLEE (+) con amikacina (91,7%), e imipenem (91,5%); y la frecuencia de mayor resistencia fue encontrada con el ácido Nalidíxico (94%), y Cefalotina (89,8%). En los BLEE negativos hubo mayor sensibilidad con la nitrofurantoína (95,5%), Cefalotina (85,1%) y gentamicina (80%), asimismo en el mismo grupo se evidenció mayor resistencia al ácido nalidíxico (64.3%). Conclusiones: La prevalencia de Escherichia coli BLEE fue del 26.5%, afectando principalmente al sexo femenino y el grupo etáreo de 35 a 64 años. En los urocultivos E. coli BLEE (+), la media de edad fue de 64 años, y en los urocultivos E. coli BLEE (-) fue 60 años. Hubo una mayor frecuencia de sensibilidad antimicrobiana de los aislados de Escherichia coli BLEE positivos con amikacina, e imipenem; y de mayor resistencia con el ácido Nalidíxico, y Cefalotina; y de los BLEE negativos hubo mayor sensibilidad con la gentamicina, Cefalotina, nitrofurantoína y mayor resistencia a ácido nalidíxico.

Con el objetivo de determinar los serotipos más frecuente de Escherichia coli Enteropatógena (EPEC) en los cuadros diarreicos agudos de niños menores de cinco años, se diseñó una investigación de tipo descriptivo, transversal, y observacional. La población de estudio estuvo constituida por 251 niños tratados en forma ambulatoria en el Hospital Nacional Docente Madre Niño “San Bartolomé” durante los meses de noviembre del 2000 a marzo del 2001. Se encontró 42 % (105 casos) de coprocultivos positivos a bacterias enteropatógenas, siendo Escherichia coli enteropatógena (EPEC) con 29,2 % (35 casos), la segunda causa más frecuente después de Shigella sp. Los serotipos de EPEC encontrados fueron: 9 casos del O119, 8 casos de O26, 6 casos del O55 y 2 casos del O86. Se concluye que lo serotipos más frecuentes de EPEC en los cuadros diarreicos agudos de niños menores de cinco años fueron el O119 y el O26.

Escherichia coli es parte de la flora normal de los intestinos de los mamíferos, sin embargo, hay cepas más agresivas denominadas patógenas debido a presencias de factores de virulencias. Este patógeno se clasifica en 6 patotipos: E. coli enterohemorrágica (EHEC), E. coli enteropatógena (EPEC), E. coli enterotoxigénica (ETEC), E. coli enteroinvasiva (EIEC), E. coli enteroagregativa (EAEC) y E. coli difusamente adherente (DAEC), responsables de cuadros diarreicos tanto en animales como en los humanos. Las alpacas, principal recurso económico de las comunidades andinas, sufren de diarreas asociadas a E. coli patógenas y son manejadas por pastores y su familia en estrecho contacto principalmente en la época de parición. El presente estudio tuvo como objetivo evaluar la presencia de cepas potencialmente patógenas en muestras fecales de alpacas neonatas y los niños dedicados a su crianza evaluando, en 7 familias, la presencia de tres patotipos de E. coli (ETEC, EHEC, EPEC). Para lo cual se analizaron muestras de heces no diarreicas en 72 alpacas neonatas y 12 niños. Al análisis de las 72 alpacas neonatas muestreadas, 40 animales presentaron al menos un patotipo de E. coli. En 33 de estos 40 animales se aislaron cepas EPEC clasificadas como típicas (n=6), atípicas (n= 18) y en 9 animales restantes se recuperaron ambos tipos de cepas típicas y atípicas. De 7/40 animales se aislaron cepas EHEC, 4 de estos 7 animales fueron cepas EHEC y de los restantes 3 se aislaron combinaciones de cepas EHEC y EPEC típicas y atípicas. En 3 de las 7 familias se detectaron 4 niños positivos a algún patotipo de E. coli. En uno de ellos se aislaron cepas EHEC y ETEC, en dos EPEC típica y uno EPEC atípica. Estos resultados