Molekulare Epidemiologie von Vancomycin-resistenten Enterokokken am Universitätsklinikum Regensburg 2018 - 2019

Vancomycin-resistente Enterokokken gehören gemäß Infektionsschutzgesetz zu den in Deutschland zu erfassenden multiresistenten Erregern. In den letzten Jahrzehnten sind trotz präventiver Maßnahmen steigende VRE-Zahlen weltweit und insbesondere auch in Deutschland zu beobachten, sodass von einer hyperendemischen Situation gesprochen werden kann. Um dieser Entwicklung entgegen zu wirken und adäquate Schutzkonzepte für Patienten in Kliniken zu entwickeln ist ein besseres Verständnis der Transmissions¬wege notwendig. Ziel dieser Arbeit war es potenzielle Transmissionsereignisse und Risikobereiche am Universitätsklinikum Regensburg (UKR) aufzuzeigen sowie Einblicke in die Populations-struktur der im hospitalen Umfeld zirkulierenden Enterokokkenstämme auf Genomebene zu erhalten. Hierfür wurden in die Studie insgesamt 331 VRE-Patienten des UKR eingeschlossen und deren stationären Aufenthalte anhand einer Linelist über den Zeitraum eines Jahres hinweg dokumentiert und ausgewertet. Zudem erfolgte eine Erfassung der den Patien¬ten zugehörigen VRE-Isolate. Hierdurch ließen sich mögliche Transmissionen und insbesondere gehäuft auftretende Erstnachweise – als Hinweise für mögliche Ausbrüche – detektieren. Es konnte so eine Risikobeurteilung einzelner Stationen und Fachab¬teilungen erfolgen. Ergänzend hierzu erfolgte für ausgewählte Isolate aus Hochrisiko¬bereichen oder im Rahmen vermuteter Ausbrüche eine Gesamtgenomsequenzierung. Für die Genotypisierung wurde das Whole Genome Sequencing angewandt, mit Untersuchung von insgesamt 1423 Zielgenen und Klassifikation anhand ihres Komplextyps (CT). Die Ergebnisse dieser Studie zeigten, dass insbesondere gastroenterologische, nephro-logische und hämatoonkologische Normalstationen sowie Intensivstationen am UKR eine hohe VRE-Last aufwiesen, sich hinsichtlich der Häufigkeit anzunehmender Trans-missionsereignisse jedoch teilweise unterschieden. So waren insbesondere für die Intensivstationen vermehrt mögliche nosokomiale Nachweise zu registrieren. Zu bemer¬ken war hier zum einen ein fehlendes systematisches Screening und zum anderen ein hoher Patientenumsatz kritisch kranker Patienten. Die Gesamtgenomsequenzierung konnte zeigen, dass es sich bei den am UKR vorkom-menden Enterokokkenstämmen um ein polyklonales Geschehen handelte, mit vielen genetisch unterschiedlichen Enterokokkenstämmen. Einzelne Stämme waren im Rahmen der analysierten Ausbrüche und in Hochrisikobereichen jedoch dominierend als Hinweis auf ein erhöhtes Ausbreitungspotential dieser Stämme. Hier ist insbesondere der Enterokokkenstamm ST80 / CT1065 zu nennen. In Kombination mit detaillierten tagegenauen Patientenbewegungen ließen sich einige direkte Transmissionsereignisse nachweisen. Dies unter anderem dank sorgfältiger Erfassung von Risikopatienten und erfolgter Screeningmaßnahmen auf den Stationen. Gleichzeitig konnte jedoch beobachtet werden, dass viele – anhand epidemiologischer Daten, der van-Klassifikation und der MLST vermutete Transmis¬sionen – sich nicht bestätigen ließen. Passend hierzu war zu beobachten, dass häufig auch bei initial negativ gescreenten Patienten ein VRE-Nachweis erfolgte, ohne dass Indexpatienten eruiert werden konnten, die Transmissionswege also ungeklärt blieben. Zu diskutieren ist hier eine mögliche Selektion einer vorbestehenden VRE-Kolonisation unter Antibiotikatherapie ebenso wie eine Transmission des vanB-Transposons von Anaerobiern auf VSE mit konsekutiver klonaler Ausbreitung eines neuen Stam¬mes. Somit ist fraglich, ob allein die Detektion und Isolierung bekannter VRE-Träger ein sinn¬volles Management zur Kontrolle der Ausbreitung darstellt. Nicht VRE per se sondern einzelne Enterokokkenstämme wie der ST80 / CT1085 scheinen ein erhöhtes Ausbrei¬tungspotential zu haben. Daher sollten auch krankenhausadaptierte VSE in hygienere¬levante Überlegungen eingeschlossen werden. Somit könnte neben einer wirkungsvol¬len Reduktion von Transmissionen auch eine unnötige Kontaktisolation bei Nachweis von VRE mit geringem Ausbreitungspotential erzielt werden. Um bestimmte Risikokons¬tellationen zu erkennen und adäquat reagieren zu können, ist ein tief gehendes Verständnis über die Phylogenetik und Populationsstruktur der Enterokokken – sowohl VSE als auch VRE – notwendig. Dies ist nur durch konsequente Nachverfolgung und Gesamtgenomsequenzierung möglich. Ein neuartiges Konzept, das als Genom-orientierte Infektionsprävention bezeichnet wird. Bereits beste¬hende Datenbanken können genutzt werden, um sowohl national als auch international gewon¬nene Informationen auszutauschen und zusammenzuführen. Hiervon sollten nicht zuletzt die Patienten profitieren, um durch kontrollierende und präventive Maßnahmen Ausbrüche zu reduzieren und unnötige Isolationen, mit einhergehenden individuellen negativen medizinischen und psychosozialen Folgen, zu verhindern

