Introduction and spread of vancomycin-resistant Enterococcus faecium (VREfm) at a German tertiary care medical center from 2004 until 2010: a retrospective whole-genome sequencing (WGS) study of the molecular epidemiology of VREfm.

BACKGROUND: In most of Europe and especially in Germany, there is currently a concerning rise in the number of hospital-acquired infections due to vancomycin-resistant Enterococcus faecium (VREfm). Therefore, there is a need to improve our understanding of the way VREfm spreads in hospitals. In this study, we investigated the molecular epidemiology of VREfm isolates from the first appearance at our university hospital in 2004 until 2010. There is only very scarce information about the molecular epidemiology of VREfm from this early time in Germany. METHODS: Our analysis includes all available first VREfm isolates of each patient at our tertiary care center collected during the years 2004-2010. If available, additional consecutive VREfm isolates from some patients were analyzed. We used multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) for the analysis and description of nosocomial transmission pathways as well as the detection of outbreaks. RESULTS: VREfm isolates from 158 patients and 76 additional subsequent patient isolates were included in the analysis. Until 2006, detections of VREfm remained singular cases, followed by a peak in the number of VREfm cases in 2007 and 2008 with a subsequent decline to baseline in 2010. MLST and cgMLST analysis show significant changes in the dominant sequence types (STs) and complex types (CTs) over the study period, with ST192 and ST17 being responsible for the peak in VREfm cases in 2007 and 2008. The four largest clusters detected during the study period are comprised of these two STs. Cluster analysis shows a focus on specific wards and departments for each cluster. In the early years of this study (2004-2006), all analyzed VREfm stemmed from clinical specimens, whereas since 2007, approximately half of the VREfm were detected by screening. Of the 234 VREfm isolates analyzed, 96% had a vanB and only 4% had a vanA resistance genotype. CONCLUSIONS: This retrospective study contributes significant knowledge about regional VREfm epidemiology from this early VREfm period in Germany. One remarkable finding is the striking dominance of vanB-positive VREfm isolates over the entire study period, which is in contrast with countrywide data. Analysis of cgMLST shows the transition from sporadic VRE cases at our institution to a sharp increase in VRE numbers triggered by oligoclonal spread and specific outbreak clusters with the dominance of ST192 and ST17.

New evidence for cofactor's amino group function in thiamin catalysis by transketolase.

Transketolase from Saccharomyces cerevisiae exhibits a rarely reported activity with a methylated analogue of the native cofactor, 4'-methylamino-thiamin diphosphate. We demonstrated the kinetic stability of the dihydroxyethyl carbanion/enamine intermediate to be dependent on the functionality of the 4'-aminopyrimidine moiety of thiamin diphosphate [R. Golbik, L.E. Meshalkina, T. Sandalova, K. Tittmann, E. Fiedler, H. Neef, S. König, R. Kluger, G.A. Kochetov, G. Schneider, G. Hübner, Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisae, FEBS J. (2005) 272 1326-1342]. This paper extends these investigations of the function of the coenzyme's aminopyrimidine in transketolase catalysis exemplified for the 4'-monomethylamino-thiamin diphosphate analogue. Here, we report near UV circular dichroism data and NMR-based analysis of reaction intermediates that give evidence for a strong destabilisation of the carbanion/enamine of DHE-4'-monomethylamino-thiamin diphosphate on the enzyme. A new negative band in near UV circular dichroism arising during turnover is attributed to the conjugate acid of the carbanion/enamine intermediate, an assignment additionally corroborated by (1)H NMR-based intermediate analysis. As opposed to the kinetically stabilized carbanion/enamine intermediate in transketolase when reconstituted with the native cofactor, DHE-4'-monomethylamino-thiamin diphosphate is rapidly released from the active centers during turnover and accumulates in the medium on a preparative scale.

Phosphorylation of serine 264 impedes active site accessibility in the E1 component of the human pyruvate dehydrogenase multienzyme complex.

