In-host evolution of Staphylococcus epidermidis in a pacemaker-associated endocarditis resulting in increased antibiotic tolerance

Treatment failure in biofilm-associated bacterial infections is an important healthcare issue. In vitro studies and mouse models suggest that bacteria enter a slow-growing/non-growing state that results in transient tolerance to antibiotics in the absence of a specific resistance mechanism. However, little clinical confirmation of antibiotic tolerant bacteria in patients exists. In this study we investigate a Staphylococcus epidermidis pacemaker-associated endocarditis, in a patient who developed a break-through bacteremia despite taking antibiotics to which the S. epidermidis isolate is fully susceptible in vitro. Characterization of the clinical S. epidermidis isolates reveals in-host evolution over the 16-week infection period, resulting in increased antibiotic tolerance of the entire population due to a prolonged lag time until growth resumption and a reduced growth rate. Furthermore, we observe adaptation towards an increased biofilm formation capacity and genetic diversification of the S. epidermidis isolates within the patient

Metabolite_measurements

Metabolite data associated to Figure S5. Data correspond to metabolite concentrations (mM) measured using high performance liquid chromatography (HPLC) and to colony forming units (CFU/ml) for Acinetobacter's bacterial growth

OD_CFU_conversion_FigS2

Data used to convert optical density (OD) values to cell density values (CFU/ml) shown in Figure S2

Batch_culture_experiments

Batch culture data associated to Figure 1, Figure 3, Figure S3 and Figure S7. Data correspond to colony-forming units per ml (CFU/ml) from batch culture experiments

Data from: The rate of environmental fluctuations shapes ecological dynamics in a two-species microbial system

Species interactions change when the external conditions change. How these changes affect microbial community properties is an open question. We address this question using a two-species consortium in which species interactions change from exploitation to competition depending on the carbon source provided. We built a mathematical model and calibrated it using single-species growth measurements. This model predicted that low frequencies of change between carbon sources lead to species loss, while intermediate and high frequencies of change maintained both species. We experimentally confirmed these predictions by growing co-cultures in fluctuating environments. These findings complement more established concepts of a diversity peak at intermediate disturbance frequencies. They also provide a mechanistic understanding for how the dynamics at the community level emerges from single-species behaviors and interspecific interactions. Our findings suggest that changes in species interactions can profoundly impact the ecological dynamics and properties of microbial systems

Growth_curves

Growth curves associated to Figure S4. Data correspond to optical density (OD) measurements from plate reader experiments. Abbreviations: "BA600" is Benzyl Alcohol 600µM; "Ben1000" is Benzoate 1000µM; "Ci1000" is Citrate 1000µM

Blunted sFasL signalling exacerbates TNF-driven neutrophil necroptosis in critically ill COVID-19 patients

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