Development of a model for the investigation of the impact of deficiency of endogenous hydrogen sulphide on resistance of multi-drug resistant bacteria (Pseudomonas aeruginosa & Klebsiella pneumonia) against antimicrobial treatment

Hydrogen sulfide (H2S) has recently been recognized as a novel gaseous transmitterwith several anti-inflammatory properties. The role of host- derived H2S in infectionsby Pseudomonas aeruginosa was investigated in clinical and mouse models. H2Sconcentrations and survival was assessed in septic patients with lung infection. Animalexperiments using a model of severe systemic multidrug-resistant P. aeruginosainfection were performed using mice with a constitutive knock-out of cystathionine-γlyase (Cse) gene (Cse-/-) and wild-type mice with a physiological expression (Cse+/+).Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bonemarrow transplantation (BMT) from a Cse+/+ donor; c) treatment with H2S synthesisinhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H2S donorsodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activitywere measured in tissue samples. The expression of quorum sensing genes (QS) wasdetermined in vivo and in vitro. Cytokine concentration was measured in serum andincubated splenocytes. Patients survivors at day 28 had significantly higher serum H2Scompared to non-survivors. A cut- off point of 5.3 μΜ discriminated survivors withsensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H2Shigher and less than 5.3 μΜ (p= 7 x 10-6). In mice expression of Cse and applicationof STS afforded protection against infection with multidrug-resistant P. aeruginosa.Cyclophosphamide pretreatment eliminated the survival benefit of Cse+/+ mice,whereas BMT increased the survival of Cse-/- mice. Cse-/- mice had increasedpathogen loads compared to Cse+/+ mice. Phagocytic activity of leukocytes from Cse-/- mice was reduced but was restored after H2S supplementation. An H2S dependentdown- regulation of quorum sensing genes of P.aeruginosa could be demonstrated invivo and in vitro. Endogenous H2S is a potential independent parameter correlatingwith the outcome of P. aeruginosa. H2S provides resistance to infection by MDRbacterial pathogens.Το υδρόθειο (H2S) έχει πρόσφατα αναγνωριστεί ως ένας νέος αέριος διαβιβαστής μεαντιφλεγμονώδεις δράσεις. Ο ρόλος του παραγόμενου από τον ξενιστή H2S σελοιμώξεις από αεριογόνο ψευδομονάδα Pseudomonas (P.) aeruginosa διερευνήθηκεσε ένα κλινικό και ζωικό μοντέλο. Μελετήθηκαν οι συγκεντρώσεις του H2S και ηεπιβίωση σηπτικών ασθενών με λοιμώξεις του κατώτερου αναπνευστικού. Στησυνέχεια αναπτύχθηκε ένα ζωικό μοντέλο σοβαρής συστηματικής λοίμωξης απόπολυανθεκτική P. aeruginosa iχρησιμοποιώντας μύες με έλλειψη του γονιδίου της γ-λυάσης της κυσταθειονίνης (Cse) (Cse-/- μύες) και μύες με φυσιολογική έκφραση τουγονιδίου (Cse+/+ μύες). Πειράματα επαναλήφθηκαν σε μύες μετά από α) θεραπεία μεκυκλοφωσφαμίδη, β) μεταμόσχευση μυελού των οστών από δότη Cse+/+, γ) θεραπείαμε τους αναστολείς της σύνθεσης H2S αμινοοξεϊκό οξύ (aminooxyacetic acid, ΑΟΑΑ)ή προπαργυλγλυκίνη (propargylglycine, PAG) και δ) τους δότες H2S θειοθειικό νάτριο(sodium thiosulfate, STS) ή το GYY3147. Το μικροβιακό φορτίο και ημυελουπεροξειδάση προσδιορίστηκαν σε δείγματα ιστών. Η έκφραση του τωνγόνιδίων του συτήματος quorum sensing (QS) της ψευδομονάδας προσδιορίσθηκε invivo και in vitro. Μετρήθηκαν οι συγκεντρώσεις κυτταροκινών στον ορό και διεγερμένασπληνοκύτταρα. Οι ασθενείς που επιβίωσαν 28 ημέρες μετά τη λοίμωξη είχανσημαντικά υψηλότερα επίπεδα H2S ορού σε σύγκριση με ασθενείς που δεν επιβίωσαν.Επίπεδα H2S ορού 5.3 μΜ διαχωρίζουν τους επιβιώσαντες με ευαισθησία 92.3%. Ηθνητότητα στις 28 ημέρες ήταν 30.9% και 93.7% σε ασθενείς με επίπεδα H2Sυψηλότερα ή χαμηλότερα από 5.3 μΜ αντίστοιχα (p= 7 x 10-6). Η φυσιολογική έκφρασητου Cse καθώς και η θεραπεία με δότη H2S δρα προστατευτικά ενάντια σε λοίμωξη μεπολυανθεκτική P. aeruginosa. Η προθεραπεία με κυκλοφωσφαμίδη εξάλειψε τοπλεονέκτημα επιβίωσης των Cse+/+ μυών, ενώ η μεταμόσχευση μυελού των οστώναύξησε την επιβίωση των Cse-/- μυών. Οι Cse-/- μύες εμφάνισαν αυξημένα μικροβιακάφορτία σε σύγκριση με τους Cse+/+ μύες. Η φαγοκυτταρική δράση των λευκοκυττάρωντων Cse-/- μυών ήταν μειωμένη ενώ αποκαταστάθηκε μετά από προσθήκη H2S. Μίασχετιζόμενη με το H2S μείωση της έκφρασης των γονιδίων του QS P.aeruginosaαναδείχθηκε in vivo και in vitro. Η ενδογενής παραγωγή H2S είναι ένας πιθανόςανεξάρτητος προστατευτικός παράγοντας για την έκβαση λοίμωξης από P.aeruginosa. Το H2S παρέχει προστασία ενάντια σε πολυανθεκτικά βακτηριακάπαθογόνα

