Colocalization of endogenous TNF with a functional intracellular splice form of human TNF receptor type 2

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

Antimicrobial coatings for environmental surfaces in hospitals: a potential new pillar for prevention strategies in hygiene

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

Antimicrobial Photodynamic Coatings Reduce the Microbial Burden on Environmental Surfaces in Public Transportation—A Field Study in Buses

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/cm2 on references and from 0–54 cfu/cm2 on AMC. The means were 13.4 ± 29.6 cfu/cm2 on references and 4.5 ± 8.4 cfu/cm2 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/cm2, 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

Examining outpatients' hand hygiene behaviour and its relation to COVID-19 infection prevention measures

BACKGROUND: The increasing demand for outpatient care is associated with a higher risk of infection transmission in these settings. However, there is limited research on infection prevention and control practices in ambulatory clinics, and none focuses on patients. AIM: This study aims to examine outpatients' hand hygiene behaviours, their determinants, and their associations with other infection prevention measures during the COVID-19 pandemic. METHODS: We observed the hand hygiene behaviour of one cohort of patients in one outpatient clinic and surveyed a separate sample in five clinics about their hand hygiene practice in outpatient facilities. A questionnaire based on the Theoretical Domains Framework (TDF) was used to examine predictors of the behaviour. Moreover, patients indicated their compliance with COVID-19 infection prevention measures, vaccination status, disease risk perception, and vaccine hesitancy. FINDINGS: Observed hand hygiene rates among 618 patients were low (12.8%), while 67.3% of the 300 surveyed patients indicated sanitising their hands upon entering the clinic. The TDF domains memory, attention, and decision processes, and emotions significantly predicted both current (today's) and general hand hygiene behaviour in outpatient clinics. Hand hygiene behaviour and compliance with COVID-19 infection prevention showed a positive association; however, no significant connection was found with patients' vaccination status, suggesting different behavioural motivators. CONCLUSION: Hand hygiene among outpatients should be improved through interventions focusing on helping patients remember to clean their hands. More research on infection prevention in outpatient facilities is needed to ensure patient safety

Dirty hands: photodynamic killing of human pathogens like EHEC, MRSA and Candida within seconds

Hand hygiene is one of the most important interventions for reducing transmission of nosocomial life-threatening microorganisms, like methicillin resistant Staphylococcus aureus (MRSA), enterohemorrhagic Escherichia coli (EHEC) or Candida albicans. All three pathogens have become a leading cause of infections in hospitals. Especially EHEC is causing severe diarrhoea and, in a small percentage of cases, haemolytic-uremic syndrome (HUS) as reported for E. coli 104:H4 in Germany 2011. We revealed the possibility to inactivate very fast and efficiently MRSA, EHEC and C. albicans using the photodynamic approach. MRSA, EHEC and C. albicans were incubated in vitro with different concentrations of TMPyP for 10 s and illuminated with visible light (50 mW cm−2) for 10 and 60 s. 1 μmol l−1 of TMPyP and an applied radiant exposure of 0.5 J cm−2 achieved a photodynamic killing of ≥99.9% of MRSA and EHEC. Incubation with higher concentrations (up to 100 μmol l−1) of TMPyP caused bacteria killing of >5 log10 (≥99.999%) after illumination. Efficient Candida killing (≥99.999%) was achieved first at a higher light dose of 12 J cm−2. Different rise and decay times of singlet oxygen luminescence signals could be detected in Candida cell suspensions for the first time, indicating different oxygen concentrations in the surrounding for the photosensitizer and singlet oxygen, respectively. This confirms that TMPyP is not only found in the water-dominated cell surrounding, but also within the C. albicans cells. Applying a water–ethanol solution of TMPyP on ex vivo porcine skin, fluorescence microscopy of histology showed that the photosensitizer was exclusively localized in the stratum corneum regardless of the incubation time. TMPyP exhibited a fast and very effective killing rate of life-threatening pathogens within a couple of seconds that encourages further testing in an in vivo setting. Being fast and effective, antimicrobial photodynamic applications might become acceptable as a tool for hand hygiene procedures and also in other skin areas

Roles for ADAM17 in TNF-R1 Mediated Cell Death and Survival in Human U937 and Jurkat Cells

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

Retrospective genome-oriented analysis reveals low transmission rate of multidrug-resistant Pseudomonas aeruginosa from contaminated toilets at a bone marrow transplant unit

