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W. Schneider-Brachert [1] R. Birngruber [1] H.J. Linde [1] S. Miehlke [2] E. Bayerdorffer [2] N. Lehn [1] [15/53] Susceptibility of Clinical Ciprofloxacin-resistant Isolates of Helicobacter pylori to new Fluoroquinolones [...]

[4/28] Identification and Characterisation of a 24 kDa Helicobacter pylori Extracellular Protein that Induces High Levels of IFN-γ in Mouse Splenocytes The immunopathogenesis of Helicobacter pylon (H.p.)-induced peptic ulceration is [...]

Acidic noncaspase proteases-like cathepsins have been introduced as novel mediators of apoptosis. A clear role for these proteases and the acidic endolysosomal compartment in apoptotic signalling is not yet defined. To understand the role and significance of noncaspases in promoting and mediating cell death, it is important to determine whether an intersection of these proteases and the caspase pathway exists. We recently identified the endolysosomal aspartate protease cathepsin D (CTSD) as a target for the proapoptotic lipid ceramide. Here, we show that tumor necrosis factor (TNF)-induced CTSD activation depends on functional acid sphingomyelinase (A-SMase) expression. Ectopic expression of CTSD in CTSD-deficient fibroblasts results in an enhanced TNF-mediated apoptotic response. Intracellular colocalization of CTSD with the proapoptotic bcl-2 protein family member Bid in HeLa cells, and the ability of CTSD to cleave directly Bid in vitro as well as the lack of Bid activation in cathepsin-deficient fibroblasts indicate that Bid represents a direct downstream target of CTSD. Costaining of CTSD and Bid with Rab5 suggests that the endosomal compartments are the common ‘meeting point’. Caspase-9 and -3 activation also was in part dependent on A-SMase and CTSD expression as revealed in the respective deficiency models. Our results link as novel endosomal intermediates the A-SMase and the acid aspartate protease CTSD to the mitochondrial apoptotic TNF pathway.Cell Death and Differentiation (2004) 11, 550-563. doi:10.1038/sj.cdd.4401382 Published online 23 January 2004 [ABSTRACT FROM AUTHOR]

Background & Aim: Esomeprazole-based triple therapy with clarithromycin and amoxicillin has recently been shown to be effective for H. pylori eradication. The aim of this study was to determine the efficacy of a 7-day triple therapy with esomeprazole, clarithromycin and metronidazole for eradication of H. pylori in the absence of antimicrobial resistance. Patients & Methods: Patients infected with H. pylori sensitive to metronidazole and clarithromycin (E-Test) were randomized to one-week triple therapy with either esomeprazole 2 × 20 mg, clarithromycin 2 × 250 mg and metronidazole 2 × 400 mg, (ECM), or omeprazole 2 × 20 mg, clarithromycin 2 × 250 mg and metronidazole 2 × 400 mg (OCM). Control endoscopy with histology and culture and/or rapid urease test was performed 4 to 8 weeks after the end of treatment. Results: 76 patients were randomized. So far, 63 patients returned to control examinations and were analyzed (23 females, 27 peptic ulcer disease, median age 43 years). All 63 patients completed their therapy as scheduled. There were no major side effects leading to discontinuation of treatment. Minor side effects occurred in 8 (23.5%) and 4 (13.8%) patients during ECM and OCM therapy, respectively. So far, 33 of 34 patients of the ECM group (97%) and 27 of 29 patients of the OCM group (93%) were cured of H. pylori infection. Post-treatment antimicrobial susceptibility testing showed resistance to both metronidazole and clarithroymcin in 2 of 3 patients. Conclusion: Our preliminary data suggest a high efficacy of esomeprazole-based triple therapy with clarithromycin and metronidazole for eradication of H. pylori in the absence of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Background & Aim: Failure of primary Helicobacter pylori therapy may lead to a high rate of post-treatment resistance to both metronidazole (MET) and clarithromycin (CLA). Due to the lack of acceptance of quadruple therapy in Germany the search for alternative salvage therapies is warranted. The aim of this study was to compare the efficacy and tolerability of a rifabutin-containing triple therapy with that of a high-dose dual therapy in eradicating H. pylori resistant to both MET and CLA. Patients & Methods: Patients with at least one previous treatment of H. pylori therapy and documented resistance to both MET & CLA (ETest) were randomized to receive either esomeprazole 2 × 20 mg, rifabutin 2 × 150 mg, and amoxicillin 2 × 1 g for 7 days (ERA), or omeprazole 3 × 40 mg and amoxicillin 3 × 1 g for 14 days (HD-OA). Control examinations were conducted 4 to 6 weeks after end of therapy. In case of persistent H. pylori infection, a cross-over therapy with the alternative regimen was performed. Results: So far, 76 patients were randomized, of those 66 have so far completed. 29 of 35 patients became H. pylori-negative after ERA therapy (82.9%), and 23 of 31 after HD-OA therapy (74.2%). Both regimens were well tolerated. Only one patient of the HD-OA group discontinued treatment prematurley due to side effects. 4 of 5 patients who failed HD-OA therapy received cross-over ERA therapy, all 4 became H. pylori-negative. 5 of 6 patients who failed ERA therapy received cross-over HD-OA therapy, 2 of them returned to the second control examination, one became H. pylori-negative. Conclusion: Our data suggest a comparable high efficacy of a 7-day triple therapy containing esomeprazole, rifabutin and amoxicillin and of the high-dose omeprazole/amoxicillin dual therapy in eradicating H. pylori resistant to both metronidazole and clarithromycin. [ABSTRACT FROM AUTHOR]

