Salmonella Typhimurium effector SseI inhibits chemotaxis and increases host cell survival by deamidation of heterotrimeric Gi proteins.

Salmonella enterica serotype Typhimurium (S. Typhimurium) is one of the most frequent causes of food-borne illness in humans and usually associated with acute self-limiting gastroenteritis. However, in immunocompromised patients, the pathogen can disseminate and lead to severe systemic diseases. S. Typhimurium are facultative intracellular bacteria. For uptake and intracellular life, Salmonella translocate numerous effector proteins into host cells using two type-III secretion systems (T3SS), which are encoded within Salmonella pathogenicity islands 1 (SPI-1) and 2 (SPI-2). While SPI-1 effectors mainly promote initial invasion, SPI-2 effectors control intracellular survival and proliferation. Here, we elucidate the mode of action of Salmonella SPI-2 effector SseI, which is involved in control of systemic dissemination of S. Typhimurium. SseI deamidates a specific glutamine residue of heterotrimeric G proteins of the Gαi family, resulting in persistent activation of the G protein. Gi activation inhibits cAMP production and stimulates PI3-kinase γ by Gαi-released Gßγ subunits, resulting in activation of survival pathways by phosphorylation of Akt and mTOR. Moreover, SseI-induced deamidation leads to non-polarized activation of Gαi and, thereby, to loss of directed migration of dendritic cells.

Vaginal Microbial Colonization after Antibiotic Treatment in Women with Preterm Premature Rupture of Membranes: An Observational Cohort Study.

Preterm premature rupture of membranes (pPROM) stands as a primary contributor to preterm deliveries worldwide, closely linked to consequential infectious peripartum complications, including chorioamnionitis and early-onset neonatal sepsis. As a prophylactic measure, individuals following pPROM routinely undergo antibiotic treatment. The aim of this study was to evaluate changes in the vaginal microbial colonization after antibiotic treatment following pPROM. Therefore, we retrospectively assessed the impact of antibiotic treatment on the maternal vaginal microbial colonization in 438 post-pPROM patients delivering before 29 gestational weeks. Vaginal samples were collected for microbiological analysis before and after antibiotic administration and analysed for seventeen pre-defined microbial groups. We observed eradication in eleven microbial groups, including beta-hemolytic streptococci group B and Gardnerella vaginalis. No significant reduction was found for the remaining groups, including Escherichia (E.) coli. Moreover, we found a notable increase in resistant bacteria after antibiotic treatment. In conclusion, broad-spectrum antimicrobial treatment exhibited substantial efficacy in eradicating the majority of pathogens in our cohort. However, certain pathogens, notably E. coli, showed resilience. Given E. coli's prominent role in infectious peripartum complications, our findings underline the challenges in antibiotic management post-pPROM and the need to establish international guidelines, particularly regarding emerging concerns about antibiotic resistances.

COVID-19 patient and personal safety - lessons learnt for pandemic preparedness and the way to the next normal.

BACKGROUND: The COVID-19 pandemic has profoundly challenged societies and healthcare systems in particular. To prevent the spread of SARS-CoV-2, infection prevention and control (IPC) strategies had to be developed on the local, national and international level. The aim of this study is to provide details of the COVID-19 experience at the Vienna General Hospital (VGH) in the context of the national and international COVID-19 response for learning and improvement. METHODS: This is a retrospective report, outlining the evolution of IPC measures and challenges encountered at the health facility (VGH), the national (Austria) and global level between February 2020 and October 2022. RESULTS: The IPC strategy at the VGH has been continuously adapted to changes in the epidemiological setting, new legal directives and Austrian by-laws. The current strategy, nationally and internationally, focuses on endemicity rather than maximum transmission risk reduction. For the VGH, this has recently resulted in an increase in COVID-19 clusters. To protect our particularly vulnerable patients, many COVID-19 precautions have been maintained. Barriers to adequate IPC implementation at the VGH and other hospitals include a lack of sufficient isolation options and non-adherence with universal face mask regulations. Globally, misinformation on COVID-19 hampered an effective response. CONCLUSIONS: This retrospective analysis of the COVID-19 response at the VGH and international reports underline the need for pandemic preparedness, readiness and response by improving future hospital design and infrastructure, conducting regular trainings for protective attire and increasing health literacy as now recently published in a concise document by WHO.

