No description available.

No description available.

Summary : Enterococcus faecium possesses a low affinity PBP5 that, like in other enterococci, is an important factor contributing to the intrinsic resistance to penicillin. The structure of PBP5 soluble form in complex with benzylpenicillin has been solved at 2.1 Å resolution. On the basis of this 3D structure, it was proposed that the I623KEKQDEKG631 turn connecting β3 and β4 strands and constituting one wall of the active site, as well as, R464 involved in a salin-bridge near the active site could be responsible for the low β-lactam affinity of PBP5fm The k2/K of sPBP5fm acylation by various b-lactams have been determined and, as expected for a low affinity PBP, they are very low. Mutants were constructed to determinate the role of the residues pointed in the 3D structure as related to the observed low affinity. For the sPBP5fm∆I7G mutant, the acylation rate constants are similar to those measured for sPBP5fm. However, when compared to the wild type, the mutants without salin-bridge show a higher affinity that could be explained as the consequence of an easier accessibility to the active site for the inactivator. This study allows mapping active site cavity and determines the importance of structural elements to understand the low affinity of PBP5. Furthermore, three news β-lactams (ceftaroline, ceftobiprole and ME1036) were tested against PBP5fm and they all exhibit inhibitory activity against the protein. Ceftaroline appears as the best inhibitor of sPBP5fm as well as the best inhibitor while tested on Enterococcus faecium cultures. Résumé : Enterococcus faecium possède une protéine PBP5 de faible affinité, qui est un facteur important de la résistance intrinsèque à la pénicilline. La structure de la forme soluble dans PBP5 complexés avec la benzylpénicilline a été résolue à 2,1 Å. Sur la base de cette structure 3D, il a été proposé que la boucle I623KEKQDEKG631 reliant brins β3 et β4 et pouvant constituer une barrière pour atteindre le site actif, ainsi que, R464 impliqué dans un pont-salin près du site actif qui pourrait être responsable de la faible affinité β-lactamines de PBP5fm. Dans un 1er temps, les k2/K de sPBP5fm sauvage par diverses β-lactamines ont été déterminées, et comme prévu, elles sont très faibles. Dans un 2nd temps, les mutants ont été produits, purifiés et caractérisés de manière à déterminer le rôle des résidus choisis dans la faible affinité. Pour le mutant sPBP5fmΔI7G, les constantes de vitesse acylation sont similaires à ceux mesurée pour sPBP5fm sauvage. Tandis que les mutants sans le pont salin R464-D481 montrent une relative plus grande affinité par rapport au type sauvage, qui pourrait être expliqué par un accès plus facile de linhibiteur au site actif. Cette étude permet une première cartographie de la cavité du site actif de PBP5fm et qui détermine limportance ou non déléments de structure pour comprendre la faible affinité. Trois nouvelles β-lactamines (ceftaroline, ceftobiprole et ME1036) ont été testés contre PBP5fm et elles présentent une activité inhibitrice de PBP5fm. La ceftaroline apparaît comme le meilleur inhibiteur de PBP5fm ainsi que le meilleur inhibiteur lors des essais sur les cultures Enterococcus faecium.

No description available.

Enterococcus faecium is a leading cause of hospital-acquired infection, disproportionally affecting immunocompromised and critically ill patients. Despite infection control measures, rates of vancomycin-resistant E. faecium (VREfm) bacteraemias have failed to decline in the United Kingdom, and Cambridge University Hospitals (CUH) report the highest numbers nationally. The aims of my PhD were to use epidemiological and genomic surveillance data to establish risk factors for acquisition and infection with E. faecium in patients at CUH, and to use a One Health approach to consider possible sources for hospital patients by relating bloodstream-associated isolates with those cultured from livestock and the environment in the same geographic region. A retrospective matched nested case control study was performed to determine risk factors for VRE bacteraemia relating to antibiotic exposure. 235 cases were matched to 220 controls for length of admission, year, specialty and ward type. Multivariable analysis demonstrated that duration of exposure to parenteral vancomycin, fluoroquinolones and meropenem were independently associated with VRE bacteraemia. This provides evidence for the importance of antimicrobial stewardship targeting high-risk antibiotics in patients at risk of VRE bacteraemia. VREfm bacteraemia may be complicated by disease recurrence. Whole genome sequencing was used to distinguish between relapse and reinfection in 14 episodes of recurrent VREfm bacteraemia. This demonstrated that 10 (71%) episodes were due to reinfection with a new strain, with reinfection being more likely with increasing time between two positive cultures. This study also evaluated 9 patients with blood cultures positive for both VREfm and vancomycin-susceptible E. faecium (VSEfm), the majority (78%) of which were found to be unrelated strains. More than half of all study isolates from these two patient groups were closely related to another isolate causing bacteraemia at CUH, suggesting that hospital acquisition of VREfm is a driver for infection and recurrence. A cross-sectional study of E. faecium in raw and treated wastewater from 20 municipal water treatment plants across the East of England revealed widespread dissemination of healthcare-associated lineages of VREfm in all sampled locations including rural areas, and environmental release in treated wastewater in 17/20 locations. Wastewater isolates were genetically intermixed with isolates causing bacteraemia at CUH, including highly related isolates indicating recent transmission between the two reservoirs. These findings are consistent with widespread distribution of healthcare-associated VREfm in community populations. A One Health approach incorporating sampling from livestock (10 pork, 10 cattle, 9 poultry farms) detected no VREfm in animals whilst 2 independent meat surveys demonstrated VREfm in 1-2% of uncooked products. Genomic comparison of >1400 E. faecium isolates from livestock, meat, wastewater and almost 800 people with bloodstream infection demonstrated that livestock and human isolates were genetically distinct. Analysis of the accessory genome added further evidence for distinct gene content associated with niche adaptation. An analysis of mobile genes encoding antibiotic resistance revealed limited evidence of sharing between human and animal populations. A prospective longitudinal study in haematology patients at CUH over 6 months revealed high rates of VREfm carriage (63% of cases) and environmental contamination (49% of samples). Genomic analysis elucidated complex colonisation dynamics with frequent loss and acquisition of subtypes, including unsuspected acquisition of new VREfm subtypes in patients already colonised with VREfm, and multiple transmission chains involving patients and the environment, including some leading to bacteraemia. These findings highlight the shortcomings of infection control and environmental cleaning and provide the basis for revised interventions.

