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Background and Objective: Staphylococcus aureus is one of the major pathogens of nosocomial infections as wells as community-acquired (CA) infections worldwide. So far, large-scale comprehensive molecular and epidemiological characterisation of S. aureus from very diverse settings has not been carried out in India. The objective of this study is to evaluate the molecular, epidemiological and virulence characteristics of S. aureus in both community and hospital settings in Chennai, southern India. Methods: S. aureus isolates were obtained from four different groups (a) healthy individuals from closed community settings, (b) inpatients from hospitals, (c) outpatients from hospitals, representing isolates of hospital-community interface and (d) HIV-infected patients to define isolates associated with the immunocompromised. Antibiotic susceptibility testing, multiplex polymerase chain reactions for detection of virulence and resistance determinants, molecular typing including Staphylococcal cassette chromosome mec (SCCmec) and agr typing, were carried out. Sequencing-based typing was done using spa and multilocus sequence typing (MLST) methods. Clonal complexes (CC) of hospital and CA methicillin-resistant S. aureus (MRSA) were identified and compared for virulence and resistance.
Results and Conclusion: A total of 769 isolates of S. aureus isolates were studied. The prevalence of MRSA was found to be 7.17%, 81.67%, 58.33% and 22.85% for groups a, b, c and d, respectively. Of the four SCCmec types (I, III, IV and V) detected, SCCmec V was found to be predominant. Panton-Valentine leucocidin toxin genes were detected among MRSA isolates harbouring SCCmec IV and V. A total of 78 spa types were detected, t657 being the most prevalent. 13 MLST types belonging to 9 CC were detected. CC1 (ST-772, ST-1) and CC8 (ST238, ST368 and ST1208) were found to be predominant among MRSA. CA-MRSA isolates with SCCmec IV and V were isolated from all study groups including hospitalised patients and were found to be similar by molecular tools. This shows that CA MRSA has probably infiltrated into the hospital settings.
Die alveoläre Echinokokkose ist eine lebensbedrohliche Erkrankung, die durch tumorartig in der Leber wachsende Larven (Metazestoden) des Fuchsbandwurms ausgelöst wird. Während Th1-dominierte Immunantworten zur Expulsion des Parasiten führen können, sind Th2-Antworten mit chronischer Infektion assoziiert. Über seine exkretorisch-sekretorischen Produkte (ESPs) nimmt Echinococcus multilocularis Einfluss auf die Polarisierung der Immunantwort. Allerdings ist bislang nur wenig über die zugrundeliegenden Mechanismen und aktiven Komponenten der ESPs bekannt.
Die Immunmodulation durch Eier des Pärchenegels Schistosoma mansoni, der wie E. multilocularis zu den Plattwürmern gehört, ist dagegen schon besser charakterisiert. Hier hat omega-1, eine Ribonuklease der T2-Familie, Aufmerksamkeit als starker Induktor von Th2-Antworten und als Hepatotoxin erregt.
Die Fragestellung dieser Arbeit war nun, ob die T2-RNase des Fuchsbandwurms (EmRNASET2) hinsichtlich ihrer Wirkungen auf Zellen des Immunsystems und der Leber Ähnlichkeiten mit omega-1 besitzt. Es konnte gezeigt werden, dass EmRNASET2 von allen Larvenstadien und auch vom adulten Wurm exprimiert wird. Der Einsatz polyklonaler Antikörper gegen rekombinant in Escherichia coli exprimierte recEmRNASET2 ermöglichte den Nachweis des Proteins in den ESPs von Primärzellen, die das frühe Stadium sich entwickelnder Metazestoden darstellen, und, wenngleich geringer ausgeprägt, in ESPs reifer Metazestoden.
Zur Untersuchung einer möglichen immunmodulatorischen Wirkung wurden dendritische Zellen (DCs) aus murinem Knochenmark generiert und mit Überständen recEmRNASET2-produzierender HEK-Zellen exponiert. Diese zeigten im Vergleich zu Überständen von mit leerem Transfektionsvektor behandelten HEK-Zellen keine signifikante Inhibition der LPS-induzierten Reifung und Interleukin-12-Produktion von DCs, wie sie für omega-1 beschrieben ist. Auch ein Pilotexperiment mit der Leberzelllinie Hep3B lieferte keinen Anhalt für eine hepatotoxische Wirkung von EmRNASET2. Somit sprechen die Ergebnisse dieser Arbeit gegen eine funktionelle Verwandtschaft von EmRNASET2 und omega-1. Unterstützt wird diese Beobachtung durch eine orientierende phylogenetische Untersuchung, in der sich EmRNASET2 näher verwandt zu einer zweiten T2-RNase von S. mansoni zeigte. Omega-1 könnte also das Resultat einer Genduplikation mit anschließender Akquirierung immunmodulatorischer Funktionen sein.