BLEACH&STAIN 15-marker Multiplexed Imaging in 3,098 Human Carcinomas Reveals Six Major PD-L1-driven Immune Phenotypes with Distinct Spatial Orchestration.

Multiplex fluorescence IHC (mfIHC) approaches were yet either limited to six markers or limited to a small tissue size that hampers translational studies on large tissue microarray cohorts. Here we have developed a BLEACH&STAIN mfIHC method that enabled the simultaneous analysis of 15 biomarkers (PD-L1, PD-1, CTLA-4, panCK, CD68, CD163, CD11c, iNOS, CD3, CD8, CD4, FOXP3, CD20, Ki67, and CD31) in 3,098 tumor samples from 44 different carcinoma entities within one week. To facilitate automated immune checkpoint quantification on tumor and immune cells and study its spatial interplay an artificial intelligence-based framework incorporating 17 different deep-learning systems was established. Unsupervised clustering showed that the three PD-L1 phenotypes (PD-L1+ tumor and immune cells, PD-L1+ immune cells, PD-L1-) were either inflamed or noninflamed. In inflamed PD-L1+patients, spatial analysis revealed that an elevated level of intratumoral M2 macrophages as well as CD11c+ dendritic cell (DC) infiltration (P < 0.001 each) was associated with a high CD3+ CD4± CD8± FOXP3± T-cell exclusion and a high PD-1 expression on T cells (P < 0.001 each). In breast cancer, the PD-L1 fluorescence intensity on tumor cells showed a significantly higher predictive performance for overall survival (OS; AUC, 0.72, P < 0.001) compared with the commonly used percentage of PD-L1+ tumor cells (AUC, 0.54). In conclusion, our deep-learning-based BLEACH&STAIN framework facilitates rapid and comprehensive assessment of more than 60 spatially orchestrated immune cell subpopulations and its prognostic relevance. IMPLICATIONS: The development of an easy-to-use high-throughput 15+1 multiplex fluorescence approach facilitates the in-depth understanding of the immune tumor microenvironment (TME) and enables to study the prognostic relevance of more than 130 immune cell subpopulations.

Provision of safe patient care during the COVID-19 pandemic despite shared patient rooms in a tertiary hospital.

BACKGROUND: The COVID-19 pandemic has resulted in the disruption of healthcare systems. Vienna General Hospital (VGH), a tertiary hospital located in Austria, ran at almost full capacity despite high levels of community SARS-CoV-2 transmission and limited isolation room capacity. To ensure safe patient care, a bundle of infection prevention and control (IPC) measures including universal pre-admission screening and serial SARS-CoV-2 testing during hospitalization was implemented. We evaluated whether testing as part of our IPC approach was effective in preventing hospital outbreaks during different stages of the pandemic. METHODS: In this retrospective single center study, we analyzed the SARS-CoV-2 PCR test results of cases admitted to VGH between a low (15/05/2020-01/08/2020) and a high incidence period (15/09/2020-18/05/2021). Outcomes were the diagnostic yield of (a) admission screening, (b) the yield of serial testing during hospitalization and (c) the occurrence of healthcare-associated COVID-19 (HA-COVID-19) and SARS-CoV-2 related hospital outbreaks. RESULTS: The admission test positivity rate was 0.2% during the low and 2.3% during the high incidence phase. Regarding test conversions, 0.04% (low incidence phase) and 0.5% (high incidence phase) of initially negative cases converted to a positive test result within 7 days after admission The HA-COVID-19 incidence rate per 100,000 patient days was 1.0 (low incidence phase) and 10.7 (high incidence phase). One COVID-19 outbreak affecting eight patients in total could be potentially ascribed to the non-compliance with our IPC protocol. CONCLUSION: Testing in conjunction with other IPC measures enabled the safe provision of patient care at a hospital with predominantly shared patient rooms despite high case numbers in the community.