Bacteria-on-a-bead: probing the hydrodynamic interplay of dynamic cell appendages during cell separation

Surface attachment of bacteria is the first step of biofilm formation and is often mediated and coordinated by the extracellular appendages, flagellum and pili. The model organism Caulobacter crescentus undergoes an asymmetric division cycle, giving rise to a motile "swarmer cell" and a sessile "stalked cell", which is attached to the surface. In the highly polarized predivisional cell, pili and flagellum, which are assembled at the pole opposite the stalk, are both activated before and during the process of cell separation. We explored the interplay of flagellum and active pili by growing predivisional cells on colloidal beads, creating a bacteria-on-a-bead system. Using this set-up, we were able to simultaneously visualize the bacterial motility and analyze the dynamics of the flagellum and pili during cell separation. The observed activities of flagellum and pili at the new cell pole of the predivisional cell result in a cooperating interplay of the appendages during approaching and attaching to a surface. Even in presence of a functioning flagellum, pili are capable of surface attachment and keeping the cell in position. Moreover, while flagellar rotation decreases the average attachment time of a single pilus, it increases the overall attachment rate of pili in a synergetic manner

Second messenger-mediated tactile response by a bacterial rotary motor

When bacteria encounter surfaces, they respond with surface colonization and virulence induction. The mechanisms of bacterial mechanosensation and downstream signaling remain poorly understood. Here, we describe a tactile sensing cascade in Caulobacter crescentus in which the flagellar motor acts as sensor. Surface-induced motor interference stimulated the production of the second messenger cyclic diguanylate by the motor-associated diguanylate cyclase DgcB. This led to the allosteric activation of the glycosyltransferase HfsJ to promote rapid synthesis of a polysaccharide adhesin and surface anchoring. Although the membrane-embedded motor unit was essential for surface sensing, mutants that lack external flagellar structures were hypersensitive to mechanical stimuli. Thus, the bacterial flagellar motor acts as a tetherless sensor reminiscent of mechanosensitive channels

In situ structure of the Caulobacter crescentus flagellar motor and visualization of binding of a CheY-homolog

