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Abstract Group A streptococcal (GAS; aka Streptococcus pyogenes) meningitis is a fulminant disease associated with high morbidity and mortality. To elucidate the mechanisms underlying the invasiveness of GAS in meningitis, we compared GAS isolates derived from five cases of meningitis to otitis and colonizing isolates. We did not observe differences in adherence to and invasion of human brain microvascular endothelial cells, virulence factors activity, or barrier disruption. Whole genome sequencing did not reveal particular invasiveness traits. Most patients previously suffered from otitis media suggesting that meningitis likely resulted from a continuous spread of the infection rather than being attributable to changes in the pathogen's virulence.

ABSTRACT Beta-lactam antibiotics are widely used to treat infections caused by the important human pathogen Staphylococcus aureus. Resistance to beta-lactams, as found in methicillin-resistant S. aureus, renders effective treatment difficult. The second messenger cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) promotes beta-lactam resistance in clinical S. aureus isolates. C-di-AMP plays a crucial role in the regulation of cellular processes such as virulence, cell wall homeostasis, and resistance to beta-lactams in many bacterial species. In S. aureus, c-di-AMP synthesis is mediated by the diadenylate cyclase DacA, while its degradation is carried out by the phosphodiesterases GdpP and Pde2. In this work, we assessed the effect of altered c-di-AMP levels due to mutations in dacA, gdpP, or gdpP/pde2 on virulence determinants. We report that a previously described growth defect in bacteria producing high c-di-AMP levels is mainly attributable to smaller cell size. High c-di-AMP levels also led to decreased survival upon oxidative stress, reduced production of the antioxidant staphyloxanthin, increased oxacillin and fosfomycin resistance, and increased cell wall thickness. While resistance to ceftaroline was not affected, high c-di-AMP levels promoted tolerance to this antibiotic. In response to cell wall stress induced by antibiotics, the three-component regulatory system VraTSR mediates an increase in cell wall synthesis via the cell wall stress stimulon (CWSS). Increased c-di-AMP levels led to an activation of the CWSS. Upon deletion of vraR, resistance to oxacillin and fosfomycin as well as cell wall thickness diminished in the ΔgdpP mutant, indicating a contribution of the VraTSR system to the cell wall related phenotypes. IMPORTANCE Antibiotic resistance and tolerance are substantial healthcare-related problems, hampering effective treatment of bacterial infections. Mutations in the phosphodiesterase GdpP, which degrades cyclic di-3′, 5′-adenosine monophosphate (c-di-AMP), have recently been associated with resistance to beta-lactam antibiotics in clinical Staphylococcus aureus isolates. In this study, we show that high c-di-AMP levels decreased the cell size and increased the cell wall thickness in S. aureus mutant strains. As a consequence, an increase in resistance to cell wall targeting antibiotics, such as oxacillin and fosfomycin as well as in tolerance to ceftaroline, a cephalosporine used to treat methicillin-resistant S. aureus infections, was observed. These findings underline the importance of investigating the role of c-di-AMP in the development of tolerance and resistance to antibiotics in order to optimize treatment in the clinical setting.

Garcia-Senosiain et al developed an in vitro opsonic phagocytosis (OP) assay using peripheral blood leukocytes that enabled quantification of the contribution of each phagocytic cell type in the OP of Plasmodium falciparum blood-stage merozoites, which have been associated with malaria protection. Using patient samples from Ghana and India, they demonstrate that peripheral blood neutrophils are the main phagocytes of P. falciparum blood-stage merozoites

COVID-19 displays diverse disease severities and symptoms including acute systemic inflammation and hypercytokinemia, with subsequent dysregulation of immune cells. Bacterial superinfections in COVID-19 can further complicate the disease course and are associated with increased mortality. However, there is limited understanding of how SARS-CoV-2 pathogenesis and hypercytokinemia impede the innate immune function against bacterial superinfections. We assessed the influence of COVID-19 plasma hypercytokinemia on the functional responses of myeloid immune cells upon bacterial challenges from acute-phase COVID-19 patients and their corresponding recovery-phase. We show that a severe hypercytokinemia status in COVID-19 patients correlates with the development of bacterial superinfections. Neutrophils and monocytes derived from COVID-19 patients in their acute-phase showed an impaired intracellular microbicidal capacity upon bacterial challenges. The impaired microbicidal capacity was reflected by abrogated MPO and reduced NETs production in neutrophils along with reduced ROS production in both neutrophils and monocytes. Moreover, we observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes, in line with suppressed autocrine and paracrine cytokine signaling. This phenotype was characterized by a high expression of CD66b, CXCR4 and low expression of CXCR1, CXCR2 and CD15 in neutrophils and low expression of HLA-DR, CD86 and high expression of CD163 and CD11b in monocytes. Furthermore, the impaired antibacterial effector function was mediated by synergistic effect of the cytokines TNF-α, IFN-γ and IL-4. COVID-19 patients receiving dexamethasone showed a significant reduction of overall inflammatory markers in the plasma as well as exhibited an enhanced immune response towards bacterial challenge ex vivo. Finally, broad anti-inflammatory treatment was associated with a reduction in CRP, IL-6 levels as well as length of ICU stay and ventilation-days in critically ill COVID-19 patients. Our data provides insights into the transient functional dysregulation of myeloid immune cells against subsequent bacterial infections in COVID-19 patients and describe a beneficial role for the use of dexamethasone in these patients.

Abstract Objectives Critically ill coronavirus disease 2019 (COVID‐19) patients are characterised by a severely dysregulated cytokine profile and elevated neutrophil counts, impacting disease severity. However, it remains unclear how neutrophils contribute to pathophysiology during COVID‐19. Here, we assessed the impact of the dysregulated cytokine profile on the regulated cell death (RCD) programme of neutrophils. Methods Regulated cell death phenotype of neutrophils isolated from critically ill COVID‐19 patients or healthy donors and stimulated with COVID‐19 or healthy plasma ex vivo was assessed by flow cytometry, time‐lapse microscopy and cytokine multiplex analysis. Immunohistochemistry of COVID‐19 patients and control biopsies were performed to assess the in situ neutrophil RCD phenotype. Plasma cytokine levels of COVID‐19 patients and healthy donors were measured by multiplex analysis. Clinical parameters were correlated to cytokine levels of COVID‐19 patients. Results COVID‐19 plasma induced a necroptosis‐sensitive neutrophil phenotype, characterised by cell lysis, elevated release of damage‐associated molecular patterns (DAMPs), increased receptor‐interacting serine/threonine‐protein kinase (RIPK) 1 levels and mixed lineage kinase domain‐like pseudokinase (MLKL) involvement. The occurrence of neutrophil necroptosis MLKL axis was further confirmed in COVID‐19 thrombus and lung biopsies. Necroptosis was induced by the tumor necrosis factor receptor 1 (TNFRI)/TNF‐α axis. Moreover, reduction of soluble Fas ligand (sFasL) levels in COVID‐19 patients and hence decreased signalling to Fas directly increased RIPK1 levels, exacerbated TNF‐driven necroptosis and correlated with disease severity, which was abolished in patients treated with glucocorticoids. Conclusion Our results suggest a novel role for sFasL signalling in the TNF‐α‐induced RCD programme in neutrophils during COVID‐19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.