Helicobacter cystein-rich protein A (HcpA) was recently shown by us to be capable of inducing high levels of interferon-γ (IFN-γ) from naive murine splenocytes. IFN-γ is the signature cytokine of the Th1 subset of helper T cells which are commonly held responsible for inflammatory response in Helicobacter pylori infected patients. We could demonstrate that IFN-γ secretion induced by H. pylori or HcpA is dependent on IL-12 secretion by antigen-presenting cells (APCs). Inhibition of IL-12 by application of anti-IL-12 antibodies leads to a decrease in total levels of IFN-γ produced by murine splenocytes. This suggests a common pathway for inducing a Th1 directed immune response for H. priori and HcpA by activating APCs as the main source of IL-12. These results could be confirmed by depletion experiments where in absence of APCs dramatically reduced IFN-γ levels were obtained. The same was also true for splenocytes depleted of NK cells indicating that they are the main producer of IFN-γ in response to H. pylori or HcpA. In silico molecular analysis of HcpA showed a pattern of cysteins and predicted helices strongly resembling HcpB, which is known to have a modular structure of four motifs of two adjacent helices linked together by disulphide bonds. We studied the importance of these motifs in HcpA and found that the carboxyterrninal part is associated with IFN-γ-secretion. A predicted coiled-coil segment at the carboxyterminus could be further shown to be relevant for the ability of HcpA to induce high levels of IFN-γ. Our recent work confirmed the immunostimulatory effects of HcpA and extents our knowledge of its molecular mechanism of action by the identification of distinct regions of HcpA to be responsible for its IFN-γ-inducing capacity. [ABSTRACT FROM AUTHOR]