Background Prevention of toilet-to-patient transmission of multidrug-resistant Pseudomonas aeruginosa (MDR PA) poses management-related challenges at many bone marrow transplant units (BMTUs). Aim To conduct a longitudinal retrospective analysis of the toilet-to-patient transmission rate for MDR PA under existing infection control (IC) measures at a BMTU with persistent MDR PA toilet colonization. Methods The local IC bundle comprised: (1) patient education regarding IC; (2) routine patient screening; (3) toilet flushing volume of 9 L; (4) bromination of toilet water tanks, and (5) toilet decontamination using hydrogen peroxide. Toilet water was sampled periodically between 2016 and 2021 (minimum every three months: 26 intervals). Upon MDR PA detection, disinfection and re-sampling were repeated until ≤3 cfu/100 mL was reached. Whole-genome sequencing (WGS) was performed retrospectively on all available MDR PA isolates (90 out of 117 positive environmental samples, 10 out of 14 patients, including nine nosocomial). Findings WGS of patient isolates identified six sequence types (STs), with ST235/CT1352/FIM-1 and ST309/CT3049/no-carbapenemase being predominant (three isolates each). Environmental sampling consistently identified MDR PA ST235 (65.5% ST235/CT1352/FIM-1), showing low genetic diversity (difference of ≤29 alleles by core-genome multi-locus sequence typing (cgMLST)). This indicates that direct toilet-to-patient transmission was infrequent although MDR PA was widespread (detection on 79 occasions, detection in every toilet). Only three MDR PA patient isolates can be attributed to the ST235/CT1352/FIM-1 toilet MRD PA population over six years. Conclusion Stringent targeted toilet disinfection can reduce the potential risk for MDR PA acquisition by patients

Nanosilver/DCOIT-containing surface coating effectively and constantly reduces microbial load in emergency room surfaces

Background Colonization of near-patient surfaces in hospitals plays an important role as a source of healthcare-associated infections. Routine disinfection methods only result in short-term elimination of pathogens. Aim To investigate the efficiency of a newly developed antimicrobial coating containing nanosilver in long-term reduction of bacterial burden in hospital surfaces to close the gap between routine disinfection cycles. Methods In this prospective, double-blinded trial, frequently touched surfaces of a routinely used treatment room in an emergency unit of a level-I hospital were treated with a surface coating (nanosilver/DCOIT-coated surface, NCS) containing nanosilver particles and another organic biocidal agent (4,5-dichloro-2-octyl-4-isothiazolin-3-one, DCOIT), whereas surfaces of another room were treated with a coating missing both the nanosilver- and DCOIT-containing ingredient and served as control. Bacterial contamination of the surfaces was examined using contact plates and liquid-based swabs daily for a total trial duration of 90 days. After incubation, total microbial counts and species were assessed. Findings In a total of 2880 antimicrobial samples, a significant reduction of the overall bacterial load was observed in the NCS room (median: 0.31 cfu/cm2; interquartile range: 0.00–1.13) compared with the control coated surfaces (0.69 cfu/cm2; 0.06–2.00; P 5 cfu/cm2) by 60% (odds ratio 0.38, P < 0.001). No significant difference in species distribution was detected between NCS and control group. Conclusion Nanosilver-/DCOIT-containing surface coating has shown efficiency for sustainable reduction of bacterial load of frequently touched surfaces in a clinical setting

An epidemic CC1-MRSA-IV clone yields false-negative test results in molecular MRSA identification assays: a note of caution, Austria, Germany, Ireland, 2020

We investigated why a clinical meticillin-resistant Staphylococcus aureus (MRSA) isolate yielded false-negative results with some commercial PCR tests for MRSA detection. We found that an epidemic European CC1-MRSA-IV clone generally exhibits this behaviour. The failure of the assays was attributable to a large insertion in the orfX/SCCmec integration site. To ensure the reliability of molecular MRSA tests, it is vital to monitor emergence of new SCCmec types and junction sites

Exploring the evolution and epidemiology of European CC1-MRSA-IV: tracking a multidrug-resistant community-associated meticillin-resistant Staphylococcus aureus clone

This study investigated the evolution and epidemiology of the community-associated and multidrug-resistant Staphylococcus aureus clone European CC1-MRSA-IV. Whole-genome sequences were obtained for 194 European CC1-MRSA-IV isolates (189 of human and 5 of animal origin) from 12 countries, and 10 meticillin-susceptible precursors (from North-Eastern Romania; all of human origin) of the clone. Phylogenetic analysis was performed using a maximum-likelihood approach, a time-measured phylogeny was reconstructed using Bayesian analysis, and in silico microarray genotyping was performed to identify resistance, virulence-associated and SCCmec (staphylococcal cassette chromosome mec) genes. Isolates were typically sequence type 1 (190/204) and spa type t127 (183/204). Bayesian analysis indicated that European CC1-MRSA-IV emerged in approximately 1995 before undergoing rapid expansion in the late 1990s and 2000s, while spreading throughout Europe and into the Middle East. Phylogenetic analysis revealed an unstructured meticillin-resistant S. aureus (MRSA) population, lacking significant geographical or temporal clusters. The MRSA were genotypically multidrug-resistant, consistently encoded seh, and intermittently (34/194) encoded an undisrupted hlb gene with concomitant absence of the lysogenic phage-encoded genes sak and scn. All MRSA also harboured a characteristic ~5350 nt insertion in SCCmec adjacent to orfX. Detailed demographic data from Denmark showed that there, the clone is typically (25/35) found in the community, and often (10/35) among individuals with links to South-Eastern Europe. This study elucidated the evolution and epidemiology of European CC1-MRSA-IV, which emerged from a meticillin-susceptible lineage prevalent in North-Eastern Romania before disseminating rapidly throughout Europe