Ανάπτυξη ενός μοντέλου για την μελέτη της επίδρασης της έλλειψης της ενδογενούς παραγωγής υδρόθειου στην ανθεκτικότητα πολυανθεκτικών μικροοργανισμών (Pseudomonas aeruginosa & Klebsiella pneumoniae) ενάντια στην αντιμικροβιακή αγωγή.

Το υδρόθειο (H2S) έχει πρόσφατα αναγνωριστεί ως ένας νέος αέριος διαβιβαστής μεαντιφλεγμονώδεις δράσεις. Ο ρόλος του παραγόμενου από τον ξενιστή H2S σελοιμώξεις από αεριογόνο ψευδομονάδα Pseudomonas (P.) aeruginosa διερευνήθηκεσε ένα κλινικό και ζωικό μοντέλο. Μελετήθηκαν οι συγκεντρώσεις του H2S και ηεπιβίωση σηπτικών ασθενών με λοιμώξεις του κατώτερου αναπνευστικού. Στησυνέχεια αναπτύχθηκε ένα ζωικό μοντέλο σοβαρής συστηματικής λοίμωξης απόπολυανθεκτική P. aeruginosa iχρησιμοποιώντας μύες με έλλειψη του γονιδίου της γ-λυάσης της κυσταθειονίνης (Cse) (Cse-/- μύες) και μύες με φυσιολογική έκφραση τουγονιδίου (Cse+/+ μύες). Πειράματα επαναλήφθηκαν σε μύες μετά από α) θεραπεία μεκυκλοφωσφαμίδη, β) μεταμόσχευση μυελού των οστών από δότη Cse+/+, γ) θεραπείαμε τους αναστολείς της σύνθεσης H2S αμινοοξεϊκό οξύ (aminooxyacetic acid, ΑΟΑΑ)ή προπαργυλγλυκίνη (propargylglycine, PAG) και δ) τους δότες H2S θειοθειικό νάτριο(sodium thiosulfate, STS) ή το GYY3147. Το μικροβιακό φορτίο και ημυελουπεροξειδάση προσδιορίστηκαν σε δείγματα ιστών. Η έκφραση του τωνγόνιδίων του συτήματος quorum sensing (QS) της ψευδομονάδας προσδιορίσθηκε invivo και in vitro. Μετρήθηκαν οι συγκεντρώσεις κυτταροκινών στον ορό και διεγερμένασπληνοκύτταρα. Οι ασθενείς που επιβίωσαν 28 ημέρες μετά τη λοίμωξη είχανσημαντικά υψηλότερα επίπεδα H2S ορού σε σύγκριση με ασθενείς που δεν επιβίωσαν.Επίπεδα H2S ορού 5.3 μΜ διαχωρίζουν τους επιβιώσαντες με ευαισθησία 92.3%. Ηθνητότητα στις 28 ημέρες ήταν 30.9% και 93.7% σε ασθενείς με επίπεδα H2Sυψηλότερα ή χαμηλότερα από 5.3 μΜ αντίστοιχα (p= 7 x 10-6). Η φυσιολογική έκφρασητου Cse καθώς και η θεραπεία με δότη H2S δρα προστατευτικά ενάντια σε λοίμωξη μεπολυανθεκτική P. aeruginosa. Η προθεραπεία με κυκλοφωσφαμίδη εξάλειψε τοπλεονέκτημα επιβίωσης των Cse+/+ μυών, ενώ η μεταμόσχευση μυελού των οστώναύξησε την επιβίωση των Cse-/- μυών. Οι Cse-/- μύες εμφάνισαν αυξημένα μικροβιακάφορτία σε σύγκριση με τους Cse+/+ μύες. Η φαγοκυτταρική δράση των λευκοκυττάρωντων Cse-/- μυών ήταν μειωμένη ενώ αποκαταστάθηκε μετά από προσθήκη H2S. Μίασχετιζόμενη με το H2S μείωση της έκφρασης των γονιδίων του QS P.aeruginosaαναδείχθηκε in vivo και in vitro. Η ενδογενής παραγωγή H2S είναι ένας πιθανόςανεξάρτητος προστατευτικός παράγοντας για την έκβαση λοίμωξης από P.aeruginosa. Το H2S παρέχει προστασία ενάντια σε πολυανθεκτικά βακτηριακάπαθογόνα.Hydrogen sulfide (H2S) has recently been recognized as a novel gaseous transmitterwith several anti-inflammatory properties. The role of host- derived H2S in infectionsby Pseudomonas aeruginosa was investigated in clinical and mouse models. H2Sconcentrations and survival was assessed in septic patients with lung infection. Animalexperiments using a model of severe systemic multidrug-resistant P. aeruginosainfection were performed using mice with a constitutive knock-out of cystathionine-γlyase (Cse) gene (Cse-/-) and wild-type mice with a physiological expression (Cse+/+).Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bonemarrow transplantation (BMT) from a Cse+/+ donor; c) treatment with H2S synthesisinhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H2S donorsodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activitywere measured in tissue samples. The expression of quorum sensing genes (QS) wasdetermined in vivo and in vitro. Cytokine concentration was measured in serum andincubated splenocytes. Patients survivors at day 28 had significantly higher serum H2Scompared to non-survivors. A cut- off point of 5.3 μΜ discriminated survivors withsensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H2Shigher and less than 5.3 μΜ (p= 7 x 10-6). In mice expression of Cse and applicationof STS afforded protection against infection with multidrug-resistant P. aeruginosa.Cyclophosphamide pretreatment eliminated the survival benefit of Cse+/+ mice,whereas BMT increased the survival of Cse-/- mice. Cse-/- mice had increasedpathogen loads compared to Cse+/+ mice. Phagocytic activity of leukocytes from Cse-/- mice was reduced but was restored after H2S supplementation. An H2S dependentdown- regulation of quorum sensing genes of P.aeruginosa could be demonstrated invivo and in vitro. Endogenous H2S is a potential independent parameter correlatingwith the outcome of P. aeruginosa. H2S provides resistance to infection by MDRbacterial pathogens