Although usually asymptomatically colonizing the human nasopharynx, the Gram-negative bacterium Neisseria meningitidis (meningococcus) can spread to the blood stream and cause invasive disease. For survival in blood, N. meningitidis evades the complement system by expression of a polysaccharide capsule and surface proteins sequestering the complement regulator factor H (fH). Meningococcal strains belonging to the sequence type (ST-) 41/44 clonal complex (cc41/44) cause a major proportion of serogroup B meningococcal disease worldwide, but they are also common in asymptomatic carriers. Proteome analysis comparing cc41/44 isolates from invasive disease versus carriage revealed differential expression levels of the outer membrane protein NspA, which binds fH. Deletion of nspA reduced serum resistance and NspA expression correlated with fH sequestration. Expression levels of NspA depended on the length of a homopolymeric tract in the nspA promoter: A 5-adenosine tract dictated low NspA expression, whereas a 6-adenosine motif guided high NspA expression. Screening German cc41/44 strain collections revealed the 6-adenosine motif in 39% of disease isolates, but only in 3.4% of carriage isolates. Thus, high NspA expression is associated with disease, but not strictly required. The 6-adenosine nspA promoter is most common to the cc41/44, but is also found in other hypervirulent clonal complexes.
Objective
The aim of this study was to determine the prevalence of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, group A Streptococcus (GAS), and Staphylococcus aureus in asymptomatic elderly people and to unravel risk factors leading to colonization.
Methods
A multi-centre cross-sectional study was conducted including 677 asymptomatic adults aged 65 years or more, living at home or in nursing homes. Study areas were Greater Aachen (North-Rhine-Westphalia) and Wuerzburg (Bavaria), both regions with medium to high population density. Nasal and oropharyngeal swabs as well as questionnaires were collected from October 2012 to May 2013. Statistical analysis included multiple logistic regression models.
Results
The carriage rate was 1.9% ([95%CI: 1.0–3.3%]; 13/677) for H. influenzae, 0.3% ([95%CI: 0–1.1%]; 2/677) for N. meningitidis and 0% ([95% CI: 0–0.5%]; 0/677) for S. pneumoniae and GAS. Staphylococcus aureus was harboured by 28.5% of the individuals ([95% CI: 25.1–32.1%]; 193/677) and 0.7% ([95% CI: 0.2–1.7%]; 5/677) were positive for methicillin-resistant S. aureus. Among elderly community-dwellers colonization with S. aureus was significantly associated with higher educational level (adjusted OR: 1.905 [95% CI: 1.248–2.908]; p = 0.003). Among nursing home residents colonization was associated with being married (adjusted OR: 3.367 [1.502–7.546]; p = 0.003).
Conclusion
The prevalence of N. meningitidis, H. influenzae, S. pneumoniae and GAS was low among older people in Germany. The S. aureus rate was expectedly high, while MRSA was found in less than 1% of the individuals.
Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs.
Neisseria meningitidis ist Auslöser invasiver Infektionen, die Sepsis und Meningitis hervorrufen. Bakterielle ADP-Ribosyltransferasen wurden als Toxine zahlreicher Bakterien wie E.coli, V. cholerae und B. pertussis beschrieben, die postranslationale Modifikationen bei eukaryotischen Proteinen mit pathologischer Wirkung für den Menschen hervorrufen. Die ADP-Ribosyltransferase NarE von Neisserien ist auf der Basis von Sequenzhomologien identifiziert worden. Die enzymatische Aktivität des Proteins wurde bereits in Studien gezeigt. Ziel dieser Arbeit war, NarE aus epidemiologischem und populationsbiologischem Blickwinkel zu betrachten.
Insgesamt wurden 576 Meningokokkenisolate (109 Isolate aus der Stammsammlung des Instituts für Hygiene und Mikrobiologie Würzburg und 467 Isolate der Meningococcus Genome Library der Meningitis Research Foundation) auf das Vorhandensein von narE sowie auf Sequenzvariationen untersucht. Das Ergebnis zeigte den Besitz des Gens bei insgesamt 247 Stämmen. Bis auf zwei Punktmutationen waren alle untersuchten narE-Sequenzen identisch. Die narE-positiven Isolate konnten neun klonalen Komplexen zugeordnet werden.
Zusätzlich wurde veranschaulicht, dass das Gen in Komplexen vorkommt, die verwandtschaftlich nicht eng miteinander verbunden sind.
Mittels Western Blot konnte bei allen narE-positiven Meningokokken die Proteinexpression bestätigt werden, wobei ein signifikanter Unterschied zwischen Stämmen des cc32 und cc41/44 festzustellen war. Auf Transkriptionsebene konnte mittels qRT-PCR kein Unterschied zwischen diesen Komplexen ermittelt werden, so dass der Expressionsunterschied auf einem posttranskriptionellen Mechanismus beruhen muss.
Neisseria gonorrhoeae ist ebenfalls im Besitz des Gens wie von Masignani et al. (2003) am Beispiel weniger Isolate beschrieben. In dieser Arbeit konnte für alle 29 getesteten Gonokokken die Insertion von vier Basenpaaren bestätigt werden, die zu einer Verschiebung im Leseraster führt, so dass NarE nicht exprimiert wird. Auch ein Neisseria sicca Stamm beinhaltet und exprimiert das narE-Gen.