Abstract Background Tumor necrosis factor (TNF) is a pleiotropic cytokine involved in a broad spectrum of inflammatory and immune responses including proliferation, differentiation, and cell death. The biological effects of TNF are mediated via two cell surface TNF receptors: p55TNFR (TNFR1; CD120a) and p75TNFR (TNFR2; CD120b). Soluble forms of these two receptors consisting of the extracellular domains are proteolytically cleaved from the membrane and act as inhibitors. A novel p75TNFR isoform generated by the use of an additional transcriptional start site has been described and was termed hicp75TNFR. We focused on the characterization of this new isoform as this protein may be involved in chronic inflammatory processes. Methods Cell lines were retroviraly transduced with hp75TNFR isoforms. Subcellular localization and colocalization studies with TNF were performed using fluorescence microscopy including exhaustive photon reassignment software, flow cytometry, and receptosome isolation by magnetic means. Biochemical properties of the hicp75TNFR were determined by affinity chromatography, ELISA, and western blot techniques. Results We describe the localization and activation of a differentially spliced and mainly intracellularly expressed isoform of human p75TNFR, termed hicp75TNFR. Expression studies with hicp75TNFR cDNA in different cell types showed the resulting protein mostly retained in the trans-Golgi network and in endosomes and colocalizes with endogenous TNF. Surface expressed hicp75TNFR behaves like hp75TNFR demonstrating susceptibility for TACE-induced shedding and NFκB activation after TNF binding. Conclusion Our data demonstrate that intracellular hicp75TNFR is not accessible for exogenously provided TNF but colocalizes with endogenously produced TNF. These findings suggest a possible intracellular activation mechanism of hicp75TNFR by endogenous TNF. Subsequent NFκB activation might induce anti-apoptotic mechanisms to protect TNF-producing cells from cytotoxic effects of TNF. In addition, the intracellular and not TACE-accessible splice form of the hp75TNFR could serve as a pool of preformed, functional hp75TNFR.

Objective: The cag PAI is considered to be one of the major virulence determining factors of H. pylori. Although the cagA gene is conventionally used as a surrogate marker for the presence of the PAI, several studies have limited the use of cagA PCR as a suitable marker. In order to determine the presence of the whole PAI we developed a PCR-based system to analyse the PAl in different clinical H. pylori strains. Methods: Based on the published sequence of H. pylori 26695 a PCRbased amplification system was developed to cover the whole PAl with overlapping amplicons of 1000-1200 bp in size. 14 clinical Hp isolates that were all shown to induce IL-8 in AGS cells were chosen to evaluate this assay system. The obtained PCR-product were visualised by gel electrophoresis. Results: Using 31 primer pairs we could successfully amplify the whole PAI in 12 of 14 strains. In two strains there were no amplicons obtained at the N- and C-terminus of HP0547, but the rest of the PAI was completely amplified. Comparison of the PCR products revealed that the PAl was highly conserved because nearly all amplicons were of equal size. When PCR products were absent with a given primer it was possible to amplify the missed region with upstream and downstream primers. This was tree for HP0527 and HP0544 that could only be amplified by long-distance PCR because of multiple gene rearrangements or sequence variations, respectively. Conclusions: Although there were differences detectable with respect to sequence variations (primer mismatch) and amplicon size in some regions the genetic variability seems to be limited. Work is in progress to determine now the sequence of the whole PAI of all 14 H. pylori strains to compare the open reading frames in more detail. [ABSTRACT FROM AUTHOR]

Objective: Helicobacter pylori CagA protein has been shown to be translocated into gastric epithelial cells and after phosphorylation to be essential for the induction of the so-called "hummingbird" phenotype. Phosphorylafion has been demonstrated to occur at tyrosine residue 972 (Y-972). Several phosphorylation motifs have been identified so fan In order to correlate these motifs with the induction of the hummingbird phenotype in AGS cells we determined the cagA Y-972 motifs in different Helicobacter pylori isolates. Methods: The complete cagA gene was amplified by PCR and sequenced from 60 H. pylori strains obtained from different clinical entities (duodenal ulcer, gastric ulcer, gastric carcinoma, and MALToma) and the Y-972 motifs were determined. AGS cells were then infected with H. pylori strains representing each motif and analysed for the induction of the hummingbird phenotype. Results: The most frequent motif found was a single SVSPEPIY in 18 strains, a single sequence repeat in 9 isolates, and one strain with 5 repeats, followed by 16 strains with a single SASPEPIY, 6 with a single repeats, and one with 3 repeats. Two strains showed a single SANHEPIY, and one 4 repeats. The motifs SASSEPIY, and SASPEEPIY were only found in one strain, respectively. 4 strains had no Y-972 motif. From each motif one strain was randomly chosen to determine the hummingbird phenotype and only the strain Ly27a harbouring 4 repeats of the motif SANHEPIY was found positive. Conclusions: The mere PCR-based determination of Y-972 does not necessarily correlate with the sequence-predicted hummingbird phenotype and supports the view that additional bacterial factors may contribute to the full virulence phenotype. Work is in progress to test all isolates for the hummingbird phenotype and, in addition, for IL-8 induction and to correlate PAI function with genotype of theY-972 motif and CagA function. [ABSTRACT FROM AUTHOR]