Bacterial flagellar motility is controlled by the binding of CheY proteins to the cytoplasmic switch complex of the flagellar motor, resulting in changes in swimming speed or direction. Despite its importance for motor function, structural information about the interaction between effector proteins and the motor are scarce. To address this gap in knowledge, we used electron cryotomography and subtomogram averaging to visualize such interactions inside Caulobacter crescentus cells. In C. crescentus, several CheY homologs regulate motor function for different aspects of the bacterial lifestyle. We used subtomogram averaging to image binding of the CheY family protein CleD to the cytoplasmic Cring switch complex, the control center of the flagellar motor. This unambiguously confirmed the orientation of the motor switch protein FliM and the binding of a member of the CheY protein family to the outside rim of the C ring. We also uncovered previously unknown structural elaborations of the alphaproteobacterial flagellar motor, including two novel periplasmic ring structures, and the stator ring harboring eleven stator units, adding to our growing catalog of bacterial flagellar diversity

Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly

Polynucleotide phosphorylase (PNPase) is an exoribonuclease that cleaves single-stranded RNA substrates with 3′–5′ directionality and processive behaviour. Its ring-like, trimeric architecture creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, but exactly how these domains help to direct the 3′ end of single-stranded RNA substrates towards the active sites is an unsolved puzzle. Insight into this process is provided by our crystal structures of RNA-bound and apo Caulobacter crescentus PNPase. In the RNA-free form, the S1 domains adopt a ‘splayed’ conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3′ end towards a constricted aperture at the entrance of the central channel. The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme. On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria

Helicobacter pylori Lipopolysaccharide Is Synthesized via a Novel Pathway with an Evolutionary Connection to Protein N-Glycosylation

Lipopolysaccharide (LPS) is a major component on the surface of Gram negative bacteria and is composed of lipid A-core and the O antigen polysaccharide. O polysaccharides of the gastric pathogen Helicobacter pylori contain Lewis antigens, mimicking glycan structures produced by human cells. The interaction of Lewis antigens with human dendritic cells induces a modulation of the immune response, contributing to the H. pylori virulence. The amount and position of Lewis antigens in the LPS varies among H. pylori isolates, indicating an adaptation to the host. In contrast to most bacteria, the genes for H. pylori O antigen biosynthesis are spread throughout the chromosome, which likely contributed to the fact that the LPS assembly pathway remained uncharacterized. In this study, two enzymes typically involved in LPS biosynthesis were found encoded in the H. pylori genome; the initiating glycosyltransferase WecA, and the O antigen ligase WaaL. Fluorescence microscopy and analysis of LPS from H. pylori mutants revealed that WecA and WaaL are involved in LPS production. Activity of WecA was additionally demonstrated with complementation experiments in Escherichia coli. WaaL ligase activity was shown in vitro. Analysis of the H. pylori genome failed to detect a flippase typically involved in O antigen synthesis. Instead, we identified a homolog of a flippase involved in protein N-glycosylation in other bacteria, although this pathway is not present in H. pylori. This flippase named Wzk was essential for O antigen display in H. pylori and was able to transport various glycans in E. coli. Whereas the O antigen mutants showed normal swimming motility and injection of the toxin CagA into host cells, the uptake of DNA seemed to be affected. We conclude that H. pylori uses a novel LPS biosynthetic pathway, evolutionarily connected to bacterial protein N-glycosylation

Tad pili play a dynamic role in Caulobacter crescentus surface colonization

Bacterial surface attachment is mediated by rotary flagella and filamentous appendages called pili. Here, we describe the role of Tad pili during surface colonization of Caulobacter crescentus. Using an optical trap and microfluidic controlled flow conditions as a mimic of natural environments, we demonstrate that Tad pili undergo repeated cycles of extension and retraction. Within seconds after establishing surface contact, pili reorient cells into an upright position promoting walking-like movements against the medium flow. Pili-mediated positioning of the flagellated pole close to the surface facilitates motor-mediated mechanical sensing and promotes anchoring of the holdfast, an adhesive substance that affords long-term attachment. We present evidence that the second messenger c-di-GMP regulates pili dynamics during surface encounter in distinct ways, promoting increased activity at intermediate levels and retraction of pili at peak concentrations. We propose a model, in which flagellum and Tad pili functionally interact and together impose a ratchet-like mechanism that progressively drives C. crescentus cells towards permanent surface attachment