sugieren a la alpaca como reservorio de cepas patógenas de E. coli con la posibilidad de transmitirlo al ser humano. Palabras clave: Escherichia coli, diarrea, niños pastores, alpaca / --- Escherichia coli is part of the normal flora of the intestine of mammals, however, there are aggressive strains denominated pathogens due to the presence of virulence factors. This pathogen is classified in 6 pathotypes: enterohemorragic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteoaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC) responsibles of diarrheas in both animals and humans. Alpacas are the principal economic resource of the Andean communities, they suffer of diarrhea associated with pathogenic E. coli strains and they are raised by shepherds and their families in close contact mainly in the breeding season. The objective of this study was to evaluate the presence of potentially pathogenic strains in stool samples from neonatal alpacas and children involved in their upbringing, in 7 families, the presence of three pathotypes of E. coli (ETEC, EHEC, EPEC). Stool samples not diarrheal were tested from 72 neonatal alpacas and 12 children. The analysis of the 72 neonatal alpacas, 40 animals had at least one pathotype of E. coli. In 33 of these 40 animals had isolated typical EPEC strains (n = 6), atypical (n = 18) and 9 remaining animals were isolated both typical and atypical strains. At 7/40 animals from EHEC, 4 strains of these 7 animals were EHEC strains and the remaining 3 were isolated EHEC strains and combinations of typical and atypical EPEC. In 3 of the 7 families were detected children positive for at least one pathotype E. coli. One child was positive to ETEC and EHEC strains, two children with typical EPEC and one child typical EPEC. These results suggest the alpaca like a reservoir of pathogenic strains of E. coli with the possibility of transmission to humans. Keywords : Escherichia coli, diarrhea, shepherd children, alpaca

In dieser Arbeit werden die Ergebnisse der molekular-epidemiologischen Analyse von Virulenzgenen im Genom von insgesamt 222 Escherichia coli (E. coli)-Isolaten dargestellt, die von Mastitis-Fällen bei Rindern isoliert wurden. Mit Hilfe der Multiplex-Polymerase-Kettenreaktion wurde die Verbreitung von 42 potentiellen Virulenzfaktor-Genen extraintestinal pathogener E. coli (ExPEC) analysiert. Neben der quantitativen Bestimmung des Vorkommens jedes Einzelgens wurde in dieser Arbeit eine differenzierte Auswertung von Genkombinationen bei E. coli Mastitis-Isolaten vorgenommen. Diese ermittelten genetischen Muster werden zur 1. Prävalenz der in der Gesamtheit der Isolate, 2. Prävalenz in den phylogenetischen ECOR-Gruppen, 3. akut klinischen und chronischen Mastitis-Episoden und 4. dem Vorkommen spezifisch tierpathogener Adhäsine korreliert. Die Mastitis-Isolate konnten aufgrund der Virulenzmarkerverteilung und Phylogenie keinem bestimmten charakteristischen Pathotyp zugeordnet werden. Die überwiegende Mehrzahl der Mastitis-Isolate zeigte aufgrund einer geringen Prävalenz Virulenz-assoziierter Gene sowie der Zugehörigkeit zu den phylogenetischen Entwicklungslinien A und B1 ein geringes Virulenzpotential extraintestinal pathogener E. coli. Die Mehrzahl der Stämme enthielt eine singuläre Virulenzdeterminante (83 Stämme; 37,4 %), eine Zweierkombination (69 Stämme; 31,1 %) oder eine Dreierkombination von Virulenzgenen (34 Stämme; 15,3 %). Vier Gene für Virulenzfaktoren in Kombination zeigten sich lediglich in sieben Stämmen (3,1 %). Insbesondere die Anwesenheit von 5 bis 18 differenten Virulenzgenen