Presents a correction to the omission of Paul Saftig and Christoph Peters as co-authors of a paper "Cathepsin D targeted by acid sphingomyelinase-derived ceramide" published in the previous issue of the "EMBO Journal".

Today, there is a continuous worldwide battle against antimicrobial resistance (AMR) and that includes vancomycin-resistant enterococci (VRE). Methods that can adequately and quickly detect transmission chains in outbreaks are needed to trace and manage this problem fast and cost-effectively. In this study, DNA-microarray-based technology was developed for this purpose. It commenced with the bioinformatic design of specific oligonucleotide sequences to obtain amplification primers and hybridization probes. Microarrays were manufactured using these synthesized oligonucleotides. A highly parallel and stringent labeling and hybridization protocol was developed and employed using isolated genomic DNA from previously sequenced (referenced) clinical VRE strains for optimal sensitivity and specificity. Microarray results showed the detection of virulence, resistance, and species-specific genes in the VRE strains. Theoretical predictions of the microarray results were also derived from the sequences of the same VRE strain and were compared to array results while optimizing protocols until the microarray result and theoretical predictions were a match. The study concludes that DNA microarray technology can be used to quickly, accurately, and economically detect specifically and massively parallel target genes in enterococci.

Introduction: Invasive pneumococcal disease is a major cause of morbidity and mortality in infectious diseases. Selective reporting of antibiotic susceptibility test results might lead to a tailored antibiotic therapy and could therefore be an important antibiotic stewardship program intervention. The aim of this study was to analyse whether a switch to selective reporting of antibiotic test results leads to a more focused antibiotic therapy in patients with a bloodstream infection with Streptococcus pneumoniae ., Methods: This study was performed as a retrospective cohort study at the University Hospital Regensburg, Germany. All blood cultures positive for Streptococcus pneumoniae between 2006 and 2021 were analysed. In 2014, a switch to selective reporting of antibiotic susceptibility test results omitting sensitivity results for agents not recommended was introduced., Results: Twenty-four hours after final antibiotic susceptibility test results were available, 20.9% before (BI) versus 15.4% after implementation (AI) of selective reporting of antibiotic test results received a narrow-spectrum penicillin, while only 2.3% BI versus 5.8% AI received a narrow-spectrum penicillin from the beginning., Conclusion: Selective reporting of antibiotic susceptibility test results without further antimicrobial stewardship interventions did not lead to a higher use of a narrow-spectrum penicillin in this study., Competing Interests: Arno Mohr received travel grants from Gilead. Bernd Salzberger received a consulting honorarium from Roche AG. All other authors declare they have no financial interests., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

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., (© 2024. The Author(s).)

