Verwendung von Licht zur Aufklärung des CRAC Kanals

Anna BlaimscheinAuch als Printexemplar in der Bibliothek verfügbarMasterarbeit Wien, FH Campus Wien 202

Successful termination of a multi-year wastewater-associated outbreak of NDM-5-carrying E. coli in a hemato-oncological center

Abstract Background In May 2018, an outbreak of NDM-5-carrying Escherichia coli (NDM-5-EC) was detected at the hemato-oncology department of a tertiary care center in Austria. This report details the outbreak investigation, control measures and the whole genome sequencing (WGS) data of the outbreak isolates. Methods A total of 15 isolates (seven clinical isolates from allogenic stem cell transplant (SCT) recipients and eight wastewater isolates recovered from patients’ toilets) were analyzed by whole genome sequencing. Results Genome based typing identified two clusters of the high risk clones ST167/CT12607 and ST617/CT2791. Long-read sequencing of selected isolates from both clusters identified two different plasmids, however with a highly similar genetic context of the bla NDM-5 containing region. Genomic analysis revealed the presence of additional resistance genes, including bla CTX-M-15, and bla OXA-1, and virulence factors. Four patients were colonized with NDM-5-EC, two patients suffered bacteremia caused by the outbreak strain and two deaths were associated with an NDM-5-EC infection. The outbreak source was traced to toilet sewage pipes, which remained persistently contaminated despite extensive cleaning and disinfection. Successful eradication of NDM-5-EC from the installations required disassembly, hot water pressure washing of the sewage pipes and complete replacement of all movable parts. Additionally, colonized patients were instructed to use wheeled commodes instead of toilets, and a pre-admission screening strategy was implemented for all patients undergoing hematologic stem cell transplantation. The outbreak was successfully terminated in November 2020. Conclusion NDM-5-EC, especially high-risk clones such as ST167 and ST617, can persist in hospital wastewater systems despite cleaning and disinfection efforts and can cause prolonged outbreaks. Therefore, a comprehensive bundle of interventions like the ones applied in our study is essential, especially in clinical settings with heavily immunosuppressed patients

New Aspects of the Virus Life Cycle and Clinical Utility of Next Generation Sequencing based HIV-1 Resistance Testing in the Genomic, the Proviral, and the Viral Reservoir of Peripheral Blood Mononuclear Cells

Background: Typically, genotypic resistance testing is recommended at the start of antiretroviral therapy and is even mandatory in cases of virologic failure. The material of choice is plasma viral RNA. However, in patients with low viremia (viral load &lt; 500 copies/ml), resistance testing by population-based sequencing is very difficult. Objective: Therefore, we aimed to investigate whether next generation sequencing (NGS) from proviral DNA and RNA could be an alternative. Material and Methods: EDTA blood samples (n = 36) from routine clinical viral load testing were used for the study. Viral loads ranged from 96 to 390,000 copies/mL, with 100% of samples having low viremia. Distribution of subtypes; A (n = 2), B (n = 16), C (n = 4), D (n = 2), G (1), CRF02 AG (n = 5), CRF01 AE (n = 5), undefined/mixed (n = 4). The extracted consensus sequences were uploaded to the Stanford HIV Drug Resistance Data Base and Geno2pheno for online analysis of drug resistance mutations and resistance factors. Results: A total of 2476 variants or drug resistance mutations (DRMs) were detected with Sanger sequencing, compared with 2892 variants with NGS. An average of 822/1008 variants were identified in plasma viral RNA by Sanger or NGS sequencing, 834/956 in cellular viral RNA, and 820/928 in cellular viral DNA. Conclusions: Both methods are well suited for the detection of HIV substitutions or drug resistance mutations. Our results suggest that cellular RNA or cellular viral DNA is an informative alternative to plasma viral RNA for variant detection in patients with low viremia, as shown by the high correlation of variants in the different viral pools. We show that by using UDS, a plus of two DRMs per patient becomes visible, which can make a big difference in the assessment of the expected resistance behavior of the virus. </jats:sec

Photocrosslinking-induced CRAC channel-like Orai1 activation independent of STIM1

AbstractCa2+ release-activated Ca2+ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca2+-sensor STIM1 in the ER membrane and the Ca2+ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca2+ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca2+ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1.</jats:p