pro Genom traten nur mit einer geringen Frequenz in zusammen 16 Isolaten (7,2 %) auf. Das absolut häufigste Virulenz-assoziierte Gen, das nachgewiesen wurde, war fimH, das für die mannosespezifische Adhäsinuntereinheit der Typ1-Fimbrien kodiert. Insgesamt gaben 88,7 % aller 222 untersuchten Stämme ein positives Signal in der Multiplex-PCR, und zwar 89,9 % der 199 klinischen Isolate sowie 85,7 % der Isolate chronischer Mastitiden. In etwa der Hälfte aller untersuchten Stämme trat auch das Gen traT auf, das Serumresistenz vermittelt (43,7 %). Die Genkombination fimH-traT wurde in wechselnden Konstellationen in insgesamt 83 Stämmen (37,3 %) gefunden. Sie ist damit die häufigste Virulenzgenkombination in den untersuchten E. coli-Genomen mit multiplen Virulenzdeterminanten. Da bei Rinder-Mastitis besonders in den schweren Fällen systemische Verläufe fördernde Faktoren wie Serumresistenz eine bedeutende Rolle spielen, könnte hier eine Selektion auf genetische Kopplung von traT mit fimH vorliegen. Deutlich geringere Prävalenzen wiesen die Virulenzgene für α-Hämolysin (hlyA, 10,8 %), den Yersiniabactinrezeptor (fyuA, 12,2 %) sowie das ebenfalls an der Serumresistenz beteiligte Gen iss (8,5 %) auf. Nur 13 (5,8 %) der 222 E. coli-Isolate besaßen keines der untersuchten Virulenzgene. Das Fehlen bekannter Virulenzgene in diesen Stämmen deutet darauf hin, dass weitere unberücksichtigte Faktoren eine Rolle bei der Virulenz von Mastitisisolaten spielen könnten oder der Status des Wirtsorganismus in diesen Fällen ausschlaggebend für eine erfolgreiche Infektion des Euters sein könnte. Offensichtlich sind die meisten der untersuchten E. coli- Virulenzfaktoren für die Pathogenese der Rindermastitis von untergeordneter Bedeutung. Von den 222 Isolaten zählten insgesamt 137 Stämme zur phylogenetischen Linie (ECOR-Gruppe) A, 62 zur ECOR-Gruppe B1, 20 zur ECOR-Gruppe B2 und 14 zur Gruppe D. Die Stämme, die zu den phylogenetischen Entwicklungslinien A, B1 und D gehören, unterschieden sich hinsichtlich der Prävalenz der Virulenzfaktormuster nicht vom Gesamtbild. Lediglich Isolate der ECOR-Gruppe B2 wiesen eine für sie typische Häufung von Virulenzgenclustern auf. Das relativ geringe Vorkommen bzw. weitgehende Fehlen (5 von 8) von Adhäsingenen spezifisch tierpathogener E. coli lässt darauf schließen, dass bislang beschriebenen Rinder-pathogenen E. coli keine Bedeutung als Verursacher einer Rindermastitis zukommt. Für die Analyse der chronischen Verlaufsform der Mastitis standen nur 21 Isolate zur Verfügung, die keinen hinreichend gesicherten Vergleich zu den Fällen mit akuter klinischer Mastitis (199 Stämme insgesamt) erlauben. Die auffällige Zunahme des Hämolysingens hlyA (23,8 %) gegenüber 9,5 % in den klinischen Isolaten (und 10,8 % in allen Stämmen) müsste in künftigen Untersuchungen invasiven Verhaltens der ExPEC beobachtet werden. Die Ergebnisse dieser Arbeit legen nahe, dass eine bovine Mastitis durch verschiedene E. coli-Varianten hervorgerufen werden kann und ein großes Potential extraintestinaler Virulenzfaktoren dazu nicht erforderlich ist. Entscheidend ist eine durch das fimH-Gen vermittelte Adhäsion, in der Hälfte der untersuchten Fälle unterstützt durch das Serumresistenz vermittelnde Gen traT. / Studies on the spread of virulence factors of extra intestinal Pathogenic Escherichia coli strains in isolates of bovine Mastitiden