Performance of laser fluorescence devices, visual and radiographic examination for the detection of occlusal caries in primary molars

The aim of this in vitro study was to compare the performance of two laser fluorescence devices (LF, LFpen), conventional visual criteria (VE), ICDAS and radiographic examination on occlusal surfaces of primary teeth. Thirty-seven primary human molars were selected from a pool of extracted teeth, which were stored frozen at -20°C until use. Teeth were assessed twice by two experienced examiners using laser fluorescence devices (LF and LFpen), conventional visual criteria, ICDAS and bitewing radiographs, with a 2-week interval between measurements. After measurement, the teeth were histologically prepared and assessed for caries extension. The highest sensitivity was observed for ICDAS at D(1) and D(3) thresholds, with no statistically significant difference when compared to the LF devices, except at the D(3) threshold. Bitewing radiographs presented the lowest values of sensitivity. Specificity at D(1) was higher for LFpen (0.90) and for VE at D(3) (0.94). When VE was combined with LFpen the post-test probabilities were the highest (94.0% and 89.2% at D(1) and D(3) thresholds, respectively). High values were observed for the combination of ICDAS and LFpen (92.0% and 80.0%, respectively). LF and LFpen showed the highest values of ICC for interexaminer reproducibility. However, regarding ICDAS, BW and VE, intraexaminer reproducibility was not the same for the two examiners. After primary visual inspection using ICDAS or not, the use of LFpen may aid in the detection of occlusal caries in primary teeth. Bitewing radiographs may be indicated only for approximal caries detection

Cohesive Properties of the Caulobacter crescentus Holdfast Adhesin Are Regulated by a Novel c-di-GMP Effector Protein

When encountering surfaces, many bacteria produce adhesins to facilitate their initial attachment and to irreversibly glue themselves to the solid substrate. A central molecule regulating the processes of this motile-sessile transition is the second messenger c-di-GMP, which stimulates the production of a variety of exopolysaccharide adhesins in different bacterial model organisms. In Caulobacter crescentus, c-di-GMP regulates the synthesis of the polar holdfast adhesin during the cell cycle, yet the molecular and cellular details of this control are currently unknown. Here we identify HfsK, a member of a versatile N-acetyltransferase family, as a novel c-di-GMP effector involved in holdfast biogenesis. Cells lacking HfsK form highly malleable holdfast structures with reduced adhesive strength that cannot support surface colonization. We present indirect evidence that HfsK modifies the polysaccharide component of holdfast to buttress its cohesive properties. HfsK is a soluble protein but associates with the cell membrane during most of the cell cycle. Coincident with peak c-di-GMP levels during the C. crescentus cell cycle, HfsK relocalizes to the cytosol in a c-di-GMP-dependent manner. Our results indicate that this c-di-GMP-mediated dynamic positioning controls HfsK activity, leading to its inactivation at high c-di-GMP levels. A short C-terminal extension is essential for the membrane association, c-di-GMP binding, and activity of HfsK. We propose a model in which c-di-GMP binding leads to the dispersal and inactivation of HfsK as part of holdfast biogenesis progression.IMPORTANCE Exopolysaccharide (EPS) adhesins are important determinants of bacterial surface colonization and biofilm formation. Biofilms are a major cause of chronic infections and are responsible for biofouling on water-exposed surfaces. To tackle these problems, it is essential to dissect the processes leading to surface colonization at the molecular and cellular levels. Here we describe a novel c-di-GMP effector, HfsK, that contributes to the cohesive properties and stability of the holdfast adhesin in C. crescentus We demonstrate for the first time that c-di-GMP, in addition to its role in the regulation of the rate of EPS production, also modulates the physicochemical properties of bacterial adhesins. By demonstrating how c-di-GMP coordinates the activity and subcellular localization of HfsK, we provide a novel understanding of the cellular processes involved in adhesin biogenesis control. Homologs of HfsK are found in representatives of different bacterial phyla, suggesting that they play important roles in various EPS synthesis systems