COVID-19 displays diverse disease severities and symptoms. Elevated inflammation mediated by hypercytokinemia induces a detrimental dysregulation of immune cells. However, there is limited understanding of how SARS-CoV-2 pathogenesis impedes innate immune signaling and function against secondary bacterial infections. We assessed the influence of COVID-19 hypercytokinemia on the functional responses of neutrophils and monocytes upon bacterial challenges from acute and corresponding recovery COVID-19 ICU patients. We show that severe hypercytokinemia in COVID-19 patients correlated with bacterial superinfections. Neutrophils and monocytes from acute COVID-19 patients showed severely impaired microbicidal capacity, reflected by abrogated ROS and MPO production as well as reduced NETs upon bacterial challenges. We observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes leading to a suppressive autocrine and paracrine signaling during bacterial challenges. Our data provide insights into the innate immune status of COVID-19 patients mediated by their hypercytokinemia and its transient effect on immune dysregulation upon subsequent bacterial infections
Background: To evaluate changes in reproductive fitness of bacteria, e.g., after acquisition of antimicrobial resistance, a low-cost high-throughput method to analyse bacterial growth on agar is desirable for broad usability, including in low-resource settings. Method: In our bacterial quantitative fitness analysis (baQFA), cultures are spotted in a predefined array on agar plates and photographed sequentially while growing. These timelapse images are analysed using a purpose-built open source software to derive normalised image intensity values for each culture spot. Subsequently, a Gompertz growth model is fitted to these optical intensity values of each culture spot and fitness is calculated from parameters of the model. For image segmentation validation, we investigated the association between normalised intensity values and colony-forming unit (CFU) counts. To represent a range of clinically important pathogenic bacteria, we used different strains of Enterococcus faecium, Escherichia coli and Staphylococcus aureus, with and without antimicrobial resistance. Relative competitive fitness (RCF) was defined as the mean fitness ratio of two strains growing competitively on one plate.Results: baQFA permitted the accurate construction of growth curves from bacteria grown on semisolid agar plates and fitting of Gompertz models: Normalised image intensity values showed a strong association with the total CFU/ml count per spotted culture (p<0.001) for all strains of the three species. Bacterial QFA showed relevant reproductive fitness differences between individual strains, suggesting substantial higher fitness of methicillin-resistant S. aureus JE2 than Cowan (RCF 1.60, p<0.001). Similarly, the vancomycin-resistant E. faecium ST172b showed higher competitive fitness than susceptible E. faecium ST172 (RCF 1.72, p<0.001). Conclusion:Our baQFA adaptation allows detection of fitness differences between our bacterial strains, and is likely to be applicable to other bacteria. In the future, baQFA may help to estimate epidemiological antimicrobial persistence or contribute to the prediction of clinical outcomes in severe infections at a low cost. 4
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