by Ling Kin Wah, Thomas. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references (leaves 103-117). / ABSTRACT --- p.i / ACKNOWLEDGMENTS --- p.v / LIST OF ABBREVIATIONS --- p.vi / TABLE OF CONTENTS --- p.viii / LIST OF TABLES --- p.xv / LIST OF FIGURES --- p.xix / INTRODUCTION --- p.1 / LITERATURE REVIEW --- p.2 / Chapter 1. --- Structure of the bacterial cell envelope --- p.2 / Chapter 2 . --- The β-lactam antibiotics --- p.4 / Chapter 3. --- Mode of action of β-lactam antibiotics --- p.5 / Chapter 4. --- Penicillin-binding proteins (PBPs) --- p.6 / Chapter 5. --- Mechanisms of bacterial resistance to β-lactam antibiotics --- p.7 / Chapter 5.1 --- Non-enzymatic resistance --- p.7 / Chapter 5.1.1 --- Alteration in cell permeability --- p.8 / Chapter 5.1.2 --- Alteration of the target site --- p.9 / Chapter 5.1.3 --- Tolerance and persistence --- p.9 / Chapter 5.2 --- Enzyme-mediated resistance --- p.12 / Chapter 6. --- Transfer of resistance --- p.13 / Chapter 7. --- β-lactamases --- p.16 / Chapter 7.1 --- History --- p.16 / Chapter 7.2 --- Classification of β-lactamases --- p.17 / Chapter 7.2.1 --- Richmond and Sykes scheme --- p.17 / Chapter 7.2.2 --- Matthew scheme --- p.18 / Chapter 7.2.3 --- Bush scheme --- p.19 / Chapter 7.3 --- β-lactamases of Gram-negative bacteria --- p.19 / Chapter 7.3.1 --- Chromosomally-mediated β-lactamases --- p.19 / Chapter 7.3.2 --- Plasmid-mediated β-lactamases --- p.20 / Chapter 7.4 --- β-lactamase inhibitors --- p.25 / Chapter 7.5 --- Regulation of β-lactamase production --- p.28 / Chapter 7.5.1 --- β-lactamase induction --- p.28 / Chapter 7.5.2 --- Mutation to constitutive enzyme production --- p.29 / Chapter 7.5.3 --- β-lactam induced β-lactamase production --- p.30 / Chapter 8. --- Emergence of resistance due to production of β-lactamases --- p.31 / Chapter 8.1 --- Resistance in staphylococci --- p.32 / Chapter 8.2 --- Resistance in haemophili and gonococci --- p.33 / Chapter 8.3 --- Resistance in Enterobacteriaceae (non E. coli) --- p.34 / Chapter 8.4 --- Distribution of β-lactamases in E. coli --- p.35 / MATERIALS AND METHODS / Chapter 1. --- Bacterial strains --- p.38 / Chapter 1.1 --- Standard organisms --- p.38 / Chapter 1.2 --- Clinical isolates --- p.38 / Chapter 2. --- Antibiotics --- p.39 / Chapter 3. --- "Media, chemicals and culture conditions" --- p.39 / Chapter 4. --- Bacterial identification and viable bacterial count --- p.39 / Chapter 5. --- Antibiotic sensitivity testing --- p.40 / Chapter 5.1 --- Disk diffusion --- p.40 / Chapter 5.2 --- Determination of minimal inhibitory concentration (MIC) --- p.40 / Chapter 6. --- Plasmid analysis --- p.41 / Chapter 6.1 --- Transfer of drug resistance plasmids --- p.41 / Chapter 6. 2 --- Molecular studies of plasmids --- p.42 / Chapter 6.2.1 --- Extraction of plasmid DNA --- p.43 / Chapter 6.2.2 --- Agarose gel electrophoresis --- p.43 / Chapter 6.2.3 --- Molecular size determination --- p.44 / Chapter 7 . --- DNA hybridization --- p.44 / Chapter 7.1 --- DNA blotting --- p.44 / Chapter 7.1.1 --- Colony blotting --- p.45 / Chapter 7.1.2 --- Southern blotting --- p.45 / Chapter 7.2 --- Labeling of oligonucleotide probe --- p.46 / Chapter 7.3 --- Hybridization --- p.47 / Chapter 7.4 --- Autoradiography --- p.47 / Chapter 7.5 --- Re-use of blots --- p.48 / Chapter 8. --- Detection and screening for classification of β-lactamases --- p.48 / Chapter 8.1 --- Detection of β-lactamases --- p.48 / Chapter 8.1.1 --- Acidimetric --- p.48 / Chapter 8.1.2 --- Chromogenic substrate --- p.49 / Chapter 8.1.2.1 --- Whole cell --- p.49 / Chapter 8.1.2.2 --- Cell extract and filtrate --- p.49 / Chapter 8.2 --- Screening for classification of β-lactamases --- p.49 / Chapter 9. --- "Preparation, purification, qualitative and quantitative analyses of the β-lactamase