Hypervirulent Klebsiella pneumoniae strains (hvKp) can cause invasive community-acquired infections in healthy patients of all ages. In this study, the prevalence of putative hvKp in a German tertiary center was investigated and hvKp were characterized by phenotypic and molecular assays. All K. pneumoniae isolates in routine microbiological diagnostics from a single center were screened by string-testing over a period of 6 months. String-test positive (≥ 0.5 mm) isolates were re-evaluated on different media and under various conditions (aerobe, anaerobe). For string-test positive isolates, genes (magA, iutA, rmpA and rmpA2) associated with hypermucoviscosity and hypervirulence were amplified by multiplex PCR. PCR-positive isolates were subjected to whole-genome sequencing and sedimentation and biofilm formation assays. From 1310 screened K. pneumoniae isolates in clinical routine 100 isolates (7.6%) were string test positive. From these, 9% (n = 9) were defined as putative hvKp (string-test+/PCR+). Highest rate of string-test-positive isolates was observed on MacConkey agar under aerobic conditions. Amongst these nine putative hvKp isolates, the international lineage ST23 carrying hvKp-plasmid pKpVP-1 was the most common, but also a rare ST86 with pKpVP-2 was identified. All nine isolates showed hypermucoviscosity and weak biofilm formation. In conclusion, 9% of string-positive, respectively 0.69% of all K. pneumoniae isolates from routine were defined as putative hypervirulent. MacConkey agar was the best medium for hvKp screening., (© 2023. The Author(s).)

Introduction: Bloodstream infections with Enterococcus faecalis are associated with relevant morbidity and mortality. Targeted antimicrobial therapy is essential. The choice of an adequate treatment may be challenging when susceptibility testing offers different options. Selective reporting of antibiotic susceptibility test results might lead to a more tailored antibiotic therapy and could therefore be an important antimicrobial stewardship program intervention. The aim of this study was to analyse whether the introduction of selective reporting of antibiotic test results leads to a more targeted antibiotic therapy in patients with bloodstream infection with Enterococcus faecalis., Methods: This study was performed as a retrospective cohort study at the University Hospital Regensburg, Germany. All patients with blood cultures positive for Enterococcus faecalis between March 2003 and March 2022 were analysed. In February 2014 selective reporting of antibiotic susceptibility test results omitting sensitivity results for agents not recommended was introduced., Results: 263 patients with blood cultures positive for Enterococcus faecalis were included. After introduction of selective reporting of antibiotic tests (AI) significantly more patients received ampicillin than before introduction of selective reporting (BI) (9.6% BI vs. 34.6% AI, p < 0.001)., Conclusion: Selective reporting of antibiotic susceptibility test results led to a significantly higher use of ampicillin., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Background: Patients with COVID-19 and severe acute respiratory failure may require veno-venous extracorporeal membrane oxygenation (VV ECMO). Yet, this procedure is resource-intensive and high mortality rates have been reported. Thus, predictors for identifying patients who will benefit from VV ECMO would be helpful., Methods: This retrospective study included 129 patients with COVID-19 and severe acute respiratory failure, who had received VV ECMO at the University Medical Center Regensburg, Germany, between 1 March 2020 and 31 December 2021. Patient-specific factors and relevant intensive-care parameters at the time of the decision to start VV ECMO were investigated regarding their value as predictors of patient survival. In addition, the intensive-care course of the first 10 days of VV ECMO was compared between survivors and patients who had died in the intensive care unit., Results: The most important parameters for predicting outcome were patient age and platelet count, which differed significantly between survivors and non-survivors (age: 52.6±8.1 vs. 57.4±10.1 years, p<0.001; platelet count before VV ECMO: 321.3±132.2 vs. 262.0±121.0 /nL, p = 0.006; average on day 10: 199.2±88.0 vs. 147.1±57.9 /nL, p = 0.002). A linear regression model derived from parameters collected before the start of VV ECMO only included age and platelet count. Patients were divided into two groups by using receiver operating characteristics (ROC) analysis: group 1: 78% of patients, mortality 26%; group 2: 22% of patients, mortality 75%. A second linear regression model included average blood pH, minimum paO2, and average pump flow on day 10 of VV ECMO in addition to age and platelet count. The ROC curve resulted in two cut-off values and thus in three groups: group 1: 25% of patients, mortality 93%; group 2: 45% of patients, mortality 31%; group 3: 30% of patients, mortality 0%., Competing Interests: Please take note of the following statement regarding Martin Kieninger’s service as Academic Editor for PLOS One: This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Kieninger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Purpose: Community-acquired methicillin-resistant Staphylococcus aureus strains (CA-MRSA) are spread worldwide and often cause recurring and persistent infections in humans. CA-MRSA strains frequently carry Panton-Valentine leukocidin (PVL) as a distinctive virulence factor. This study investigates the molecular epidemiology, antibiotic resistance and clinical characteristics of PVL-positive MRSA strains in Northern Bavaria, Germany, isolated over an eight-year period., Methods: Strains were identified by MALDI-TOF MS and antibiotic susceptibility was tested by automated microdilution (VITEK 2) or disk diffusion. PVL-encoding genes and mecA were detected by PCR. MRSA clonal complexes (CC) and lineages were assigned by genotyping via DNA microarray and spa -typing., Results: In total, 131 PVL-positive MRSA were collected from five hospital sites between 2009 and 2016. Predominant lineages were CC8-MRSA-[IV+ACME], USA300 (27/131; 20.6%); CC30-MRSA-IV, Southwest Pacific Clone (26/131; 19.8%) and CC80-MRSA-IV (25/131; 19.1%). Other CCs were detected less frequently. Resistance against erythromycin and clindamycin was prevalent, whereas all strains were sensitive towards vancomycin and linezolid. In total, 100 cases (76.3%) were causally linked to an infection. The majority (102/131; 77.9%) of isolates were detected in skin swabs or swabs from surgical sites., Conclusions: During the sample period we found an increase in the PVL-positive MRSA lineages CC30 and CC1. Compared to less-abundant lineages CC1 or CC22, the predominant lineages CC8, CC30 and CC80 harbored a broader resistance spectrum. Furthermore, these lineages are probably associated with a travel and migration background. In the spatio-temporal setting we investigated, these were arguably drivers of diversification and change in the landscape of PVL-positive MRSA.