Routine Surveillance of Healthcare-Associated Infections Misses a Significant Proportion of Invasive Aspergillosis in Patients with Severe COVID-19.

Rates of invasive aspergillosis (IA) among COVID-19 ICU patients seem to reach over 30% in certain settings. At Vienna General Hospital (VGH), all rooms in COVID-19 ICUs were put under negative pressure as a protective measure, thus increasing the risk of exposure to environmental pathogens for patients. Even though all ICU patients are surveilled for healthcare-associated infections (HAI), there were concerns that the routine protocol might not be sufficient for IA detection. We reviewed the electronic patient charts of all patients with COVID-19 admitted to ICUs between 1 March 2020 and 31 July 2021 for fungal co- or superinfections, comparing four diagnostic algorithms based on different recommendations for the diagnosis of IA (according to EORTC/MSG, BM-AspICU, IAPA and CAPA) to our routine surveillance protocol. We found that out of 252 patients who were admitted to the ICU during the study period, 25 (9.9%) fulfilled the criteria of probable or possible IA of at least one algorithm. The IAPA definitions detected 25 and the CAPA definition 23 probable and 2 possible cases, out of which only 16 were classified as hospital-acquired IA by routine surveillance. In conclusion, adjustment of the routine protocol using a classification system especially designed for respiratory viral illness seems useful for the surveillance of IA in a highly vulnerable patient cohort.

The effectiveness of surface disinfectants and a micellic H2O2 based water disinfectant on Candida auris.

BACKGROUND: As infection control faces new challenges from emerging, multidrug resistant strains of the yeast Candida auris, this study was conducted in order to examine the efficacy of hospital surface disinfectants and a new water disinfectant against C. auris biofilm forms. METHODS: We tested four reference strains of C. auris (NCPF8971, NCPF8977, NCPF8984, DSM21092) and one C. albicans strain (ATCC10231) against disinfectants based on ethanol (ETH), quaternary ammonium (QAC), a combination of glutaraldehyde, quaternary ammonium and surfactant (ALD) and potassium peroxymonosulfate (PP) as well as 3.4% H2O2 and 4.25% H2O2 alone. In addition, a micellic-based formulation containing 17% v/v hydrogene peroxide disinfectant (mH2O2) was tested. The efficacy of the disinfectants was measured in a 96-well plate using tetrazolium salt reduction (XTT) and the log10 reduction assay. RESULTS: ETH and QAC displayed in clinically recommended concentrations more than 5log10 reduction and more than 80% XTT activity reduction for all of the Candida biofilms and planktonic cells. Only biofilms of C. auris NCPF8984 were additionally sensitive to all remaining disinfectants. All tested C. auris biofilms were sensitive to PP disinfectant and showed more than 5log10 reduction. However, the XTT assay showed a reduction of less than 80% for the PP disinfectant, indicating the presence of active but non-culturable cells. The 25% mH2O2 (corresponding to 4.25% H2O2) killed Candida biofilms after 1 minute.

Engineering Photorhabdus luminescens toxin complex (PTC) into a recombinant injection nanomachine.

Engineering delivery systems for proteins and peptides into mammalian cells is an ongoing challenge for cell biological studies as well as for therapeutic approaches. Photorhabdus luminescens toxin complex (PTC) is a heterotrimeric protein complex able to deliver diverse protein toxins into mammalian cells. We engineered the syringe-like nanomachine for delivery of protein toxins from different species. In addition, we loaded the highly active copepod luciferase Metridia longa M-Luc7 for accurate quantification of injected molecules. We suggest that besides the probable size limitation, the charge of the cargo also influences the efficiency of packing and transport into mammalian cells. Our data show that the PTC constitutes a powerful system to inject recombinant proteins, peptides, and potentially, other molecules into mammalian cells. In addition, in contrast to other protein transporters based on pore formation, the closed, compact structure of the PTC may protect cargo from degradation.