from transconjugants TU117, TB117 and the recipient K12" --- p.51 / Chapter 9.1 --- Large scale preparation of enzyme --- p.51 / Chapter 9.2 --- Gel filtration --- p.52 / Chapter 9.3 --- Preparative isoelectric focusing (PIEF) --- p.53 / Chapter 9.4 --- Protein determination --- p.55 / Chapter 9.5 --- Qualitative analyses and characterization of β-lactamases --- p.56 / Chapter 9.5.1 --- Analytical isoelectric focusing --- p.56 / Chapter 9.5.1.1 --- Semi-quantitative determination of β-lactamases --- p.56 / Chapter 9.5.1.2 --- Polyacrylamide gel preparation --- p.57 / Chapter 9.5.1.3 --- Isoelectric focusing --- p.58 / Chapter 9.5.1.4 --- pH measurement --- p.58 / Chapter 9.5.1.5 --- Gel development and recording --- p.59 / Chapter 9.5.1.5.1 --- Nitrocefin staining --- p.59 / Chapter 9.5.1.5.2 --- Silver staining --- p.59 / Chapter 9.5.1.6 --- Isoelectric point (pI) determination --- p.60 / Chapter 9.5.2 --- Spectrophotometric assay of β-lactam substrates --- p.60 / Chapter 9.5.2.1 --- Absorption spectra of β-lactam antibiotics --- p.60 / Chapter 9.5.2.2 --- The molar extinction coefficient of β-lactam substrates --- p.60 / Chapter 9.5.2.3 --- Measurement of β-lactamase hydrolytic activities --- p.61 / Chapter 9.5.2.4 --- Determination of enzyme kinetics --- p.61 / Chapter 9.5.3 --- Molecular weight determination of proteins --- p.62 / Chapter 9.5.3.1 --- SDS-polyacrylamide gel preparation --- p.62 / Chapter 9.5.3.1.1 --- Resolving gel --- p.62 / Chapter 9.5.3.1.2 --- Stacking gel --- p.63 / Chapter 9.5.3.2 --- Electrophoresis --- p.63 / Chapter 9.5.3.3 --- Staining and recording --- p.64 / Chapter 9.5.3.4 --- Molecular weight determination --- p.64 / RESULTS / Chapter 1. --- Collection of organisms --- p.65 / Chapter 2 . --- Identification of organisms --- p.65 / Chapter 3. --- Antibiotic sensitivity testing --- p.66 / Chapter 4. --- Genetic and molecular studies of ampicillin- resistant plasmids --- p.68 / Chapter 4.1 --- Transfer of ampicillin-resistant factor --- p.68 / Chapter 4.1.1 --- E. coli K12 14R525 as recipient --- p.68 / Chapter 4.1.2 --- other Enterobacteriaceae --- p.68 / Chapter 4.2 --- Plasmid studies of E. coli --- p.69 / Chapter 5. --- Detection and identification of β-lactamases --- p.69 / Chapter 5.1 --- Analytical IEF --- p.70 / Chapter 5.2 --- DNA hybridization --- p.70 / Chapter 5.2.1 --- Colony blot hybridization --- p.70 / Chapter 5.2.2 --- Southern blot hybridization --- p.71 / Chapter 6. --- Characterization of TEM-1 producing E. coli --- p.71 / Chapter 6.1 --- Susceptibility testing --- p.71 / Chapter 6.2 --- Enzyme kinetic study --- p.72 / Chapter 6.2.1 --- Absorption spectra and molar extinction coefficient of β-lactam antibiotics --- p.72 / Chapter 6.2.2 --- Comparison of the substrate profiles --- p.73 / Chapter 6.3 --- Correlation of MICs to β-lactamase activities --- p.73 / Chapter 7. --- "Isolation, quantitation and characterization of β-lactamases isolated from three E. coli strains" --- p.74 / Chapter 7.1 --- Preparation of β-lactamases --- p.75 / Chapter 7. 2 --- Purification of β-lactamases --- p.76 / Chapter 7.2.1 --- Gel-filtration chromatography --- p.76 / Chapter 7.2.2 --- Preparative isoelectric focusing --- p.77 / Chapter 7.3 --- Characterization of the purified β-lactamases --- p.78 / Chapter 7.3.1 --- Isoelectric point --- p.78 / Chapter 7.3.2 --- Molecular weight assessment --- p.79 / Chapter 7.3.3 --- Enzyme kinetic study --- p.79 / DISCUSSION / Chapter 1. --- Epidemiology of ampici11in (or amoxycillin)- resistant E. coli --- p.81 / Chapter 2. --- Distribution of β-lactamases