Experimental Intestinal Stenosis Alters Crohn's Disease-Like Intestinal Inflammation in Ileitis-Prone Mice

Background Clinical observations indicate that mechanical factors contribute to the expression or recurrence of Crohn's disease. We investigated whether the creation of an intestinal stenosis could alter the severity of the expected Crohn-like ileitis, in a Crohn's disease animal model, the TNF Delta are/+ mouse. Methods Thirty-six, 6-weeks-old TNF Delta are/+ mice, were divided into 3 intervention groups: triple suture, single suture and sham. In the terminal ileum, in the first group, a triple suture stenosis was created, whereas, in the second, a loose suture was placed. Same triple-suture stenosis was performed on twelve wild type mice. All animals were sacrificed at 6 weeks post-operatively and the ileum parts were evaluated histopathologically. A summative total ileitis score was applied in each sample using a bespoke semiquantitative histological scoring system for the Crohn-like changes. Results The triple suture stenosis induced significant muscular hypertrophy proximal to interventional site which was more prominent in TNF Delta are/+ than wild type mice. In triple suture group, the total ileitis score was significantly increased proximal to the intervention as compared to the single suture (P: 0.004) and the sham groups (P: 0.013). The total ileitis score distally, was unaffected, regardless of the experimental intervention. Intestinal stenosis did not induce intestinal inflammation in wild type mice. Conclusion The creation of a stenosis in the terminal ileum of TNF Delta are/+ mice alters Crohn-like inflammation. We assume that mechanical forces, such as intraluminal pressure, may contribute as important co-factors to the pathophysiology of Crohn's disease in genetically predisposed subjects

Efficacy of tigecycline alone or in combination for experimental infections by KPC carbapenemase-producing Klebsiella pneumoniae