At the junction of glycolysis and the Krebs cycle in cellular metabolism, the pyruvate dehydrogenase multienzyme complex (PDHc) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. In mammals, PDHc is tightly regulated by phosphorylation-dephosphorylation of three serine residues in the thiamin-dependent pyruvate dehydrogenase (E1) component. In vivo, inactivation of human PDHc correlates mostly with phosphorylation of serine 264, which is located at the entrance of the substrate channel leading to the active site of E1. Despite intense investigations, the molecular mechanism of this inactivation has remained enigmatic. Here, a detailed analysis of microscopic steps of catalysis in human wild-type PDHc-E1 and pseudophosphorylation variant Ser264Glu elucidates how phosphorylation of Ser264 affects catalysis. Whereas the intrinsic reactivity of the active site in catalysis of pyruvate decarboxylation remains nearly unaltered, the preceding binding of substrate to the enzyme's active site via the substrate channel and the subsequent reductive acetylation of the E2 component are severely slowed in the phosphorylation variant. The structure of pseudophosphorylation variant Ser264Glu determined by X-ray crystallography reveals no differences in the three-dimensional architecture of the phosphorylation loop or of the active site, when compared to those of the wild-type enzyme. However, the channel leading to the active site is partially obstructed by the side chain of residue 264 in the variant. By analogy, a similar obstruction of the substrate channel can be anticipated to result from a phosphorylation of Ser264. The kinetic and thermodynamic results in conjunction with the structure of Ser264Glu suggest that phosphorylation blocks access to the active site by imposing a steric and electrostatic barrier for substrate binding and active site coupling with the E2 component. As a Ser264Gln variant, which carries no charge at position 264, is also selectively deficient in pyruvate binding and reductive acetylation of E2, we conclude that mostly steric effects account for inhibition of PDHc by phosphorylation.

Direct kinetic evidence for half-of-the-sites reactivity in the E1 component of the human pyruvate dehydrogenase multienzyme complex through alternating sites cofactor activation.

Recent kinetic and structural studies on various thiamin-dependent enzymes, including the bacterial E1 component of the pyruvate dehydrogenase complex (PDHc), suggested an active center communication between the cofactors in these multimeric enzymes. This regulatory mode has been inferred from the dissymmetry of active sites in proteolytic patterns and X-ray structures and from a complex macroscopic kinetic behavior not being consistent with independently working active sites. Here, direct microscopic kinetic evidence for this hypothesis is presented for the alpha2beta2-type E1 component of the human pyruvate dehydrogenase complex. Only one of the two thiamin molecules bound to the two active sites is in a chemically activated state exhibiting an apparent C2 ionization rate constant of approximately 50 s(-1) at pH 7.6 and 30 degrees C, whereas the thiamin in the "inactive site" ionizes with a rate that is at least 3 orders of magnitude smaller. The chemical nonequivalence is also exhibited in the ability to bind the substrate analogue methyl acetylphosphonate and in the catalytic turnover of the substrate pyruvate in the E1-only reaction. In the activated active site, pyruvate is rapidly bound and decarboxylated with apparent forward rate constants of covalent pyruvate binding of 2 s(-1) and decarboxylation of the formed 2-lactyl-thiamin intermediate of 5 s(-1). In the dormant site, these steps are as slow as 0.03 s(-1). Under the conditions that were used, only the heterotetramer can be detected by analytical ultracentrifugation, thus ruling out the possibility that multiple oligomeric species with different reactivities cause the observed kinetic effects. The results are consistent with the recently suggested model of an active site synchronization in PDHc-E1 via a proton wire that keeps the two active sites in an alternating activation state [Frank, R. A., et al. (2004) Science 306, 872]. Kinetic studies on the related thiamin enzymes transketolase, pyruvate oxidase, and bacterial pyruvate decarboxylase are not consistent with a chemical and/or functional nonequivalence of the active sites as observed in the E1 component of hsPDHc. We hypothesize that the alternating sites reaction in PDHc-E1 aids in the synchronized acyl transfer to the E2 component in the highly organized multienzyme complex.