in ampicillin- resistant E. coli --- p.84 / Chapter 3. --- Correlation between level of resistance and β-lactamase activity --- p.86 / Chapter 4. --- Plasmid-mediated TEM-1 β-lactamase --- p.89 / Chapter 4.1 --- Transfer of resistance --- p.89 / Chapter 4 .2 --- Identification of β-lactamases by DNA hybridization --- p.91 / Chapter 5. --- Mechanism of high-level resistance --- p.93 / Chapter 5.1 --- Selection of resistant strains --- p.93 / Chapter 5.2 --- β-lactamases preparation and purification --- p.95 / Chapter 5.3 --- Hyperproduction of β-lactamase --- p.97 / SUMMARY AND CONCLUSIONS --- p.102 / REFERENCES --- p.103 / APPENDICES / Chapter 1. --- TABLES --- p.118 / Chapter 2. --- FIGURES --- p.153

Orientador: Antonio Fernando Pestana de Castro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-23T07:46:51Z (GMT). No. of bitstreams: 1 Sousa_Adrianade_M.pdf: 8565896 bytes, checksum: 90d1693edefb33884de5361360364d81 (MD5) Previous issue date: 1997 / Resumo: Foram estudadas 190 amostras não-toxigênicas (L T-I, L T-II, STa, STb, SL T-I, SL T-II, CNF1, CNF2) de Escherichia colí isoladas de diarréia em suínos. Pelo teste de aderência em células HeLa, 47 amostras foram identificadas com diferentes perfis de adesão, a saber: agregativa (EaggEC) 18/47 (38,3%),"cover slip"(CS) 7/47 (14,9%); difusa (DA) 2/47 (4,2%) e não característica (NC) 20/47 (42,5%). Não foram encontradas amostras com adesão localizada (AL). Ao contrário do que ocorre em amostras de origem humana, quando o mesmo teste foi realizado em células HEp-2, houve correspondência de apenas 17% dos resultados, principalmente entre as amostras com perfil NC. O teste de Microhemaglutinação Manose-Resistente (MHMR) não revelou títulos significantes com nenhuma das hemácias testadas ( ovina, bovina, equina, humana, de cobaio e galinha ). A sorologia para detecção de fatores de colonização já descritos identificou a presença das adesinas K99, K88, F41, F42, F165, 987P, F107, 8199 e 2134P entre as amostras estudadas, as quais não estão entre as que apresentaram resultados relevantes em testes posteriores. O teste de FAS para avaliar a polimerização da actina, foi positivo (++) com apenas 07 (14,8%), das amostras aderentes em HeLa, a saber: 817, E3, 2a2, 2d2, 2374c, 2386a, 2387 a, o que poderia indicar presença do gene eaeA. Testes realizados com sonda específica e PC R, contudo, foram negativos, o que foi confirmado pelo teste de Western blot usando-se soro anti-intimina específico. O exame de cultivos de HEp-2 infectados com algumas amostras FAS+, ( E3, 2d2 e 2374c ) e FAS - ( C9), por microscopia eletrônica, revelou lesões "attaching and effacing"{AlE), típicas de amostras eaeA+. Duas destas amostras ( E3 e 2d2 ) testadas em alça-ligada de leitões recém-nascidos, não produziram lesões semelhantes, ou pelo menos, com a mesma intensidade que a amostra E2348/69, padrão EPEC. A descrição da presença do gene eaeA em amostras do sorogrupo 045"K65" isoladas de diarréia em suínos encaminhou-nos na realização de sorotipagem das nossas amostras, sendo encontradas 4 amostras pertencentes a este sorogrupo, as quais não apresentaram resultados relevantes nos demais testes realizados. Conclusivamente, os resultados obtidos permitem supor a existência nos colibacilos não toxigênicos isolados de suíno com diarréia estudados, de alguma estrutura, eventualmente também responsável pelos padrões de adesão encontrados, que à semelhança da intimina de EPEC e/ou EHEC, causariam o fenômeno de AlE na célula hospedeira / Abstract: Not informed. / Mestrado / Microbiologia / Mestre em Ciências Biológicas