Activation of Gq signaling by Pasteurella multocida toxin inhibits the osteoblastogenic-like actions of Activin A in C2C12 myoblasts, a cell model of fibrodysplasia ossificans progressiva.

The human disease fibrodysplasia ossificans progressiva (FOP) is a rare and highly disabling disorder of extensive heterotopic bone growth that is caused by a point mutation (R206H) in the activation domain of Alk2, a BMP (bone morphogenic protein) type 1 receptor. The mutation leads to extensive BMP-signaling induced by Activin A, which is normally an antagonist for wildtype receptors, resulting in excessive and uncontrolled bone formation. Here, we studied the effects of Pasteurella multocida toxin (PMT), which activates osteoclasts and inhibits osteoblast activity, in C2C12 myoblasts expressing the mutant Alk2(R206H) receptor as model of FOP. In our study, we mainly used alkaline phosphatase (ALP) activity as marker to determine osteoblast differentiation. BMP-4 stimulated an increase in ALP activity in C2C12-Alk2wt and C2C12-Alk2(R206H) cells. By contrast, Activin A only induced ALP activity in C2C12-Alk2(R206H) cells. In both cases, PMT acted as a potent inhibitor of ALP activity. PMT-induced inhibition of ALP activity was paralleled by a constitutive activation of the heterotrimeric Gq protein. Expression of a permanently active Gαq blocked Activin A/Alk2(R206H)-dependent increase in ALP activity. Inactivation of Gq by specific inhibitor FR900359 blocked the PMT effect. Similarly, canonical second messengers and effectors of Gαq (e.g. ionophore A23187-induced increase in intracellular Ca2+ and activation of PKC by PMA (phorbol 12-myristate 13-acetate)) inhibited Alk2(R206H)-mediated induction of ALP activity. Notably, Activin A-induced increase in ALP activity in C2C12-Alk2(R206H) cells was also inhibited by stimulation of the α1A-adrenoceptor, which couples to Gαq, by phenylephrine. PMT did not alter tail phosphorylation of the major downstream effectors of the Alk2 receptor, Smad1/5/9; neither did the toxin affect nuclear translocation of the Smad-complex. However, PMT diminished BMP responsive element-induced gene expression. The data indicate that PMT potently inhibits the induction of osteoblast markers in a FOP model via activation of G proteins. Moreover, our findings indicate that activation of G protein-coupled receptors and of G protein signaling might be a rationale for pharmacological therapy of FOP.

Involvement of Osteocytes in the Action of Pasteurella multocida Toxin.

Pasteurella multocida toxin (PMT) causes progressive atrophic rhinitis with severe turbinate bone degradation in pigs. It has been reported that the toxin deamidates and activates heterotrimeric G proteins, resulting in increased differentiation of osteoclasts and blockade of osteoblast differentiation. So far, the action of PMT on osteocytes, which is the most abundant cell type in bone tissue, is not known. In MLO-Y4 osteocytes, PMT deamidated heterotrimeric G proteins, resulting in loss of osteocyte dendritic processes, stress fiber formation, cell spreading and activation of RhoC but not of RhoA. Moreover, the toxin caused processing of membrane-bound receptor activator of NF-κB ligand (RANKL) to release soluble RANKL and enhanced the secretion of osteoclastogenic TNF-α. In a co-culture model of osteocytes and bone marrow cells, PMT-induced osteoclastogenesis was largely increased as compared to the mono-culture model. The enhancement of osteoclastogenesis observed in the co-culture was blocked by sequestering RANKL with osteoprotegerin and by an antibody against TNF-α indicating involvement of release of the osteoclastogenic factors from osteocytes. Data support the crucial role of osteocytes in bone metabolism and osteoclastogenesis and identify osteocytes as important target cells of PMT in progressive atrophic rhinitis.