Recent reports provide evidence that contaminated healthcare environments represent major sources for the acquisition and transmission of pathogens. Antimicrobial coatings (AMC) may permanently and autonomously reduce the contamination of such environmental surfaces complementing standard hygiene procedures. This review provides an overview of the current status of AMC and the demands to enable a rational application of AMC in health care settings. Firstly, a suitable laboratory test norm is required that adequately quantifies the efficacy of AMC. In particular, the frequently used wet testing (e.g. ISO 22196) must be replaced by testing under realistic, dry surface conditions. Secondly, field studies should be mandatory to provide evidence for antimicrobial efficacy under real-life conditions. The antimicrobial efficacy should be correlated to the rate of nosocomial transmission at least. Thirdly, the respective AMC technology should not add additional bacterial resistance development induced by the biocidal agents and co- or cross-resistance with antibiotic substances. Lastly, the biocidal substances used in AMC should be safe for humans and the environment. These measures should help to achieve a broader acceptance for AMC in healthcare settings and beyond. Technologies like the photodynamic approach already fulfil most of these AMC requirements.

Millions of people use public transportation daily worldwide and frequently touch surfaces, thereby producing a reservoir of microorganisms on surfaces increasing the risk of transmission. Constant occupation makes sufficient cleaning difficult to achieve. Thus, an autonomous, permanent, antimicrobial coating (AMC) could keep down the microbial burden on such surfaces. A photodynamic AMC was applied to frequently touched surfaces in buses. The microbial burden (colony forming units, cfu) was determined weekly and compared to equivalent surfaces in buses without AMC (references). The microbial burden ranged from 0-209 cfu/cm 2 on references and from 0-54 cfu/cm 2 on AMC. The means were 13.4 ± 29.6 cfu/cm 2 on references and 4.5 ± 8.4 cfu/cm 2 on AMC ( p < 0.001). The difference in microbial burden on AMC and references was almost constant throughout the study. Considering a hygiene benchmark of 5 cfu/cm 2 , the data yield an absolute risk reduction of 22.6% and a relative risk reduction of 50.7%. In conclusion, photodynamic AMC kept down the microbial burden, reducing the risk of transmission of microorganisms. AMC permanently and autonomously contributes to hygienic conditions on surfaces in public transportation. Photodynamic AMC therefore are suitable for reducing the microbial load and closing hygiene gaps in public transportation.

