Medical Microbiology and Immunology最新文献

Betacoronaviruses are emerging pathogens with pandemic potential, as shown by the recent COVID-19 pandemic caused by SARS-CoV-2. The replication of SARS-CoV-2 in monocytes and macrophages triggers the production of cytokines and chemokines, leading to a persistent inflammatory environment associated with increased disease severity. Dasatinib (DASA) is a broad-spectrum tyrosine kinase inhibitor that targets a wide range of tyrosine kinases, including ABL, SRC, c-KIT, PDGFR-α and ß, involved in the pathophysiology of various malignancies. Studies have reported additional mechanisms of action for DASA beyond the oncological context, including anti-inflammatory and antiviral effects. We investigated the potential of DASA as a promising therapeutic approach against betacoronavirus infections. Using an in vitro model of infection of RAW 264.7 cells with MHV-3, a betacoronavirus that mimics severe COVID-19 in murine models, we observed that both pre and post-infection treatment with DASA significantly reduced viral titers and pro-inflammatory mediators, such as IL-6, TNF, and CXCL2. Pre-treatment with DASA interfered with the early stages of the viral cycle in macrophages, such as viral adsorption and internalization, reducing viral titers. We demonstrated that SRC tyrosine kinase signaling is activated during MHV-3 infection. Post-infection treatment with DASA negatively modulated the SRC-MAPK-NF-ĸB signaling pathway, reducing the release of pro-inflammatory mediators by macrophages. Our data suggest the potential use of DASA as a promising adjuvant therapeutic strategy for treating coronavirus infections by negatively modulating SRC-mediated signaling pathways involved in inflammation and reducing MHV-3 replication. The results demonstrated that SRC signaling could be a target for interventions in controlling coronavirus infections.

Staphylococcus aureus (S. aureus) is a major opportunistic pathogen, causing acute and chronic infections including urinary tract infection (UTI). S. aureus relies on the Accessory gene regulator (Agr), a central quorum sensing (QS) system and the urease genes ureABCEFGD, for the regulation of urease expression and its pathogenicity. Urease is a key virulence factor for S. aureus, modulating immune responses by altering the local urinary pH and impairing immune defenses in the hostile urinary environment. QS is regulated by the external stimuli and in urological disease, the role of different urinary metabolite conditions and pH on the urease expression and Agr-QS regulation remains poorly understood. In this study, we explored the growth, biofilm formation, and urease activity under various simulated urinary metabolic and pH environments to study the response of S. aureus strains isolated from patients with urological diseases. The expression levels of QS genes and urease genes were compared in different urinary conditions that included the conditions from which the strains were isolated. A correlation analysis was used to study the associations between growth, pH changes, urease activity, biofilm formation, and the expression of agr and ure genes to predict their regulatory relationships. Our results demonstrated significant differences across glycosuria, haematuria, creatininuria, and albuminuria in growth, biofilm formation, and urease activity in S. aureus strains (p < 0.001). Significantly higher growth and urease activity were noted in glycosuria and haematuria-originated strains under the similar simulated conditions (p < 0.001). However, all the simulated conditions increased the expression levels of agr and ure genes; in the pre-adapted environment, favoring their survival highlighting niche adaptation. The simulated conditions with acidic pH significantly overexpressed the agr and ure genes compared to alkaline pH (p < 0.001). Increased expression profile of the QS system under the disease specific urine metabolic conditions, suggests its role in promoting bacterial fitness in the urinary environment and also forms the basis of managing UTI with targeted approach.

Bathing strategies with antiseptic agents, such as Chlorhexidine and Octenidine, have been widely adopted to mitigate infection risks in intensive care units (ICU). However, concerns exist regarding their long-term effects on skin microbiome structures and potential unintended consequences, including antibiotic cross-resistance. This longitudinal study characterized the compositional changes of the skin microbiome of ICU patients upon these two antiseptic bathing strategies when compared to standard water and soap bathing. Samples were collected in a three-armed cluster randomized decolonization trial (registration number DRKS00010475). Skin swabs from 5 different sites and three time points were analyzed by culture-based methods, 16S rRNA-gene amplicon sequencing and multiplex Taq-Man assays for detection of antimicrobial resistance genes (ARG). Our results show that Chlorhexidine bathing led to a sustained reduction of the bacterial biomass on different skin sites, as measured by both molecular and culture-based methods. Thereby, the microbial structures remained largely unaltered both in their diversity and their taxonomic composition. However, the loss of microbiome site-specificity observed on the skin of ICU patients remained unchanged independently from the bathing strategy applied and persisted even after discharge. None of the antiseptic bathing strategies led to an increase or accumulation of antibiotic-resistance determinants on any of the skin sites investigated in this study. Thus, this study suggests that daily patient bathing with 2% Chlorhexidine impregnated cloths or 0.08% Octenidine wash mitts does not impact skin microbiome structures and antibiotic resistance gene accumulation in ICU patients when compared to non-antiseptic water and soap bathing routine.