Although in vitro data suggest that tigecycline is active against Klebsiella pneumoniae carbapenemaseproducing K. pneumoniae (KPC-Kp), experimental and clinical data are limited. We studied the effect of tigecycline alone or in combination for experimental infections by KPC-Kp. A total of 540 male C57BL/6 mice were infected with three genetically diverse KPC-Kp isolates susceptible to tigecycline with meropenem minimum inhibitory concentrations (MICs) of 4, 16 and 256 mu g/mL, respectively. Mice were randomly treated with water for injection, tigecycline, meropenem and colistin alone, and double or triple combinations of tigecycline, colistin and meropenem. Mouse survival was recorded for 14 days. In separate experiments, mice were sacrificed 6 h and 24 h after bacterial challenge for quantitative culture of tissues and serological analysis. Time-kill curves were performed. Tigecycline, colistin and meropenem concentrations were measured in tissues and serum by high-performance liquid chromatography (HPLC). Survival was significantly prolonged when mice were treated with tigecycline alone and tigecycline-containing regimens compared with control mice and mice treated with tigecycline-sparing regimens. Tigecycline-sparing regimens were active only against the isolate with a meropenem MIC of 4 mu g/mL. Mortality was associated with progression to multiple organ failure. Tigecycline and tigecyclinecontaining regimens achieved a rapid decrease of bacterial loads both in tissues and in vitro. Tigecycline concentrations in tissues were negatively correlated with tissue bacterial load. Tigecycline alone or in combination with meropenem and/or colistin achieves effective treatment of experimental KPC-Kp infections irrespective of the meropenem MIC. (c) 2021 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved

Extracorporeal Membrane Oxygenation (ECMO)-Associated Coagulopathy in Adults

Extracorporeal membrane oxygenation (ECMO) is used for the management of severe respiratory and cardiac failure and as a bridge to achieve definite treatment or transplantation. ECMO-associated coagulopathy (EAC) is a frequent complication leading to high rates of thrombosis or severe haemorrhage, contributing to morbidity and mortality among patients. Understanding the pathophysiology of EAC is substantial for effectively managing patients on ECMO. We analyse the underlying mechanism of EAC and discuss the monitoring of the coagulation profile, combining the viscoelastic point-of-care assays with the conventional coagulation laboratory tests

Association of Vitamin D with severity and outcome of COVID-19: Clinical and Experimental Evidence

Introduction: The role of vitamin in COVID-19 remains controversial. We investigated the association between endogenous vitamin D and the severity of COVID-19 as well as the mechanisms of action of vitamin D supplementation. Methods: 25(OH)D3 in serum was associated with disease severity and outcome in 190 COVID-19 patients. In a COVID-19 animal model using intravenous injection of plasma from patients with COVID-19 ARDS into C57/BL6 mice, mice were treated with 0.25μg human 1,25(OH)D3 or vehicle. Mice were sacrificed on day 4. Cytokines and myeloperoxidase (MPO) in tissues were measured. Changes in gene expression after vitamin D supplementation were measured. Results: Vitamin D deficiency and insufficiency were associated with increased severity and unfavourable outcome after 28 days. Vitamin D levels were negatively associated with biomarkers of COVID-19 severity. Vitamin D supplementation after challenge of mice with COVID-19 plasma led to reduced levels of TNFα, IL-6, IFNγand MPO in the lung, as well as down-regulation of pro-inflammatory pathways. Conclusion: Normal levels of endogenous Vitamin D are associated with reduced severity and risk of unfavourable outcome in COVID-19, possibly through attenuation of tissue-specific hyperinflammation

Growth on Carbohydrates from Carbonaceous Meteorites Alters the Immunogenicity of Environment-Derived Bacterial Pathogens

The last decade has witnessed a renewed interest in space exploration. Public and private institutions are investing considerable effort toward the direct exploration of the Moon and Mars, as well as more distant bodies in the solar system. Both automated and human-crewed spacecraft are being considered in these efforts. As inevitable fellow travelers on the bodies of astronauts, spaceships, or equipment, terrestrial microorganisms will undoubtedly come into contact with extraterrestrial environments, despite stringent decontamination. These microorganisms could eventually adapt and grow in their new habitats, where they might potentially recolonize and lead to the infection of the human space travelers. In this article, we demonstrate that clinically relevant bacterial species found in the environment are able to grow in minimal media with sugar compounds identified in extraterrestrial carbon sources. As a surrogate model, we used carbohydrates previously isolated from carbonaceous meteorites. The bacteria underwent an adaptation process that caused structural modifications in the cell envelope that sparked changes in pathogenic potential, both in vitro and in vivo. Understanding the adaptation of microorganisms exposed to extraterrestrial environments, with subsequent changes in their immunogenicity and virulence, requires a comprehensive analysis of such scenarios to ensure the safety of major space expeditions in the decades to come