Metastasis is the major cause of death in cancer patients. Circulating tumor cells need to migrate through the endothelial layer of blood vessels to escape the hostile circulation and establish metastases at distant organ sites. Here, we identified the membrane-bound metalloprotease ADAM17 on endothelial cells as a key driver of metastasis. We show that TNFR1-dependent tumor cell-induced endothelial cell death, tumor cell extravasation, and subsequent metastatic seeding is dependent on the activity of endothelial ADAM17. Moreover, we reveal that ADAM17-mediated TNFR1 ectodomain shedding and subsequent processing by the γ-secretase complex is required for the induction of TNF-induced necroptosis. Consequently, genetic ablation of ADAM17 in endothelial cells as well as short-term pharmacological inhibition of ADAM17 prevents long-term metastases formation in the lung. Thus, our data identified ADAM17 as a novel essential regulator of necroptosis and as a new promising target for antimetastatic and advanced-stage cancer therapies., Competing Interests: Disclosures: I. Sagi reported a patent (no. US 10,933,122 B2) issued. D. Schmidt-Arras reported personal fees from Mestag Therapeutics Ltd. outside the submitted work. No other disclosures were reported., (© 2021 Bolik et al.)

In the context of microarray-based epidemiological typing of the clonal organism Staphylococcus aureus /MRSA , a strain was identified that did not belong to known clonal complexes. The molecular analysis by microarray-based typing yielded signals suggesting that it was a mosaic or hybrid strain of two lineages. To verify this result, the isolate was sequenced with both, short-read Illumina and long-read Nanopore technologies and analysed in detail. This supported the hypothesis that the genome of this strain, ST6610-MRSA-IVg comprised of segments originating from two different clonal complexes (CC). While the backbone of the strain's genome, i.e., roughly 2 megabases, belongs to CC8, a continuous insert of 894 kb (approx. 30% of the genome) originated from CC140. Beside core genomic markers in the normal succession and orientation, this insert also included the mecA gene, coding for PbP2a and causing methicillin resistance, localised on an SCC mec IVg element. This particular SCC mec type was also previously observed in CC140 MRSA from African countries. A second conspicuous observation was the presence of the trimethoprim resistance gene dfrG within on a prophage that occupied an attachment site normally used by Panton-Valentine Leucocidin phages. This observation could indicate a role of large-scale chromosomal recombination in the evolution of S. aureus as well as a role of phages in the dissemination of antibiotic resistance genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Burgold-Voigt, Monecke, Simbeck, Holzmann, Kieninger, Liebler-Tenorio, Braun, Collatz, Diezel, Müller, Schneider-Brachert and Ehricht.)

Signaling via death receptor family members such as TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Pro-survival signaling is mediated via TNF-R1 complex I at the cellular plasma membrane. Cell death induction requires complex IIa/b or necrosome formation, which occurs in the cytoplasm. In many cell types, full apoptotic or necroptotic cell death induction requires the internalization of TNF-R1 and receptosome formation to properly relay the signal inside the cell. We interrogated the role of the enzyme A disintegrin and metalloprotease 17 (ADAM17)/TACE (TNF-α converting enzyme) in death receptor signaling in human hematopoietic cells, using pharmacological inhibition and genetic ablation. We show that in U937 and Jurkat cells the absence of ADAM17 does not abrogate, but rather increases TNF mediated cell death. Likewise, cell death triggered via DR3 is enhanced in U937 cells lacking ADAM17. We identified ADAM17 as the key molecule that fine-tunes death receptor signaling. A better understanding of cell fate decisions made via the receptors of the TNF-R1 superfamily may enable us, in the future, to more efficiently treat infectious and inflammatory diseases or cancer.