The Enterobacter cloacae complex (ECC) is a prevalent nosocomial pathogen associated with various human infections, which currently comprises several species, including Enterobacter cloacae and Enterobacter hormaechei. Strains capable of producing biofilm on various biotic and abiotic surfaces pose a particular threat. Therefore, we focused on three E. hormaechei strains in whose genomes the presence of the biofilm-related genes: fimA, csgA, csgD, and sdiA was confirmed. Kinetic of biofilm formation by these strains on urological catheters depended on the catheter material (silicon or latex), temperature (24 °C or 37 °C) and incubation time. The ability of phages to disrupt biofilm formation was assessed and found to be the most effective when phages were applied at an early stages of this process. Moreover, destruction of existing biofilm by bacteriophages and/or silver or copper nanoparticles was strain-dependent. Incubation with Enterobacter-specific bacteriophages enabled, in some cases, almost complete eradication of three-day biofilms attached to urological catheters. In genomes of two Enterobacter-specific bacteriophages the presence of regions encoding proteins with lytic activity were identified (6 regions in Entb_43 phage and 4 regions in Entb_45 phage genomes, respectively). These results highlight the threat of biofilm-related infections, but also indicate the multifaceted anti-biofilm activity of bacteriophages, which should be considered for useful in clinical practice.

Fosfomycin (FOF) exhibits broad-spectrum antimicrobial activity, and is mainly used in combination therapy. Previous in vitro studies have shown synergistic effects of FOF in combination with flucloxacillin (FLX) against Staphylococcus aureus isolates. This study aims to validate these findings in vitro and investigate the synergistic effect in an in vivo Galleria mellonella model. Five methicillin- and FOF-susceptible isolates (ATCC-29213 & 4 clinical isolates); one methicillin- and FOF-resistant strain (DSMZ-23622) and four methicillin-resistant and FOF-susceptible strains (ATCC-33592 & 3 clinical isolates) were tested with checkerboard assays to assess synergism. Time-kill curves were generated for two MSSA (ATCC 29213 and 231/20) and two MRSA strains (ATCC 33592 and DSMZ 23622). The in vivo efficacy of FOF and/or FLX was evaluated by a G. mellonella survival assay and by determining the total bacterial count (TBC) in hemolymph. Checkerboard assays revealed additive or indifferent effects, with some indicating synergism. Time-kill curves demonstrated higher reduction in TBC with combination therapy compared to monotherapy. In vivo, the combination therapy showed the greatest reduction of TBC in larval haemolymph compared to monotherapy, and the survival assay showed highly synergistic activity of FLX plus FOF against MRSA (ATCC-33592) and MSSA (ATCC 6538), resulting in an average reduction in mortality of 48 and 40%, respectively, compared to monotherapies. Therefore, FOF plus FLX could be an alternative for the calculated or definitive treatment of S. aureus infections without antimicrobial susceptibility results or even for salvage therapy of MRSA infections after treatment failure or necessary discontinuation of classical MRSA drugs.

Enterovirus A71 (EV-A71) infection is known to cause hand-foot-mouth disease, which may develop severe symptoms such as encephalitis, herpangina, and paralysis, leading to pulmonary edema and even death in children under five years old. Existing animal models for EV-A71 pathogenesis have limitations, necessitating novel models to study human-relevant disease mechanisms. Using glycoproteomic profiling to identify EV-A71-interacting proteins, we previously discovered human nucleolin (hNCL) as an attachment molecule that enhances viral binding and infection in vitro. Here, we developed human nucleolin transgenic (hNCL-Tg) mice to investigate EV-A71 pathogenesis in vivo. Compared to wild-type (WT) mice, EV-A71-infected hNCL-Tg mice exhibited higher clinical scores, progressive limb paralysis, and increased mortality. Six days post-infection, hNCL-Tg mice showed elevated viral loads in the spinal cord and skeletal muscle, with pronounced EV-A71 VP1 expression in these tissues and the brainstem. Histopathology revealed severe skeletal muscle damage and significant pulmonary edema, characterized by lung congestion, hemorrhage, and erythrocyte infiltration into alveoli. Infected hNCL-Tg mice also displayed elevated levels of encephalitis- and pulmonary edema-associated proinflammatory cytokines (IL-1β, IL-6, IL-13). These findings establish the hNCL-Tg mouse as a robust model for studying EV-A71 pathogenesis and evaluating preclinical therapeutics.

Hepatitis E virus (HEV) has attracted increasing attention in transfusion medicine in recent years. Mandatory testing regimes in Europe have resulted in not only ensuring the safety of blood products, but also providing information on the spread and immunology of HEV infections. We tracked a cohort of 497 donors identified as HEV RNA-positive during blood donation. Several follow-up samples were collected and serologically analyzed for 370 of them, up to five years after the index donation. In addition to the expected increase in immunoglobulins M (IgM) and G (IgG) titers at the beginning and the decrease over the years, we observed a proportion of 7.3% with positive anti-HEV IgM (long-term IgM-positive) and 9.1% with negative anti-HEV IgG (seroreversion) in five-year follow-ups, determined by serological tests from three different manufacturers. Both phenomena have an impact on the assessment of the correlation between incidence and seroprevalence. They are dependent on the sensitivity and specificity of serologic assays used and have a sex bias, which indicates a stronger, longer-lasting humoral immune response in women. These data offer new insights into the long-term development of immunity to HEV and thus complement short-term epidemiological data on the incidence and seroprevalence that have been obtained so far.