Zugl.: Berlin, Freie Univ., Diss., 2004 / Dateiformat: zip, Dateien im PDF-Format

Freie Universiẗat, Diss., 2004--Berlin. / Dateiformat: zip, Dateien im PDF-Format.

Universiẗat, Diss., 2003--München.

Freie Universiẗat, Diss., 2005--Berlin. / Dateiformat: zip, Dateien im PDF-Format. _ ERscheinungsjahr an der Haupttitelstelle: 2005.

Summary Objectives and hypothesis: This thesis concerns the study of plasmids of vancomycin- resistant enterococci isolated from feces of American crows in the years 2012 - 2013 period. The hypothesis is that, in various environments, there is one or more types of epidemiologically significant vanA gene-carrying plasmids that are capable of horizontally spread. Methods: Based on PFGE method the number and size of plasmids were detected in selected isolates of vancomycin-resistant E. faecium. Using PCR method the isolates were subjected to detection of genes of replicases, relaxases and toxin-antitoxin system of plasmid-bound resistance genes. Using 19 primers were characterized types of Tn1546. Results: Of the 12 tested vancomycin-resistant isolates of E. faecium the following number and size of plasmids was proven using PFGE method: 2 isolates contained two plasmids (17%), 3 isolates contained three plasmids (25 %), 5 isolates contained four plasmids (42 %) and 2 isolates contained five plasmids (17 %). All isolates (n = 12) were then subjected to the detection of genes of replicases, relaxases and toxin-antitoxin system for typing of plasmids from each plasmid families. RepA_N family of plasmids: genes characterizing plasmids related to pRUM: rep17 in 11 isolates (92 %), gene Axe-Txe was detected in 5 isolates (42 %) genes characterizing plasmids related to pLG1: rep20 in 7 isolates (58 %) genes characterizing plasmids related to pAD1: relpAD1 gene was detected in one isolate (8 %) Inc18 family of plasmids: genes characterizing plasmids related to pIL501: rep1 gene detected in one case (8 %) genes characterizing plasmids related to pRES25: rep2 gene in 2 isolates (17 %) genes characterizing plasmids related to pEF1: relpEF1 detected in 11 isolates (92 %) pHTB family of plasmids: genes characterizing plasmids related to pHTB: rep22 gene was detected in 4 isolates (33%) and in 2 isolates gene relpHTB was detected (17%) RCR family of plasmids: genes characterizing plasmids related to pRI: positive detection of Rep14 gene in 8 isolates (67%) and in 4 isolates relpRI gene was detected Small theta-replicating plasmids: genes characterizing plasmids related to pEF418 plasmids: rep18a gene in 2 isolates (17%) genes characterizing plasmids related to pB82: rep18b gene was detected in one isolate (8%) genes characterizing plasmids related to pCIZ2: relpCIZ2 gene was detected in 9 isolates tested (75%) Types of transposon Tn1546 Using the PCR method types of Tn1546 were characterized. In 4 isolates (n = 12; 33 %) Tn1546 was characterized as a F3 type. In one isolate (8 %) Tn1546 was characterized as a type F5, in one isolate (8 %) as a type PP-16. In 6 isolates Tn1546 was untypeable. Most likely these are new, yet unknown types. Conclusion: This is the first study of plasmids of vancomycin-resistant isolates E. faecium isolated from feces of American crows. These results emphasize not only a high proportion of plasmids in individual isolates, but also a high proportion of genes with horizontal transfer.