Background: Opening schools and keeping children safe from SARS-CoV-2 infections at the same time is urgently needed to protect children from direct and indirect consequences of the COVID-19 pandemic. To achieve this goal, a safe, efficient, and cost-effective SARS-CoV-2 testing system for schools in addition to standard hygiene measures is necessary. Methods: We implemented the screening WICOVIR concept for schools in the southeast of Germany, which is based on gargling at home, pooling of samples in schools, and assessment of SARS-CoV-2 by pool rRT-PCR, performed decentralized in numerous participating laboratories. Depooling was performed if pools were positive, and results were transmitted with software specifically developed for the project within a day. Here, we report the results after the first 13 weeks in the project. Findings: We developed and implemented the proof-of-concept test system within a pilot phase of 7 weeks based on almost 17,000 participants. After 6 weeks in the main phase of the project, we performed >100,000 tests in total, analyzed in 7,896 pools, identifying 19 cases in >100 participating schools. On average, positive children showed an individual CT value of 31 when identified in the pools. Up to 30 samples were pooled (mean 13) in general, based on school classes and attached school staff. All three participating laboratories detected positive samples reliably with their previously established rRT-PCR standard protocols. When self-administered antigen tests were performed concomitantly in positive cases, only one of these eight tests was positive, and when antigen tests performed after positive pool rRT-PCR results were already known were included, 3 out of 11 truly positive tests were also identified by antigen testing. After 3 weeks of repetitive WICOVIR testing twice weekly, the detection rate of positive children in that cohort decreased significantly from 0.042 to 0.012 ( p = 0.008). Interpretation: Repeated gargle pool rRT-PCR testing can be implemented quickly in schools. It is an effective, valid, and well-received test system for schools, superior to antigen tests in sensitivity, acceptance, and costs., Competing Interests: TWa has invested in a company that performs PCR pool tests for companies. RR, FS, and DW are employed by NOVOGENIA, a commercial PCR test laboratory. JN and PP are employed by Maganamed GmbH, a commercial software company. DATEV eG is a software company which provides a non-profit test center for the project. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kheiroddin, Schöberl, Althammer, Cibali, Würfel, Wein, Kulawik, Buntrock-Döpke, Weigl, Gran, Gründl, Langguth, Lampl, Judex, Niggel, Pagel, Schratzenstaller, Schneider-Brachert, Gastiger, Bodenschatz, Konrad, Levchuk, Roth, Schöner, Schneebauer, Rohrmanstorfer, Dekens, Brandstetter, Zuber, Wallerstorfer, Burkovski, Ambrosch, Wagner and Kabesch.)

Abstract: Antibiotic stewardship (ABS) programs intend to improve outcomes of nosocomial infections and to counteract the emergence of further antimicrobial resistances. At the anesthesiologic-neurosurgical intensive care unit (ICU) of the University Medical Center Regensburg (Germany) we implemented a standard operating procedure (SOP) with clear instructions for the preanalytical handling and storage of microbiological samples. We intended to find out whether the instructions given in the SOP led to a higher rate of ideal material being sent to the laboratory and to overall better quality of the received results.We retraced retrospectively all samples taken in cases of suspected pneumonia, urinary tract infection, bloodstream infection, catheter infection associated with a central venous or arterial catheter and ventriculitis due to external ventricular drainage as well as all smears taken for the screening for multi-resistant bacteria within a time period of 1 year before to 1 year after the implementation of the SOP.In the case of suspected pneumonia and urinary tract infection, large amounts of ideal material were sent to the microbiological laboratory. A remarkable improvement after the implementation of the SOP, however, could only be observed regarding the number of urine samples taken from older urinary catheters, which was significantly lower in the "SOP group". Samples for microbiological diagnostics were taken much more often in the daytime, although storage of the probes did not lead to worse results.Concrete instructions enable adequate preanalytical handling of microbiological probes. However, we could not recognize substantial improvements probably due to a preexisting high process quality on the ICU. Microbiological diagnostics during the night shift has to be improved., Competing Interests: The authors have no funding and conflicts of interests to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)