Background
Tapeworms lack a canonical piRNA-pathway, raising the question of how they can silence existing mobile genetic elements (MGE). Investigation towards the underlying mechanisms requires information on tapeworm transposons which is, however, presently scarce.
Methods
The presence of densovirus-related sequences in tapeworm genomes was studied by bioinformatic approaches. Available RNA-Seq datasets were mapped against the Echinococcus multilocularis genome to calculate expression levels of densovirus-related genes. Transcription of densovirus loci was further analyzed by sequencing and RT-qPCR.
Results
We herein provide evidence for the presence of densovirus-related elements in a variety of tapeworm genomes. In the high-quality genome of E. multilocularis we identified more than 20 individual densovirus integration loci which contain the information for non-structural and structural virus proteins. The majority of densovirus loci are present as head-to-tail concatemers in isolated repeat containing regions of the genome. In some cases, unique densovirus loci have integrated close to histone gene clusters. We show that some of the densovirus loci of E. multilocularis are actively transcribed, whereas the majority are transcriptionally silent. RT-qPCR data further indicate that densovirus expression mainly occurs in the E. multilocularis stem cell population, which probably forms the germline of this organism. Sequences similar to the non-structural densovirus genes present in E. multilocularis were also identified in the genomes of E. canadensis, E. granulosus, Hydatigera taeniaeformis, Hymenolepis diminuta, Hymenolepis microstoma, Hymenolepis nana, Taenia asiatica, Taenia multiceps, Taenia saginata and Taenia solium.
Conclusions
Our data indicate that densovirus integration has occurred in many tapeworm species. This is the first report on widespread integration of DNA viruses into cestode genomes. Since only few densovirus integration sites were transcriptionally active in E. multilocularis, our data are relevant for future studies into gene silencing mechanisms in tapeworms. Furthermore, they indicate that densovirus-based vectors might be suitable tools for genetic manipulation of cestodes.
Bacterial meningitis is a serious life threatening infection of the CNS. To cause meningitis, blood–borne bacteria need to interact with and penetrate brain endothelial cells (BECs) that comprise the blood–brain barrier. BECs help maintain brain homeostasis and they possess an array of efflux transporters, such as P-glycoprotein (P-gp), that function to efflux potentially harmful compounds from the CNS back into the circulation. Oftentimes, efflux also serves to limit the brain uptake of therapeutic drugs, representing a major hurdle for CNS drug delivery. During meningitis, BEC barrier integrity is compromised; however, little is known about efflux transport perturbations during infection. Thus, understanding the impact of bacterial infection on P-gp function would be important for potential routes of therapeutic intervention. To this end, the meningeal bacterial pathogen, Streptococcus agalactiae, was found to inhibit P-gp activity in human induced pluripotent stem cell-derived BECs, and live bacteria were required for the observed inhibition. This observation was correlated to decreased P-gp expression both in vitro and during infection in vivo using a mouse model of bacterial meningitis. Given the impact of bacterial interactions on P-gp function, it will be important to incorporate these findings into analyses of drug delivery paradigms for bacterial infections of the CNS.
The central nervous system (CNS) barriers are highly specialized cellular barriers that promote brain homeostasis while restricting pathogen and toxin entry. The primary cellular constituent regulating pathogen entry in most of these brain barriers is the brain endothelial cell (BEC) that exhibits properties that allow for tight regulation of CNS entry. Bacterial meningoencephalitis is a serious infection of the CNS and occurs when bacteria can cross specialized brain barriers and cause inflammation. Models have been developed to understand the bacterial – BEC interaction that lead to pathogen crossing into the CNS, however, these have been met with challenges due to these highly specialized BEC phenotypes. This perspective provides a brief overview and outlook of the in vivo and in vitro models currently being used to study bacterial brain penetration, and opinion on improved models for the future.
Here, we present the unique case of a 51‐year‐old German patient with multiple myeloma excreting Ascaris lumbricoides in his stool five weeks after allogeneic hematopoietic stem cell transplantation. Stool analysis remained negative for the presence of eggs, and there was no eosinophilia in the peripheral blood at any time around stem cell transplantation. The patient was commenced on a three‐day treatment with mebendazole, which was well tolerated. No serious interactions with the concomitant post‐transplant medication or negative effects on the hematopoiesis were observed, and the myeloma still is in complete remission. To our knowledge, this is the first report on excretion of A lumbricoides in the context of allogeneic stem cell transplantation. The case is remarkable with view to the fact that the parasite has supposedly survived all courses of myeloma treatment including autologous and allogeneic conditioning. Parasitosis with A lumbricoides has a worldwide prevalence of about a billion and is extremely rare in northern Europe. Possibly the patient got infected during a trip to Egypt years before multiple myeloma was diagnosed.